Covalently crosslinked polysaccharides and methods of use thereof

Abstract

Described herein are hydrogel capsules (e.g., alginate hydrogel capsules) comprising a polysaccharide polymer capable of covalently cross-linking with another portion, such as another polysaccharide polymer; and related compositions and uses thereof.

Description

[0001] Priority Claim

[0002] This application claims priority to 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 incorporated herein by reference in its entirety. Background of the Invention

[0003] The function of implanted devices depends to a large extent on the recipient's biological immune response pathways (Anderson et al., Semin. Immunol. 20:86–100 (2008); Langer, Adv. Mater. 21:3235–3236 (2009)). Modulation of the immune response can have a beneficial effect on the fidelity and function of these devices. Accordingly, there is a need in the art for new compounds, compositions, and devices that achieve this goal. Summary of the Invention

[0004] Described herein are polymers (such as polysaccharide polymers) covalently crosslinked to another moiety, such as another polymer, and related compositions, hydrogel capsules comprising the polysaccharide polymers, and their uses. In one embodiment, the polymer (e.g., a polysaccharide polymer) is crosslinked by one of the following methods: (i) a thiol-ene photoclick reaction; (ii) a Michael addition reaction; or (iii) an inverse electron demand Diels Alder reaction. In one embodiment, the polysaccharide polymer comprises a crosslinking moiety (such as 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. Incorporating a crosslinking agent into the polysaccharide polymer and then into the hydrogel capsule incorporating the polysaccharide polymer can allow for the modulation of certain properties of the hydrogel capsule, including capsule diameter, stability, and integrity.

[0005] Details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the detailed description, drawings, examples, and claims. Brief Description of the Drawings

[0006] Figures 1A - 1C is a graph comparing the average burst strength of exemplary double-crosslinked hydrogel capsules (e.g., hydrogel capsules comprising a covalently crosslinked moiety and an ionically crosslinked moiety) described herein with ionically crosslinked hydrogel capsules.

[0007] Figure 2 is a schematic diagram depicting an exemplary architecture of the polymers and related hydrogel capsules described herein. Detailed Description

[0008] The present disclosure provides a polysaccharide polymer comprising a crosslinked portion and a compound of formula (I), and related compositions, hydrogel capsules comprising the polysaccharide polymer, and methods of making and uses thereof.

[0009] Abbreviations and Definitions

[0010] To facilitate a better understanding of the present invention, 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 commonly understood by one of ordinary skill in the art to which this invention pertains.

[0011] Unless the context clearly dictates otherwise, as used herein (including in the appended claims), singular terms such as "a / an" and "the" include their corresponding plural referents.

[0012] "About" or "approximately" as used herein means modifying a parameter defined by a numerical value (e.g., physical descriptions of a hydrogel capsule such as diameter, sphericity, number of cells encapsulated therein, number of capsules in a formulation), and refers to the recited numerical value being within an acceptable functional range of the defined parameter determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., limitations of the measuring system, including the acceptable error range of the measuring system. For example, "about" can mean a range of 20% above and below the recited numerical value. As a 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 to 1.8 mm and can encapsulate 4M to 6M cells. As another non-limiting example, a formulation of about 100 devices (e.g., hydrogel capsules) includes a formulation having 80 to 120 devices. In some embodiments, the term "about" means that the modified parameter can vary up to 15%, 10%, or 5% above and below the stated numerical value of the parameter. Alternatively, particularly with respect to certain characteristics of the devices described herein, such as cell productivity, or density of a CBP or defibrillation compound, the term "about" can mean within an order of magnitude of the recited value, e.g., within 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold.

[0013] As used herein, the term "acquire" or "acquiring" refers to the process of obtaining a value (e.g., a numerical value), an image, or a physical entity (e.g., a sample) by "directly acquiring" or "indirectly acquiring" the value or physical entity. "Directly acquiring" means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. "Indirectly acquiring" refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquires the physical entity or value). Directly acquiring a value or physical entity includes performing a process that involves a physical change of a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, such as using a fluorescence microscope to obtain fluorescence microscopy data.

[0014] As used herein, "administer", "administering", or "administration" refers to implanting, absorbing, ingesting, injecting, or otherwise introducing into a subject, or providing to a subject for administration, an entity described herein (e.g., a device or a formulation of a device).

