Covalently Crosslinked Polysaccharides and Methods of Use Thereof

Abstract

The regulation of immune responses can have beneficial effects on the fidelity and function of implant devices. Therefore, there is a need in the art for novel compounds, compositions, and devices that achieve this goal. 【Solution】Hydrogel capsules (e.g., alginate hydrogel capsules) containing a polysaccharide polymer capable of covalent cross-linking to another site such as another polysaccharide polymer, as well as related compositions and uses thereof, are described herein.

Description

Technical Field

[0001] Claim of Priority This application claims the priority of U.S. Application No. 63 / 357,894 filed on July 1, 2022 and U.S. Application No. 63 / 452,091 filed on March 14, 2023. The disclosure of each of the foregoing applications is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] The function of implant devices depends largely on the recipient's biological immune response pathways (Anderson et al., Semin. Immunol. 20:86 - 100 (2008), Langer, Adv. Mater. 21:3235 - 3236 (2009)). Regulation of the immune response can have a beneficial effect on the fidelity and function of these devices. Therefore, new compounds, compositions, and devices that achieve this goal are needed in the art.

Summary of the Invention

[0003] Covalently crosslinked polymers (e.g., polysaccharide polymers) to other sites such as another polymer, and related compositions, hydrogel capsules containing the same, and their uses are described herein. In one embodiment, the polymer (e.g., polysaccharide polymer) is crosslinked through one of the following methods: (i) thiolene photoclick reaction, (ii) Michael addition reaction, or (iii) inverse electron demand Diels - Alder reaction. In one embodiment, the polysaccharide polymer contains both a crosslinking site (e.g., a compound of formula (IV) or (V)) and a compound of formula (I), or a pharmaceutically acceptable salt thereof. These polysaccharide polymers can be incorporated into hydrogel capsules capable of encapsulating cells. By including a crosslinking agent in the polysaccharide polymer and then in the hydrogel capsule incorporating the polysaccharide polymer, it may be possible to adjust certain properties of the hydrogel capsule, including the diameter, stability, and integrity of the capsule.

[0004] Details of one or more embodiments of the present invention are described herein. Other features, objects, and advantages of the present invention will become apparent from the embodiments, drawings, examples, and claims for carrying out the invention.

Brief Description of Drawings

[0005]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0006] The present disclosure provides a polysaccharide polymer containing a crosslinking site and a compound of formula (I), and related compositions, hydrogel capsules containing the same, and methods for their production and use.

[0007] Abbreviations and Definitions To make the present invention more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning generally understood by those skilled in the art to which the present invention pertains.

[0008] As used herein, including in the appended claims, singular terms such as, for example, "a," "an," and "the" include their corresponding plural references unless the context clearly indicates otherwise.

[0009] As used herein, the terms "about" or "approximately" when used to modify a numerically defined parameter (e.g., a physical description of a hydrogel capsule, e.g., diameter, sphericity, number of cells encapsulated therein, number of capsules in a preparation), mean that the recited numerical value is within an acceptable functional range for a defined parameter determined by one of ordinary skill in the art that depends in part on how the measurement, or determination, was made, including the acceptable error range for the measurement system. For example, "about" can mean a range of plus or minus 20% of the recited numerical value. By way of non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells can have a diameter of 1.2 - 1.8 mm and can encapsulate 4M - 6M cells. By way of another non-limiting example, a preparation of about 100 devices (e.g., hydrogel capsules) includes preparations having 80 - 120 devices. In some embodiments, the term "about" means that the modified parameter can vary by as much as 15%, 10%, or 5% above or below the numerical value described for that parameter. Alternatively, particularly with respect to certain properties of the devices described herein, such as cell productivity, or density of CBP or non-fibrous compounds, the term "about" can mean within an order of magnitude above and below the recited value, e.g., within 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold.

[0010] "Obtaining", or "obtain", as used herein, refers to getting an item of value, such as a numerical value, an image, or a physical entity (e.g., a sample), either "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" means performing a process to obtain the value or physical entity (e.g., performing an analytical method or protocol). "Indirectly obtaining" refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes processes that involve physical changes to physical substances or the use of machines or devices. Examples of directly obtaining a value include obtaining a sample from a human subject. Directly obtaining a value includes performing a process that uses a machine or device, such as using a fluorescence microscope to obtain fluorescence microscopy data.

[0011] "Administering", "administer", or "administration", as used herein, refers to implanting, absorbing, ingesting, injecting, or otherwise introducing into a subject the entities described herein (e.g., a device or a preparation of a device), or providing such an entity to a subject for administration.

[0012] "Non-fibrous," as used herein, means a compound, or material that mitigates the foreign body reaction (FBR). For example, the amount of FBR in a tissue induced by implantation of a device (e.g., a hydrogel capsule) containing a non-fibrous compound (e.g., a hydrogel capsule containing a polymer covalently modified with a compound listed in Table 3) is lower than that induced by implantation of a non-fibrous null reference device that lacks any non-fibrous compound but has substantially the same composition (e.g., the same CBP-polymer, same cell type(s)) and structure (e.g., size, shape, number of compartments). In one embodiment, the degree of FBR is evaluated by using an assay known in the art, such as that described in WO2017 / 075630, or one or more of the assays / methods described in Vegas, A., et al., Nature Biotechnol (supra) (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining of macrophages (CD68 or F4 / 80) and confocal microscopy, myofibroblasts (alpha smooth muscle actin, SMA) or general cell deposition, quantification of RNA sequences of 79 known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophil cells on a device (e.g., a capsule) recovered 14 days later in the intraperitoneal space of an immunocompetent mouse, for example) to assess the immunological response in a tissue containing an implanted device (e.g., a hydrogel capsule), which may include protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis. In one embodiment, FBR is evaluated by measuring the levels in a tissue of one or more biomarkers of the immune response, such as cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4, including their embedding.In some embodiments, the FBR induced by the device of the present invention (e.g., a hydrogel capsule comprising a non-fibrous compound disposed on its outer surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR null reference device, e.g., a device that is substantially identical to the device being tested or claimed, except that it lacks means for reducing FBR (e.g., a hydrogel capsule that does not contain a non-fibrous compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or later.

[0013] As used herein, "cell" refers to a cell that has been manipulated or a cell that has not been manipulated. In one embodiment, the cell is an immortalized cell or a manipulated cell derived from an immortalized cell. In one embodiment, the cell is a viable cell and is viable, for example, as measured by any technique described herein or known in the art.

[0014] "Cell-binding peptide (CBP)", as used herein, means a linear or cyclic peptide comprising an amino acid sequence derived from the cell-binding domain of a ligand of a cell adhesion molecule (CAM) (e.g., mediating cell-matrix connections or cell-cell connections). CBP is less than 50, 40, 30, 25, 20, 15, or 10 amino acids in length. In one embodiment, CBP is between 3 and 12 amino acids in length, 4 to 10 amino acids in length, or 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. The CBP amino acid sequence can be identical to a naturally occurring binding domain sequence or a conservatively substituted variant thereof. In one embodiment, the CAM ligand is a mammalian protein. In one embodiment, the CAM ligand is a human protein selected from the group of proteins listed in Table 1 below. In one embodiment, CBP comprises a cell-binding sequence listed in Table 1 below, or a conservatively substituted variant thereof. In one embodiment, CBP comprises at least one of the cell-binding sequences listed in Table 1 below. In one embodiment, CBP consists essentially of a cell-binding sequence listed in Table 1 below. In one embodiment, CBP is an RGD peptide, which means that the peptide contains the amino acid sequence RGD (SEQ ID NO: 43) and optionally contains one or more additional amino acids located at one or both of the N-terminus and C-terminus. In one embodiment, CBP is a cyclic peptide containing RGD (SEQ ID NO: 43), e.g., one of the cyclic RGD peptides described in Vilaca, H. et al., Tetrahedron 70(35):5420-5427(2014). In one embodiment, CBP is a linear peptide containing RGD (SEQ ID NO: 43) and is less than 6 amino acids in length. In one embodiment, CBP is a linear peptide consisting essentially of RGD (SEQ ID NO: 43) or RGDSP (SEQ ID NO: 59). [Table 1]

[0015] As used herein, "CBP-polymer" means a polymer comprising at least one cell-binding peptide molecule covalently attached to the polymer via a linker. In one embodiment, the polymer is not a peptide or polypeptide. In one embodiment, the polymer in the CBP-polymer does not contain amino acids. In one embodiment, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as alginate, such as sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., consisting essentially of a single amino acid or a peptide of several identical or different amino acids), which is attached to the N-terminus or C-terminus of the CBP via a peptide bond. In one embodiment, the C-terminus of the amino acid linker is attached to the N-terminus of the CBP, and the N-terminus of the amino acid linker is attached to at least one pendant carboxyl group in the polysaccharide via an amide bond. In one embodiment, the linker-CBP structure is expressed as G (1-4) -CBP, meaning that the linker has one, two, three, or four glycine residues ("G (1-4) " is disclosed as SEQ ID NO: 70). In one embodiment, one or more of the monosaccharide sites in the CBP-polysaccharide, such as CBP-alginate), are not modified by the CBP. For example, the unmodified site has a free carboxyl group or lacks a pendant carboxyl group that can be modified. In one embodiment, the number of polysaccharide sites having covalently attached CBP is less than any of the following values: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%.

[0016] In one embodiment, the density of CBP modification in the CBP-polymer is estimated by combustion analysis with respect to the nitrogen percentage. In one embodiment, the CBP-polymer is an RGD-polymer (e.g., RGD-alginate), which is a linker-RGD molecule (e.g., a peptide consisting essentially of GRGD (SEQ ID NO: 62), or GRGDSP (SEQ ID NO: 60)), and the density of linker-RGD molecule modification (e.g., conjugation density), when determined using the assays described herein, is about 0.05% nitrogen (N) to 1.00% N, about 0.10% N to 0.75% N, about 0.20% N to about 0.50% N, or about 0.30% N to about 0.40% N. In one embodiment, the conjugation density of linker-RGD modification in RGD-alginate (e.g., MMW alginate covalently modified with GRGDSP (SEQ ID NO: 60)) contains 0.1 to 1.0, 0.2 to 0.8, 0.3 to 0.7, 0.3 to 0.6, 0.4 to 0.6 micromoles of linker-RGD sites per gram of RGD-polymer in a solution having a viscosity of 80 to 120 cP, as determined by any assay capable of quantifying the amount of peptide conjugated to the polymer (e.g., the quantitative peptide conjugation assay described herein). Unless specifically stated otherwise or readily apparent from the context, the specifically described numerical concentrations, concentration ranges, densities, or density ranges of CBP in the CBP-polymer refer to the concentration or density of conjugated CBP molecules, i.e., excluding residual free (e.g., unconjugated) CBP that may be present in the CBP-polymer.

[0017] "Cell-binding polypeptide (CBPP)", as used herein, means a polypeptide that is at least 50, at least 75, or at least 100 amino acids in length and that includes the amino acid sequence of the cell-binding domain of a CAM ligand, or a conservatively substituted variant thereof. In one embodiment, the CAM ligand is a mammalian protein. In one embodiment, the CBPP amino acids include the naturally occurring amino acid sequence of a full-length CAM ligand, such as one of the proteins listed in Table 1, or a conservatively substituted variant thereof. "CBP density", as used herein, unless otherwise expressly stated herein, refers to the amount or concentration of the linker-CBP moiety in a CBP-polymer, such as G 1-3 RGD (SEQ ID NO: 63) or G 1-3 refers to the amount or concentration of the linker-CBP moiety in an alginate modified with RGDSP (SEQ ID NO: 64).

[0018] "Cell-binding substance (CBS)", as used herein, means any chemical, biological, or other type of substance (e.g., small organic compound, peptide, polypeptide) that is capable of mimicking the activity of at least one of the ligands of a cell-adhesion molecule (CAM) or other cell-surface molecule that mediates cell-matrix connections or cell-cell connections or other receptor-mediated signaling. In one embodiment, when present in a polymeric composition encapsulating cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells. In one embodiment, the CBS facilitates the interaction between two or more viable cells encapsulated in the polymeric composition. In one embodiment, the presence of the CBS in a polymeric composition encapsulating a plurality of cells (e.g., viable cells) correlates with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are transplanted into a subject, such as a mouse. In one embodiment, the CBS is physically bound to one or more polymer molecules in the polymeric composition. In one embodiment, the CBS is a cell-binding peptide or cell-binding polypeptide as defined herein.

[0019] A "conservatively modified variant" or "conservative substitution", as used herein, refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except that it has one or more conservative amino acid substitutions in its amino acid sequence. In one embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. Conservative amino acid substitutions refer to amino acid substitutions with amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and have minimal impact on the biological activity of the resulting substituted peptide or polypeptide. Tables of conservative substitutions of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are shown in Table 2 below.

Table 2

[0020] "Consists essentially of", and variations thereof, such as "consist essentially of" or "consisting essentially of", when used throughout this specification and the claims, indicate the inclusion of any recited element or group of elements, and any inclusion of other elements of the same or a different nature that do not materially change the basic or novel characteristics of the specified molecule, composition, device, or method. By way of non-limiting example, a cell-binding peptide or therapeutic protein that consists essentially of a recited amino acid sequence may also include one or more amino acids of one or more amino acid residues that do not significantly affect the relevant biological activity of the cell-binding peptide or therapeutic protein, respectively, including substitutions within the recited amino acid sequence. As another non-limiting example, a cell-binding peptide that consists essentially of a recited amino acid sequence may contain one or more covalently attached moieties (e.g., a radiolabel or a fluorescent label) that do not substantially alter the relevant biological activity of the cell-binding peptide, such as the ability to increase the viability or productivity of the encapsulated cells described herein.

[0021] "Cross-linked", and variations thereof such as "cross-link" or "x-linked", when used throughout this specification, refer to a chemical bond (e.g., an ionic bond, e.g., a covalent bond) between two polymers. In some embodiments, where more than one chemical bond is present, cross-linking refers to a mixture of both covalent and ionic bonds. In some embodiments, where more than one chemical bond is present, cross-linking refers to different types of covalent bonds (e.g., covalent bonds containing different or orthogonal functional groups). In some embodiments, where more than one chemical bond is present, cross-linking refers to the same type of covalent bond (e.g., a covalent bond containing the same functional group). In some embodiments, where more than one chemical bond is present, cross-linking refers to the same type of ionic bond (e.g., an ionic bond containing the same ion, e.g., Ba 2+ including ionic bonds).

[0022] "Derived from", as used herein with respect to cells, refers to cells obtained from a tissue, cell line, or cell, and optionally then induced to produce cells that have been cultured, passaged, differentiated, induced, etc. For example, mesenchymal stem cells are derived from mesenchymal tissue and can then be differentiated into various cell types.

[0023] "Device", as used herein, refers to any implantable object (e.g., particle, hydrogel capsule, implant, medical device) described herein. In some embodiments, the device contains cells (e.g., viable cells) that can express a therapeutic agent after implantation of the device and has a configuration that supports cell viability by allowing cell nutrients to enter the device. In some embodiments, the device allows for the release of metabolic by-products and / or therapeutic agents produced by viable cells from the device.

[0024] "Differential volume", as used herein, refers to the volume of one compartment within a device described herein, excluding the space occupied by another compartment(s). For example, the differential volume of a second (e.g., outer) compartment within a two-compartment device having an inner compartment and an outer compartment refers to the volume within the second compartment excluding the space occupied by the first (inner) compartment.

[0025] As used herein, "effective amount" refers to the amount of a device, device composition, or component of a device or device compound, such as a plurality of hydrogel capsules, that is sufficient to elicit a biological response, e.g., sufficient to treat a disease, disorder, or condition, including agents produced by cells, e.g., engineered cells, or cells, e.g., engineered RPE cells. In some embodiments, the term "effective amount" refers to the amount of a component of a device, e.g., the number of cells in a device, the density of a non-fibrous compound disposed on the surface and / or barrier compartment of a device, the density of CBS in a cell-containing compartment. As will be appreciated by those of skill in the art, the effective amount can vary depending on the desired biological endpoint, the pharmacokinetics of the therapeutic agent, composition or device (e.g., capsule, particle), the condition being treated, the mode of administration, and factors such as the age and health of the subject. Effective amounts include both therapeutic and prophylactic treatments. For example, to reduce FBR, an effective amount of a compound of formula (I) can reduce fibrosis or halt the growth or spread of fibrous tissue on or near the implanted device. The effective amount of a device, composition, or component, e.g., a non-fibrous compound, can be determined by techniques known in the art or by any technique described herein.

[0026] As used herein, "endogenous nucleic acid" refers to a nucleic acid that is naturally present in a target cell.

[0027] As used herein, "endogenous polypeptide" refers to a polypeptide that is naturally present in a target cell.

[0028] "Engineered cell", as used herein, is a cell having a modification that does not occur naturally and typically contains a nucleic acid sequence (e.g., DNA or RNA) (e.g., an exogenous nucleic acid sequence) or a polypeptide that is not present (or is present at a different level) in otherwise similar cells under otherwise unengineered conditions. In one embodiment, the engineered cell contains an exogenous nucleic acid (e.g., a vector or a modified chromosomal sequence). In one embodiment, the engineered cell contains an exogenous polypeptide. In one embodiment, the engineered cell contains an exogenous nucleic acid sequence, e.g., a sequence such as DNA or RNA that is not present in otherwise similar unengineered cells. In one embodiment, the exogenous nucleic acid sequence is chromosomal, e.g., the exogenous nucleic acid sequence is an exogenous sequence located within an endogenous chromosomal sequence. In one embodiment, the exogenous nucleic acid sequence is chromosomal or extrachromosomal, e.g., a non-integrating vector. In one embodiment, the exogenous nucleic acid sequence contains an RNA sequence, e.g., mRNA. In one embodiment, the exogenous nucleic acid sequence contains a chromosomal or extrachromosomal exogenous nucleic acid sequence that contains RNA, e.g., mRNA, or a sequence that expresses as a regulatory RNA. In one embodiment, the exogenous nucleic acid sequence contains a chromosomal or extrachromosomal nucleic acid sequence that encodes a polypeptide or expresses as a polypeptide. In one embodiment, the exogenous nucleic acid sequence contains a first chromosomal or extrachromosomal exogenous nucleic acid sequence that regulates the conformation or expression of a second nucleic acid sequence, and the second amino acid sequence can be exogenous or endogenous. For example, the engineered cell can contain an exogenous nucleic acid that controls the expression of an endogenous sequence. In one embodiment, the engineered cell contains a polypeptide that is present at a level or distribution different from that found in otherwise similar unengineered cells. In one embodiment, the engineered cell contains RPE that has been engineered to produce RNA or a polypeptide. For example, the engineered cell can contain an exogenous nucleic acid sequence that contains a chromosomal or extrachromosomal exogenous nucleic acid sequence that contains RNA, e.g., mRNA, or a sequence that expresses as a regulatory RNA.In one embodiment, the engineered cell (e.g., RPE cell) contains an exogenous nucleic acid sequence, which contains a chromosomal or extrachromosomal nucleic acid sequence encoding a polypeptide or expressing as a polypeptide. In one embodiment, the polypeptide is encoded by a codon-optimized sequence to achieve a higher expression of the polypeptide than the naturally occurring coding sequence. The codon-optimized sequence can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or Java® Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl_2, pp.W526-W531 (2005)). In one embodiment, the engineered cell (e.g., RPE cell) contains an exogenous nucleic acid sequence that regulates the conformation or expression of an endogenous sequence. In one embodiment, the engineered cell (e.g., RPE cell) is cultured from a population of stably transfected cells or from a monoclonal cell line.

[0029] "Exogenous nucleic acid" as used herein is a nucleic acid that does not naturally occur in the subject cell.

[0030] "Exogenous polypeptide" as used herein is a polypeptide that does not naturally occur in the subject cell, e.g., the engineered cell. Reference to an amino acid position of a particular sequence means the position of the amino acid in a reference amino acid sequence, e.g., the sequence of the full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations at other positions in the reference amino acid sequence, e.g., deletions, insertions, and / or substitutions.

[0031] "Factor VII protein", or "FVII protein", as used herein, unless otherwise specified, means a polypeptide comprising the amino acid sequence of a naturally occurring Factor VII protein, or a variant thereof having FVII biological activity (e.g., promoting blood clotting), as determined by assays recognized in the art. Naturally occurring FVII exists as a single-chain zymogen, a zymogen-like double-stranded polypeptide, and a fully activated double-stranded form (FVIIa). In some embodiments, reference to FVII includes the single-chain and its double-stranded forms (including zymogen-like and FVIIa). FVII proteins that can be produced by the devices described herein (e.g., devices containing engineered RPE cells) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions. In some embodiments, variant FVII proteins can be activated to a fully activated double-stranded form (Factor VIIa) having at least 50%, 75%, 90% or more (>100% including) of the activity of wild-type Factor VIIa. Variants of FVII and FVIIa are known, e.g., Marzeptacog Alfa (activated) (MarzAA), and the variants described in European Patent No. 1373493, U.S. Patent No. 7771996, U.S. Patent No. 9476037, and U.S. Published Application No. US20080058255. The biological activity of Factor VII can be quantified by assays recognized in the art, unless otherwise specified. For example, the FVII biological activity in a sample of biological fluid, such as plasma, can be determined by (i) measuring the amount of Factor Xa and Factor X generated in a system containing tissue factor (TF) embedded in a lipid membrane (Persson et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) measuring Factor X hydrolysis in an aqueous system; (iii) measuring its physical binding to TF using an apparatus based on surface plasmon resonance (Persson, FEBS Letts. 413:359-363, 1997); or (iv) measuring the hydrolysis of a synthetic substrate; and / or (v) measuring thrombin generation in a TF-independent in vitro system. In one embodiment, FVII activity is evaluated by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuville sur Oise, France) in which a biological sample containing FVII is mixed with thromboplastin calcium, Factor X, and SXa-11 (a chromogenic substrate specific for Factor Xa).

[0032] "Factor VIII protein", or "FVIII protein", as used herein, unless otherwise specified, means a polypeptide comprising the amino acid sequence of a naturally occurring Factor VIII polypeptide, or a variant thereof, that has FVIII biological activity, e.g., coagulant activity, as determined by assays recognized in the art. FVIII proteins that can be produced by the devices described herein (e.g., devices containing engineered RPE cells) include wild-type primate (e.g., human), porcine, canine, and murine proteins, and variants of such wild-type proteins, including fragments, mutants, variants having one or more amino acid substitutions and / or deletions, B-domain deleted (BDD) variants, single-chain variants, and fusions of any of the foregoing wild-type or variant proteins with a half-life extending polypeptide. In one embodiment, the cell comprises an exogenous sequence encoding a precursor Factor VIII polypeptide (e.g., having a signal sequence) having a complete or partial deletion of the B domain. In one embodiment, the cell is engineered to encode a single-chain Factor VIII polypeptide comprising a variant FVIII protein, the variant FVIII protein preferably having at least 50%, 75%, 90%, or more (>100% including) of the coagulant activity of the corresponding wild-type Factor VIII. Assays for measuring the coagulant activity of FVIII proteins include one-step or two-step coagulation assays (Rizza et al., 1982, Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or chromogenic substrate FVIII:C assays (Rosen, S. 1984. Scand J Haematol 33:139-145, suppl.).