[0015] As used herein, "defibrotic" refers to a compound or material that reduces the foreign body response (FBR). For example, the amount of FBR in a tissue induced by implanting a device (e.g., a hydrogel capsule) containing a defibrotic compound (e.g., a hydrogel capsule containing a polymer covalently modified with a compound listed in Table 3) into the biological tissue is lower than the FBR induced by implanting a reference device that is fibrotic-null, i.e., lacks any defibrotic compound but has substantially the same composition (e.g., the same CBP-polymer, one or more of the same cell types) and structure (e.g., size, shape, number of compartments). In one embodiment, one or more assays / methods known in the art, such as those described in WO 2017 / 075630 or one or more of the assays / methods described in Vegas, A. et al., Nature Biotechnol (supra) (e.g., subcutaneous cathepsin measurement of the implanted capsule, Masson's trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining of macrophages (CD68 or F4 / 80), myofibroblasts (α-smooth muscle actin, SMA), or general cell deposition, and confocal microscopy, quantification of 79 RNA sequences of known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophils on a device (e.g., a capsule) retrieved 14 days later from the peritoneal cavity of a suitable test subject (e.g., an immunocompetent mouse)) are used to evaluate the extent of FBR by the immune response in the tissue containing the implanted device (e.g., a hydrogel capsule), which immune response may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis. In one embodiment, FBR is evaluated by measuring the levels of one or more immune response biomarkers (e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4) in the tissue containing the implant. In some embodiments, the FBR induced by the device of the present invention (e.g., a hydrogel capsule containing a defibrotic 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 a reference device that is FBR-null, e.g., a device that is substantially identical to the tested or claimed device except for lacking a device for reducing FBR (e.g., a hydrogel capsule that does not contain a defibrotic compound but is otherwise substantially identical to the claimed capsule). In some embodiments, FBR (e.g., the level of one or more biomarkers) is measured at 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 longer.

[0016] As used herein, "cell" refers to an engineered or non-engineered cell. In one embodiment, the cell is an immortalized cell or an engineered cell derived from an immortalized cell. In one embodiment, the cell is a living cell, e.g., viable as measured by any technique described herein or known in the art.

[0017] As used herein, "cell-binding peptide (CBP)" means a linear or cyclic peptide comprising an amino acid sequence of a cell-binding domain that contains a ligand derived from a cell adhesion molecule (CAM) (e.g., a cell adhesion molecule that mediates cell-matrix or cell-cell junctions). The length of the CBP is less than 50, 40, 30, 25, 20, 15, or 10 amino acids. In one embodiment, the length of the CBP is between 3 and 12 amino acids, between 4 and 10 amino acids, or is 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The CBP amino acid sequence can be identical to the naturally occurring binding domain sequence or can be 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, the CBP comprises a cell-binding sequence listed in Table 1 below or a conservatively substituted variant thereof. In one embodiment, the CBP comprises at least one of the cell-binding sequences listed in Table 1 below. In one embodiment, the CBP consists essentially of the cell-binding sequences listed in Table 1 below. In one embodiment, the CBP is an RGD peptide, meaning the peptide contains the amino acid sequence RGD (SEQ ID NO:43) and optionally contains one or more additional amino acids at one or both of the N-terminus and / or C-terminus. In one embodiment, the 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, the CBP is a linear peptide containing RGD (SEQ ID NO:43) and has a length less than 6 amino acids. In one embodiment, the CBP is a linear peptide consisting essentially of RGD (SEQ ID NO:43) or RGDSP (SEQ ID NO:59).

[0018] Table 1: Exemplary CAM Ligand Proteins and Cell-Binding Sequences

[0019]

[0020]

[0021] 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 any 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., substantially composed of a single amino acid, or a peptide of several identical or different amino acids), and the linker is connected 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 connected to the N-terminus of the CBP, and the N-terminus of the amino acid linker is connected to at least one pendant carboxyl group in the polysaccharide via an amide bond. In one embodiment, the structure of the linker-CBP is represented as G (1-4) -CBP, which means that the linker has one, two, three, or four glycine residues ("G (1-4) " disclosed as SEQ ID NO:70). In one embodiment, one or more of the monosaccharide moieties in the monosaccharide moiety of the CBP-polysaccharide (e.g., CBP-alginate) are unmodified by the CBP, e.g., the unmodified moiety has a free carboxyl group or lacks a pendant carboxyl group that can be modified. In one embodiment, the number of polysaccharide moieties 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%.

[0022] In one embodiment, the density of CBP modification in the CBP-polymer is estimated by combustion analysis of the nitrogen percentage. In one embodiment, the CBP-polymer is an RGD-polymer (e.g., RGD-alginate), which is a polymer (e.g., alginate) covalently modified with a linker-RGD molecule (e.g., a peptide consisting essentially of GRGD (SEQ ID NO:62) or GRGDSP (SEQ ID NO:60)), and as determined using the assays described herein, the density of linker-RGD molecule modification (e.g., conjugation density) is from about 0.05% nitrogen (N) to 1.00% N, from about 0.10% N to about 0.75% N, from about 0.20% N to about 0.50% N, or from about 0.30% N to about 0.40% N. In one embodiment, as determined by any assay capable of quantifying the amount of peptide conjugated to the polymer, such as the quantitative peptide conjugation assay described herein, the conjugation density of linker-RGD modification in RGD-alginate (e.g., MMW alginate covalently modified with GRGDSP (SEQ ID NO:60)) is 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 moiety per gram of RGD-polymer (e.g., saline solution) having a viscosity of 80 to 120 cP in solution. Unless otherwise expressly stated or obvious from the context, the specifically recited 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., it does not include any residual free (e.g., unconjugated) CBP that may be present in the CBP-polymer.

[0023] As used herein, "cell-binding polypeptide (CBPP)" means a polypeptide having a length of at least 50, at least 75, or at least 100 amino acids and comprising 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 comprise 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.

[0024] As used herein, "CBP-density" refers to the amount or concentration of linker-CBP moiety in a CBP-polymer (e.g., alginate modified with G 1-3 RGD (SEQ ID NO:63) or G 1-3 RGDSP (SEQ ID NO:64)), unless expressly stated otherwise herein.

[0025] As used herein, "cell-binding substance (CBS)" means any chemical, biological, or other type of substance (e.g., small organic compound, peptide, polypeptide) that can mimic at least one activity of a ligand of a cell adhesion molecule (CAM) or other cell surface molecule that mediates cell-matrix junctions or cell-cell junctions or other receptor-mediated signal transduction. In one embodiment, when present in a polymeric composition encapsulating cells, the CBS is capable of forming a temporary or permanent bond or contact with one or more of the cells. In one embodiment, the CBS promotes interaction between two or more live cells encapsulated in the polymeric composition. In one embodiment, the presence of CBS in a polymeric composition encapsulating a plurality of cells (e.g., live cells) is associated with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are implanted into a test subject (e.g., a mouse). In one embodiment, the CBS physically attaches 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.

[0026] As used herein, "conservatively modified variant" or "conservative substitution" refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for having one or more conservative amino acid substitutions in the 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 are those in which an amino acid is replaced with an amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.) and that has a minimal effect 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 listed in Table 2 below.

[0027] Table 2. Exemplary groups of conservative amino acid substitutions.

[0028]

[0029]

[0030] As used throughout the specification and claims, "consists essentially of" and variations such as "consist essentially of" or "consisting essentially of" mean including any recited element or group of elements, and optionally including other elements of similar or different nature that do not materially alter the basic or novel characteristics of the specified molecule, composition, device, or method. As a non-limiting example, a cell-binding peptide or therapeutic protein consisting essentially of the recited amino acid sequence may also contain one or more amino acids (including substitutions of one or more amino acid residues in the recited amino acid sequence) of one or more amino acid residues that do not materially affect the relevant biological activity of the cell-binding peptide or therapeutic protein. As another non-limiting example, a cell-binding peptide consisting essentially of the recited amino acid sequence may contain one or more covalently attached moieties (e.g., radioactive or fluorescent labels) that do not materially alter the relevant biological activity of the cell-binding peptide, e.g., its ability to increase the viability or productivity of encapsulated cells as described herein.

[0031] As used throughout this specification, "crosslinked" and variations thereof; such as "crosslinking" or "x-linked", refer to a chemical bond (e.g., an ionic bond, e.g., a covalent bond) between two polymers. In some embodiments, when two or more chemical bonds are present, crosslinking refers to a mixture of covalent and ionic bonds. In some embodiments, when two or more chemical bonds are present, crosslinking refers to different types of covalent bonds (e.g., covalent bonds containing different or orthogonal functional groups). In some embodiments, when two or more chemical bonds are present, crosslinking refers to the same type of covalent bond (e.g., covalent bonds containing the same functional group). In some embodiments, when two or more chemical bonds are present, crosslinking refers to the same type of ionic bond (e.g., an ionic bond containing the same ion (e.g., Ba 2+ ))).

[0032] As used herein with respect to cells, "derived from" means a cell obtained from a tissue, cell line, or cell, and then optionally the cell is cultured, passaged, differentiated, induced, etc. to produce a derived cell. For example, mesenchymal stem cells can be derived from mesenchymal tissue and then differentiated into multiple cell types.

[0033] As used herein, "device" refers to any implantable object described herein (e.g., particle, hydrogel capsule, implant, medical device). In some embodiments, the device contains cells (e.g., living cells) capable of expressing 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 metabolic by-products and / or therapeutic agents produced by living cells to be released from the device.

[0034] As used herein, "differential volume" refers to the volume of a compartment within a device described herein, which does not include the space occupied by one or more additional compartments. For example, the differential volume of the second compartment (e.g., outer compartment) in a two-compartment device having an inner compartment and an outer compartment refers to the volume within the second compartment, which does not include the space occupied by the first compartment (inner compartment).

[0035] As used herein, "effective amount" refers to an amount of a device, device composition, or component of a device or device composition sufficient to elicit a biological response, e.g., to treat a disease, disorder, or condition, such as the amount of a plurality of hydrogel capsules containing cells (e.g., engineered cells) or an agent (e.g., therapeutic agent) produced by 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 the device, the density of a defibrosis compound disposed on the surface of and / or in a barrier compartment of the device, the density of CBS in a cell-containing compartment. As will be understood by one of ordinary skill in the art, the effective amount can vary depending on factors such as 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 the age and health status of the subject. The effective amount encompasses both therapeutic and prophylactic treatments. By way of example, to mitigate FBR, an effective amount of a compound of formula (I) can reduce fibrosis on or near an implanted device or prevent the growth or spread of fibrotic tissue on or near an implanted device. The effective amount of a device, composition, or component (e.g., defibrosis compound) can be determined by any technique known in the art or described herein.

[0036] As used herein, "endogenous nucleic acid" is a nucleic acid that naturally occurs in a subject's cells.

[0037] As used herein, "endogenous polypeptide" is a polypeptide that naturally occurs in a subject's cells.