[0033] A number of FVIII-BDD variants are known, including, for example, those disclosed in U.S. Patent No. 4,868,112 (e.g., column 2, line 2 to column 19, line 21 and Table 2); No. 5,112,950 (e.g., column 2, lines 55-68, FIG. 2, and Example 1); No. 5,171,844 (e.g., column 4, line 22 to column 5, line 36); No. 5,543,502 (e.g., column 2, lines 17-46); No. 5,595,886; No. 5,610,278; No. 5,789,203 (e.g., column 2, lines 26-51 and Examples 5-8); No. 5,972,885 (e.g., column 1, line 25 to column 2, line 40); No. 6,048,720 (e.g., column 6, lines 1-22 and Example 1); No. 6,060,447; No. 6,228,620; No. 6,316,226 (e.g., column 4, line 4 to column 5, line 28 and Examples 1-5); No. 6,346,513; No. 6,458,563 (e.g., column 4, lines 25-53) and No. 7,041,635 (e.g., column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39), including variants having complete or partial B-domain deletions. In some embodiments, the FVIII-BDD protein produced by the devices described herein (e.g., expressed by engineered cells contained in the devices) has one or more of the following deletions of amino acids in the B domain: (i) most of the B domain excluding the amino-terminal B domain sequence essential for intracellular processing of the major translation product into two polypeptide chains (WO91 / 09122); (ii) amino acids 747-1638 (Hoeben R.C., et al. J. Biol. Chem. 265(13):7318-7323(1990)); amino acids 771-1666 or amino acids 868-1562 (Meulien P., et al. Protein Eng. 2(4):301-6(1988)); amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942(1986)); amino acids 797-1562 (Eaton et al., Biochemistry 25:8343-8347(1986)); 741-1646 (Kaufman, WO87 / 04187)), 747-1560 (Sarver et al., DNA 6:553-564(1987)); amino acids 741-1648 (Pasek, WO88 / 00831)), amino acids 816-1598 or 741-1689 (deletions of Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597); any of the specific deletions described in column 10, line 65 to column 11, line 36 of U.S. Patent No. 9,956,269, including deletions that include one or more residues in the furin protease recognition sequence, e.g., LKRHQR at amino acids 1643-1648 (e.g., SEQ ID NO:65).

[0034] In other embodiments, the FVIII-BDD protein retains any of the following B-domain amino acids or amino acid sequences: (i) one or more N-linked glycosylation sites in the B-domain, such as residues 757, 784, 828, 900, 963, or optionally 943, the first 226 amino acids, or the first 163 amino acids (Miao, H.Z., et al., Blood 103(a):3412-3419 (2004), Kasuda, A., et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, S.W., et al., J. Thromb. Haemost. 9:2235-2242 (2011)).

[0035] In some embodiments, the FVIII-BDD protein is a single-chain variant generated by substitution of one or more amino acids in the furin protease recognition sequence (LKRHQR at amino acids 1643-1648 (SEQ ID NO: 65)) that prevents proteolytic cleavage at this site (including any of the substitutions at positions R1645 and / or R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353, and 9,670,267).

[0036] In some embodiments, any of the above FVIII-BDD proteins may further include one or more of the following variations: the F309S substitution to improve expression of the FVIII-BDD protein (Miao, H.Z., et al., Blood 103(a):3412-3419 (2004)); albumin fusion (WO2011 / 020866); and Fc fusion (WO04 / 101740).

[0037] All FVIII-BDD amino acid positions referred to herein refer to positions in full-length human FVIII unless otherwise specified.

[0038] The "Factor IX protein" or "FIX protein", as used herein, unless otherwise specified, means a polypeptide comprising the amino acid sequence of a naturally occurring Factor IX protein or its variant having FIX biological activity, such as coagulation activity, as determined by assays recognized in the art. FIX is produced as an inactive zymogen that is converted to the active form by cleavage of the activation peptide by Factor XIa and is held together by one or more disulfide bonds to produce a heavy chain and a light chain. The FIX proteins that can be produced by the devices described herein (e.g., devices containing engineered RPE cells) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, variants having one or more amino acid substitutions and / or deletions, and fusions of any of the foregoing wild-type or variant proteins with a half-life extending polypeptide. In one embodiment, the cells are engineered to encode a full-length wild-type human Factor IX polypeptide (e.g., having a signal sequence) or a functional variant thereof. Variant FIX proteins preferably have at least 50%, 75%, 90% or more (>100% including) of the coagulation activity of wild-type Factor VIX. Assays for measuring the coagulation activity of FIX proteins include the Biophen Factor IX assay (Hyphen BioMed) and one-stage clotting assays (activated partial thromboplastin time (aPTT) (e.g., as described in EP2 032 607), thrombin generation time assay (TGA) and rotational thromboelastometry (e.g., as described in WO2012 / 006624).

[0039] Several functional FIX variants are known and can be expressed by engineered cells encapsulated in the devices described herein, including any of the functional FIX variants described in the following international patent publications: WO02 / 040544, page 4, lines 9-30 and page 15, lines 6-31; WO03 / 020764, Tables 2 and 3, pages 14-24, and page 12, lines 1-27; WO2007 / 149406, page 4, line 1 - page 19, line 11; WO2007 / 149406A2, page 19, line 12 - page 20, line 9; WO08 / 118507, page 5, line 14 - page 6, line 5; WO09 / 051717, page 9, line 11 - page 20, line 2; WO09 / 137254, page 2, paragraph

[0006] - page 5, paragraph

[0011] and page 16, paragraph

[0044] - page 24, paragraph

[0057] ; WO09 / 130198A2, page 4, line 26 - page 12, line 6; WO09 / 140015, page 11, paragraph

[0043] - page 13, paragraph

[0053] ; WO2012 / 006624; WO2015 / 086406.

[0040] In certain embodiments, the FIX polypeptide comprises a wild-type or variant sequence fused to a heterologous polypeptide or non-polypeptide moiety that extends the half-life of the FIX protein. Exemplary half-life extension moieties include Fc, albumin, PAS sequences, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) having its four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, or any combination thereof. An exemplary FIX polypeptide is the rFIXFc protein described in WO2012 / 006624, which is a FIXFc single-chain (FIXFc-sc) and an Fc single-chain (Fc-sc) joined together via two disulfide bonds in the hinge region of Fc.

[0041] FIX variants also include the gain and loss of functional variants. An example of the gain of a functional variant is the "Padua" variant of human FIX, which has L (leucine) instead of R (arginine) at position 338 of the mature protein (corresponding to amino acid position 384 of SEQ ID NO: 2) and has higher catalytic and coagulant activities compared to wild-type human FIX (Chang et al., J. Biol. Chem., 273:12089-94 (1998)). An example of a loss-of-function variant is alanine substituted for lysine at the fifth amino acid position from the start of the mature protein, which results in a protein with reduced binding to collagen IV (e.g., loss of function). The term "interleukin-2 protein" or "IL-2 protein" as used herein, unless otherwise specified, means a polypeptide comprising the amino acid sequence of a naturally occurring IL-2 protein or a variant thereof having IL-2 biological activity, e.g., activating IL-2 receptor signaling in Treg cells as determined by assays recognized in the art. IL-2 proteins that can be produced by the devices described herein, e.g., devices containing engineered RPE cells, include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Variant IL-2 proteins preferably have at least 50%, 75%, 90%, or more (>100% including) of the biological activity of the corresponding wild-type IL-2. Assays for the biological activity of IL-2 proteins are described in U.S. Patent No. 10,035,836 and include, for example, measuring the level of phosphorylated STAT5 protein in Treg cells compared to CD4+CD25- / low T cells or NK cells. Variant IL-2 proteins that can be produced by the devices of the present disclosure (e.g., devices containing engineered RPE cells) include proteins having one or more of the following amino acid substitutions: N88R, N88I, N88G, D20H, Q126L, Q126F, and C125S or C125A.

[0042] "Pancreatic islet cells", as used herein, include any cells that are naturally occurring, synthetically produced, or modified, and that partially or wholly repeat, mimic, or otherwise express the function of the cells of the pancreatic islets of Langerhans. The term "pancreatic islet cells" includes stem cells, such as glucose-responsive insulin-producing cells derived from induced pluripotent stem cell lines.

[0043] "Mannitol", as used herein, refers to D-mannitol unless otherwise expressly stated.

[0044] "Medium molecular weight alginate" or "MMW-Alg", as used herein, means an alginate having an approximate molecular weight of 75 kDa to 150 kDa.

[0045] The terms "mesenchymal stem functional cells", or "MSFC", as used herein, refer to cells that are derived from mesodermal cells or have at least one characteristic specific to those cells, and MSFC are i) not in a final state of differentiation and ii) can differentiate into one or more cell types. MSFC do not include cells derived from endothelial cells, such as intestinal cells, or cells derived from ectodermal cells, such as skin, CNS, or neurons. In one embodiment, MSFC are pluripotent. In one embodiment, MSFC are not totipotent. In one embodiment, MSFC include one or more of the following characteristics. a) It includes cells directly isolated from mesenchymal stem cells (MSC) or cells derived therefrom (including cells derived from primary cell cultures of MSC), naturally occurring MSC, such as cells directly isolated from MSC from humans or other mammals (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division from isolation), cells directly isolated from transformed, pluripotent, immortalized, or long-term (e.g., more than 5 or 10 passages or rounds of cell division) MSC cultures (e.g., less than 5 or 10 passages or rounds of cell division since isolation). b) It is a cell obtained from a low-differentiated cell, such as a cell that has been grown, programmed, or reprogrammed (e.g., in vitro) into an MSC, or a naturally occurring MSC excluding any genetic manipulation, or a cell from a primary or long-term culture of an MSC, or one or more of the cells described in a) above. Examples of low-differentiated cells from which the MSFC can be derived include iPS cells, embryonic stem cells, or other totipotent or pluripotent cells. See, for example, Chen, Y.S. et al (2012) Stem Cells Transl Med 1(83-95), Frobel, J et al (2014) Stem Cell Reports 3(3):414-422, Zou, L et al (2013) Sci Rep 3:2243. c) It is pluripotent, as measured, for example, by microscopy, in any assay capable of providing information regarding cell pluripotency. d) It exhibits a characteristic mononuclear ovoid, star-shaped, or spindle-shaped form with a circular to oval nucleus. The elongated oval nucleus may have prominent nucleoli, as well as a mixture of heterochromatin and euchromatin. The MSFC (e.g., MSC) may have a small cytoplasm but may have many thin processes that appear to extend from the nucleus. e) It is capable of cell division, as measured, for example, by microscopy, in any assay capable of providing information regarding cell division. In one embodiment, the MSFC is capable of cell division in culture (e.g., before being encapsulated or incorporated into a device). In one embodiment, it is capable of cell division after being encapsulated, for example, as described herein, or after being incorporated into a device (e.g., a device described herein). In one embodiment, it is not capable of cell division after reaching confluence. f) It is capable of differentiating into mesenchymal cell lineages, such as osteoblasts, chondroblasts, adipocytes, or fibroblasts. g) It expresses one, two, three, four, five, or all of the mesenchymal cell markers, such as CD105, CD106, CD73, CD90, Stro-1, CD49a, CD29, CD44, CD146, CD166, TNAP+, THY-1+, Stro-2, Stro-4, and alkaline phosphatase. h) It does not express one, two, three, or any significant level of CD34, CD31, VE-cadherin, CD45, HLA-DR, CD11b, and glycophorin or leukocyte differentiation antigens, such as CD14, CD33, CD3, and CD19. i) It expresses one, two, or all of CD75, CD90, and CD105 and does not express one, two, or either of CD45, CD34, and CD14. j) It is anti-inflammatory or immunosuppressive, for example, as measured by any method capable of providing information regarding inflammation, such as in vivo inhibition of T cell proliferation. k) It can be adherent, for example, plastic adherent, as determined, for example, by visual inspection, or l) It can grow three-dimensionally, as determined, for example, by visual inspection.

[0046] "Parathyroid hormone", or "PTH", as used herein, means a polypeptide, or peptide, that has PTH biological activity, and that comprises the amino acid sequence of a naturally occurring parathyroid hormone polypeptide, or peptide, or a variant thereof, as determined, for example, by an assay recognized in the art. PTH polypeptides and peptides that can be expressed by the encapsulated cells described herein include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Such PTH polypeptides and peptides include, but are not limited to, preproPTH polypeptide (115 amino acids), proPTH polypeptide (90 amino acids), mature 84 amino acid peptide (PTH(1-84)), and biologically active variants thereof, such as truncated variant peptide PTH(1-34). PTH peptide variants having one or more amino acid substitutions in the human wild-type sequence are described, for example, in U.S. Patent Nos. 7,410,948 and 8,563,513, and U.S. Patent Application Publication No. 2013 / 0217630. PTH variants preferably have at least 50%, 75%, 90%, or more (>100% including) of the biological activity of the corresponding wild-type PTH. Assays for detecting a given PTH variant by tandem mass spectrometry are described in U.S. Patent No. 8,383,417. The biological activity assay of PTH peptide variants - the stimulation of adenylate cyclase determined by measuring cAMP levels - is described in U.S. Patent No. 7,410,948.

[0047] "Poloxamer", as used herein, refers to the standard general term for a class of nonionic triblock linear copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two polyoxyethylene (poly(ethylene oxide)) moieties. "Poloxamer 188" or "P188", as used herein, refers to a poloxamer having an approximate molecular weight of 1800 g / mol for the polyoxypropylene core and an oxyethylene content of about 80 wt%, for example, 79.0 to 83.7 percent. In one embodiment, poloxamer 188 has an average molecular weight of 8350 g / mol. In one embodiment, poloxamer 188 has an average molecular weight of 7680 g / mol to 9510 g / mol and an oxyethylene content of 81.8 ± 1.9 wt%, as determined by, for example, size exclusion chromatography. In one embodiment, each polyoxyethylene chain in poloxamer 188 has 75 to 85 (e.g., 80) ethylene oxide monomers, and the polyoxypropylene core has 25 to 30 (e.g., 27) propylene oxide monomers. In one embodiment, the poloxamer 188 used in the processes described herein substantially meets the specifications described in the monograph of the poloxamer published by the United States Pharmacopeia - National Formulary (USP-NF) or the European Pharmacopoeia (Ph.Eur.) that is official at the time the process is carried out.

[0048] "Polymer composition", as used herein, is a composition (e.g., solution, mixture) comprising one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, block copolymers, and can be both natural and synthetic. A homopolymer contains one type of building block, or monomer, while a copolymer contains two or more types of monomers.

[0049] "Polypeptide", as used herein, is a polymer that contains amino acid residues linked via peptide bonds and has at least 2, and in some embodiments, at least 10, 50, 75, 100, 150, or 200 amino acid residues.

[0050] "Prevention", "prevent", and "preventing", as used herein, refer to administering or applying a therapy prior to the onset of a disease, disorder, or medical condition in order to prevent the physical signs of the disease, disorder, or medical condition, e.g., administering a composition of a device encapsulating cells (e.g., as described herein). In some embodiments, "prevention", "prevent", and "preventing" require that the signs or symptoms of the disease, disorder, or medical condition have not occurred or have not been observed. In some embodiments, the treatment includes prevention, and in other embodiments, it does not include prevention.

[0051] "Replacement therapy", or "replacement protein", is a therapeutic protein, or a functional fragment thereof, that replenishes or enhances the beneficial function of a protein that is decreased, present in insufficient amounts, altered (e.g., mutated), or lacking in a subject having a disease or medical condition associated with the decreased, altered, or absent protein. Examples include a given blood clotting factor in a given blood clotting disorder or a given lysosomal enzyme in a given lysosomal storage disorder. In one embodiment, the replacement therapy, or replacement protein, provides the function of an endogenous protein. In one embodiment, the replacement therapy, or replacement protein, has the same amino acid sequence as a naturally occurring variant of the protein being replaced, e.g., a wild-type allele or an allele not associated with the disorder. In one embodiment, the replacement therapy, or replacement protein, has an amino acid sequence that differs from a naturally occurring variant, e.g., wild-type, allele or allele not associated with the disorder, e.g., the allele the subject carries, by about 1, 2, 3, 4, 5, 10, 15, or 20% or less of the amino acid residues.

[0052] As used herein with respect to the claimed device (e.g., a hydrogel capsule), a "reference device" means a device (e.g., a hydrogel capsule) that (i) lacks a particular feature, e.g., an FBR alleviating means (e.g., a barrier compartment (e.g., an RGD polymer) containing a non-fibrous compound (as defined herein) or a CBS (as defined herein)), and (ii) encapsulates approximately the same amount of cells of the same cell type(s) in a cell-containing compartment as the claimed device, and (iii) has a polymer composition and structure that is substantially similar to the claimed device, except that it lacks a particular feature (e.g., a non-fibrous compound or a CBS). In one embodiment, the number of viable cells in the cell-containing compartment of the reference device is within 80% - 120%, or within 90% - 110%, of the number of viable cells in the cell-containing compartment of the claimed device. In one embodiment, the cells in the reference device and the claimed device are obtained from the same cell culture. In one embodiment, substantially similar polymer compositions means that all polymers in the reference and claimed devices, which include any CBP-polymer and non-fibrous polymer components, are of the same chemical and molecular weight class (e.g., an alginate having a high G content and the same molecular weight range), where applicable. For example, in one embodiment, the cell-containing compartment of a CBP null reference device is formed from an unmodified version of the polymer (e.g., an alginate) in the CBP-polymer used to form the cell-containing compartment of the claimed device. In some embodiments where the claimed two-compartment hydrogel microcapsule has (i) an inner compartment formed from a CBP-polymer encapsulating a plurality of cells and (ii) an outer compartment formed from a mixture of a chemically modified polymer (e.g., CM-LMW-alginate as described herein) and an unmodified polymer (e.g., U-HMW-alginate as described herein), the outer compartments of the reference capsule and the claimed capsule are formed from the same polymer mixture, while the inner compartment of the reference capsule is formed from a suspension of cells in the same polymer mixture used for the outer compartment.In one embodiment, substantially similar structures means that the reference device and the claimed device have the same number of compartments (e.g., one, two, three, etc.) and have approximately the same size and shape. "RPE cells", as used herein, refers to cells having one or more of the following characteristics: a) retinal pigment epithelial cells (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC® CRL-2302™)) or cells derived therefrom, such as cells cultured from the ARPE-19 cell line and stably transfected with an exogenous sequence encoding a therapeutic protein or otherwise manipulated to express an exogenous protein or other exogenous substance, cells derived from a primary cell culture of RPE cells, naturally occurring RPE cells, e.g., cells isolated directly from a human or other mammal (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division from isolation), cells derived from transformed, immortalized, or long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell cultures; b) cells obtained from cells that have developed, been programmed, or reprogrammed (e.g., in vitro) to be substantially similar to one or more of undifferentiated cells, e.g., RPE cells or cells from a primary or long-term culture of naturally occurring RPE cells or RPE cells (e.g., the cells may be derived from iPS cells), excluding any genetic manipulation; or c) cells having one or more of the following properties: i) expressing one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) not expressing one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) naturally found in the retina and forming a monolayer over the choroidal blood vessels in Bruch's membrane; iv) responsible for epithelial transport, light absorption, secretion, and immune regulation in the retina; or v) synthetically generated or modified from naturally occurring cells to have the same or substantially the same gene content, and optionally the same or substantially the same epigenetic content, as an immortalized RPE cell line (e.g., the ARPE-19 cell line (ATCC® CRL-2302™)).In one embodiment, the RPE cells described herein are engineered, for example, to have new properties, e.g., the cells are engineered to express a therapeutic agent when encapsulated in a polymer composition comprising CBP or CBS. In other embodiments, the RPE cells are not engineered.

[0053] As used herein, "physiological saline solution" means normal saline, i.e., water containing 0.9% NaCl, unless otherwise specified.

[0054] "Sequence identity," or "identity rate," as used herein to refer to two nucleotide sequences, or two amino acid sequences, means that the two sequences are identical within a specified region, or that the two sequences have identical nucleotides, or amino acids, at a specified percentage of nucleotide, or amino acid positions, within the specified region when the two sequences are compared and aligned for maximum correspondence over a comparison window, or specified region. Sequence identity can be determined using standard techniques known in the art, including, but not limited to, any of the algorithms described in U.S. Patent Application Publication No. 2017 / 02334455. In one embodiment, the specified percentage of identical nucleotides, or amino acid positions, is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0055] As used herein, "spherical" means, for example, a device (e.g., a hydrogel capsule, or other particle) having a curved surface that forms a sphere (e.g., a perfectly round ball), or a spheroid shape, which may have waves and undulations on its surface. Spheres and spheroid-like objects can be mathematically defined by the rotation of a circle, ellipse, or combination around each of the three orthogonal axes a, b, and c. In a sphere, the three axes are of the same length. Usually, a spheroid-like shape is an ellipsoid having semi-major axes within 10%, or 5%, or 2.5% of each other (for its averaged surface). The diameter of a sphere or spheroid-like shape is the average diameter, e.g., the average of the semi-major axes.

[0056] As used herein when the term "ellipsoid" refers to a device (e.g., a hydrogel capsule, or other particle), it means that the device (i) has a complete or classical oblate or prolate ellipsoid shape, or (ii) has a surface that generally forms an ellipsoid, e.g., may have waves and undulations, and / or may be an ellipse (for its averaged surface) having semi-major axes within 100% of each other.

[0057] As used herein, "subject" refers to a human or non-human animal. In one embodiment, the subject is, for example, a human of any age group (i.e., male or female), a pediatric subject (e.g., infancy, childhood, adolescence) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult). In one embodiment, the subject is a non-human animal, e.g., a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey, or a rhesus monkey)). In one embodiment, the subject is a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, e.g., a chicken, a duck, a goose, or a turkey). In a given embodiment, the animal is a mammal. The animal can be male or female and can be of any developmental stage. The non-human animal can be a transgenic animal.

[0058] As used herein, "gross volume" refers to the volume within one compartment of a multi-compartment device, including the space occupied by another compartment. For example, the gross volume of the second (e.g., outer) compartment of a two-compartment device refers to the volume within the second compartment, including the space occupied by the first compartment. "Treat", "treating", and "treatment" as used herein refer to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of the symptoms of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of the signs of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of the underlying cause of a disease, disorder, or condition. In some embodiments, "treat", "treating", and "treatment" require that the symptoms or signs of a disease, disorder, or condition be present or observed. In other embodiments, treatment can be administered, for example, in a prophylactic treatment, in the absence of signs or symptoms of a disease or condition. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., taking into account a history of symptoms and / or from the perspective of genetic or other susceptibility factors). Treatment can also be continued after the symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment includes prevention, and in other embodiments, prevention is not included. "Von Willebrand factor protein" or "VWF protein", as used herein, unless otherwise specified, means a polypeptide containing the amino acid sequence of a naturally occurring VWF polypeptide, or a variant thereof, that has VWF biological activity, such as FVIII binding activity, as determined by assays recognized in the art. VWF proteins that can be produced by the devices described herein (e.g., expressed by engineered cells contained in the device) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. The encapsulated cells can be engineered to encode any of the following VWF polypeptides: a 2813 amino acid precursor VWF, a VWF lacking a 22 amino acid signal peptide and optionally a 741 amino acid prepropeptide, a 2050 amino acid mature VWF protein, and cleavage variants thereof such as VWF fragments sufficient to stabilize the endogenous FVIII levels in VWF-deficient mice, e.g., a cleavage variant containing the D´D3 region (amino acids 764-1247) or the D1D2D´D3 region; and VWF variants having one or more amino acid substitutions within the D´ region, such as those described in U.S. Patent No. 9,458,223. Variant VWF proteins preferably have at least 50%, 75%, 90%, or more (>100% including) of the biological activity of the corresponding wild-type VWF protein. Assays recognized in the art for determining the biological activity of VWF include ristocetin cofactor activity (Federici A B et al. 2004. Haematologica 89:77-85), binding of VWF to GP Ibα of the platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), and collagen binding (Kallas & Talpsepp. 2001. Annals of Hematology 80:466-471). In some embodiments, the VWF protein produced by the devices of the present disclosure comprises a naturally occurring or variant VWF amino acid sequence fused to a heterologous polypeptide or non-polypeptide moiety that extends the half-life of the VWF protein. Exemplary half-life extending moieties include Fc, albumin, PAS sequences, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) having its four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, or any combination thereof.