[0038] As used herein, an "engineered cell" is a cell that has non-naturally occurring alterations and typically contains a nucleic acid sequence (e.g., DNA or RNA) or polypeptide that is not present (or present at a different level) in other similar cells under similar conditions in which they have not been engineered (exogenous nucleic acid sequence). In one embodiment, the engineered cell contains an exogenous nucleic acid (e.g., a vector or an altered chromosomal sequence). In one embodiment, the engineered cell contains an exogenous polypeptide. In one embodiment, the engineered cell contains an exogenous nucleic acid sequence, such as a sequence that is not present in similar non-engineered cells, such as DNA or RNA. In one embodiment, the exogenous nucleic acid sequence is chromosomal, e.g., the exogenous nucleic acid sequence is an exogenous sequence placed within an endogenous chromosomal sequence. In one embodiment, the exogenous nucleic acid sequence is chromosomal or extrachromosomal, such as a non-integrated vector. In one embodiment, the exogenous nucleic acid sequence contains an RNA sequence, such as mRNA. In one embodiment, the exogenous nucleic acid sequence contains a chromosomal or extrachromosomal exogenous nucleic acid sequence that contains a sequence expressed as RNA, such as mRNA or regulatory RNA. In one embodiment, the exogenous nucleic acid sequence contains a chromosomal or extrachromosomal nucleic acid sequence that contains a sequence encoding a polypeptide or expressed 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, wherein the second amino acid sequence can be exogenous or endogenous. For example, an 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 present at a level or distribution different from that found in similar non-engineered cells. In one embodiment, the engineered cell includes an RPE that has been engineered to produce RNA or a polypeptide. For example, an engineered cell can contain an exogenous nucleic acid sequence that contains a chromosomal or extrachromosomal exogenous nucleic acid sequence that contains a sequence expressed as RNA, such as mRNA or regulatory RNA. In one embodiment, the engineered cell (e.g., an RPE cell) contains an exogenous nucleic acid sequence that contains a chromosomal or extrachromosomal nucleic acid sequence that contains a sequence encoding a polypeptide or expressed as a polypeptide. In one embodiment, the polypeptide is encoded by a codon-optimized sequence to achieve higher polypeptide expression than the naturally occurring coding sequence. The codon-optimized sequence can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene TM(GenScript, Piscataway, NJ USA), (ATUM, Newark, CA USA) or generated by the Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol. 33, Suppl. Issue 2, pp. W526 - W531 (2005)). In one embodiment, the engineered cell (e.g., RPE cell) comprises an exogenous nucleic acid sequence that modulates 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.

[0039] As used herein, "exogenous nucleic acid" is a nucleic acid that does not naturally occur in the cells of a subject.

[0040] As used herein, "exogenous polypeptide" is a polypeptide that does not naturally occur in the cells of a subject (e.g., engineered cells). Reference to an amino acid position in a particular sequence refers to the position of the amino acid in a reference amino acid sequence, e.g., the sequence of a full - length mature (after signal peptide cleavage) wild - type protein (unless otherwise specified), and does not exclude the presence of variations (e.g., deletions, insertions, and / or substitutions) at other positions in the reference amino acid sequence.

[0041] Unless otherwise indicated, as used herein, "Factor VII protein" or "FVII protein" refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VII protein or a variant thereof, as determined by assays well known in the art, said polypeptide having FVII biological activity, such as promoting blood clotting. Naturally occurring FVII exists as a single-chain zymogen, an enzymatically-like double-chain polypeptide, and a fully activated double-chain form (FVIIa). In some embodiments, reference to FVII includes its single-chain and double-chain forms, including the enzymatically-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, variants having one or more amino acid substitutions and / or deletions. In some embodiments, variant FVII proteins are capable of being activated into a fully activated double-chain form (Factor VIIa) having at least 50%, 75%, 90%, or more (including >100%) of the activity of wild-type Factor VIIa. Variants of FVII and FVIIa are known, such as marzeptacog α (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. US 20080058255. Unless otherwise indicated, Factor VII biological activity can be quantified by assays well known in the art. For example, the FVII biological activity in a sample of biological fluid (e.g., plasma) can be measured by: (i) measuring the amount of Factor Xa produced in a system comprising tissue factor (TF) and Factor X 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 a surface plasmon resonance-based instrument (Persson, FEBS Letts. 413:359-363, 1997); or (iv) measuring the hydrolysis of a synthetic substrate; and / or (v) measuring thrombin production in a TF-independent in vitro system. In one embodiment, FVII activity is evaluated by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuvillesur Oise, France), in which a biological sample containing FVII is mixed with prothrombin kinase calcium, Factor X, and SXa-11 (a chromogenic substrate specific for Factor Xa).

[0042] Unless otherwise indicated, as used herein, "Factor VIII protein" or "FVIII protein" refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VIII polypeptide or a variant thereof, as determined by assays recognized in the art, said polypeptide having FVIII biological activity, such as coagulant activity. FVIII proteins that can be expressed 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, B domain deleted (BDD) variants, single-chain variants, and fusions of any of the foregoing wild-type or variant proteins with a polypeptide having an extended half-life. In one embodiment, the cells are engineered to encode a precursor Factor VIII polypeptide having a complete or partial deletion of the B domain (e.g., having a signal sequence). In one embodiment, the cells are engineered to encode a single-chain Factor VIII polypeptide that contains a variant FVIII protein, preferably having at least 50%, 75%, 90% or more (including >100%) of the coagulant activity of the corresponding wild-type Factor VIII. Assays for measuring the coagulant activity of FVIII proteins include one-stage or two-stage 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.).

[0043] Many FVIII-BDD variants are known and include, for example, variants having a complete or partial deletion of the B domain disclosed in any of the following U.S. Patent Nos.: 4,868,112 (e.g., column 2, line 2 to column 19, line 21 and Table 2); 5,112,950 (e.g., column 2, lines 55-68, Figure 2and U.S. Patent Nos. 5,171,844 (e.g., column 4, lines 22 to column 5, line 36); 5,543,502 (e.g., column 2, lines 17-46); 5,595,886; 5,610,278; 5,789,203 (e.g., column 2, lines 26-51 and Examples 5-8); 5,972,885 (e.g., column 1, line 25 to column 2, line 40); 6,048,720 (e.g., column 6, lines 1-22 and Example 1); 6,060,447; 6,228,620; 6,316,226 (e.g., column 4, line 4 to column 5, line 28 and Examples 1-5); 6,346,513; 6,458,563 (e.g., column 4, lines 25-53); and 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).In some embodiments, the FVIII-BDD protein produced by the devices described herein (e.g., expressed by engineered cells contained within the devices) has one or more of the following amino acid deletions in the B-domain: (i) most of the B-domain except for the amino-terminal B-domain sequence necessary for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 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, WO 87 / 04187)); amino acids 747-1560 (Sarver et al., DNA 6:553-564 (1987)); amino acids 741-1648 (Pasek, WO 88 / 00831)); deletions of amino acids 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597); including deletions of one or more residues in the furin recognition sequence, such as the deletion of LKRHQR (SEQ ID NO:65) located at amino acids 1643-1648, including any of the specific deletions cited in column 10, line 65 to column 11, line 36 of U.S. Patent No. 9,956,269.

[0044] 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).

[0045] In some embodiments, the FVIII-BDD protein is a single-chain variant generated by substitution of one or more amino acids in the furin recognition sequence, LKRHQR (SEQ ID NO:65) at amino acids 1643-1648, where the substitution or deletion prevents proteolytic cleavage at that site, including any substitution at positions R1645 and R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353, and 9,670,267.

[0046] In some embodiments, any of the above FVIII-BDD proteins may further comprise 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 fusions (WO 2011 / 020866); and Fc fusions (WO 04 / 101740).

[0047] Unless otherwise indicated, all FVIII-BDD amino acid positions referred to herein are positions in full-length human FVIII.

[0048] Unless otherwise indicated, as used herein, "Factor IX protein" or "FIX protein" refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor IX protein or a variant thereof, as determined by assays well recognized in the art, said polypeptide having FIX biological activity, such as coagulation activity. FIX is produced as an inactive zymogen, which is converted to the active form by excision of the activation peptide by Factor XIa, resulting in a heavy chain and a light chain linked together by one or more disulfide bonds. 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 polypeptide having an extended half-life. 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 (including >100%) 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 the one-stage clotting assay (activated partial thromboplastin time (aPTT) (e.g., as described in EP 2 032 607), thrombin generation assay (TGA), and rotational thromboelastometry (e.g., as described in WO 2012 / 006624).

[0049] Many 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: WO 02 / 040544, pages 4, lines 9-30 and page 15, lines 6-31; WO 03 / 020764, Tables 2 and 3, pages 14-24 and page 12, lines 1-27; WO2007 / 149406, page 4, line 1 to page 19, line 11; WO 2007 / 149406 A2, page 19, line 12 to page 20, line 9; WO 08 / 118507, page 5, line 14 to page 6, line 5; WO 09 / 051717, page 9, line 11 to page 20, line 2; WO 09 / 137254, page 2, paragraph

[006] to page 5, paragraph

[011] and page 16, paragraph

[044] to page 24, paragraph

[057] ; WO 09 / 130198A2, page 4, line 26 to page 12, line 6; WO 09 / 140015, page 11, paragraph

[0043] to page 13, paragraph

[0053] ; WO2012 / 006624; WO 2015 / 086406.

[0050] 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 extending moieties include Fc, albumin, PAS sequences, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) having 4 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 WO 2012 / 006624, which is a FIXFc single chain (FIXFc-sc) and an Fc single chain (Fc-sc) joined together by two disulfide bonds in the hinge region of Fc.

[0051] FIX variants also include gain-of-function and loss-of-function variants. An example of a gain-of-function variant is the "Padua" variant of human FIX, which has an L (leucine) instead of an R (arginine) at position 338 of the mature protein (corresponding to amino acid position 384 of SEQ ID NO: 2) and has greater catalytic and coagulation activity 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 the substitution of lysine with alanine at the fifth amino acid position starting from the mature protein, which results in a protein with reduced binding to collagen IV (e.g., loss of function). Unless otherwise indicated, as used herein, "interleukin-2 protein" or "IL-2 protein" means a polypeptide comprising the amino acid sequence of a naturally occurring IL-2 protein or a variant thereof, as determined by assays well known in the art, said polypeptide having IL-2 biological activity, such as activation of IL-2 receptor signaling in Treg cells. 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), suid, 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 higher (including >100%) 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.