[0059] Selected chemical definitions Specific functional groups, and definitions of chemical terms, are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (inside front cover), and specific functional groups are generally defined as described therein. Further, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999, Smith and March, March’s Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0060] The abbreviations used in this specification have their conventional meanings in the chemical and biological arts. The chemical structures and formulas shown in this specification are constructed in accordance with the standard rules of chemical valency known in the chemical art. When a range of values is recited, it is intended to include each value within the range and sub-ranges subsumed therein. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0061] As used herein, "alkyl" refers to a group of straight or branched chain saturated hydrocarbon groups having from 1 to 24 carbon atoms ("C1-C 24 alkyl"). In some embodiments, the alkyl group has from 1 to 12 carbon atoms ("C1-C 12 alkyl"), from 1 to 10 carbon atoms ("C1-C 12"(alkyl)", having from 1 to 8 carbon atoms ("C1-C8 alkyl"), from 1 to 6 carbon atoms ("C1-C6 alkyl"), from 1 to 5 carbon atoms ("C1-C5 alkyl"), from 1 to 4 carbon atoms ("C1-C4 alkyl"), from 1 to 3 carbon atoms ("C1-C3 alkyl"), from 1 to 2 carbon atoms ("C1-C2 alkyl"), or 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has from 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanil (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Each example of an alkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted by one or more substituents, e.g., for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0062] As used herein, "alkenyl" refers to a group of a straight-chain or branched-chain hydrocarbon group having from 2 to 24 carbon atoms and one or more carbon-carbon double bonds and no triple bonds ("C2-C 24 alkenyl"). In some embodiments, the alkenyl group has from 2 to 10 carbon atoms ("C2-C 10"(an alkenyl group), having 2 to 8 carbon atoms ("C2-C8 alkenyl"), 2 to 6 carbon atoms ("C2-C6 alkenyl"), 2 to 5 carbon atoms ("C2-C5 alkenyl"), 2 to 4 carbon atoms ("C2-C4 alkenyl"), 2 to 3 carbon atoms ("C2-C3 alkenyl"), or 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds can be internal (e.g., those in 2-butenyl) or terminal (e.g., those in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. Examples of C2-C6 alkenyl groups include the aforementioned C 2-4 alkenyl groups and pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each example of an alkenyl group is independently optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkenyl").

[0063] As used herein, the term "alkynyl" refers to a group of a straight-chain or branched-chain hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 alkenyl"). In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("C2-C 10"(alkynyl"), having 2 to 8 carbon atoms ("C2-C8 alkynyl"), 2 to 6 carbon atoms ("C2-C6 alkynyl"), 2 to 5 carbon atoms ("C2-C5 alkynyl"), 2 to 4 carbon atoms ("C2-C4 alkynyl"), 2 to 3 carbon atoms ("C2-C3 alkynyl"), or 2 carbon atoms ("C2 alkynyl"). One or more carbon-carbon triple bonds can be internal (e.g., as in 2-butynyl) or terminal (e.g., as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. Each example of an alkynyl group is independently optionally substituted, i.e., unsubstituted ("unsubstituted alkynyl") or substituted by one or more substituents, e.g., for example 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkynyl").

[0064] As used herein, the term "heteroalkyl" refers to an acyclic stable straight chain, or branched chain, or a combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The O, N, P, S, and Si heteroatom(s) can be located at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms can be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3, etc. When "heteroalkyl" is recited followed by a specific heteroalkyl group, for example, -CH2O, -NR C R D etc. are recited, it is understood that the terms heteroalkyl and -CH2O or -NR C R D are not redundant and not mutually exclusive. Rather, the specific heteroalkyl group is recited for clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups such as, for example, -CH2O, -NR C R D etc. Each example of a heteroalkyl group is independently optionally substituted, i.e., unsubstituted ( "unsubstituted heteroalkyl") or substituted by one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or one substituent ( "substituted heteroalkyl").

[0065] The terms "alkylene", "alkenylene", "alkynylene", or "heteroalkylene", alone or as part of another substituent, each mean a divalent group derived from alkyl, alkenyl, alkynyl, or heteroalkyl, respectively, unless otherwise stated. Alkylene, alkenylene, alkynylene, or heteroalkylene groups can be described, for example, as C1-C6 member alkylene, C2-C6 member alkenylene, C2-C6 member alkynylene, or C1-C6 member heteroalkylene, and the term "member" refers to non-hydrogen atoms within the moiety. In the case of heteroalkylene groups, the heteroatom(s) can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not indicated by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- can represent both -C(O)2R'- and -R'C(O)2-.

[0066] As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system having 6 to 14 ring carbon atoms and 0 heteroatoms provided by an aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) (a "C6-C 14 aryl"). In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C 10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 aryl"; e.g., anthracyl). An aryl group can be, for example, C6-C 10It can be described as a member aryl, and the term "member" refers to a non-hydrogen ring atom within a moiety. Examples of aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each example of an aryl group can be independently optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted by one or more substituents ("substituted aryl").

[0067] As used herein, "heteroaryl" is a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having ring carbon atoms and 1 to 4 ring heteroatoms provided by an aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In a heteroaryl group containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom when the valence permits. A heteroaryl bicyclic ring system can contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes a ring system in which the heteroaryl ring defined above is fused to one or more aryl groups and the point of attachment is on either the aryl or heteroaryl ring; in such a case, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. For a bicyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment is on either ring, i.e., on the ring bearing the heteroatom (e.g., 2-indolyl) or on the ring not containing a heteroatom (e.g., 5-indolyl). A heteroaryl group can be described, for example, as 6- to 10-membered heteroaryl, and the term "member" refers to a non-hydrogen ring atom within a moiety.

[0068] In some embodiments, the heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms provided by the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 10-membered heteroaryl"), a 5- to 10-membered aromatic ring system. In some embodiments, the heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms provided by the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heteroaryl"), a 5- to 8-membered aromatic ring system. In some embodiments, the heteroaryl group is a 5- to 6-membered aromatic ring system having ring carbon atoms provided by the aromatic ring system and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heteroaryl"), a 5- to 6-membered aromatic ring system. In some embodiments, the 5- to 6-membered heteroaryl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each example of the heteroaryl group may independently be optionally substituted, i.e., unsubstituted (a "non-substituted heteroaryl") or substituted by one or more substituents (a "substituted heteroaryl").

[0069] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-fused bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Representative 6,6-fused bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme, and heme derivatives.

[0070] As used herein, the terms “arylene” and “heteroarylene” each mean a divalent group derived from an aryl and a heteroaryl, respectively, alone or as part of another substituent.

[0071] As used herein, "cycloalkyl" refers to a group of non-aromatic cyclic hydrocarbon groups having 3 to 10 ring carbon atoms ("C3-C 10 cycloalkyl") and 0 heteroatoms in a non-aromatic ring system. In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C 10 cycloalkyl"). The cycloalkyl group can be described, for example, as a C4-C7 membered cycloalkyl, and the term "membered" refers to non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrieneyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), etc. Exemplary C3-C 10 cycloalkyl groups include, but are not limited to, the aforementioned C3-C8 cycloalkyl groups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10) etc. may be mentioned. As shown in the foregoing examples, in a predetermined embodiment, the cycloalkyl group is monocyclic ("monocyclic cycloalkyl"), or contains a fused, bridged, or spiro ring system, for example, a bicyclic system ("bicyclic cycloalkyl"), and may be saturated or may be partially unsaturated. "Cycloalkyl" also includes a ring system in which the cycloalkyl ring defined above is fused to one or more aryl groups and the point of attachment is on the cycloalkyl ring. In such a case, the number of carbon atoms continues to refer to the number of carbon atoms in the cycloalkyl ring system. Each example of the cycloalkyl group may independently be optionally substituted, that is, unsubstituted ("unsubstituted cycloalkyl") or substituted by one or more substituents ("substituted cycloalkyl").

[0072] "Heterocyclyl", as used herein, is a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In a heterocyclyl group containing one or more nitrogen atoms, the point of attachment can be a carbon or a nitrogen atom when the valence permits. The heterocyclyl group can be monocyclic ("monocyclic heterocyclyl"), or any of a fused, bridged, or spiro ring system, such as a bicyclic system ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. The bicyclic heterocyclyl ring system can contain one or more heteroatoms in one or both rings. "Heterocyclyl" includes a ring system in which the heterocyclyl ring defined above is fused to one or more cycloalkyl groups and the point of attachment is on either the cycloalkyl ring or the heterocyclyl ring, or a ring system in which the heterocyclyl ring defined above is fused to one or more aryl groups or heteroaryl groups and the point of attachment is on the heterocyclyl ring. In such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. The heterocyclyl group can be described, for example, as 3- to 7-membered heterocyclyl, and the term "member" refers to non-hydrogen ring atoms within the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each example of heterocyclyl can be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted by one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0073] In some embodiments, the heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (a "5- to 10-membered heterocyclyl"), a 5- to 10-membered non-aromatic ring system. In some embodiments, the heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"), a 5- to 6-membered non-aromatic ring system. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiirenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrol-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholin-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiacanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0074] "Amino", as used herein, refers to the group -NR 70 R 71 (wherein R 70 and R 71 are each independently hydrogen, C1-C8 alkyl, C3-C 10 cycloalkyl, C4-C 10 heterocyclyl, C6-C 10 aryl, and C5-C 10 heteroaryl). In some embodiments, amino refers to NH2.

[0075] As used herein, "cyano" refers to the group -CN.

[0076] As used herein, "halo", or "halogen", independently or as part of another substituent, unless otherwise stated, means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0077] As used herein, "hydroxy" refers to the group -OH.

[0078] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, when defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl, or “substituted” or “unsubstituted” heteroaryl group). Generally, the term “substituted” means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom), whether or not preceded by the term “optionally,” is replaced by a substituent that results in a stable compound (e.g., a compound that does not undergo spontaneous transformation by rearrangement, cyclization, elimination, or other reactions) by substitution. Unless otherwise indicated, a “substituted” group has substituents at one or more substitutable positions of the group, and when two or more positions in any given structure are substituted, the substituents are the same or different at each position. The term “substituted” is intended to include substitution with any of the acceptable substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any such combinations to arrive at a stable compound. For the purposes of the present disclosure, a heteroatom such as nitrogen may have a hydrogen substituent and / or any suitable substituent described herein that satisfies the valence of the heteroatom and results in the formation of a stable moiety.

[0079] Two or more substituents may optionally combine to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically found attached to the cyclic basic structure, but not necessarily so. In one embodiment, the ring-forming substituent is attached to adjacent members of the basic structure. For example, two ring-forming substituents attached to adjacent members of the cyclic basic structure produce a fused ring structure. In another embodiment, the ring-forming substituent is attached to a single member of the basic structure. For example, two ring-forming substituents attached to a single member of the cyclic basic structure produce a spirocyclic structure. In yet another embodiment, the ring-forming substituent is attached to non-adjacent members of the basic structure.

[0080] The compounds of formula (I) described herein may contain one or more asymmetric centers and, thus, may exist in various isomeric forms, such as enantiomers, and / or diastereomers. For example, the compounds described herein may be in the form of the individual enantiomers, diastereomers, or geometric isomers, or in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers can be isolated from the mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), as well as the formation and crystallization of chiral salts, or the preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen et al., Tetrahedron 33:2725 (1977), Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers and, alternatively, as mixtures of various isomers.

[0081] As used herein, a pure enantiomeric compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., is enantiomerically pure). In other words, the "S" form of the compound is substantially free of the "R" form of the compound and is thus enantiomerically pure with respect to the "R" form. The terms "enantiomerically pure" or "pure enantiomer" indicate that the compound contains greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0082] The compounds of formula (I) described herein may also contain one or more isotope substitutions. For example, H can be 1 H, 2 H (D or deuterium), and 3 H (T or tritium) in any isotopic form, C can be 12 C, 13 C, and 14 C in any isotopic form, O can be 16 O and 18 O in any isotopic form, and so on. The term "pharmaceutically acceptable salts" means salts of the active compounds prepared with relatively non-toxic acids or bases, depending on certain substituents found on the compounds described herein. When the compounds of formula (I) used to prepare the devices of the present disclosure contain relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds used in the present disclosure contain relatively basic functionality, the acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as alginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). The specific compounds used in the devices of the present disclosure (e.g., particles, hydrogel capsules) contain both basic and acidic functionality such that the compounds can be converted to either a base or an acid addition salt. These salts can be prepared by methods known to those of skill in the art. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for use in the present disclosure.

[0083] "Polysaccharide", as used herein, refers to a polymer of monosaccharides, or disaccharide carbohydrates, joined together by glycosidic bonds. Polysaccharides may be linear or branched. Exemplary monosaccharides include glucose, galactose, mannose, allose, altrose, talose, idose, gulose, fructose, ribose, arabinose, lyxose, xylose, rhamnose, glucuronic acid, galacturonic acid, mannuronic acid, and guluronic acid. Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, amylopectin, glycogen, gelatin, cellulose, amylose, chitin, chitosan, or derivatives or variants thereof (e.g., those described in Laurienzo (2010), Mar Drugs 9:2435-65).

[0084] The devices of the present disclosure may contain a compound of formula (I) in prodrug form. A prodrug is one of those compounds that readily undergoes a chemical change under physiological conditions to provide a compound useful for preparing a device in the present disclosure. Additionally, a prodrug can be converted to a useful compound of formula (I) by chemical or biochemical means in an ex vivo environment.

[0085] Certain compounds of formula (I) described herein may exist in unsolvated forms, as well as solvated forms including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of formula (I) described herein may exist in polymorphic or amorphous forms. Usually, all physical forms are equivalent for the intended uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0086] The term "solvate" usually refers to the form of a compound associated with a solvent through a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates, and further include both stoichiometric solvates and non-stoichiometric solvates.

[0087] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is within a limited ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R·x H2O, where R is the compound and x is a number greater than 0.

[0088] As used herein, the term "tautomer" means a compound structure in interchangeable forms, where there is a change in the replacement of a hydrogen atom and an electron. Thus, the two structures can be in equilibrium through the movement of π electrons and an atom (usually H). For example, enol and ketone are tautomers because they are rapidly interconverted by treatment with either an acid or a base. Tautomeric forms may be related to the achievement of the optimal chemical reactivity and biological activity of the target compound.

[0089] As used herein, the symbol "

Chem.

Chem.

[0090] The terms "covalent bond", "covalent bond", and "covalent bond", as used herein, refer to a type of chemical bond involving the sharing of electrons between two adjacent atoms. Examples of covalent bonds include those formed between carbon and hydrogen (C-H bond), carbon atoms (C-C bond), carbon atoms and oxygen atoms (C-O bond), and carbon and nitrogen (C-N bond). Depending on the identity of the atoms, the covalent bond can be a single bond, a double bond, or a triple bond. That is, the covalent bond can involve the sharing of one pair, two pairs, or three pairs of electrons.

[0091] The terms "ionic", "ionic bond", and "ionic bond", as used herein, refer to a type of chemical bond involving the Coulombic attraction between adjacent atoms of opposite charge (i.e., ions).

[0092] Modified polysaccharide polymer The polysaccharide polymers described herein are covalently modified at the covalent crosslinker sites. In one embodiment, the polysaccharide polymer can be a linear, branched, or crosslinked polysaccharide polymer, or a polysaccharide polymer of a selected molecular weight range, degree of polymerization, viscosity, or melt flow rate. Branched polysaccharide polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendrimerized polymers, graft co(polymers), ladder type, and dendrimers. The polysaccharide polymer can be a thermoresponsive polymer, for example, a gel (e.g., which becomes solid or liquid upon exposure to heat or a predetermined temperature), or a photocrosslinkable polymer. In some embodiments, the polysaccharide polymer may be biodegradable, for example, may contain labile bonds, or may be dissociated by an enzyme, such as a lyase. In some embodiments, the polysaccharide polymer is composed of a single type of repeating monomer unit. In other embodiments, the polysaccharide polymer is composed of different types of repeating monomer units (e.g., two types of repeating monomer units, three types of repeating monomer units, e.g., a polymer blend). In some embodiments, the polysaccharide can be composed of mannuronic acid and guluronic acid monomers.

[0093] In some embodiments, the polymer is a naturally occurring or synthetic polymer. In some embodiments, the polymer is a naturally occurring polysaccharide or a synthetic polysaccharide. In one embodiment, the polysaccharide polymer is cellulose, for example, carboxymethyl cellulose. In one embodiment, the polysaccharide polymer is polylactide, polyglycoside, or polycaprolactone. In one embodiment, the polysaccharide polymer is hyaluronate, for example, sodium hyaluronate. In one embodiment, the polymer is collagen, elastin, or gelatin. In one embodiment, the polymer is chitin.

[0094] In some embodiments, the polysaccharide polymer is a hydrogel-forming polymer. A hydrogel-forming polymer includes a hydrophilic structure and, thus, is capable of retaining a large amount of water within the three-dimensional network. The hydrogel-forming polymer can include polymers that form homopolymer hydrogels, copolymer hydrogels, or multipolymer interpenetrating polymer hydrogels and can be, for example, essentially amorphous, semi-crystalline, or crystalline, as described in Ahmed (2015) J Adv Res 6:105-121. Exemplary hydrogel-forming polymers include proteins (e.g., collagen), gelatin, polysaccharides (starch, alginate, hyaluronate, agarose), and synthetic polysaccharides.

[0095] Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, amylopectin, glycogen, gelatin, cellulose, amylose, chitin, chitosan, or derivatives or variants thereof (e.g., as described in Laurienzo (2010), Mar Drugs 9:2435-65). The polysaccharide polymer can include heparin, chondroitin sulfate, dermatan, dextran, or carboxymethyl cellulose. In some embodiments, the polysaccharide polymer is a cross-linked polymer. In some embodiments, the polysaccharide polymer is a cell surface polysaccharide.

[0096] In some embodiments, the polysaccharide polymer is alginate. Alginate is a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate is high guluronic acid (G) alginate and contains about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the alginate is high mannuronic acid (M) alginate and contains about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, more than 90%, or more mannuronic acid (M). In some embodiments, the M:G ratio is about 1. In some embodiments, the M:G ratio is less than 1. In some embodiments, the M:G ratio is greater than 1. In some embodiments, the alginate has an approximate molecular weight of <75 kDa and optionally a G:M ratio of ≧1.5. In some embodiments, the alginate has an approximate molecular weight of 75 kDa to 150 kDa and optionally a G:M ratio of ≧1.5. In some embodiments, the alginate has an approximate molecular weight of 150 to 250 kDa and optionally a G:M ratio of ≧1.5.

[0097] A polysaccharide polymer (e.g., any of the polymers described herein, e.g., any of the alginates described herein) comprising a sugar moiety having the structure of formula (I), or a pharmaceutically acceptable salt thereof, may be modified in one or more monomer units. In some embodiments, at least 0.5 percent of the sugar monomers of the polysaccharide polymer have the structure of formula (I) (e.g., at least 1, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 percent, or more, of the sugar monomers have the structure of formula (I)). In some embodiments, 0.5 to 50%, 10 to 90%, 10 to 50%, or 25 to 75% of the sugar monomers of the polysaccharide polymer have the structure of formula (I). In some embodiments, 1 to 20% of the sugar monomers of the polysaccharide polymer have the structure of formula (I). In some embodiments, 1 to 10% of the sugar monomers of the polysaccharide polymer have the structure of formula (I). In some embodiments, 1 to 50% of the sugar monomers have the structure of formula (I).

[0098] In some embodiments, the polysaccharide polymer (when including sugar monomers having the structure of formula (I)) comprises, on a weight basis, an increase in %N of at least 0.1, 0.2, 0.5, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10% N (when compared to the unmodified polymer), where %N is determined by elemental analysis and corresponds to the amount of the compound of formula (I) in the modified polymer.

[0099] In some embodiments, the polysaccharide polymer (when including sugar monomers having the structure of formula (I)) comprises, on a weight basis, an increase in %N of 0.1 to 10% N (when compared to the unmodified polymer), where %N is determined by elemental analysis and corresponds to the amount of the compound of formula (I) in the modified polymer.

[0100] In some embodiments, the polysaccharide polymer (when including sugar monomers having the structure of formula (I)) contains, on a weight basis, an increase in %N (when compared to the unmodified polymer) of 0.1 to 2% N, where %N is determined by elemental analysis and corresponds to the amount of the compound of formula (I) in the modified polymer.

[0101] In some embodiments, the polysaccharide polymer (when including sugar monomers having the structure of formula (I)) contains, on a weight basis, an increase in %N (when compared to the unmodified polymer) of 2 to 4% N, where %N is determined by elemental analysis and corresponds to the amount of the compound of formula (I) in the modified polymer.

[0102] In some embodiments, the polysaccharide polymer (when including sugar monomers having the structure of formula (I)) contains, on a weight basis, an increase in %N (when compared to the unmodified polymer) of 4 to 8% N, where %N is determined by elemental analysis and corresponds to the amount of the compound of formula (I) in the modified polymer.

[0103] In some embodiments, any of the polysaccharide polymers described herein (e.g., alginate) contain sugar monomers having one or more of formulae (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), or a pharmaceutically acceptable salt thereof. In some embodiments, the polysaccharide polymer contains sugar monomers having the structure of formula (II-a). In some embodiments, the polymer is modified with a compound of formula (II-b). In some embodiments, the polysaccharide polymer contains sugar monomers having the structure of formula (II-c). In some embodiments, the polysaccharide polymer contains sugar monomers having the structure of formula (II-d). In some embodiments, the polysaccharide polymer contains sugar monomers having the structure of formula (II-e). In some embodiments, the polysaccharide polymer contains sugar monomers having the structure of formula (II-f).

[0104] In some embodiments, the polymer (e.g., alginate) is modified with the compounds shown in Table 3.

[0105] In some embodiments, the polymer (e.g., alginate) modified with a compound of formula (I) is not a modified polymer described in any one of WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 187225, WO2016 / 019391, WO2017 / 075630, WO2017 / 075631, WO2018 / 067615, WO2019 / 169333, and US2016-0030359.

[0106] Non-fibrous compound In some embodiments, the polymers described herein comprise at least one non-fibrous compound of formula (I),

Chemical formula

[0107] In some embodiments, the compound of formula (I) is a compound of formula (I-a):

Chemical formula

[0108] In some embodiments, for formulas (I) and (I-a), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-, -N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, or -N(R C) - is as follows. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C ) - is as follows. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C ) - is as follows. In some embodiments, A is alkyl, -O-, -C(O)O-, -C(O)-, -OC(O), or -N(R C ) - is as follows. In some embodiments, A is -N(R C )C(O)-, -N(R C )C(O)(C1-C6-alkylene)-, or -N(R C )C(O)(C1-C6-alkenylene)-. In some embodiments, A is -N(R C ) - is as follows. In some embodiments, A is -N(R C ) - is as follows, and R C and R D are independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, and the alkylene is substituted by R 1 . In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, and R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)C(CH3)2-. In some embodiments, A is -N(R C )C(O)(methylene)-, and R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0109] In some embodiments, for formulas (I) and (I-a), L 1is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1 is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 is C1-C6 alkyl. In some embodiments, L 1 is -CH2-, -CH(CH3)-, -CH2CH2CH2, or -CH2CH2-. In some embodiments, L 1 is -CH2- or -CH2CH2-.

[0110] In some embodiments, for formulas (I) and (I-a), L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C1-C 12 alkyl. In some embodiments, L 3 is C1-C6 alkyl. In some embodiments, L 3 is -CH2-. In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is optionally substituted by one or more R 2 (e.g., oxo) C1-C 12 heteroalkyl. In some embodiments, L 3 is optionally substituted by one or more R 2 (e.g., oxo) C1-C6 heteroalkyl. In some embodiments, L 3 is -C(O)OCH2-, -CH2(OCH2CH2)2-, -CH2(OCH2CH2)3-, CH2CH2O-, or -CH2O-. In some embodiments, L 3 is -CH2O-.

[0111] In some embodiments, for formulas (I) and (I-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some embodiments, M is -CH2-. In some embodiments, M is heteroalkyl (e.g., C1-C6 heteroalkyl). In some embodiments, M is (-OCH2CH2-)z, where z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, (-OCH2CH2-)4, or (-OCH2CH2-)5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, or (-OCH2CH2-)4. In some embodiments, M is (-OCH2CH2-)3. In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is [Chemical formula] is. In some embodiments, M is R 7 (e.g., phenyl substituted by one R 7 ). In some embodiments, M is [Chemical formula] is. In some embodiments, R 7 is CF3.

[0112] In some embodiments, for formula (I) and (I-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for formula (I) and (I-a), P is tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is monocyclic heteroaryl. In some embodiments, P is nitrogen-containing heteroaryl. In some embodiments, P is monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is 5-membered heteroaryl. In some embodiments, P is 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl, or thiazolyl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is

Chemical formula

Chemical formula

[0113] In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is

Chemical formula

[0114] In some embodiments, for formula (I) or (I-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl. In some embodiments, Z is an oxygen-containing heterocyclyl. In some embodiments, Z is a 4-membered heterocyclyl, 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [Chemical formula] is. In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is [Chemical formula] is.