[0052] As used herein, "islet cell" means a cell comprising any naturally occurring or any synthetically produced or modified cell that is intended to reproduce, mimic, or otherwise express in part or in whole the function of a part or all of the Langerhans islet cells. The term "islet cell" includes glucose-responsive insulin-producing cells derived from stem cells (e.g., derived from an induced pluripotent stem cell line).

[0053] Unless otherwise expressly stated, "mannitol" as used herein refers to D-mannitol.

[0054] As used herein, "medium molecular weight alginate" or "MMW-Alg" means an alginate having an approximate molecular weight of from 75 kDa to 150 kDa.

[0055] As used herein, the term "mesenchymal stem function cell" or "MSFC" refers to a cell derived from a mesodermal lineage or a cell having at least one characteristic unique to cells of a mesodermal lineage, and wherein the MSFC: i) is not in a terminally differentiated state; and ii) can terminally differentiate into one or more cell types. MSFC does not include cells of endodermal origin, such as enterocytes, or cells of ectodermal origin, such as cells derived from skin, CNS, or neurons. In one embodiment, the MSFC is multipotent. In one embodiment, the MSFC is not totipotent. In one embodiment, the MSFC comprises one or more of the following characteristics:

[0056] a) It comprises a mesenchymal stem cell (MSC) or a cell derived therefrom, including: cells from a primary cell culture of an MSC; cells directly isolated (without long-term culture, e.g., fewer than 5 or 10 passages or cell division rounds since isolation) from a naturally occurring MSC, such as from a human or other mammal; cells derived from a transformed, pluripotent, immortalized, or long-term (e.g., more than 5 or 10 passages or cell division rounds) MSC culture.

[0057] b) It comprises cells obtained from less differentiated cells, such as cells developed, programmed or reprogrammed (e.g., in vitro) into MSCs, or cells substantially similar to one or more of naturally occurring MSCs or cells from primary or long-term MSC cultures, except for any genetic engineering, or cells described in a) above. Examples of less differentiated cells from which MSFCs can be derived include IPS cells, embryonic stem cells, or other full-potency or enriched-potency cells; see, e.g., Chen, YS 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;

[0058] c) it is pluripotent, e.g. as measured by any assay capable of providing information about the pluripotency of a cell (e.g. microscopy);

[0059] d) It presents a characteristic mononuclear oval, star, or spindle shape with a round to oval nucleus. The oval elongated nucleus may have a prominent nucleolus and a mixture of heterochromatin and euchromatin. MSFCs (e.g., MSCs) may have a small amount of cytoplasm but with many thin processes that appear to extend from the nucleus;

[0060] e) it is capable of cell division, e.g., as measured by any assay capable of providing information about cell division (e.g., microscopy). 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 (e.g., encapsulated as described herein) or incorporated into a device (e.g., a device described herein). In one embodiment, after reaching confluence, it is incapable of cell division;

[0061] f) it is able to differentiate into mesenchymal cell lineages, such as osteoblasts, chondroblasts, adipocytes, or fibroblasts;

[0062] g) it expresses mesenchymal cell markers, such as one, two, three, four, five or all of CD105, CD106, CD73, CD90, Stro-1, CD49a, CD29, CD44, CD146, CD166, TNAP+, THY-1+, Stro-2, Stro-4 and alkaline phosphatase;

[0063] h) It does not express one, two, three, or any of CD34, CD31, VE-cadherin, CD45, HLA-DR, CD11b, and glycophorin or leukocyte differentiation antigens (such as CD14, CD33, CD3, and CD19) at a significant level;

[0064] i) It expresses one, two, or all of CD75, CD90, and CD105, and does not express one, two, or any of CD45, CD34, and CD14;

[0065] j) It has an anti-inflammatory or immunosuppressive effect, such as measured by any method capable of providing information about inflammation (such as in vivo inhibition of T cell proliferation);

[0066] k) It is capable of adhesion, such as plastic adhesion, as determined, for example, by visual inspection; or

[0067] l) It can grow in three-dimensional space, as determined, for example, by visual inspection.

[0068] As used herein, "parathyroid hormone" or "PTH" means a polypeptide or peptide comprising the amino acid sequence of a naturally occurring parathyroid hormone polypeptide or peptide or a variant thereof, which, as determined by assays generally recognized in the art, has PTH biological activity. PTH polypeptides and peptides that can be expressed by the encapsulated cells described herein include wild-type primate (such as human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Such PTH polypeptides and peptides can consist essentially of the wild-type human sequences of preproPTH polypeptide (115 amino acids), proPTH polypeptide (90 amino acids), mature 84-amino acid peptide (PTH(1-84)), and its biologically active variants (such as truncated variant peptide PTH(1-34)). PTH peptide variants having one or more amino acid substitutions in the human wild-type sequence have been 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 higher (including >100%) of the biological activity of the corresponding wild-type PTH. U.S. Patent No. 8,383,417 describes an assay for detecting certain PTH variants by tandem mass spectrometry. U.S. Patent No. 7,410,948 describes a biological activity assay for PTH peptide variants - determination of the stimulation of adenylate cyclase by measuring cAMP levels.

[0069] As used herein, "poloxamer" is a standard generic term referring to a class of nonionic triblock linear copolymers consisting of a central hydrophobic chain of polyoxypropylene (polypropylene oxide) flanked by two polyoxyethylene (polyethylene oxide) moieties on either side. As used herein, "poloxamer 188" or "P 188" refers to a poloxamer having an approximate molecular weight of the polyoxypropylene core of 1800 g / mol and an ethylene oxide content of about 80% by weight (e.g., 79.0% to 83.7%). In one embodiment, the average molecular weight of poloxamer 188 is 8350 g / mol. In one embodiment, poloxamer 188 has an average molecular weight of 7680 g / mol to 9510 g / mol, as determined, for example, by size exclusion chromatography, and an ethylene oxide content of 81.8 ± 1.9% by weight. In one embodiment, each polyoxyethylene chain in poloxamer 188 has 75 - 85 (e.g., 80) ethylene oxide monomers, and the polyoxypropylene core has 25 - 30 (e.g., 27) propylene oxide monomers. In one embodiment, the poloxamer 188 used in the processes described herein substantially meets the specifications set forth in the poloxamer monographs published in the United States Pharmacopeia - National Formulary (USP - NF) or the European Pharmacopoeia (Ph.Eur.), which are officially published at the time of carrying out the process.

[0070] As used herein, "polymer composition" 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 natural and synthetic. A homopolymer contains one type of structural unit or monomer, while a copolymer contains more than one type of monomer.

[0071] As used herein, "polypeptide" refers to a polymer comprising amino acid residues linked by peptide bonds and having at least two, and in some embodiments at least 10, 50, 75, 100, 150 or 200 amino acid residues.

[0072] As used herein, "Prevention", "prevent" and "preventing" refer to including administering or applying a therapy before the onset of a disease, disorder or affliction, e.g., administering a composition of an encapsulated cell device (e.g., as described herein) to preclude the physical manifestation of the disease, disorder or affliction. In some embodiments, "Prevention", "prevent" and "preventing" require that the signs or symptoms of the disease, disorder or affliction have not yet developed or been observed. In some embodiments, the treatment includes prevention, and in other embodiments, it does not include prevention.

[0073] "Alternative therapy" or "alternative protein" is a therapeutic protein or a functional fragment thereof that replaces or enhances the beneficial function of a protein that is reduced, present in insufficient amounts, altered (e.g., mutated), or lacking in a subject having a disease or disorder associated with a reduced, altered, or absent protein. Examples are certain blood clotting factors in certain blood clotting disorders or certain lysosomal enzymes in certain lysosomal storage diseases. In one embodiment, the alternative therapy or alternative protein provides the function of an endogenous protein. In one embodiment, the alternative therapy or alternative 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 alternative therapy or alternative protein differs from a naturally occurring variant (e.g., a wild-type allele or an allele not associated with the disorder, such as an allele carried by the subject) at no more than about 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20% of the amino acid residues in the amino acid sequence.

[0074] As used herein, a "reference device" as used with respect to a claimed device (e.g., a hydrogel capsule) is a device (e.g., a hydrogel capsule) that: (i) lacks a specific feature, e.g., an FBR mitigation means (e.g., a barrier compartment containing a defibrillation compound (as defined herein) or a CBS (as defined herein) (e.g., an RGD polymer)), (ii) encapsulates in its cell-containing compartment approximately the same number of cells of one or more of the same cell types as in the claimed device, and (iii) has a polymer composition and structure that is substantially similar to that of the claimed device except for lacking that specific feature (e.g., defibrillation compound or CBS). In one embodiment, the number of live cells in the cell-containing compartment of the reference device is within 80% to 120%, or 90% to 110%, of the number of live 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 composition means that all polymers in the reference device and the claimed device (including the polymer components of any CBP polymers and defibrillation polymers, if applicable) have the same chemical and molecular weight classes (e.g., alginate having a high G content and the same molecular weight range). For example, in one embodiment, the cell-containing compartment of a CBP (CBP-null) reference device is formed from the unmodified form of the polymer (e.g., 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, and the inner compartment of the reference capsule is formed from a suspension of cells in the same polymer mixture as the polymer mixture used for the outer compartment. In one embodiment, substantially similar structure means that the reference device and the claimed device have the same number of compartments (e.g., one, two, three, etc.) and approximately the same size and shape. As used herein, "RPE cells" are cells having one or more of the following characteristics: a) they contain retinal pigment epithelial cells (RPE) (e.g., using the ARPE-19 cell line ( CRL-2302 TM) cells cultured from or derived therefrom (e.g., cells cultured from the ARPE-19 cell line stably transfected with an exogenous sequence encoding a therapeutic protein, or such cultured ARPE-19 cells otherwise engineered to express an exogenous protein or other exogenous substance), cells from a primary cell culture of RPE cells, cells directly isolated from naturally occurring RPE cells (e.g., from a human or other mammal) without long-term culture (e.g., less than 5 or 10 passages or rounds of cell division since isolation), cells from a transformed, immortalized or long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) cells obtained from less differentiated cells, such as cells developed, programmed or reprogrammed (e.g., in vitro) into RPE cells, or cells that are substantially similar to one or more of naturally occurring RPE cells or cells from a primary or long-term culture of RPE cells apart from any genetic engineering (e.g., the cells may be derived from iPS cells); c) cells having one or more of the following characteristics: 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 occurring in the retina and forming a monolayer above 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 produced or modified from naturally occurring cells to have the same or substantially the same genetic content and optionally the same or substantially the same epigenetic content as an immortalized RPE cell line (e.g., the ARPE-19 cell line ( CRL-2302 TM ))). In one embodiment, the RPE cells described herein are engineered, for example, to have new properties, such as 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 unengineered.