[0115] In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydride. In some embodiments, Z is a sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxidyl. In some embodiments, Z is [Chemical formula] It is. In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is

Chemical formula

[0116] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic nitrogen-containing heterocyclyl optionally substituted by one or more R 5 s. In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl. In some embodiments, Z is

Chemical formula

Chemical formula

[0117] In some embodiments, for formula (I) and (I-a), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is monosubstituted phenyl (for example, having one R 5 ). In some embodiments, Z is monosubstituted phenyl where one R 5 is a nitrogen-containing group. In some embodiments, Z is monosubstituted phenyl where one R 5 is NH2. In some embodiments, Z is monosubstituted phenyl where one R 5 is an oxygen-containing group. In some embodiments, Z is monosubstituted phenyl where one R 5is a monosubstituted phenyl that is an oxygen-containing heteroalkyl. In some embodiments, Z is one R 5 is a monosubstituted phenyl where it is OCH3. In some embodiments, Z is one R 5 is a monosubstituted phenyl where it is in the ortho position. In some embodiments, Z is one R 5 is a monosubstituted phenyl where it is in the meta position. In some embodiments, Z is one R 5 is a monosubstituted phenyl where it is in the para position.

[0118] In some embodiments, for formulas (I) and (I-a), Z is alkyl. In some embodiments, Z is C1-C 12 alkyl. In some embodiments, Z is C1-C 10 alkyl. In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is C1-C8 alkyl substituted by 1 to 5 R 5 s. In some embodiments, Z is C1-C8 alkyl substituted by one R 5 . In some embodiments, Z is C1-C8 alkyl substituted by one R 5 , and R 5 is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , or -N(R C1 )(R D1 ). In some embodiments, Z is C1-C8 alkyl substituted by one R 5 , and R 5 is -OR A1 or -C(O)OR A1 . In some embodiments, Z is C1-C8 alkyl substituted by one R 5 , and R 5 is -OR A1 or -C(O)OH. In some embodiments, Z is -CH3.

[0119] In some embodiments, for formulas (I) and (I-a), Z is heteroalkyl. In some embodiments, Z is C1-C 12 heteroalkyl. In some embodiments, Z is C1-C 10 heteroalkyl. In some embodiments, Z is C1-C8 heteroalkyl. In some embodiments, Z is C1-C6 heteroalkyl. In some embodiments, Z is nitrogen-containing heteroalkyl optionally substituted with one or more R 5 s. In some embodiments, Z is nitrogen and sulfur-containing heteroalkyl substituted by 1-5 R 5 s. In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethan-1-aminyl.

[0120] In some embodiments, Z is -OR A or -C(O)OR A . In some embodiments, Z is -OR A (e.g., -OH or -OCH3). In some embodiments, Z is -OCH3. In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).

[0121] In some embodiments, Z is hydrogen.

[0122] In some embodiments, L 2 is a bond, and P and L 3 are independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0123] In some embodiments, the compound of formula (I) is a compound of formula (I-b): [Chemistry] or a pharmaceutically acceptable salt thereof, wherein ring M 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by 1-5 R 3 ; ring Z 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl optionally substituted by 1-5 R 5 ; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or each of R 2a and R 2b or each of R 2c and R 2d together form an oxo group; X is absent, N(R 10 )(R 11 ), O, or S; R C is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted by 1-6 R 6 ; each of R 3 , R 5 , and R 6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), SRE1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, and R 10 and R 11 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 s, and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and each m and n is independently 1, 2, 3, 4, 5, or 6, and "

Chemical formula

[0124] In some embodiments, the compound of formula (I-b) is a compound of formula (I-b-i):

Chemical formula

Chem.

[0125] In some embodiments, the compound of formula (I-b-i) is the compound of formula (I-b-ii):

Chem.

Chemical formula

[0126] In some embodiments, the compound of formula (I) is a compound of formula (I-c):

Chemical formula

Chemical formula

[0127] In some embodiments, the compound of formula (I) is a compound of formula (I-d):

Chemical formula

Chemical formula

[0128] In some embodiments, the compound of formula (I) is a compound of formula (I-e):

Chemical formula

Chemical formula

[0129] In some embodiments, the compound of formula (I) is a compound of formula (I-f):

Chemical formula

Chemical formula

[0130] In some embodiments, the compound of formula (I) is a compound of formula (II):

Chemical formula

Chem.

[0131] In some embodiments, the compound of formula (II) is a compound of formula (II-a):

Chem.

Chemical Structure

[0132] In some embodiments, the compound of formula (I) is a compound of formula (III):

Chemical Structure

Chem.

[0133] In some embodiments, the compound of formula (III) is a compound of formula (III-a):

Chem.

Chemical formula

[0134] In some embodiments, the compound of formula (III-a) is a compound of formula (III-b):

Chemical formula

Chemical formula

[0135] In some embodiments, the compound of formula (III-a) is a compound of formula (III-c):

Chemical formula

Chemical formula

[0136] In some embodiments, the compound of formula (III-c) is a compound of formula (III-d):

Chemical formula

Chemical formula

[0137] In some embodiments, the compound of formula (I) is a compound of formula (III-e):

Chemical formula

Chemical formula

[0138] In some embodiments, the compound of formula (I) is a compound of formula (III-f):

Chemical formula

Chemical formula

[0139] In some embodiments, the compound of formula (I) is a compound of formula (III-g):

Chem.

Chem.

[0140] In some embodiments, the compound of formula (I) is a compound of formula (III-h):

Chemical formula

Chemical formula

[0141] In some embodiments, the compound of formula (I) is a compound of formula (III-i):

Chemical formula

Chemical formula

[0142] In some embodiments, the compound is a compound of formula (I). In some embodiments, L 2 is a bond, and P and L 3 are independently absent.

[0143] In some embodiments, the compound is a compound of formula (I-a). In some embodiments of formula (II-a), L 2 is a bond, P is heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl. In some embodiments, L 2 is a bond, and P and L 3 are independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0144] In some embodiments, the compound is a compound of formula (I-b). In some embodiments, P is absent, L 1 is -NHCH2, L 2 is a bond, M is aryl (e.g., phenyl), L 3is -CH2O, and Z is heterocyclyl (e.g., nitrogen-containing heterocyclyl, e.g., thiomorpholinyl-1,1-dioxide).

[0145] In some embodiments of formula (I-b), P is absent, and L 1 is -NHCH2, and L 2 is a bond, M is absent, and L 3 is a bond, and Z is heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).

[0146] In some embodiments, the compound is a compound of formula (I-b-i). In some embodiments of formula (I-b-i), R 2a and R 2b each is independently hydrogen or CH3, R 2c and R 2d each is independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M 2 is phenyl optionally substituted by one or more R 3 , R 3 is -CF3, and Z 2 is heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).

[0147] In some embodiments, the compound is a compound of formula (I-b-ii). In some embodiments of formula (I-b-ii), R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, q is 0, p is 0, m is 1, and Z 2 is heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranyl).

[0148] In some embodiments, the compound is a compound of formula (I-c). In some embodiments of formula (I-c), R 2c and R 2d each is independently hydrogen, m is 1, p is 1, q is 0, R 5 is -CH3, and Z is heterocyclyl (e.g., nitrogen-containing heterocyclyl, e.g., piperazinyl).

[0149] In some embodiments, the compound is a compound of formula (I-d). In some embodiments of formula (I-d), R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).

[0150] In some embodiments, the compound is a compound of formula (I-f). In some embodiments of formula (I-f), R 2a and R 2b each is independently hydrogen, n is 1, M is -CH2-, P is nitrogen-containing heteroaryl (e.g., imidazolyl), L 3 is -C(O)OCH2-, and Z is CH3.

[0151] In some embodiments, the compound is a compound of formula (II-a). In some embodiments of formula (II-a), R 2a and R 2b each is independently hydrogen, n is 1, q is 0, L 3 is -CH2(OCH2CH2)2, and Z is -OCH3.

[0152] In some embodiments of formula (II-a), R 2a and R 2b each is independently hydrogen, n is 1, L3 is either a bond or -CH2, and Z is either hydrogen or -OH.

[0153] In some embodiments, the compound is a compound of formula (III). In some embodiments of formula (III), R 2a , R 2b , R 2c , and R 2d are each independently hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 1 is heteroalkyl optionally substituted by R 5 (e.g., -N(CH3)(CH2CH2)S(O)2CH3).

[0154] In some embodiments, the compound is a compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d are each independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is aryl (e.g., phenyl) substituted by one R 5 (e.g., -NH2).

[0155] In some embodiments, the compound is a compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d are each independently hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 2 is heterocyclyl (e.g., nitrogen-containing heterocyclyl, e.g., nitrogen-containing spiroheterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl).

[0156] In some embodiments, the compound is a compound of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c , and R 2d are each independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments of formula (III-d), R 2a and R 2b are each independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2.

[0157] In some embodiments, the compound is a compound of formula (I-b), (I-d), or (I-e). In some embodiments, the compound is a compound of formula (I-b), (I-d), or (II). In some embodiments, the compound is a compound of formula (I-b), (I-d), or (I-f). In some embodiments, the compound is a compound of formula (I-b), (I-d), or (III).

[0158] In some embodiments, the compound of formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO2017 / 075630, US2012-0213708, US2016-0030359, or US2016-0030360.

[0159] In some embodiments, the compound of formula (I) comprises a compound shown in Table 3, or a pharmaceutically acceptable salt thereof. In some embodiments, the outer surface and / or one or more compartments within the device described herein comprise a small molecule compound shown in Table 3, or a pharmaceutically acceptable salt thereof.

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

[0160] Conjugation of any of the compounds of Table 3 with a polymer (e.g., alginate) can be carried out as described in Example 2 of WO2019 / 195055 or by any other suitable chemical reaction.

[0161] In some embodiments, the compound is a compound of formula (I) (e.g., formula (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (II), (II-a), (III), (III-a), (III-b), (III-c), (III-d), (III-e), (III-f), (III-g), (III-h), or (III-i)), or a pharmaceutically acceptable salt thereof,

Chemical formula

[0162] In some embodiments, the polysaccharide polymers or devices described herein (e.g., hydrogel capsules) are [Chemical] It includes a compound of formula (I) or a pharmaceutically acceptable salt of any of the compounds.

[0163] In some embodiments, the compound of formula (I) (e.g., compound 101 in Table 3), as described in WO2020 / 069429, when determined by combustion analysis for nitrogen percentage, has a conjugation density of at least 2.0% and less than 9.0%, or 3.0% - 8.0%, 4.0 - 7.0, 5.0 - 7.0, or 6.0 - 7.0 or about 6.8 and is covalently bound to alginate (e.g., alginate with an approximate MW < 75 kDa and a G:M ratio ≧ 1.5).

[0164] Crosslinking site In some embodiments, the crosslinking site can undergo a thiol - ene click reaction. In some embodiments, the crosslinking site contains a thiol. In some embodiments, the thiol includes an alkylthiol or an arylthiol. In some embodiments, the click crosslinking agent may contain two or more thiol groups. In some embodiments, the click crosslinking agent may contain two, three, four, five, or six thiol groups. In some embodiments, the thiol is a compound of formula (IV): [Chemical] Or a pharmaceutically acceptable salt or tautomer thereof, wherein Q is O, NR 33 , or C(R 34a )(R 34b ), and each of R 33 , R 34a , R 34b , R 60a , R 60b , R 61a , R 61b , and R 62 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )(C(O)R B1 , -C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 , and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl.

[0165] In some embodiments, the compound of formula (IV) is a compound of formula (IV-a): [Chemical formula] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 60a , R 60b , R 61a , R 61b , R 63a , and R 63b are each independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )(C(O)RB1 ,-C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 , and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl.

[0166] In some embodiments, the compound of formula (IV) is a compound of formula (IV-b):

Chemical formula

[0167] In some embodiments, the compound of formula (IV) is a compound of formula (IV-c):

Chemical formula

[0168] In some embodiments, the crosslinker moiety comprises an alkenyl group. In some embodiments, the crosslinker comprises a cyclyl or heterocyclyl group. In some embodiments, the crosslinker comprises a norbornenyl moiety. In some embodiments, the crosslinker is a compound of formula (V): [Chemical formula] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T and U is independently O, NR 33 , or C(R 34a )(R 34b ), and R 33 , R 34a , R 34b , R 65a , R 65b , R 65c , R 65d , R 65e , R 65f , R 65g , and R 66 are each independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1, -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )(C(O)R B1 , -C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 , and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl.

[0169] In some embodiments, the crosslinker of formula (V) is a compound of formula (V-a): [Chemical formula] or a pharmaceutically acceptable salt or tautomer thereof, wherein U is O, NR 33 , or C(R 34a )(R 34b ), and R 33 , R 34a , R 34b , R 65a , R 65b , R 65c , R 65d , R 65e , R 65f , R 65g , R 66 , R 67a , R 67b each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )(C(O)R B1 , -C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, each R A1 , R B1 , R C1 , R D1 , and R E1 is, independently, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 s, and each R 7 is, independently, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl.

[0170] In some embodiments, the crosslinking agent of formula (V) is a compound of formula (V-b):

Chemical formula

[0171] In some embodiments, the click crosslinking agent is a compound of formula (VI):

Chemical formula

[0172] In some embodiments, the crosslinking agent of formula (VI) is a compound of formula (VI-a):

Chemical formula

[0173] In some embodiments, the crosslinking agent of formula (VI) is a compound of formula (VI-b):

Chemical formula

[0174] In some embodiments, the crosslinking agent comprises an aryl or heteroaryl group. In some embodiments, the click crosslinking agent is a compound of formula (VII):

Chemical formula

[0175] In some embodiments, the crosslinker of formula (VII) is a compound of formula (VII-a):

Chemical formula

[0176] In some embodiments, the crosslinking agent of formula (VII) is a compound of formula (VII-b):

Chemical formula

[0177] In some embodiments, the crosslinking agent of formula (VII) is a compound of formula (VII-c):

Chemical formula

[0178] In some embodiments, the crosslinker moiety comprises a tetrazinyl moiety.

[0179] In some embodiments, the compound of any one of formulas (IV), (V), (VI), and (VII) is selected from the compounds in Table 4. [Table 11]

[0180] A polymer modified with a crosslinker The crosslinker can be covalently attached to a polysaccharide, such as alginate. A modified polysaccharide polymer, such as a modified alginate polymer, can be crosslinked to another polymer. In one embodiment, the polysaccharide polymer is modified with two or more types of crosslinkers.

[0181] In one embodiment, the modified polysaccharide is a compound of formula (VIII): [Chemical formula] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T and U is independently C(R 40 )(R 41 ), O, or N(R 42 ), and R 38a , R 38b , R 39a , R 39b , R 40 , R41 and R 42 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each R 32 and R 35 are hydrogen, alkyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each R A1 , R B1 , R C1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1 to 6 R 7 s, and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and the click crosslinking agent has the structures of formula (IV), (IV-a), (IV-b), (IV-c), (V), (V-a), (V-b), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), and (VII-c).

[0182] A polysaccharide polymer modified with a click crosslinking agent and a non-fibrous small molecule compound In one embodiment, the polysaccharide polymer has the structure of formula (IX):

Chemical formula

[0183] In one embodiment, the modified polysaccharide polymer is a compound selected from Table 5. [Table 12] [Table 13] [Table 14] [Table 15] or a pharmaceutically acceptable salt thereof.

[0184] The polysaccharide polymers described herein may be modified with any suitable functional group (e.g., a carboxyl group or a hydroxyl group). In one embodiment, the polysaccharide polymer is modified with a single type of functional group. In one embodiment, the polysaccharide polymer is modified with two or more types of functional groups. In one embodiment, the polysaccharide polymers described herein may be modified on one or more functional groups by a compound of formula (I) and / or a compound of formula (IV)-(VII).

[0185] In one embodiment, the degree of modification (i.e., the percent of functional groups of the polymer modified with the photoactive crosslinking agent) is greater than 99%. In one embodiment, the degree of modification of the polymer is less than 99%. In one embodiment, the degree of modification is greater than about 95%. In one embodiment, the degree of modification is about 50%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 99%. In one embodiment, the degree of modification is about 1% to about 80%. In one embodiment, the degree of modification is about 1% to about 75%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 70%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 65%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 60%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 55%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 50%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 45%. In a preferred embodiment, not all of the functional groups (e.g., carboxyl groups) of the modified polysaccharide are substituted, which allows for both ionic and covalent bonding.

[0186] In one embodiment, the polysaccharide polymers described herein are modified with one, two, three, or more unique compounds. In one embodiment, the polysaccharide polymers described herein are modified with a photoactive crosslinking agent (e.g., a compound of formula (IV), a compound of formula (I), and a cell adhesion molecule (e.g., RGD)). In one embodiment, the polysaccharide polymers described herein are modified with a photoactive crosslinking agent. In one embodiment, the polysaccharide polymers described herein are modified with a compound of formula (I). In one embodiment, the polysaccharide polymers described herein are modified with a cell adhesion molecule. In one embodiment, the polysaccharide polymers described herein are modified with both a photoactive crosslinking agent and a cell adhesion molecule. In a preferred embodiment, the polysaccharide polymers described herein are modified with both a photoactive crosslinking agent and a compound of formula (I). In one embodiment, the polysaccharide polymers described herein are modified with both a cell adhesion molecule and a compound of formula (I).

[0187] In one embodiment, the degree of modification (i.e., the percent of functional groups of the polymer modified with the clickable crosslinker) is greater than 99%. In one embodiment, the degree of modification of the polymer is less than 99%. In one embodiment, the degree of modification is greater than about 95%. In one embodiment, the degree of modification is about 50%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 99%. In one embodiment, the degree of modification is about 1% to about 80%. In one embodiment, the degree of modification is about 1% to about 75%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 70%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 65%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 60%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 55%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 50%. In one embodiment, the polysaccharide polymer has a degree of modification of about 1% to about 45%. In a preferred embodiment, not all of the functional groups (e.g., carboxyl groups) of the modified polysaccharide are substituted, which allows for both ionic and covalent bonds.

[0188] In one embodiment, the degree of modification (i.e., the percent of functional groups of the polymer modified with the clickable crosslinker) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, the degree of modification (i.e., the percent of functional groups of the polymer modified with the clickable crosslinker) is greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, the degree of modification (i.e., the percent of functional groups of the polymer modified with the clickable crosslinker) is less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0189] In some embodiments, the polysaccharide polymers described herein retain unreacted carboxylic acid groups sufficient to enable ionic crosslinking, e.g., when the polymer is used to prepare hydrogel capsules having double crosslinks. In some embodiments, the polysaccharide polymers described herein do not include a degree of modification greater than 10% of the carboxylic acid groups. In some embodiments, the polysaccharide polymers described herein do not include a degree of modification greater than 5% of the carboxylic acid groups. In some embodiments, the polysaccharide polymers described herein do not include a degree of modification greater than 5%, 6%, 7%, 8%, 9%, or 10% of the carboxylic acid groups.

[0190] Characteristics of Hydrogel Capsules The present disclosure is further characterized by hydrogel capsules comprising the polysaccharide polymers described herein. The hydrogel capsules can be produced, for example, by crosslinking (i.e., covalently bonding) crosslinking groups or by ionic crosslinking, for example, in the presence of a divalent cation (e.g., Ba2+). In one embodiment, the hydrogel capsules described herein are produced by reacting a crosslinking agent. In one embodiment, the hydrogel capsules described herein are produced by covalent crosslinking and ionic crosslinking. Those skilled in the art will recognize that it is possible to initiate polymerization by other methods, including heat, ultrasound, and gamma rays, in the presence of a suitable initiator.

[0191] The hydrogel capsules described herein are formed by crosslinking of one or more types of polysaccharide polymers. In one embodiment, the hydrogel capsule comprises only a polysaccharide polymer. In one embodiment, the hydrogel capsule comprises the same type of polysaccharide polymer, for example, an alginate polymer. In one embodiment, the hydrogel capsule is formed by polymerization of two identical polysaccharides. In one embodiment, the hydrogel capsule is formed by polymerization of two different polysaccharides. In one embodiment, the hydrogel capsule comprises a plurality of polymers, for example, a plurality of polysaccharide polymers. In one embodiment, the hydrogel capsule comprises one polysaccharide polymer and a non-polysaccharide polymer.

[0192] The hydrogel capsules described herein may be homogeneous, i.e., they may not contain a non-polysaccharide polymer. In one embodiment, the hydrogel capsules described herein do not contain a polymer selected from polyacrylamide, poly(vinyl alcohol), poly(ethylene oxide), polyethylene glycol (PEG), and polyphosphazene. In one embodiment, the hydrogel capsule does not contain poly(vinyl alcohol). In one embodiment, the hydrogel capsule does not contain poly(ethylene oxide). In one embodiment, the hydrogel capsule does not contain polyethylene glycol (PEG). In one embodiment, the hydrogel capsule does not contain polyphosphazene.

[0193] In one embodiment of the present invention, the hydrogel capsule is a two-compartment hydrogel capsule. In a preferred embodiment of the present invention, the hydrogel capsule consists of an inner compartment and an outer compartment. In one embodiment, the two compartments are formed from the same type of modified polysaccharide. In one embodiment, the two compartments are formed from different types of modified polysaccharides.

[0194] In some embodiments, the first and second compartments comprise a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment comprises a blend of polymers. In some embodiments, the second (outer) compartment comprises a blend of polymers. In some embodiments, the first and second compartments comprise the same blend of polymers. In some embodiments, the first and second compartments comprise different blends of polymers.

[0195] In some embodiments, the first compartment comprises a blend of polymers and the second compartment does not comprise a blend of polymers. In some embodiments, the first compartment comprises a blend of polymers and the second compartment comprises a blend of polymers.

[0196] In some embodiments, the first compartment does not comprise a blend of polymers and the second compartment comprises a blend of polymers. In some embodiments, the first compartment comprises a single type of polymer and the second compartment comprises a blend of polymers.

[0197] In some embodiments, the first and second compartments comprise a blend of alginate polymers. In some embodiments, the first compartment comprises a blend of alginate polymers. In some embodiments, the second compartment comprises a blend of alginate polymers.

[0198] In some embodiments of the present invention, the first and second compartments comprise a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the first compartment comprises a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the second compartment comprises a blend of VLVG alginate and SLG100 alginate.

[0199] In some embodiments, the hydrogel capsule comprises (i) an inner compartment comprising VLVG alginate, wherein the VLVG alginate comprises a compound of formula (IV), and SLG100 alginate, wherein the SLG100 alginate comprises a compound of formula (V); and (ii) an outer compartment comprising a blend of VLVG and SLG100 alginates, wherein the VLVG alginate comprises a compound of formula (IV) and the SLG100 alginate comprises a compound of formula (V).

[0200] In some embodiments of the present invention, the first and second compartments comprise a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the first compartment comprises a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the second compartment comprises a blend of VLVG alginate and SLG100 alginate.

[0201] In some embodiments, the hydrogel capsule comprises (i) an inner compartment comprising a blend of VLVG and SLG100 alginates, wherein the VLVG alginate comprises a compound of formula (IV) and the SLG100 alginate comprises a compound of formula (V); and (ii) an outer compartment comprising a blend of VLVG and SLG100 alginates, wherein the VLVG alginate comprises a compound of formula (IV) and the SLG100 alginate comprises a compound of formula (V).

[0202] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment comprising a blend of VLVG and SLG100 alginates, wherein the VLVG alginate comprises the compounds of formula (I) and formula (IV), and the SLG100 alginate comprises the compound of formula (V); and (ii) an outer compartment comprising a blend of VLVG and SLG100 alginates, wherein the VLVG alginate comprises the compounds of formula (I) and formula (IV), and the SLG100 alginate comprises the compound of formula (V).

[0203] The modified polymer comprising a crosslinking site can undergo further polymerization, for example, by reacting with compatible functional groups on the same or different polymers. In one embodiment, a crosslinked polymer can be formed by reacting a first polymer modified to contain a thiol group with a second polymer modified to contain an alkene group. In some embodiments, the hydrogel is formed by crosslinking of unsaturated functional groups by a chain growth polymerization process. In other embodiments, the hydrogel is formed by crosslinking of unsaturated functional groups by a step growth polymerization process. The step growth polymerization process preferably includes a reaction between one or more unsaturated functional groups (e.g., alkenyl groups) of one polysaccharide chain and a thiolated functional group of another polymer chain. In some embodiments, step growth polymerization, thiol-ene click reaction.

[0204] The present disclosure features a double-crosslinked polysaccharide for encapsulating mammalian cells. The double-crosslinked polysaccharide hydrogel contains at least one cell-binding substance (CBS) (as defined herein). The cells are capable of expressing a therapeutic agent when the hydrogel is transplanted into a subject, such as a human or other mammalian subject. Further, the device includes at least one means (as defined herein) for reducing FBR.

[0205] In some embodiments, the unmodified polymer is unmodified alginate. In some embodiments, the alginate is high guluronic acid (G) alginate and contains about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the alginate is high mannuronic acid (M) alginate and contains about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, over 90%, or more mannuronic acid (M). In some embodiments, the M:G ratio is about 1. In some embodiments, the M:G ratio is less than 1. In some embodiments, the M:G ratio is greater than 1. In one embodiment, the unmodified alginate has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of ≧1.5.

[0206] In some embodiments, the non-fibrous polymer comprises alginate chemically modified with a compound of formula (I). The alginate in the modified non-fibrous polymer can be the same as or different from any unmodified alginate present in the device. In one embodiment, the density of the compound of formula (I) in the non-fibrous alginate (e.g., the amount of conjugation) is between about 4.0% and about 8.0%, between about 5.0% and about 7.0%, or between about 6.0% and about 7.0% nitrogen (e.g., as determined by combustion analysis for nitrogen content). In one embodiment, the amount of compound 101 results in an increase in %N of about 0.5% - 2%, 2% - 4% N, about 4% - 6% N, about 6% - 8%, or about 8% - 10% N (when compared to unmodified alginate), where %N is determined by combustion analysis and corresponds to the amount of compound 101 in the modified alginate.

[0207] The hydrogel capsules described herein can be porous or non-porous. The pores of the polysaccharide hydrogel (e.g., alginate hydrogel) function as a membrane selectively permeable to small proteins and molecules, while larger, undesirable molecules such as immunoglobulins are prevented from accessing the encapsulated cells. In preferred embodiments, the hydrogels and hydrogel capsules described herein are porous. In one embodiment, the hydrogel capsule has an average pore diameter of about 10 nm to about 50 nm. In some embodiments, the average pore diameter is about 10 nm to 40 nm. In some embodiments, the average pore diameter is about 10 nm to 30 nm. In some embodiments, the average pore diameter is 10 - 20 nm.

[0208] The physical properties of the hydrogel capsules described herein (e.g., as described in the examples) control the release of the encapsulated molecules (e.g., as determined by the dextran permeability assay). In some embodiments, the average molecular weight permeability is about 50 kDa to about 400 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 400 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 350 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 300 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 250 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 200 kDa. In some embodiments, the average molecular weight permeability is about 100 kDa to about 150 kDa. In a preferred embodiment, the average molecular weight permeability is about 125 kDa to about 175 kDa.

[0209] The hydrogel capsules described herein can be porous or non-porous. The pores of polysaccharide hydrogel capsules (e.g., formed from alginate hydrogels) function as membranes selectively permeable to small proteins and molecules, while larger, undesirable molecules such as immunoglobulins are prevented from accessing the encapsulated cells. In preferred embodiments, the hydrogels and hydrogel capsules described herein are porous. In some embodiments, the average pore diameter is about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm. In some embodiments, the average pore diameter is greater than about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm. In some embodiments, the average pore diameter is less than about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm.

[0210] In some embodiments, the average pore diameter of the first compartment and the average pore diameter of the second compartment of the particle (e.g., hydrogel capsule) are substantially the same. In some embodiments, the average pore diameter of the first compartment and the average pore diameter of the second compartment of the particle differ by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, the average pore diameter of the particle (e.g., the average pore diameter of the first compartment and / or the average pore diameter of the second compartment) depends on a number of factors, such as the material(s) within each compartment, the presence and density of the photoactive crosslinker, and the presence and density of the compound of formula (I).

[0211] The hydrogel capsules described herein should not have pores with a diameter sufficient to permit the migration of cells (e.g., immune cells, e.g., dendritic cells) through the hydrogel. In some embodiments, the diameter of the pores is small enough to prevent the migration of antibodies through the hydrogel. In some embodiments, the hydrogel capsules described herein do not have a pore diameter greater than 75 μm. In some embodiments, the hydrogel capsules described herein do not have a pore diameter of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, or greater than 75 μm.

[0212] In some embodiments, the hydrogel capsules described herein can be characterized by an absolute breaking strength (e.g., crushing strength) determined by using a texture analyzer. In some embodiments, the absolute breaking strength is from 50 to 800 g. In some embodiments, the hydrogel capsules described herein have an absolute strength of about 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, 300 g, 310 g, 320 g, 330 g, 340 g, 350 g, 360 g, 370 g, 380 g, 390 g, 400 g, 410 g, 420 g, 430 g, 440 g, 450 g, 460 g, 470 g, 480 g, 490 g, 500 g, 510 g, 520 g, 530 g, 540 g, 550 g, 560 g, 570 g, 580 g, 590 g, 600 g, 610 g, 620 g, 630 g, 640 g, 650 g, 660 g, 670 g, 680 g, 690 g, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or 800 g. In some embodiments, the hydrogel capsules described herein have an absolute strength greater than about 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, 300 g, 310 g, 320 g, 330 g, 340 g, 350 g, 360 g, 370 g, 380 g, 390 g, 400 g, 410 g, 420 g, 430 g, 440 g, 450 g, 460 g, 470 g, 480 g, 490 g, 500 g, 510 g, 520 g, 530 g, 540 g, 550 g, 560 g, 570 g, 580 g, 590 g, 600 g, 610 g, 620 g, 630 g, 640 g, 650 g, 660 g, 670 g, 680 g, 690 g, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or 800 g.In some embodiments, the hydrogel capsules described herein have an absolute strength of less than about 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, 300 g, 310 g, 320 g, 330 g, 340 g, 350 g, 360 g, 370 g, 380 g, 390 g, 400 g, 410 g, 420 g, 430 g, 440 g, 450 g, 460 g, 470 g, 480 g, 490 g, 500 g, 510 g, 520 g, 530 g, 540 g, 550 g, 560 g, 570 g, 580 g, 590 g, 600 g, 610 g, 620 g, 630 g, 640 g, 650 g, 660 g, 670 g, 680 g, 690 g, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or 800 g.

[0213] The present disclosure features particles (e.g., hydrogel capsules) comprising a first compartment, a second compartment, a crosslinking site (e.g., a compound of formula (IV) or (V)) described herein, and optionally a compound of formula (I).

[0214] The photoactive crosslinking site is covalently bonded to a polysaccharide polymer present in the first and / or second compartment. The particles (e.g., hydrogel capsules) can be spherical or can have any other shape. The particles (e.g., hydrogel capsules) can comprise materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The particles (e.g., hydrogel capsules) can be made entirely of one type of material or can contain a number of other materials within the second (outer) compartment and the first (inner) compartment.

[0215] In some embodiments, the first compartment is modified with a compound of formula (I). In some embodiments, the second compartment is modified with a compound of formula (I). In some embodiments, both the first compartment and the second compartment are independently modified with a compound of formula (I).

[0216] In some embodiments, the particles (e.g., hydrogel capsules) have a maximum linear dimension (LLD), such as an average diameter or diameter, that is greater than 1 millimeter (mm), preferably about 1.5 mm or more. In some embodiments, the particles (e.g., hydrogel capsules) can be on the order of 10 mm in diameter or size. For example, the particles (e.g., hydrogel capsules) described herein can have a diameter or size in the range of 0.5 mm to 10 mm, 1 mm to 10 mm, 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 mm, 3 mm to 7 mm, 3 mm to 6 mm, 3 mm to 5 mm, 3 mm to 4 mm, 3.5 mm to 8 mm, 3.5 mm to 7 mm, 3.5 mm to 6 mm, 3.5 mm to 5 mm, 3.5 mm to 4 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, 4 mm to 5 mm, 4.5 mm to 8 mm, 4.5 mm to 7 mm, 4.5 mm to 6 mm, 4.5 mm to 5 mm, 5 mm to 8 mm, 5 mm to 7 mm, 5 mm to 6 mm, 5.5 mm to 8 mm, 5.5 mm to 7 mm, 5.5 mm to 6 mm, 6 mm to 8 mm, 6 mm to 7 mm, 6.5 mm to 8 mm, 6.5 mm to 7 mm, 7 mm to 8 mm, or 7.5 mm to 8 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size between about 1 mm and about 8 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size between about 1 mm and about 4 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size between about 1 mm and about 2 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size between about 1.5 mm and about 2 mm.

[0217] In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter, or diameter, that is the largest linear dimension (LLD), e.g., 1 millimeter (mm) or less. In some embodiments, the particles (e.g., hydrogel capsules) are in a diameter range of 0.3 mm to 1 mm, 0.4 mm to 1 mm, 0.5 mm to 1 mm, 0.6 mm to 1 mm, 0.7 mm to 1 mm, 0.8 mm to 1 mm, or 0.9 mm to 1 mm.

[0218] In one embodiment, the second (outer) compartment completely surrounds the first (inner) compartment, and the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between the two compartments. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably greater than 100 nm, and can be on the order of 1 mm. For example, the thickness of the outer compartment in the particles described herein is 10 nanometers to 1 millimeter, 100 nanometers to 1 millimeter, 500 nanometers to 1 millimeter, 1 micrometer (μm) to 1 millimeter, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness of the outer compartment is between 100 nanometers and 1 millimeter, between 1 μm and 1 mm, between 1 μm and 500 μm, or between 5 μm and 1 mm.

[0219] In some embodiments, both the first compartment and the second compartment contain the same polymer. In some embodiments, the first compartment and the second compartment contain different polymers. In some embodiments, the first compartment contains alginate. In some embodiments, the second compartment contains alginate. In some embodiments, both the first compartment and the second compartment contain alginate. In some embodiments, the alginate in the first compartment is different from the alginate in the second compartment. In some embodiments, the first compartment contains alginate and the second compartment contains a different polymer (e.g., a polysaccharide, e.g., hyaluronate, or chitosan). In some embodiments, the second compartment contains alginate and the first compartment contains a different polymer (e.g., a polysaccharide, e.g., hyaluronate or chitosan).

[0220] Both the first compartment and the second compartment can contain a single component (e.g., one polymer) or two or more components (e.g., a blend of polymers). In some embodiments, the first compartment contains only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and a chemically modified alginate). In some embodiments, the second compartment contains only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and a chemically modified alginate). In some embodiments, both the first and second compartments independently contain only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and a chemically modified alginate).

[0221] In some embodiments, the first and second compartments comprise a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment comprises a blend of polymers. In some embodiments, the second (outer) compartment comprises a blend of polymers. In some embodiments, the first and second compartments comprise the same blend of polymers. In some embodiments, the first and second compartments comprise different blends of polymers. In some embodiments, at least one polymer in the blend comprising the outer compartment is covalently modified with a photoactive crosslinking agent (e.g., a compound of formula (IV), (V), or (VI)) described herein. In some embodiments, at least one polymer in the blend comprising the second (outer) compartment is covalently modified with a non-fibrous compound (e.g., a compound of formula (I)) described herein. In some embodiments, at least one polymer in the blend comprising the second (outer) compartment is covalently modified with both a photoactive crosslinking agent and a non-fibrous compound.

[0222] In some embodiments, the first compartment comprises a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers. In some embodiments, the first compartment comprises a blend of two polymers. In some embodiments, the first compartment comprises a blend of three polymers. In some embodiments, the first compartment comprises a blend of four polymers. In some embodiments, the first compartment comprises a blend of five polymers. In some embodiments, the first compartment comprises a blend of six polymers. In some embodiments, the first (inner) compartment comprises a blend of seven polymers. In some embodiments, the first (inner) compartment comprises a blend of eight polymers. In some embodiments, the first (inner) compartment comprises a blend of nine polymers. In some embodiments, the first (inner) compartment comprises a blend of ten polymers.

[0223] In some embodiments, the second (outer) compartment comprises a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers. In some embodiments, the second (outer) compartment comprises a blend of two polymers. In some embodiments, the second (outer) compartment comprises a blend of three polymers. In some embodiments, the second (outer) compartment comprises a blend of four polymers. In some embodiments, the second (outer) compartment comprises a blend of five polymers. In some embodiments, the second (outer) compartment comprises a blend of six polymers. In some embodiments, the second (outer) compartment comprises a blend of seven polymers. In some embodiments, the second (outer) compartment comprises a blend of eight polymers. In some embodiments, the second (outer) compartment comprises a blend of nine polymers. In some embodiments, the second (outer) compartment comprises a blend of ten polymers.

[0224] In some embodiments, the first compartment comprises a blend of polymers and the second compartment does not comprise a blend of polymers. In some embodiments, the first compartment comprises a blend of polymers and the second compartment comprises a blend of polymers.

[0225] In some embodiments, the first compartment does not comprise a blend of polymers and the second compartment comprises a blend of polymers. In some embodiments, the first compartment comprises a single type of polymer and the second compartment comprises a blend of polymers.

[0226] In some embodiments, the first and second compartments comprise a blend of polymers, and the polymers of the blend are any two miscible polymers.

[0227] In some embodiments, the first and second compartments comprise a blend of polymers, and the polymers are selected from the group consisting of alginate, hyaluronate, and chitosan.

[0228] In some embodiments, the first and second compartments comprise a blend of polymers, the polymers being selected from the group consisting of alginate, hyaluronate, and chitosan. In some embodiments, the first compartment comprises a blend of polymers, the polymers being selected from the group consisting of alginate, hyaluronate, and chitosan. In some embodiments, the second compartment comprises a blend of polymers, the polymers being selected from the group consisting of alginate, hyaluronate, and chitosan.

[0229] In some embodiments, the first and second compartments comprise a blend of alginate polymers. In some embodiments, the first compartment comprises a blend of alginate polymers. In some embodiments, the second compartment comprises a blend of alginate polymers.

[0230] In some embodiments, the first and second compartments comprise a blend of alginate polymers, the alginate polymers being selected from high guluronic acid alginate and high mannuronic acid alginate. In some embodiments, the first compartment comprises a blend of alginate polymers, the alginate polymers being selected from high guluronic acid alginate and high mannuronic acid alginate. In some embodiments, the second compartment comprises a blend of alginate polymers, the alginate polymers being selected from high guluronic acid alginate and high mannuronic acid alginate.

[0231] In some embodiments, the first and second compartments comprise a blend of alginate polymers, the alginate polymers being selected from low molecular weight alginate, medium molecular weight alginate, high molecular weight alginate, and ultra-high molecular weight alginate. In some embodiments, the first compartment comprises a blend of alginate polymers, the alginate polymers being selected from low molecular weight alginate, medium molecular weight alginate, high molecular weight alginate, and ultra-high molecular weight alginate. In some embodiments, the second compartment comprises a blend of alginate polymers, the alginate polymers being selected from low molecular weight alginate, medium molecular weight alginate, high molecular weight alginate, and ultra-high molecular weight alginate.

[0232] In some embodiments, the first and second compartments include a blend of alginate polymers, and the alginate polymers are selected from Kimica Algin IL-2, Kimica Algin IL-6, Kimica Algin I-1, Kimica Algin I-3, Kimica Algin I-5, Kimica Algin I-8, Kimica Algin LZ-2, Kimica Algin ULV-L3, Kimica Algin ULV-L5, Kimica Algin ULV-1G, Kimica Algin ULV-5G, Kimica Algin ULV IL-6G, Pronova UP VLVM, Pronova UP LVM, Pronova UP MVM, Pronova UP VLVG, Pronova UP MVG, Pronova UP LVG, Pronova SLM20, Pronova SLM100, Pronova SLG20, and Pronova SLG100. In some embodiments, the first compartment includes a blend of alginate polymers, and the alginate polymers are selected from Kimica Algin IL-2, Kimica Algin IL-6, Kimica Algin I-1, Kimica Algin I-3, Kimica Algin I-5, Kimica Algin I-8, Kimica Algin LZ-2, Kimica Algin ULV-L3, Kimica Algin ULV-L5, Kimica Algin ULV-1G, Kimica Algin ULV-5G, Kimica Algin ULV IL-6G, Pronova UP VLVM, Pronova UP LVM, Pronova UP MVM, Pronova UP VLVG, Pronova UP MVG, Pronova UP LVG, Pronova SLM20, Pronova SLM100, Pronova SLG20, and Pronova SLG100.In some embodiments, the second compartment comprises a blend of alginate polymers, and the alginate polymers are selected from Kimica Algin IL-2, Kimica Algin IL-6, Kimica Algin I-1, Kimica Algin I-3, Kimica Algin I-5, Kimica Algin I-8, Kimica Algin LZ-2, Kimica Algin ULV-L3, Kimica Algin ULV-L5, Kimica Algin ULV-1G, Kimica Algin ULV-5G, Kimica Algin ULV IL-6G, Pronova UP VLVM, Pronova UP LVM, Pronova UP MVM, Pronova UP VLVG, Pronova UP MVG, Pronova UP LVG, Pronova SLM20, Pronova SLM100, Pronova SLG20, and Pronova SLG100.

[0233] In some embodiments, the first and second compartments contain a blend of two alginate polymers in any ratio. In some embodiments, the ratio of the two alginate polymers in the blend is about 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, or 50:50. In some embodiments, the ratio of the two alginate polymers in the blend is about 99:1. In some embodiments, the ratio of the two alginate polymers in the blend is about 95:5. In some embodiments, the ratio of the two alginate polymers in the blend is about 90:10. In some embodiments, the ratio of the two alginate polymers in the blend is about 85:15. In some embodiments, the ratio of the two alginate polymers in the blend is about 80:20. In some embodiments, the ratio of the two alginate polymers in the blend is about 75:25. In some embodiments, the ratio of the two alginate polymers in the blend is about 70:30. In some embodiments, the ratio of the two alginate polymers in the blend is about 65:35. In some embodiments, the ratio of the two alginate polymers in the blend is about 60:40. In some embodiments, the ratio of the two alginate polymers in the blend is about 55:45. In some embodiments, the ratio of the two alginate polymers in the blend is about 50:50.

[0234] In some embodiments, the first and second compartments comprise a blend of two alginate polymers in any ratio. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, or greater than 50:50. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 99:1. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 95:5. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 90:10. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 85:15. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 80:20. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 75:25. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 70:30. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 65:35. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 60:40. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 55:45. In some embodiments, the ratio of the two alginate polymers in the blend is greater than about 50:50.

[0235] In some embodiments, the polymer of the first compartment of the particle (e.g., hydrogel capsule) is modified with one compound of formula (I), and the polymer of the second compartment of the particle (e.g., hydrogel capsule) is modified with a different compound of formula (I). In some embodiments, the particle (e.g., hydrogel capsule) comprises a mixture of a polymer modified with a compound of formula (I) and an unmodified polymer (e.g., a polymer not modified with a compound of formula (I)). In some embodiments, the first compartment comprises a mixture (i.e., blend) of a polymer modified with a compound of formula (I) and an unmodified polymer (e.g., a polymer not modified with a compound of formula (I)). In some embodiments, the second compartment comprises a mixture (i.e., blend) of a polymer modified with a compound of formula (I) and an unmodified polymer (e.g., a polymer not modified with a compound of formula (I)).

[0236] The polymers of the particles (e.g., hydrogel capsules) described herein can be modified with a compound of formula (I), or a pharmaceutically acceptable salt thereof, at one or more monomers of the polymer. The modified polymer of the particle (e.g., hydrogel capsule) can be present in the first (inner) compartment of the particle, the second (outer) compartment of the particle, or both the first (inner) and second (outer) compartments of the particle. In some embodiments, the modified polymer is present only within the second compartment (including the outer surface of the particle). In some embodiments, at least 0.5% of the monomers of the polymer are modified with a compound of formula (I) (e.g., at least 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the monomers of the polymer are modified with a compound of (I)). In some embodiments, 0.5% to 50%, 10% to 90%, 10% to 50%, or 25 to 75% of the monomers of the polymer are modified with a compound of formula (I). In some embodiments, 1% to 20% of the monomers of the polymer are modified with a compound of formula (I). In some embodiments, 1% to 10% of the monomers of the polymer are modified with a compound of formula (I).

[0237] In some embodiments, a polymer (e.g., alginate) (when modified with a compound of formula (I), e.g., compound 101 of Table 3) has any of the following values: (i) at least 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% N by weight, (ii) 0.1% - 10% N by weight, (iii) 0.1% - 2% N by weight, (iv) 2% - 4% N by weight, (v) 4% - 8% N by weight, (vi) 5% - 9% N by weight, (vii) 6% - 9% N by weight, (viii) 6% - 8% N by weight, (ix) 7% - 9% N by weight, (x) 8% - 9% N by weight (in each case, %N is determined by combustion analysis (e.g., as described in Example 2 herein) and corresponds to the amount of the compound of formula (I) in the modified polymer), and includes an increase in %N (compared to an unmodified polymer, e.g., alginate).

[0238] Particles (e.g., hydrogel capsules) (e.g., a first compartment, or a second compartment therein) can contain a compound of formula (I) in an amount that imparts a particular characteristic to the particle. For example, the particle surface (e.g., the outside of the outer compartment) can include a concentration, or density, of the compound of formula (I) such that the particle is non-fibrous in a subject (i.e., reduces a foreign body reaction). In one embodiment, the particle surface includes an alginate chemically modified with an effective amount of compound 101 for non-fibrosis. In one embodiment, an effective amount of compound 101 for non-fibrosis results in an increase in %N of about 0.5% - 2%, 2% - 4% N, about 4% - 6% N, about 6% - 8%, or about 8% - 10% N (compared to unmodified alginate), and %N is determined by combustion analysis (e.g., as described in Example 2 herein) and corresponds to the amount of compound 101 in the modified alginate.

[0239] In one embodiment, a mechanical test of the hydrogel capsule is performed using a 5 mm probe attached to a 5 kg load cell on a TA.XT plus Texture Analyzer (Stable Micro Systems, Surrey, United Kingdom). Individual capsules are placed on the platform and compressed from above by the probe at a fixed rate of 0.5 mm / second. When a repulsive force of 1 g is measured, contact between the probe and the capsule is detected. The probe then continues to move 90% of the distance between the contact height of the probe and the platform, compressing the capsule to the point of rupture. The resistance of the probe to the compression force is measured and can be plotted as a function of the probe displacement (force vs. displacement curve). Typically, the capsule ruptures slightly before complete rupture, and the force applied to the probe decreases by a small amount. An analysis macro can be programmed to detect the first occurrence of a 0.25 - 0.5 g decrease in the force vs. displacement curve. The force applied by the probe when this occurs is called the initial rupture force. In one embodiment, the desired mechanical strength of the particles (e.g., two - compartment hydrogel capsules) described herein has an initial rupture force of greater than 1, 1.5, 2, 2.5, or 3 grams, or at least 2 grams.

[0240] In one embodiment, the desired mechanical strength of the particles (e.g., hydrogel capsules) is the ability to remain intact at a desired time point after transplantation into a subject. For example, both the outer and inner compartments of the hydrogel capsule removed from the subject are visually intact when observed by light microscopy, e.g., by bright - field imaging as described in the examples herein, after recovery from immunocompetent mice.

[0241] In one embodiment, the particle surface comprises an alginate chemically modified with Compound 101 at a concentration, or density, of the Compound 101 in the modified alginate that provides both non-fibrous characteristics and a desired mechanical strength to the particle, e.g., an N% increase (compared to unmodified alginate) of any of the following values: (i) from 1% to 3% by weight, (ii) an N of from 2% to 4% by weight, (iii) an N of from 4% to 8% by weight, (iv) an N of from 5% to 9% by weight, (v) an N of from 6% to 9% by weight, (vi) an N of from 6% to 8% by weight, (vii) an N of from 7% to 9% by weight, and (ix) an N of from 8% to 9% by weight (in each case, %N is determined by combustion analysis (e.g., as described in Example 2 herein) and corresponds to the amount of the Compound of Formula (I) in the modified alginate).

[0242] The particle (e.g., a first compartment and a second compartment therein) comprises alginate, which can be chemically modified with a compound of Formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid sites can be activated to couple to one or more amine-functionalized compounds to achieve an alginate modified with a compound of Formula (I). The alginate polymer can be dissolved in water (30 mL / gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 equivalent) and N-methylmorpholine (1 equivalent). A solution (0.3 M) of the compound of Formula (I) in a buffer or solvent, e.g., acetonitrile, can be added to this mixture. The reaction can be heated to 55° C. for 16 hours, then cooled to room temperature and concentrated via rotary evaporation. The residue can then be dissolved in a buffer or solvent, e.g., water. The mixture can then be filtered, e.g., through a cyano-modified silica gel (Silicycle) bed, and the filter cake can be washed with water. The resulting solution can then be exchanged with a buffer or water at least once, at least twice, at least three times, or more than that and dialyzed against a buffer or water for 24 hours (10,000 MWCO membrane). The resulting solution can be concentrated, e.g., via lyophilization, to provide the desired chemically modified alginate.

[0243] In some embodiments, the particles described herein comprise cells. In some embodiments, the cells are engineered to produce a therapeutic agent (e.g., a protein, or polypeptide, such as an antibody, protein, enzyme, or growth factor). In some embodiments, the cells are disposed with a first compartment. In some embodiments, the cells are disposed with a second compartment. In some embodiments, the cells are disposed within a first compartment and the second compartment is cell-free. The particles (e.g., hydrogel capsules) can include active or inactive fragments of a protein, or polypeptide, such as glucose oxidase (e.g., for a glucose sensor), kinase, phosphatase, oxygenase, hydrogenase, or reductase, etc.

[0244] The particles (e.g., hydrogel capsules) described herein can be configured to release a therapeutic agent, e.g., an exogenous substance, e.g., a therapeutic agent described herein. In some embodiments, the therapeutic agent is a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutic agent is a biological substance. In some embodiments, the therapeutic agent is a nucleic acid (e.g., RNA or DNA), protein (e.g., hormone, enzyme, antibody, antibody fragment, antigen, or epitope), small molecule, lipid, drug, vaccine, or any derivative thereof.

[0245] Particles (e.g., hydrogel capsules) (e.g., as described herein) can be provided as a preparation, or composition, for implantation, or administration to a subject. In some embodiments, at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the particles (e.g., hydrogel capsules) in the preparation, or composition, have the properties described herein, such as an average diameter, or average pore size.

[0246] Cells and Therapeutic Agents The hydrogel capsules of the present disclosure can contain a wide variety of different cell types (e.g., human cells), including, but not limited to, adipocytes, epidermal cells, epithelial cells, endothelial cells, fibroblasts, pancreatic islet cells, mesenchymal stem cells, pericytes, subtypes of any of the foregoing, cells derived from any of the foregoing, cells derived from induced pluripotent stem cells, and mixtures of one or more of the foregoing. Exemplary cell types include those described in WO2017 / 075631 and WO2019 / 195055. In one embodiment, the hydrogel capsules described herein contain a plurality of cells. In one embodiment, the plurality of cells are in the form of a cell suspension prior to being encapsulated within the hydrogel capsules described herein. The cells in the suspension can be in the form of single cells (e.g., from a monolayer cell culture), or in another form, for example, disposed on a microcarrier (e.g., beads, or matrix), or provided as three-dimensional aggregates of cells (e.g., cell clusters, or spheroids). The cell suspension can contain a plurality of cell clusters (e.g., as spheroids), or microcarriers. In some embodiments, the hydrogel capsules do not contain any pancreatic islet cells and do not contain any cells capable of producing insulin in response to glucose.

[0247] The hydrogel capsules of the present disclosure reduce the adhesion of immune cells as compared to an untreated control. In one embodiment, the hydrogel capsules reduce the adhesion of macrophages as compared to an untreated control. In one embodiment, the reduction in macrophage adhesion is between about 1-fold and 10-fold less than that of the untreated control. In one embodiment, the reduction in macrophage adhesion is between about 1-fold and 8-fold less than that of the untreated control. In one embodiment, the reduction in macrophage adhesion is between about 1-fold and 7-fold less than that of the untreated control. In one embodiment, the reduction in macrophage adhesion is between about 1-fold and 6-fold less than that of the untreated control. In one embodiment, the reduction in macrophage adhesion is between about 1-fold and 5-fold less than that of the untreated control.

[0248] The hydrogels or hydrogel capsules of the present disclosure enable the encapsulated cells (e.g., engineered cells) to retain viability (e.g., as determined by a cell viability assay). In some embodiments, the hydrogel or hydrogel capsule enables the encapsulated cells to retain viability for at least 7 days, at least 1 month, or at least 1 year.

[0249] The present disclosure features cells that produce, or are capable of producing, a therapeutic agent for the prevention or treatment of a disease, disorder, or condition described herein. In one embodiment, the cells are engineered cells. In one embodiment, the cells are engineered to sense a stimulus, such as a chemical signal, and express a therapeutic agent in response to the stimulus. The therapeutic agent can be any biological substance, such as a nucleic acid (e.g., nucleotide, DNA, or RNA), polypeptide, lipid, sugar (e.g., monosaccharide, disaccharide, oligosaccharide, or polysaccharide), or small molecule (each of which is described in further detail below). Exemplary therapeutic agents include the agents listed in WO2017 / 075631 and WO2019 / 195055.

[0250] In some embodiments, the cells (e.g., engineered cells) produce a nucleic acid. The nucleic acids produced by the cells described herein can be of various sizes and can contain one or more nucleosides or nucleotides, e.g., 2, 3, 4, 5, 10, 25, 50 or more nucleosides or nucleotides. In some embodiments, the nucleic acid is, for example, a short fragment of RNA or DNA and can be used, for example, as a reporter or for diagnostic purposes. Exemplary nucleic acids include single nucleosides or nucleotides (e.g., adenosine, thymidine, cytidine, guanosine, uridine monophosphate, inosine monophosphate), RNA (e.g., mRNA, siRNA, miRNA, RNAi), and DNA (e.g., vectors, chromosomal DNA). In some embodiments, the nucleic acid has an average molecular weight (kD) of about 0.25, 0.5, 1, 1.5, 2, 2.5, 5, 10, 25, 50, 100, 150, 200 or more.

[0251] In some embodiments, the therapeutic agent is a peptide or polypeptide (e.g., a protein) such as a hormone, an enzyme, a cytokine (e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine), a growth factor, a coagulation factor, or a lipoprotein. A peptide or polypeptide (e.g., a protein, e.g., a hormone, a growth factor, a blood coagulation factor or a coagulation factor, an antibody molecule, an enzyme, a cytokine, a cytokine receptor, or a chimeric protein comprising a cytokine or a cytokine receptor) produced by cells within the implantable element can have a naturally occurring amino acid sequence or can include variants of a naturally occurring sequence. The variant can be a naturally occurring or non-naturally occurring amino acid substitution, mutation, deletion, or addition compared to a naturally occurring reference sequence. The naturally occurring amino acid sequence can be a polymorphic variant. The naturally occurring amino acid sequence can be a human or non-human amino acid sequence. In some embodiments, the naturally occurring amino acid sequence, or a naturally occurring variant thereof, is a human sequence. Further, a peptide, or polypeptide (e.g., a protein) for use in the present invention can be modified in any way, for example, by chemical or enzymatic modification (e.g., glycosylation, phosphorylation). In some embodiments, the peptide has about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the protein has an average molecular weight (kD) of 5, 10, 25, 50, 100, 150, 200, 250, 500 or more.

[0252] In some embodiments, the protein is a hormone. Exemplary hormones include antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone-releasing hormone (GHRH), thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), thyroxine, calcitonin, parathyroid hormone, aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone. In some embodiments, the protein is insulin (e.g., insulin A-chain, insulin B-chain, or proinsulin). In some embodiments, the protein is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone. In some embodiments, the protein is not insulin (e.g., insulin A-chain, insulin B-chain, or proinsulin).

[0253] In some embodiments, the protein is a growth factor, such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), and insulin-like growth factors -I and -II (IGF-I and IGF-II).

[0254] In some embodiments, the protein is a clotting factor, or coagulation factor, for example, a blood clotting factor, or a blood coagulation factor. In some embodiments, the protein is a protein involved in clotting, i.e., the process by which blood is converted from a liquid to a solid, or a gel. Exemplary clotting factors and coagulation factors include Factor I (e.g., fibrinogen), Factor II (e.g., prothrombin), Factor III (e.g., tissue factor), Factor V (e.g., proaccelerin, labile factor), Factor VI, Factor VII (e.g., stable factor, proconvertin), Factor VIII (e.g., antihemophilic factor A), Factor VIIIC, Factor IX (e.g., antihemophilic factor B), Factor X (e.g., Stuart - Power factor), Factor XI (e.g., plasma thromboplastin antecedent), Factor XII (e.g., Hageman factor), Factor XIII (e.g., fibrin - stabilizing factor), von Willebrand factor, prekallikrein, heparin cofactor II, high - molecular - weight kininogen (e.g., Fitzgerald factor), antithrombin III, and fibronectin. In some embodiments, the protein is an anticoagulant factor, for example, protein C.

[0255] In some embodiments, the protein is an antibody molecule. As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain, or a fragment thereof, that includes, for example, at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, monoclonal antibodies (including full - length antibodies having an immunoglobulin Fc region). In one embodiment, the antibody molecule includes a full - length antibody, or a full - length immunoglobulin chain. In one embodiment, the antibody molecule includes an antigen - binding fragment, or a functional fragment, of a full - length antibody, or a full - length immunoglobulin chain. In one embodiment, the antibody molecule is a monospecific antibody molecule that binds to a single epitope, for example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each of which binds to the same epitope. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, the same protein (or a subunit of a multimeric protein). In one embodiment, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0256] Cells within the implantable element described herein can generate various types of antibody molecules, including synthetic proteins that contain at least all classes of immunoglobulins, fragments thereof, and antigen-binding variable domains of antibodies. Antibody molecules can be antibodies, such as IgG antibodies like IgG1, IgG2, IgG3, or IgG4. Antibody molecules can be in the form of antigen-binding fragments, including Fab fragments, F(ab’)2 fragments, single-chain variable regions, etc. Antibodies can be polyclonal or monoclonal (mAb). Monoclonal antibodies include "chimeric" antibodies (where a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species, or an antibody belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody derived from another species, or an antibody belonging to another antibody class or subclass, as long as it specifically binds to the target antigen and / or exhibits the desired biological activity) and fragments of such antibodies, as long as they specifically bind to the target antigen and / or exhibit the desired biological activity. In some embodiments, the antibody molecule is a single-domain antibody (e.g., nanobody). The described antibodies can also be modified by recombinant means, such as by amino acid deletion, addition, or substitution, to enhance the effectiveness of the antibody in mediating the desired function. Exemplary antibodies include anti-beta-galactosidase, anti-collagen, anti-CD14, anti-CD20, anti-CD40, anti-HER2, anti-IL-1, anti-IL-4, anti-IL6, anti-IL-13, anti-IL17, anti-IL18, anti-IL-23, anti-IL-28, anti-IL-29, anti-IL-33, anti-EGFR, anti-VEGF, anti-CDF, anti-flagellin, anti-IFN-α, anti-IFN-β, anti-IFN-γ, anti-mannose receptor, anti-VEGF, anti-TLR1, anti-TLR2, anti-TLR3, anti-TLR4, anti-TLR5, anti-TLR6, anti-TLR9, anti-PDF, anti-PD1, anti-PDL-1, or anti-nerve growth factor antibodies. In some embodiments, the antibody is an anti-nerve growth factor antibody (e.g., fulranumab, fasinumab, tanezumab).

[0257] In some embodiments, the protein is a cytokine, or a cytokine receptor, or a chimeric protein comprising a cytokine, or their receptors, for example, tumor necrosis factor alpha, and beta, their receptors, and their derivatives, renin, lipoprotein, colchicine, corticotropin, vasopressin, somatostatin, repressin, pancreatozymin, leuprolide, alpha-1-antitrypsin, atrial natriuretic factor, pulmonary surfactant, plasminogen activators other than tissue-type plasminogen activator (t-PA), for example, urokinase, bombesin, thrombin, enkephalinase, RANTES (usually, its expression and secretion are controlled when T cells are activated), human macrophage inflammatory protein (MIP-1-alpha), serum albumin, for example, human serum albumin, Mullerian duct inhibitor, relaxin A-chain, relaxin B-chain, prorelaxin, mouse gonadotropin-related peptide, chorionic gonadotropin, microbial proteins, for example, beta-lactamase, DNase, inhibin, activin, receptors for hormones, or growth factors, integrin, protein A, or D, rheumatoid factor, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF), for example, TGF-alpha, and TGF-beta (including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5), insulin-like growth factor-I, and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins, CD proteins, for example, CD-3, CD-4, CD-8, and CD-19, erythropoietin, bone-inductive factors, antitoxins, interferons, for example, interferon-alpha (for example, interferon alpha 2A), -beta, -gamma, -lambda, and consensus interferon, colony-stimulating factors (CSF), for example, M-CSF, GM-CSF, and G-CSF, interleukins (IL), for example, IL-1 to IL-10, superoxide dismutase, T cell receptor, surface membrane proteins, degradation promoting factors, transport proteins, homing receptors, addressins, contraceptives, for example, prostaglandins, fertility promoters,Controlled proteins, antibodies (including fragments thereof), and chimeric proteins, such as immunoadhesins, precursors, derivatives, prodrugs, and analogs of these compounds, and pharmaceutically acceptable salts of these compounds, or their precursors, derivatives, prodrugs, and analogs are included. Suitable proteins or peptides can be native or recombinant and include, for example, fusion proteins.

[0258] Examples of polypeptides (e.g., proteins) produced by cells in the implantable elements described herein also include CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (KC), CXCL2 (SDF1a), CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNFB (LTA), TNFC (LTB), TNFSF4, TNFSF5 (CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL21, IL3, IL5, IL6, IL11, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL16, IL32, IL34, IL10, IL22, IL19, IL20, IL24, IL26, IL29, IFNL1, IFNL2, IFNL3, IL28, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNB1, IFN K, IFN W1, IFNG, IL1A (IL1F1), IL1B (IL1F2), IL1Ra (IL1F3), IL1F5 (IL36RN), IL1F6 (IL36A), IL1F7 (IL37), IL1F8 (IL36B), IL1F9 (IL36G), IL1F10 (IL38), IL33 (IL1F11), IL18 (IL1G), IL17, KITLG, IL25 (IL17E), CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), SPP1, TGFB1, TGFB2,TGFB3, CCL3L1, CCL3L2, CCL3L3, CCL4L1, CCL4L2, IL17B, IL17C, IL17D, IL17F, AIMP1(SCYE1), MIF, Areg, BC096441, Bmp1, Bmp10, Bmp15, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, C1qtnf4, Ccl21a, Ccl27a, Cd70, Cer1, Cklf, Clcf1, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlf1, Ctf2, Ebi3, Edn1, Fam3b, Fasl, Fgf2, Flt3l, Gdf10, Gdf11, Gdf15, Gdf2, Gdf3, Gdf5, Gdf6, Gdf7, Gdf9, Gm12597, Gm13271, Gm13275, Gm13276, Gm13280, Gm13283, Gm2564, Gpi1, Grem1, Grem2, Grn, Hmgb1, Ifna11, Ifna12, Ifna9, Ifnab, Ifne, Il17a, Il23a, Il25, Il31, Iltifb, Inhba, Lefty1, Lefty2, Mstn, Nampt, Ndp, Nodal, Pf4, Pglyrp1, Prl7d1, Scg2, Scgb3a1, Slurp1, Spp1, Thpo, Tnfsf10, Tnfsf11, Tnfsf12, Tnfsf13, Tnfsf13b, Tnfsf14, Tnfsf15, Tnfsf18, Tnfsf4, Tnfsf8, Tnfsf9, Tslp, Vegfa, Wnt1, Wnt2, Wnt5a, Wnt7a, Xcl1, Epinephrine, Melatonin, Triiodothyronine, Prostaglandin, Leukotriene, Prostacyclin, Thromboxane, Pancreatic Amylin, Müllerian Inhibiting Factor or Hormone, Adiponectin, Corticotropin, Angiotensin, Vasopressin, Arginine Vasopressin, Atriopeptin, Brain Natriuretic Peptide, Calcitonin, Cholecystokinin, Cortistatin, Enkephalin, Endothelin, Erythropoietin, Follicle-Stimulating Hormone, Galanin, Gastric Inhibitory Polypeptide, Gastrin, Ghrelin, Glucagon, Glucagon-Like Peptide-1, Gonadotropin-Releasing Hormone, Hepcidin, Human Chorionic Gonadotropin,Human placental lactogen, inhibin, somatomedin, leptin, lipotropin, melanocyte-stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, pituitary adenylate cyclase-activating peptide, relaxin, renin, secretin, somatostatin, thrombopoietin, thyrotropin, thyrotropin-releasing hormone, vasoactive intestinal peptide, androgen, alpha-glucosidase (also known as acid maltase), glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine palmitoyltransferase, carnitine, and myoadenylate deaminase are included.

[0259] In some embodiments, the protein is a replacement therapy or a replacement protein. In some embodiments, the replacement therapy or replacement protein is a clotting factor or coagulation factor, for example, factor VIII (e.g., including the naturally occurring human factor VIII amino acid sequence or a variant thereof) or factor IX (e.g., including the naturally occurring human factor IX amino acid sequence or a variant thereof).

[0260] In some embodiments, the cell is engineered to express factor VIII, for example, recombinant factor VIII. In some embodiments, the cell is derived from human tissue and is engineered to express factor VIII, for example, recombinant factor VIII. In some embodiments, the recombinant factor VIII is a B-domain deleted recombinant factor VIII (FVIII-BDD).

[0261] In some embodiments, the cells are derived from human tissue and engineered to express Factor IX, such as recombinant Factor IX. In some embodiments, the cells are engineered to express Factor IX, such as wild-type human Factor IX (FIX), or a polymorphic variant thereof. In some embodiments, the cells are engineered to express a gain-of-function (GIF) variant of wild-type FIX protein (FIX-GIF), and the GIF variant has a higher specific activity than the corresponding wild-type FIX.

[0262] In some embodiments, the replacement therapy, or replacement protein, is an enzyme, such as alpha-galactosidase, alpha-L-iduronidase (IDUA), or N-sulphoglucosamine sulphonohydrolase (SGSH). In some embodiments, the replacement therapy, or replacement protein, is an enzyme, such as alpha-galactosidase A (including, for example, the naturally occurring human alpha-galactosidase A amino acid sequence, or a variant thereof). In some embodiments, the replacement therapy, or replacement protein, is a cytokine, or an antibody.

[0263] In some embodiments, the therapeutic agent is a sugar such as a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. In some embodiments, the sugar comprises a triose, tetrose, pentose, hexose, or heptose moiety. In some embodiments, the sugar comprises a linear monosaccharide or a cyclic monosaccharide. In some embodiments, the sugar comprises a glucose, galactose, fructose, rhamnose, mannose, arabinose, glucosamine, galactosamine, sialic acid, mannosamine, glucuronic acid, galacturonic acid, mannuronic acid, or guluronic acid moiety. In some embodiments, the sugar is bound to a protein (e.g., an N-linked glycan or an O-linked glycan). Exemplary sugars include glucose, galactose, fructose, mannose, rhamnose, sucrose, ribose, xylose, sialic acid, maltose, amylose, inulin, fructooligosaccharide, galactooligosaccharide, mannan, lectin, pectin, starch, cellulose, heparin, hyaluronic acid, chitin, amylopectin, or glycogen. In some embodiments, the therapeutic agent is a sugar alcohol.

[0264] In some embodiments, the therapeutic agent is a lipid. The lipid can be hydrophobic or amphiphilic and can form a tertiary structure such as a liposome, vesicle, or membrane, or an insert into a liposome, vesicle, or membrane. The lipid can include a fatty acid, glycerolipid, glycerophospholipid, sterol lipid, prenol lipid, sphingolipid, glycolipid, polyketide, or sphingolipid. Examples of lipids produced by the cells described herein include anandamide, docosahexaenoic acid, prostaglandin, leukotriene, thromboxane, eicosanoid, triglyceride, cannabinoid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, ceramide, sphingomyelin, cerebroside, ganglioside, estrogen, androsterone, testosterone, cholesterol, carotenoid, quinone, hydroquinone, or ubiquinone.

[0265] In some embodiments, the therapeutic agent is a small molecule. Small molecules may include natural products produced by cells. In some embodiments, the small molecule has low availability or does not conform to Lipinski's rules of five (a set of guidelines used to estimate the likelihood that a small molecule will be an orally active drug in humans, see, e.g., Lipinski, C.A. et al (2001) Adv Drug Deliv 46:2-36). Exemplary small molecule natural products include antibacterial agents (e.g., carmonam, daptomycin, fidaxomicin, fosfomycin, isepamicin, micronomicin sulfate, myocamycin, mupirocin, netilmicin sulfate, teicoplanin, thienamycin, rifamycin, erythromycin, vancomycin), antiparasitic agents (e.g., artemisinin, ivermectin), anticancer agents (e.g., doxorubicin, aclarubicin, aminolevulinic acid, argrabin, omacetaxine mepesuccinate, paclitaxel, pentostatin, peplomycin, romidepsin, trabectdin, actinomycin D, bleomycin, chromomycin A, daunorubicin, leucovorin, neocarzinostatin, streptozocin, trabectedin, vinblastine, vincristine), antidiabetic agents (e.g., voglibose), central nervous system agents (e.g., L-dopa, galantamine, ziconotide), statins (e.g., mevastatin), antifungal agents (e.g., fumagillin, cyclosporine), 1-deoxynojirimycin, and theophylline, steroids (cholesterol, estrogen, testosterone). For additional small molecule natural products, see Newman, D.J. and Cragg, M. (2016) J Nat Prod 79:629-661 and Butler, M.S. et al (2014) Nat Prod Rep 31:1612-1661.

[0266] In some embodiments, the cells are engineered to synthesize small non-protein or non-peptide molecules. For example, in one embodiment, the cells can produce statins (e.g., taurastatin, pravastatin, fluvastatin, or atorvastatin).

[0267] In some embodiments, the therapeutic agent is an antigen (e.g., a viral antigen, a bacterial antigen, a fungal antigen, a plant antigen, an environmental antigen, or a tumor antigen). Antigens are recognized by those skilled in the art as being immunostimulatory, i.e., capable of stimulating an immune response or conferring effective immunity against the organism or molecule from which they are derived. Antigens can be nucleic acids, peptides, proteins, sugars, lipids, or combinations thereof.

[0268] Cells, e.g., engineered cells, e.g., the engineered cells described herein, can produce a single therapeutic agent or multiple therapeutic agents. In some embodiments, the cells produce a single therapeutic agent. In some embodiments, a cell cluster comprises cells that produce a single therapeutic agent. In some embodiments, at least about 1 percent, or about 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, or 99 percent of the cells in the cluster produce a single therapeutic agent (e.g., a therapeutic agent described herein). In some embodiments, the cells produce multiple therapeutic agents, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 therapeutic agents. In some embodiments, a cell cluster comprises cells that produce multiple therapeutic agents. In some embodiments, at least about 1 percent, or about 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, or 99 percent of the cells in the cluster produce multiple therapeutic agents (e.g., a therapeutic agent described herein).

[0269] These therapeutic agents may be related or may form complexes. In some embodiments, the therapeutic agent is secreted or released from the cell in an active form. In some embodiments, the therapeutic agent is secreted or released from the cell in an inactive form, for example, as a prodrug. In the latter case, the therapeutic agent can be activated by a downstream agent such as an enzyme. In some embodiments, the therapeutic agent is not secreted or released from the cell, but is maintained within the cell. For example, the therapeutic agent can be an enzyme involved in the detoxification or metabolism of unwanted substances, and the detoxification or metabolism of the unwanted substances occurs intracellularly.

[0270] In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 1 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 2 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 3 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 4 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 5 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 6 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 7 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 8 M mL -1 thereof. In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 9 M mL-1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 10M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 15M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 20M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 25M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 30M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 35M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 40M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 45M mL -1 contains mammalian cells at a concentration of. In some embodiments, the hydrogel capsules described herein are about 50M mL -1 contains mammalian cells at a concentration of.

[0271] In some embodiments, the hydrogel capsules described herein are about 1 - 50M mL -1 , 1 - 45M mL -1 , 1 - 40M mL -1 , 1 - 35M mL -1 , 1 - 30M mL -1 , 1 - 25M mL -1 , 1 - 20M mL -1 , 1 - 15M mL -1 , 1 - 10M mL -1 , 1 - 5M mL -1 , 5 - 50M mL -1 , 5 - 45M mL -1 , 5 - 40M mL -1 , 5 - 35M mL-1 , 5 to 30 M mL -1 , 5 to 25 M mL -1 , 5 to 20 M mL -1 , 5 to 15 M mL -1 , 5 to 10 M mL -1 , 10 to 50 M mL -1 , 10 to 45 M mL -1 , 10 to 40 M mL -1 , 10 to 35 M mL -1 , 10 to 30 M mL -1 , 10 to 25 M mL -1 , 10 to 20 M mL -1 , 10 to 15 M mL -1 , 15 to 50 M mL -1 , 15 to 45 M mL -1 , 15 to 40 M mL -1 , 15 to 35 M mL -1 , 15 to 30 M mL -1 , 15 to 25 M mL -1 , 15 to 20 M mL -1 , 20 to 50 M mL -1 , 20 to 45 M mL -1 , 20 to 40 M mL -1 , 20 to 35 M mL -1 , 20 to 30 M mL -1 , or 20 to 25 M mL -1 contains mammalian cells at a concentration of...

[0272] Therapeutic method Described herein is a method for preventing or treating a disease, disorder, or condition in a subject through administration or transplantation of a hydrogel capsule comprising (i) a polysaccharide polymer described herein and (ii) pancreatic islet cells. In some embodiments, the methods described herein directly or indirectly reduce or alleviate at least one symptom of a disease, disorder, or condition (e.g., type 1 diabetes). In some embodiments, the methods described herein prevent or delay the onset of a disease, disorder, or condition (e.g., type 1 diabetes). In some embodiments, the subject is human.

[0273] In some embodiments, the disease, disorder, or condition affects a bodily system, such as, for example, the nervous system (e.g., the peripheral nervous system, or the central nervous system), the vascular system, the skeletal system, the respiratory system, the endocrine system, the lymphatic system, the genital system, or the gastrointestinal tract. In some embodiments, the disease, disorder, or condition affects a part of the body, such as blood, the eye, the brain, the skin, the lung, the stomach, the mouth, the ear, the leg, the foot, the hand, the liver, the heart, the kidney, the bone, the pancreas, the spleen, the large intestine, the small intestine, the spinal cord, the muscle, the ovary, the uterus, the vagina, or the penis.

[0274] In some embodiments, the disease, disorder, or condition is an autoimmune disease. In some embodiments, the disease, disorder, or condition is diabetes (type 1 or type 2).

[0275] The present disclosure further includes a method for identifying a subject having or suspected of having a disease, disorder, or condition (e.g., type 1 diabetes) described herein, and in response to such identification, administering to the subject (i) a polysaccharide polymer described herein, and (ii) a hydrogel capsule containing pancreatic islet cells, wherein the hydrogel capsule is optionally modified with a compound of formula (I) or a composition thereof. In one embodiment, the subject has or is diagnosed with diabetes (e.g., type 1 diabetes). The subject may have any biomarker or other diagnostic criterion associated with diabetes, such as, for example, elevated blood glucose levels (e.g., greater than 300 mg / dL, greater than 400 mg / dL), or elevated hemoglobin A1C levels (e.g., greater than 5.9% hemoglobin A1C level, greater than 6.5% hemoglobin A1C level, greater than 7% hemoglobin A1C level). In one embodiment, the subject is human. In one embodiment, the subject is an adult. In one embodiment, the subject is a pediatric subject (e.g., a subject less than 21 years old, less than 18 years old, less than 15 years old, less than 12 years old, less than 10 years old, or less than 6 years old).

[0276] Method for producing particles The present disclosure further includes a method of making the particles described herein, such as particles comprising a first compartment, a second compartment, and a compound of formula (I). In some embodiments where the particles are hydrogel capsules, the method of making the particles comprises contacting a plurality of droplets comprising a first and a second polymer solution (e.g., each comprising a hydrogel-forming polymer) with an aqueous crosslinking solution. The droplets can be formed using any technique known in the art.

[0277] Each compartment of the particles described herein can include an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinking agent, or a blend thereof. Briefly, when performing a method of preparing particles configured as two-compartment hydrogel capsules, the volume of a first polymer solution (e.g., including an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinking agent, or a blend thereof, and optionally containing cells) is filled into a first syringe connected to the lumen of a coaxial needle. The first syringe can then be connected to a syringe pump oriented vertically above a container containing an aqueous crosslinking solution including a crosslinking agent, a buffer, and an osmotic pressure regulator. The volume of a second polymer solution (e.g., including an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinking agent, or a blend thereof, and optionally containing cells) is filled into a second syringe connected to the outer lumen of the coaxial needle. The second syringe can then be connected to a syringe pump oriented horizontally with respect to the container containing the crosslinking solution. Next, a high-voltage power generator can be connected to the top and bottom of the needle. Next, using the syringe pumps and the power generator, the first and second polymer solutions can be extruded through the syringes at settings determined to achieve a desired droplet rate of the polymer solutions into the crosslinking solution. One of ordinary skill in the art can readily determine various combinations of needle lumen size, voltage range, flow rate, droplet rate, and droplet distance to produce a two-compartment hydrogel capsule composition, where most (e.g., at least 80%, 85%, 90%, or more) of the capsules are within 10% of the target size and have a spherical-like shape. After discharging the first and second volumes of the polymer solution, the droplets can be crosslinked in the crosslinking solution for a period of time, e.g., about 5 minutes.

[0278] Exemplary process parameters for preparing a composition of millicapsules (e.g., millicapsules with a diameter of 1.5 mm) include the following. Place the coaxial needle above the surface of the crosslinking solution at a distance sufficient to provide a drop distance from the needle tip to the solution surface. In one embodiment, the distance between the needle tip and the solution surface is between 1 and 5 cm. In one embodiment, the first and second polymer solutions are extruded through the needle at a total flow rate between 0.05 mL / min and 5 mL / min, or between 0.05 mL / min and 2.5 mL / min, or between 0.05 mL / min and about 1 mL / min, or between 0.05 mL / min and 0.5 mL / min, or between 0.1 mL / min and 0.5 mL / min. In one embodiment, the first and second polymer solutions are extruded through the needle at a total flow rate of about 0.05 mL / min, 0.1 mL / min, 0.15 mL / min, 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, or 0.5 mL / min. In one embodiment, the flow rates of the first and second polymer solutions through the needle are substantially the same. In one embodiment, the flow rates of the first and second polymer solutions through the needle are different.

[0279] In one embodiment, the voltage of the device is between 1 kV and 20 kV, or between 1 and 15 kV, or between 1 kV and 10 kV, or between 5 kV and 10 kV. The voltage may be adjusted until the desired droplet velocity is reached. In one embodiment, the droplet velocity of the device is between 1 droplet / 10 seconds and 50 droplets / 10 seconds, or between 1 droplet / 10 seconds and 25 droplets / 10 seconds.

[0280] In one embodiment, the number of non-particle fragments on the surface of the crosslinking solution is determined. Subsequently, the particles that have fallen to the bottom of the crosslinking vessel can be collected, for example, by transferring the crosslinking solution containing the particles to another vessel and leaving the non-particle fragments on the solution surface of the original crosslinking vessel. Thereafter, the removed particles can be sedimented, the crosslinking solution can be removed, and then the particles can be washed one or more times with a buffer solution (e.g., HEPES buffer solution). In one embodiment, one or more aliquots of the resulting particle composition (e.g., a preparation of the particles) are examined by microscopy to evaluate the quality of the composition, e.g., particle defects and the number of satellite particles.

[0281] In some embodiments, the crosslinking solution further comprises a processing additive (e.g., a hydrophilic, non-ionic surfactant). The processing additive can reduce the surface tension of the crosslinking solution. Agents useful as processing additives in the present disclosure include polysorbate-type surfactants, copolymers of polyethylene oxide (PEO) and polypropylene oxide (PPO), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, and non-ionic surfactants such as Tween® 20, Tween® 80, Triton™ X-100, IGEPAL® CA-630, poloxamer 188, or poloxamer 407, or surfactants having substantially the same chemical and physical properties as those listed in the exemplary surfactant table immediately below. [Table 16]

[0282] In some embodiments, the processing additive is a non-ionic surfactant. In one embodiment, the processing additive comprises two or more surfactants, for example, two or more hydrophilic surfactants. In some embodiments, the processing additive does not contain Tween® 20 (polysorbate 20) or Triton™ X-100. In one embodiment, the processing additive is IGEPAL® CA-630 (polyethylene glycol sorbitan monooleate). In some embodiments, the processing additive is poloxamer 188.

[0283] In some embodiments, the processing additive (e.g., surfactant) is present in the crosslinking solution at a concentration of at least 0.0001% or more. In some embodiments, the crosslinking solution contains at least 0.001%, 0.01%, or 0.1% of the processing additive. In some embodiments, the processing additive is present at a concentration selected from between about 0.001% and about 0.1%, about 0.005% and about 0.05%, about 0.005% and about 0.01%, and about 0.01% and about 0.5%. In one embodiment, the processing additive is a surfactant and is present at a concentration below the critical micelle concentration for the surfactant.

[0284] In some embodiments, the crosslinking agent is a single type of divalent cation or a mixture of different types, for example, containing one or more of Ba 2+ , Ca 2+ , Sr 2+ . In some embodiments, the crosslinking agent is, for example, BaCl2 at a concentration of 1 mM to 100 mM or 7.5 mM to 20 mM. In some embodiments, the crosslinking agent is, for example, CaCl2 at a concentration of 50 mM to 100 mM. In some embodiments, the crosslinking agent is, for example, SrCl2 at a concentration of 37.5 mM to 100 mM. In some embodiments, the crosslinking agent is a mixture of BaCl2 (e.g., 5 mM to 20 mM) and CaCl2 (e.g., 37.5 mM to 12.5 mM), or a mixture of BaCl2 (e.g., 5 mM to 20 mM) and SrCl2 (e.g., 37.5 mM to 12.5 mM).

[0285] In some embodiments, the crosslinking agent is SrCl2, and the processing additive is Tween® 80 (or a surfactant having substantially the same chemical and physical properties as those listed in the exemplary surfactant table) at a concentration of less than 0.1%, for example, about 0.005% to 0.05%, about 0.005% to about 0.01%. In some embodiments, the concentration of SrCl2 is about 50 mM. In some embodiments, the crosslinking agent is SrCl2, and the processing additive is poloxamer 188 at a concentration of 1%.

[0286] The type and concentration of the buffer in the aqueous crosslinking solution are selected to maintain the solution pH at approximately neutral, for example, about 6.5 to about 7.5, about 7.0 to about 7.5, or about 7.0. In one embodiment, the buffer is compatible with the particle, for example, the biologically derived material encapsulated in the cell. In some embodiments, the buffer in the aqueous crosslinking solution contains HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0287] The osmotic concentration regulator in the aqueous crosslinking solution is selected to maintain the osmotic pressure of the solution at a value similar to that of the polymer solution (including, in some embodiments, the cell suspension), for example, or with a variation of up to 20%, 10%, or 5% higher or lower. In some embodiments, the osmotic regulator is mannitol at a concentration of 0.1 M to 0.3 M.

[0288] In some embodiments, the crosslinking solution contains 25 mM HEPES buffer, 20 mM BaCl2, 0.2 M mannitol, and 0.01% poloxamer 188.

[0289] In some embodiments, the crosslinking solution contains 50 mM strontium chloride hexahydrate, 0.165 M mannitol, 25 mM HEPES, and 0.01% surfactant having substantially the same chemical and physical properties as those listed in the exemplary surfactant table of Tween 80.

[0290] In one embodiment, the processing additive is poloxamer 188, which is present in the particle composition (e.g., a preparation of particles) in a detectable amount after the washing step. Poloxamer 188 can be detected by any technique known in the art, for example, by partially or completely dissolving the particles in an aliquot of the composition by sodium sulfate precipitation and analyzing the supernatant by LC / MS.

[0291] The reduction in the surface tension of the crosslinking solution can be evaluated by any method known in the art, for example, by using a contact angle goniometer or a tensiometer, for example, via the Du Nouy ring method (see, for example, Davarci et al (2017) Food Hydrocolloids 62:119 - 127).

[0292] Exemplary recited embodiments 1. A polysaccharide polymer comprising: (i) a crosslinking site; and (ii) a compound of formula (I):

Chemical formula

[0293] 2. The polysaccharide polymer according to embodiment 1, wherein the cross-linking site is covalently bonded to a sugar monomer in the polysaccharide polymer.

[0294] 3. The polysaccharide polymer according to embodiment 2, wherein the cross-linking site is bonded to a carboxylate site in the sugar monomer.

[0295] 4. The polysaccharide polymer according to any one of embodiments 1 to 3, wherein the cross-linking site comprises an alkyl, alkenyl, alkynyl, ester, ketone, amine, thiol, cycloalkyl, heterocyclyl, aryl, or heteroaryl group.

[0296] 5. The polysaccharide polymer according to any one of embodiments 1 to 4, wherein the cross-linking site is capable of reacting with a second cross-linking site upon activation, for example, by heat, acid, or a catalyst.

[0297] 6. The polysaccharide polymer according to any one of embodiments 1 to 5, wherein the cross-linking site is present on the polysaccharide polymer at a density of at least about 1%, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more, when determined by comparison with a reference standard.

[0298] 7. When the crosslinked site is determined, for example, by comparison with a reference standard, it is present on the polysaccharide polymer at a density of 1% to 10%, for example, 1% to 8%, 1% to 6%, or 1% to 4%, the polysaccharide polymer according to any one of Embodiments 1 to 6.

[0299] 8. The polysaccharide polymer according to any one of Embodiments 1 to 7, wherein the polysaccharide polymer is selected from alginate, hyaluronate, and chitosan.

[0300] 9. The polysaccharide polymer according to any one of Embodiments 1 to 8, wherein the polysaccharide polymer is alginate.

[0301] 10. The polysaccharide polymer according to Embodiment 9, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

[0302] 11. The crosslinked site has the structure of formula (IV):

Chemical formula

[0303] 12. The polysaccharide polymer according to Embodiment 11, wherein the crosslinking site contains a thiol site.

[0304] 13. The crosslinking site has the structure of formula (V):

Chemical formula

[0305] 14. The polysaccharide polymer according to Embodiment 13, wherein the crosslinking site contains a norbornenyl site.

[0306] 15. The crosslinking site has the structure of formula (VI):

Chemical formula

[0307] 16. The polysaccharide polymer according to Embodiment 15, wherein the crosslinking site contains a maleimide site.

[0308] 17. The crosslinking site has the structure of formula (VII):

Chemical formula

[0309] 18. The polysaccharide polymer according to embodiment 17, wherein the crosslinked site contains a tetrazinyl site.

[0310] 19. The polysaccharide polymer according to any one of embodiments 1 to 18, wherein the crosslinked site has a structure selected from Table 4 or a pharmaceutically acceptable salt thereof.

[0311] 20. The polysaccharide polymer according to any one of embodiments 1 to 19, wherein the polysaccharide polymer contains one of the compounds of formula (IV), (V), (VI), or (VII) or a pharmaceutically acceptable salt thereof.

[0312] 21. The polysaccharide polymer according to any one of embodiments 1 to 20, wherein the polysaccharide polymer contains two of the compounds of formula (IV), (V), (VI), or (VII) or a pharmaceutically acceptable salt thereof.

[0313] 22. The polysaccharide polymer according to any one of embodiments 1 to 21, wherein the compound of formula (I) has a structure selected from Table 3 or a pharmaceutically acceptable salt thereof.

[0314] 23. The polysaccharide polymer according to any one of embodiments 1 to 22, wherein the compound of formula (I) is selected from compound 100, compound 101, compound 110, compound 112, compound 113, compound 114, compound 122, and compound 123 or a pharmaceutically acceptable salt thereof.

[0315] 24. The polysaccharide polymer according to any one of embodiments 1 to 23, wherein the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

[0316] 25. The polysaccharide polymer according to any one of embodiments 1 to 24, wherein the polysaccharide polymer is alginate, the crosslinked site is selected from the compounds listed in Table 4 or a pharmaceutically acceptable salt thereof, and the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

[0317] 26. A composition comprising the polysaccharide polymer according to any one of Embodiments 1 to 25.

[0318] 27. A hydrogel capsule comprising the polysaccharide polymer according to any one of Embodiments 1 to 25.

[0319] 28. The hydrogel capsule according to Embodiment 27, wherein the hydrogel capsule comprises a single compartment containing the polysaccharide polymer (for example, the polysaccharide polymer described herein).

[0320] 29. The hydrogel capsule according to Embodiment 27, wherein the hydrogel capsule comprises a plurality of compartments, and one of the compartments contains the polysaccharide polymer (for example, the polysaccharide polymer described herein).

[0321] 30. The hydrogel capsule according to Embodiment 29, wherein the hydrogel capsule comprises an inner compartment and an outer compartment.

[0322] 31. The inner compartment contains a first polysaccharide polymer containing the cross-linking site, The hydrogel capsule according to Embodiment 30, wherein the outer compartment contains a second polysaccharide polymer containing the cross-linking site.

[0323] 32. A hydrogel capsule, (i) a compound of formula (I):

Chemical formula

[0324] 33. The hydrogel capsule according to embodiment 32, wherein the polysaccharide polymer (for example, the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from alginate, hyaluronate, and chitosan.

[0325] 34. The hydrogel capsule according to any one of embodiments 32 to 33, wherein the polysaccharide polymer (for example, the first polysaccharide polymer and / or the second polysaccharide polymer) is alginate.

[0326] 35. The hydrogel capsule according to embodiments 32 to 34, wherein the first polysaccharide polymer is alginate.

[0327] 36. The hydrogel capsule according to embodiments 32 to 35, wherein the second polysaccharide polymer is alginate.

[0328] 37. The hydrogel capsule according to any one of Embodiments 32 to 36, wherein the alginate is guluronic acid (G)-rich alginate or mannuronic acid (M)-rich alginate.

[0329] 38. The crosslinked site has the structure of formula (IV):

Chemical formula

[0330] 39. The crosslinked site has a structure of formula (V):

Chemical formula

[0331] 40. The crosslinking site has the structure of formula (VI):

Chemical formula

[0332] 41. The crosslinking site has the structure of formula (VII):

Chemical formula

[0333] 42. The hydrogel capsule according to any one of Embodiments 38 to 41, wherein the compound of the formula (IV), (V), (VI), or (VII) is selected from the compounds in Table 4 or a pharmaceutically acceptable salt thereof.

[0334] 43. The hydrogel capsule according to any one of Embodiments 32 to 42, wherein the compound of the formula (I) has a structure selected from Table 3 or a pharmaceutically acceptable salt thereof.

[0335] 44. The hydrogel capsule according to any one of Embodiments 32 to 43, wherein the compound of the formula (I) is selected from Compound 100, Compound 101, Compound 110, Compound 112, Compound 113, Compound 114, Compound 122, and Compound 123, or a pharmaceutically acceptable salt thereof.

[0336] 45. The hydrogel capsule according to any one of Embodiments 32 to 44, wherein the compound of the formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

[0337] 46. The hydrogel capsule according to any one of Embodiments 32 to 45, having a diameter of 0.1 mm to 5 mm.

[0338] 47. The hydrogel capsule according to any one of Embodiments 32 to 46, having a diameter of 1 mm to 5 mm.

[0339] 48. The hydrogel capsule according to any one of Embodiments 32 to 47, having a diameter of 1 mm to 2.5 mm.

[0340] 49. The hydrogel capsule according to any one of Embodiments 32 to 48, encapsulating cells.

[0341] 50. The hydrogel capsule according to Embodiment 49, wherein the cells produce a therapeutic agent.

[0342] 51. The hydrogel capsule according to Embodiment 50, wherein the therapeutic agent is a protein, such as a hormone, a blood coagulation factor, an antibody, or an enzyme.

[0343] 52. The hydrogel capsule according to any one of Embodiments 32 to 51, formulated for transplantation into a subject (e.g., transplantation into the intraperitoneal (IP) space, peritoneal cavity, omentum, omental bursa, subcutaneous fat).

[0344] 53. The hydrogel capsule according to any one of Embodiments 32 to 52, wherein the transplantable element is formulated for transplantation into the IP space of the subject.

[0345] 54. A composition comprising the hydrogel capsule according to any one of Embodiments 32 to 53.

[0346] 55. A method for producing a hydrogel capsule comprising the polysaccharide polymer according to any one of Embodiments 1 to 24.

[0347] 56. A method for increasing the stability of a hydrogel capsule containing a polysaccharide polymer, the method comprising providing both means of ionically crosslinking the polysaccharide polymer and covalently crosslinking the polysaccharide polymer.

[0348] 57. The means of ionically crosslinking the polysaccharide polymer according to embodiment 56, wherein the means comprises the use of a divalent cation (e.g., Ba 2+ , Ca 2+ , Sr 2+ ).

[0349] 58. The method according to any one of embodiments 56 to 57, wherein the means of covalently crosslinking the polysaccharide polymer comprises the use of a crosslinking site.

[0350] 59. The polysaccharide polymer according to any one of embodiments 1 to 25, wherein the polysaccharide retains additional carboxylic acid groups after addition of a clickable crosslinking agent.

[0351] 60. The polysaccharide polymer according to any one of embodiments 1 to 25 or 59, which is not reduced (e.g., not treated with a reducing agent) before being modified with a clickable crosslinking agent.

[0352] 61. The polysaccharide polymer according to any one of embodiments 1 to 25 or 59, which is not oxidized (e.g., not treated with an oxidizing agent) before being modified with a clickable crosslinking agent.

[0353] 62. The polysaccharide polymer according to any one of embodiments 1 to 25 or 59 to 61, wherein the clickable crosslinking agent is not hydrolyzable.

[0354] 63. The polysaccharide polymer according to any one of embodiments 1 to 25 or 59 to 62, wherein the compound of formula (I) is not hydrolyzable.

[0355] 64. The polysaccharide polymer according to any one of embodiments 1 to 25 or 59 to 63, wherein the clickable crosslinking agent is not a thiol or vinyl sulfone.

[0356] 65. A method for treating a disease, disorder, or medical condition in a subject, the method comprising administering to the subject a hydrogel capsule according to any one of embodiments 32 to 53.

[0357] 66. The method according to embodiment 65, wherein the disease, disorder, or medical condition is diabetes (e.g., type 1 diabetes).

[0358] 67. The method according to embodiment 65, wherein the disease, disorder, or medical condition is not diabetes (e.g., type 1 diabetes).

[0359] 68. The method according to any one of embodiments 65 to 67, wherein the subject is a human.

Examples

[0360] The following examples are provided so that the invention described herein can be more fully understood. The synthetic and biological examples described in this application are provided to illustrate the compounds, compositions, devices, and methods provided herein and should in no way be construed as limiting their scope.

[0361] The compounds, modified polymers, implantable elements, and their compositions provided herein can be prepared from readily available starting materials using modifications to the specific synthetic protocols shown below, which will be well known to those skilled in the art. Typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are shown, but it should be understood that other process conditions can be used unless otherwise stated. Optimal reaction conditions can vary depending on the specific reactants or solvents used, but such conditions can be determined by routine optimization procedures by those skilled in the art.

[0362] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent undesired reactions of certain functional groups. The selection of suitable protecting groups for specific functional groups, as well as the conditions suitable for protection and deprotection, are well known in the art. For example, numerous protecting groups and their introduction and removal are described in Greene et al., Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991 and the references cited therein.

[0363] Exemplary compounds, modified polymers, implantable elements, and compositions of the present invention can be prepared using any of the strategies described below.

[0364] Example 1: Synthesis of Sodium Alginate Modified with Exemplary Thiols and Compounds of Formula (I)

Chemical Formula

[0365] After purification, the solution is concentrated to a refractive index value of 1.3360 and refilled into a 400 mL EasyMax reactor. Stirring is set at 300 rpm and the batch temperature is adjusted to 25 °C. To add the thiol crosslinker, follow the exemplary reaction conditions. In a separate sterile container, weigh mesithionine hydrochloride (0.91 g, 5.28 mmol), dissolve it in 1 M MES pH 7.0 buffer (7.5 mL), and then fill the reactor. Weigh 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) in a sterile container, dissolve it in 1 M MES pH 7.0 buffer (10 mL), and then add it to the reactor over 2 minutes. When the filling is complete, heat the reaction mixture to 35 °C in 1 hour, hold it at 35 °C for 15 hours, and then cool it to 25 °C. Purify the reaction mixture via tangential flow filtration (10 kDa molecular weight cut-off) against 10 volume exchanges with physiological saline. After purification, concentrate the solution to a refractive index value of 1.3380.

[0366] Example 2: Synthesis of Sodium Alginate Modified with Exemplary Maleimide [Chemical formula] In this example, an alginate polymer containing a maleimide crosslinking agent may be synthesized according to the exemplary procedure outlined below. Nova Matrix PRONOVA™ UP LG20 (300 g; 1.25% w / w in water, 3.75 g of sodium alginate) is weighed into a 400 mL EasyMax reactor equipped with overhead stirring. In a separate sterile container, 1-(2-aminoethyl)-maleimide hydrochloride (0.93 g, 5.28 mmol) is weighed and dissolved in 1 M MES pH 7.0 buffer (7.5 mL), and then charged into the reactor. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) is weighed in a sterile container, dissolved in 1 M MES pH 7.0 buffer (10 mL), and then added to the reactor over 2 minutes. Once the charging is complete, the reaction mixture is heated to 35 °C over 1 hour, held at 35 °C for 15 hours, and then cooled to 25 °C. The reaction mixture is purified via tangential flow filtration (10 kDa molecular weight cut-off) against 10 volume exchanges with physiological saline. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0367] Example 3: Synthesis of a Double-Crosslinked Alginate Polymer by Michael Addition Reaction

Chemical Structure

[0368] Example 4: Synthesis of Sodium Alginate Modified with Exemplary Tetrazine and Compound of Formula (I) [Chemical formula] In this example, an alginate polymer containing a thiol crosslinking agent and a compound of formula (I) is synthesized. Nova Matrix PRONOVA (trademark) UP LG20 (300 g; 1.25% w / w in water, 3.75 g of sodium alginate) is weighed into a 400 mL EasyMax reactor equipped with overhead stirring. In a separate 150 mL sterile container, 4-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)thiomorpholine 1,1-dioxide (5.77 g, 14.74 mmol) is combined with endotoxin-free water (20 g) and mixed on a shaker at 300 rpm until completely dissolved. Once dissolved, the pH is adjusted to pH 7.0 with 6N and 1N hydrochloric acid and charged into the EasyMax reactor. The stirring is set at 300 rpm and the batch temperature is adjusted to 25 °C. In a separate 150 mL container, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.88 g, 14.02 mmol) is combined with endotoxin-free water (45 g) and manually mixed until completely dissolved. The solution is added to the EasyMax reactor over a 2-minute period. Once the addition is complete, the batch is heated to 35 °C over 1 hour, held at 35 °C for 15 hours, and then cooled to 25 °C. Once the reaction is complete, the batch is filtered through a pad of cyano-silica before being purified via tangential flow filtration (10 kDa molecular weight cut-off). The solution is first purified against 10 volume exchanges with physiological saline, followed by 10 volume exchanges with endotoxin-free water.

[0369] After purification, the solution is concentrated to a refractive index value of 1.3360 and refilled into a 400 mL EasyMax reactor. Stirring is set at 300 rpm and the batch temperature is adjusted to 25 °C. To add the thiol crosslinker, follow the exemplary reaction conditions. In a separate sterile container, weigh 1-[4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine (1.18 g, 5.28 mmol), dissolve it in 1 M MES pH 7.0 buffer (7.5 mL), and then fill the reactor. Weigh 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) in a sterile container, dissolve it in 1 M MES pH 7.0 buffer (10 mL), and then add it to the reactor over 2 minutes. When the filling is complete, heat the reaction mixture to 35 °C in 1 hour, hold it at 35 °C for 15 hours, and then cool it to 25 °C. Purify the reaction mixture via tangential flow filtration (10 kDa molecular weight cut-off) against 10 volume exchanges with physiological saline. After purification, concentrate the solution to a refractive index value of 1.3380.

[0370] Example 5: Synthesis of Sodium Alginate Modified with Exemplary Norbornene

Chemical formula

[0371] Example 6: Synthesis of a Double-Crosslinked Alginate Polymer by Retro-Electron Demand Diels–Alder Reaction [Chemical formula] To form a covalently crosslinked alginate, a thiol crosslinking agent and a maleimide crosslinking agent may be coupled together according to the exemplary protocol outlined herein: An alginate polymer containing compound 303 and an alginate polymer containing compound 305 are dissolved in 1 M MES buffer and incubated at 20 - 30 °C for 4 - 12 hours. The reaction mixture is purified via tangential flow filtration (10 kDa molecular weight cut-off) against 10 volume exchanges with saline. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0372] Example 7: Synthesis of double-crosslinked alginate polymer by thiol-ene photoclick reaction To form a covalently crosslinked alginate, a thiol crosslinker and a norbornene crosslinker may be coupled together according to the exemplary protocol outlined herein: an alginate polymer containing compound 301 or 300 and an alginate polymer containing compound 305 (1:2 molar ratio), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator are dissolved in 1 M MES buffer and irradiated with UV light for 0.5 h with stirring. The reaction mixture is purified via tangential flow filtration (10 kDa molecular weight cut-off) against 10 volume exchanges with saline. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0373] Example 8: Synthesis of exemplary double-crosslinked alginate hydrogel capsules Before preparing the 1-compartment and 2-compartment alginate hydrogel capsules, the buffer solution and the alginate solution were sterilized using a sterile process by filtration through a 0.2-μm filter. To prepare particles configured as 2-compartment hydrogel capsules with a diameter of approximately 1.5 mm, an electrostatic droplet generator was set up as follows: An ES series 0-100-kV, 20-watt high-voltage generator (EQ series, Matsusada, NC, USA) was connected to the top and bottom of a coaxial needle (inner lumen 22G, outer lumen 18G, Paragon). The inner lumen was attached to a first 5-ml Luer-lock syringe (BD, NJ, USA) connected to a vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA). The outer lumen was connected via a Luer coupling to a second 5-ml Luer-lock syringe connected to a horizontally oriented second syringe pump (Pump 11 Pico Plus). The two syringe pumps moved the first and second alginate solutions through both lumens of the coaxial needle from the syringes, and droplets containing both alginate solutions were extruded one by one from the needle into a glass dish containing a crosslinking solution. The settings of each Pico Plus syringe pump were 12.06 mm in diameter, and in the following examples, the flow rates of each pump were adjusted to achieve various test flow rates, but the total flow rate was maintained at 10 ml / hour.

[0374] For the preparation of both 2-compartment and 1-compartment double-crosslinked alginate hydrogel capsules, after extrusion of the desired volume of the alginate solution, the alginate droplets were ionically crosslinked for 5 minutes in a crosslinking solution containing 25 mM HEPES buffer, 20 mM BaCl2, and 0.2 M mannitol. In some experiments, the crosslinking solution also contained 0.01% poloxamer 188. The capsules that dropped to the bottom of the crosslinking container were collected into a conical tube by pipetting. After the capsules had sedimented in the tube, the crosslinking buffer was removed and the capsules were washed. The capsules were washed 4 times with HEPES buffer and resuspended therein.

[0375] In some experiments, the quality of the capsules in the two-compartment, or single-compartment capsule compositions was examined. An aliquot containing at least 200 capsules was taken from the composition, transferred to a well plate, and the entire aliquot was examined for quality by light microscopy by counting the number of spherical capsules from the total number. In some experiments, the mechanical strength of the capsules in the two-compartment capsule composition was examined using a texture analyzer to determine the initial breaking force as described above in this specification.

[0376] Example 9: Preparation of a two-compartment hydrogel containing a modified polysaccharide polymer Using the method of Example 8, a two-compartment hydrogel may be synthesized from the alginate polymers described in Examples 3, 6, and 7. As a non-limiting example, a two-compartment hydrogel containing both the inner and outer compartments of the double-crosslinked alginate of Example 3 may be synthesized according to this method. As another example, a two-compartment hydrogel containing the inner compartment of the double-crosslinked alginate of Example 6 and the outer compartment containing the double-crosslinked alginate of Example 7 may be synthesized.

[0377] Example 10: Synthesis of an exemplary double-crosslinked alginate hydrogel capsule The destruction or mechanical strength of the particles (e.g., hydrogel capsules) can be determined after manufacture but prior to implantation by performing a destruction test using a texture analyzer. In one embodiment, a mechanical test of the hydrogel capsules is performed using a 5 mm probe attached to a 5 kg load cell with a TA.XT plus Texture Analyzer (Stable Micro Systems, Surrey, United Kingdom). Individual capsules are placed on a platform and compressed from above by the probe at a fixed rate of 0.5 mm / second. When a repulsive force of 1 g is measured, contact between the probe and the capsule is detected. The probe then continues to move 90% of the distance between the contact height of the probe and the platform and compresses the capsule to the breaking point. The resistance of the probe to the compression force is measured and can be plotted as a function of the probe displacement (force vs. displacement curve). Typically, the capsule will break slightly before completely rupturing and the force applied to the probe will decrease by a small amount. An analysis macro can be programmed to detect the first instance where a decrease of 0.25 - 0.5 g occurs in the force vs. displacement curve. The force applied by the probe when this occurs is called the initial breaking force. In one embodiment, the breaking force of a capsule preparation manufactured using the apparatus described herein is the average of the initial breaking forces of at least 10, 20, 30, or 40 capsules.

[0378] In this example, three exemplary alginate hydrogel capsule formulations were prepared and their breaking strengths analyzed according to the protocols outlined in Examples 3, 6, and 7. The architecture of the hydrogel capsules is as described in Table 6. As shown in FIGS. 1A - 1C, the double-crosslinked alginate hydrogel exhibits increased average breaking strength compared to a hydrogel consisting of only ionic (e.g., Ba 2+ mediated) crosslinking. In FIGS. 1A - 1C, (1), (3), and (5) refer to ionic crosslinked alginate hydrogels and (2), (4), and (6) refer to hydrogel capsules 1, 2, and 3 as shown in Table 6. [Table 17]

[0379] Example 11: Encapsulation of Mammalian Cells in a Hydrogel Containing a Modified Polysaccharide Polymer Exemplary mammalian cells can be encapsulated in the hydrogel described in Example 10. The cells may be added to the inner and / or outer layers of the two-compartment hydrogel (e.g., at a density of 5-8×10 6 cells / mL -1 ).

[0380] Equivalents and Ranges This application references various issued patents, published patent applications, journal articles, and other publications, all of which are hereby incorporated by reference in their entirety. In the event of any conflict between any of the incorporated references and this specification, this specification shall control. Further, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Such embodiments are considered to be known to those of ordinary skill in the art and may be excluded even if the exclusion is not explicitly stated herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0381] Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above specification, drawings, or examples, but rather is as set forth in the appended claims. Those of ordinary skill in the art will understand that various changes and modifications to this specification can be made without departing from the spirit or scope of the present disclosure as defined in the following claims.

Claims

1. It is a polysaccharide polymer, (i) Cross-linking site and (ii) Compounds of formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, in the formula, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R Ｃ ), -N(R Ｃ )(O)-, -C(O)N(R Ｃ )-, -N(R Ｃ )(R Ｄ )-, -NCN-, -N(R Ｃ )(O)(C １ -C ６ -alkylene)-, -N(R Ｃ )(O)(C ２ -C ６ -alkenylene)-, -C(=N(R Ｃ )(R Ｄ ))O-, -S-, -S(O ｘ )-, -OS(O ｘ )-, -N(R Ｃ )(O)-, -S(O ｘ )(R ｘ )-, -S(O Ｃ )(R Ｆ ) ｙ -, -Si(OR Ａ ) ２ -, -Si(R Ｇ )(OR Ａ )-, -B(OR Ａ )-, or a metal, each of which is optionally linked to a linking group (e.g., a linking group described herein), optionally substituted by one or more R １ ; L １ and L ３ Each of them is independently a bond, alkyl, or heteroalkyl, and each alkyl and heteroalkyl is one or more R ２ It is optionally replaced by, L ２ However, it is a combination, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of these is one or more R ３ It is optionally replaced by, P is one or more R ４ A heteroaryl that is optionally substituted by, Z is an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of these is one or more R ５ It is optionally replaced by, Each R Ａ , R Ｂ , R Ｃ , R Ｄ , R Ｅ , R Ｆ , and R Ｇ However, independently, these are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R ６ It is optionally replaced by, Or R Ｃ and R Ｄ However, together with the nitrogen atom to which they are bonded, one or more R ６ It forms a ring (e.g., a 5- to 7-membered ring) which is optionally substituted by Each R １ , R ２ , R ３ , R ４ , R ５ , and R ６ However, independently, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , S(O) ｘ R Ｅ１ , -OS(O) ｘ R Ｅ１ , -N(R Ｃ１ ) S(O) ｘ R Ｅ１ , -S(O) ｘ N(R) Ｃ１ ) (Caution Ｄ１ ), -P(R Ｆ１ ) ｙ , cycloalkyl, heterocyclyl, aryl, heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R ７ It is optionally replaced by, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , R Ｅ１ , and R Ｆ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is one or more R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. x is 1 or 2, The polysaccharide polymer comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein y is 2, 3, or 4.

2. (a) The crosslinked portion is covalently bonded to a sugar monomer in the polysaccharide polymer, and optionally the crosslinked portion is bonded to a carboxylate portion in the sugar monomer. (b) The crosslinking site comprises an alkyl, alkenyl, alkynyl, ester, ketone, amine, thiol, cycloalkyl, heterocyclyl, aryl, or heteroaryl group. (c) The crosslinking site is capable of reacting with a second crosslinking site, for example, with heat, acid, base, or catalyst, when activated. (d) When the crosslinking sites are determined, for example, by comparison with a reference standard, they are present on the polysaccharide polymer at a density of at least about 1%, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or higher, or (e) When the crosslinked portion is determined, for example, by comparison with a reference standard, it is present on the polysaccharide polymer at a density of 1% to 10%, for example, 1% to 8%, 1% to 6%, or 1% to 4%. The polysaccharide polymer according to claim 1.

3. (a) The polysaccharide polymer is selected from alginate, hyaluronate, and chitosan, or (b) The polysaccharide polymer is an alginate, and optionally the alginate is a high guluronic acid (G) alginate or a high mannuronic acid (M) alginate. The polysaccharide polymer according to claim 1.

4. (a) The crosslinked portion has the structure of formula (IV): 【Chemistry 2】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, Q is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ 、 R ３４ａ 、 R ３４ｂ 、 R ６０ａ 、 R ６０ｂ 、 R ６１ａ 、 R ６１ｂ 、 and R ６２ each of which is, independently, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ 、 -C(O)OR Ａ１ 、 -C(O)R Ｂ１ 、 -OC(O)R Ｂ１ 、 -N(R Ｃ１ )(R Ｄ１ ), -N(R Ｃ１ )(O)C(R Ｂ１ ), -C(O)N(R Ｃ１ ), SR Ｅ１ 、 cycloalkyl, heterocyclyl, aryl, or heteroaryl, and Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, the crosslinking site is an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and optionally, the crosslinking site includes a thiol site. (b) The bridging portion has the structure of formula (V): 【Transformation 3】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T and U are independently O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ , R ３４ａ , R ３４ｂ , R ６５ａ , R ６５ｂ , R ６５ｃ , R ６５ｄ , R ６５ｅ , R ６５ｆ , R ６５ｇ , and R ６６ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, the crosslinking site is an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and optionally, the crosslinking site includes a norborneyl site. (c) The bridging portion has the structure of formula (VI): 【Chemistry 4】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T, Y １ , and Y ２ Each of these independently is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ , R ３４ａ , R ３４ｂ , R ６９ , and R ７０ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, the crosslinking site is an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and optionally, the crosslinking site includes a maleimide site. (d) The bridging portion has the structure of formula (VII): 【Transformation 5】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and Ring M is a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring, each of which has 1 to 6 R groups. ７ It is optionally replaced by, R ３３ , R ３４ａ , R ３４ｂ , and R ７４ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, the crosslinking site is an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl, and optionally, the crosslinking site includes a tetradinyl site, or (e) The crosslinked portion has a structure selected from Table 4, or a pharmaceutically acceptable salt thereof. The polysaccharide polymer according to claim 1.

5. (a) The polysaccharide polymer comprises one of the compounds of formula (IV), (V), (VI), or (VII), or a pharmaceutically acceptable salt thereof. (b) The polysaccharide polymer comprises two of the compounds of formula (IV), (V), (VI), or (VII), or a pharmaceutically acceptable salt thereof. (c) The compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof. (d) The compound of formula (I) is selected from compound 100, compound 101, compound 110, compound 112, compound 113, compound 114, compound 122, and compound 123, or a pharmaceutically acceptable salt thereof. (e) The compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof, (f) The polysaccharide polymer is an alginate, the crosslinking site is selected from the compounds listed in Table 4 or their pharmaceutically acceptable salts, and the compound of formula (I) is compound 101 or its pharmaceutically acceptable salt. The polysaccharide polymer according to claim 1.

6. A composition comprising the polysaccharide polymer described in any one of claims 1 to 5.

7. (a) comprising the polysaccharide polymer described in any one of claims 1 to 5, or (b)(i) Compound of formula (I): 【Transformation 6】 An inner compartment comprising a first polysaccharide polymer comprising a pharmaceutically acceptable salt thereof, wherein, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R Ｃ )-,-N(R Ｃ )C(O)-, -C(O)N(R Ｃ )-,-N(R Ｃ )N(R Ｄ )-, -NCN-, -N(R Ｃ ) C(O)(C １ -C ６ -Alkilen)-, -N(R Ｃ ) C(O)(C ２ -C ６ -Alkenylene)-, -C(=N(R Ｃ ) (Caution Ｄ )) O-, -S-, -S(O) ｘ -, -OS(O) ｘ -, -N(R Ｃ ) S(O) ｘ -, -S(O) ｘ N(R) Ｃ )-,-P(R Ｆ ) ｙ -, -Si( OR Ａ ) ２ -, -Si(R Ｇ ) ( OR Ａ )-,-B(OR Ａ ) - or a metal, each of which is optionally linked to a bonding group (e.g., a bonding group described herein), and one or more R １ It is optionally replaced by, L １ and L ３ Each of them is independently a bond, alkyl, or heteroalkyl, and each alkyl and heteroalkyl is one or more R ２ It is optionally replaced by, L ２ However, it is a combination, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of these is one or more R ３ It is optionally replaced by, P is one or more R ４ A heteroaryl that is optionally substituted by, Z is an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of these is one or more R ５ It is optionally replaced by, Each R Ａ , R Ｂ , R Ｃ , R Ｄ , R Ｅ , R Ｆ , and R Ｇ However, independently, these are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R ６ It is optionally replaced by, Or R Ｃ and R Ｄ However, together with the nitrogen atom to which they are bonded, one or more R ６ It forms a ring (e.g., a 5- to 7-membered ring) which is optionally substituted by Each R １ , R ２ , R ３ , R ４ , R ５ , and R ６ However, independently, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , S(O) ｘ R Ｅ１ , -OS(O) ｘ R Ｅ１ , -N(R Ｃ１ ) S(O) ｘ R Ｅ１ , -S(O) ｘ N(R) Ｃ１ ) (Caution Ｄ１ ), -P(R Ｆ１ ) ｙ , cycloalkyl, heterocyclyl, aryl, heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R ７ It is optionally replaced by, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , R Ｅ１ , and R Ｆ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is one or more R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. x is 1 or 2, The inner compartment where y is 2, 3, or 4, (ii) A hydrogel capsule comprising an outer compartment containing a second polysaccharide polymer including a crosslinking site.

8. (a) The hydrogel capsule contains a single compartment comprising the polysaccharide polymer (for example, the polysaccharide polymer described herein), or (b) The hydrogel capsule comprises a plurality of compartments, one of which comprises the polysaccharide polymer (for example, the polysaccharide polymer described herein), and optionally, The hydrogel capsule comprises an inner compartment and an outer compartment, and further optionally, The inner compartment contains a first polysaccharide polymer including the crosslinked portion, The outer compartment contains a second polysaccharide polymer including the crosslinked portion. A hydrogel capsule according to claim 7(a).

9. (a) The polysaccharide polymer (for example, the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from alginate, hyaluronate, and chitosan. (b) The polysaccharide polymer (for example, the first polysaccharide polymer and / or the second polysaccharide polymer) is an alginate. (c) The first polysaccharide polymer is an alginate. (d) The second polysaccharide polymer is an alginate. (e) The alginate is a high guluronic acid (G) alginate or a high mannuronic acid (M) alginate. (f) The crosslinked portion has the structure of formula (IV): 【Transformation 7】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, Q is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ , R ３４ａ , R ３４ｂ , R ６０ａ , R ６０ｂ , R ６１ａ , R ６１ｂ , and R ６２ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. (g) The crosslinked portion has the structure of formula (V): 【Transformation 8】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T and U are independently O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ , R ３４ａ , R ３４ｂ , R ６５ａ , R ６５ｂ , R ６５ｃ , R ６５ｄ , R ６５ｅ , R ６５ｆ , R ６５ｇ , and R ６６ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. (h) The bridging portion has the structure of formula (VI): 【Chemistry 9】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T, Y １ , and Y ２ Each of these independently is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and R ３３ , R ３４ａ , R ３４ｂ , R ６９ , and R ７０ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. (i) The bridging portion has the structure of formula (VII): 【Chemistry 10】 or having a pharmaceutically acceptable salt or tautomer thereof, wherein the formula, T is O, NR ３３ , or C(R ３４ａ ) (Caution ３４ｂ ) and Ring M is a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring, each of which has 1 to 6 R groups. ７ It is optionally replaced by, R ３３ , R ３４ａ , R ３４ｂ , and R ７４ Each of these independently consists of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (Caution Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), SR Ｅ１ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , and R Ｅ１ However, independently, they are hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl has 1 to 6 R ７ It is optionally replaced by, Each R ７ However, independently, they are alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl. (j) The compound of formula (IV), (V), (VI), or (VII) is selected from the compounds in Table 4 or their pharmaceutically acceptable salts. (k) The compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof. (l) The compound of formula (I) is selected from compound 100, compound 101, compound 110, compound 112, compound 113, compound 114, compound 122, and compound 123, or a pharmaceutically acceptable salt thereof, (m) The compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof. A hydrogel capsule according to claim 7(b).

10. (a) The hydrogel capsule has a diameter of 0.1 mm to 5 mm, (b) The hydrogel capsule has a diameter of 1 mm to 5 mm, (c) The hydrogel capsule has a diameter of 1 mm to 2.5 mm. (d) The hydrogel capsule encapsulates cells, and optionally the cells produce a therapeutic agent, and optionally the therapeutic agent is a protein, such as a hormone, blood coagulation factor, antibody, or enzyme. (e) The hydrogel capsule is formulated for implantation into a subject (e.g., implantation into the intraperitoneal (IP) space, peritoneal cavity, retina, omental bursa, or subcutaneous fat), (f) The transplantable elements are formulated for transplantation into the target IP space. The hydrogel capsule according to claim 7(b).

11. A composition comprising the hydrogel capsule described in claim 7.

12. A method for producing a hydrogel capsule containing a polysaccharide polymer according to any one of claims 1 to 5.

13. A method for increasing the stability of a hydrogel capsule containing a polysaccharide polymer, comprising providing means for both ionically crosslinking the polysaccharide polymer and covalently crosslinking the polysaccharide polymer.

14. (a) The means for ion crosslinking the polysaccharide polymer is a divalent cation (for example, Ba ２＋ Ca ２＋ , Sr ２＋ ) including the use and / or (b) The means for covalently crosslinking the polysaccharide polymer includes the use of crosslinking sites, The method according to claim 13.

15. A composition according to claim 11 for use in a method for treating a disease, disorder, or condition in a subject, wherein the method comprises administering the composition to the subject, thereby treating the disease, disorder, or condition in the subject, optionally, (a) The disease, disorder, or condition is diabetes (for example, type 1 diabetes), (b) The disease, disorder, or condition is not diabetes (e.g., type 1 diabetes), (c) The subject is a human being. The aforementioned composition.

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