[0075] As used herein, "salt solution" means physiological saline, i.e., water containing 0.9% NaCl, unless otherwise specified.

[0076] As used herein when referring to two nucleotide sequences or two amino acid sequences, "sequence identity" or "percent identity" means that when two sequences are compared and aligned to obtain maximum correspondence over a comparison window or specified region, the two sequences are identical in the specified region, or have the same nucleotide or amino acid at a specified percentage of nucleotide or amino acid positions within the 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 nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0077] As used herein, "spherical" means 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 sphere-like shape, which may, for example, have waves and undulations on its surface. Spheres and sphere-like objects can be mathematically defined by rotating a circle, an ellipse, or a combination thereof about each of three perpendicular axes a, b, and c. For a sphere, the lengths of the three axes are the same. Generally, a sphere-like shape is an ellipsoid (with respect to its average surface) in which the semi-major axes differ from each other by no more than 10%, 5%, or 2.5%. The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-major axes.

[0078] The term "spheroid" as used herein when referring to a device (e.g., a hydrogel capsule or other particle) means that the device has (i) a perfect or classical oblate or prolate spheroid shape, or (ii) a surface that generally forms a spheroid, which may, for example, have corrugations and undulations and / or may be an ellipsoid (with respect to its average surface) in which the semi-major axes are within 100% of each other.

[0079] As used herein, "subject" refers to a human or non-human animal. In one embodiment, the subject is a human (i.e., male or female), such as a person of any age group, a pediatric subject (e.g., an infant, a child, an adolescent), or an adult subject (e.g., a young person, a middle-aged person, or an elderly person). In one embodiment, the subject is a non-human animal, such as 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, such as a chicken, a duck, a goose, or a turkey). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0080] As used herein, "total volume" refers to the volume within one compartment of a multi-compartment device, which includes the space occupied by another compartment. For example, the total volume of the second compartment (e.g., the outer compartment) of a two-compartment device refers to the volume within the second compartment, which includes the space occupied by the first compartment. As used herein, the terms "treatment", "treat", and "treating" refer to alleviating, reversing, mitigating one or more of the symptoms, manifestations, or underlying causes of a disease, disorder, or affliction, delaying its onset, or inhibiting its progression. In one embodiment, treatment includes alleviating, reversing, mitigating the symptoms of a disease, disorder, or affliction, delaying its onset, or inhibiting its progression. In one embodiment, treatment includes alleviating, reversing, mitigating the manifestations of a disease, disorder, or affliction, delaying its onset, or inhibiting its progression. In one embodiment, treatment includes alleviating, reversing, mitigating, reducing the underlying cause of a disease, disorder, or affliction, or delaying its onset. In some embodiments, "treatment" requires that signs or symptoms of a disease, disorder, or affliction have appeared or have been observed. In other embodiments, treatment can be administered in the absence of signs or symptoms of a disease or affliction, such as in prophylactic treatment. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., considering a history of symptoms and / or based on genetic or other susceptibility factors). Treatment can also continue after symptoms have resolved, for example to delay or prevent their recurrence. In some embodiments, treatment includes prophylaxis, and in other embodiments, it does not include prophylaxis. Unless otherwise indicated, as used herein, "von Willebrand factor protein" or "VWF protein" means a polypeptide comprising the amino acid sequence of a naturally occurring VWF polypeptide or a variant thereof, as determined by assays recognized in the art, which polypeptide has VWF biological activity, such as FVIII binding activity. VWF proteins that can be produced by the devices described herein (e.g., expressed by engineered cells contained within the device) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Encapsulated cells can be programmed to encode any of the following VWF polypeptides: the 2813 amino acid precursor VWF, VWF lacking the 22 amino acid signal peptide and optionally the 741 amino acid propeptide, the 2050 amino acid mature VWF protein and its truncated variants (such as VWF fragments sufficient to stabilize endogenous FVIII levels in VWF-deficient mice, e.g., truncated variants containing the D'D3 region (amino acids 764-1247) or the D1D2D'D3 region); and VWF variants having one or more amino acid substitutions (e.g., in the D' region described in U.S. Patent No. 9,458,223). Variant VWF proteins preferably have at least 50%, 75%, 90% or higher (including >100%) 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 AB et al., 2004. Haematologica 89:77 - 85), the 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).

[0081] 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 sequence, transferrin, CTP (the 28 - amino acid C - terminal peptide (CTP) of human chorionic gonadotropin (hCG) with 4 O - glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin - binding polypeptides, albumin - binding small molecules, or any combination thereof.

[0082] The chemical definitions selected

[0083] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the CAS version of the Periodic Table, Handbook of Chemistry and Physics, 75th Edition, inside front cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry and specific functional moieties and reactivity are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5th 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, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0084] The abbreviations used herein have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas set forth herein are constructed according to the standard rules of valence known in the art of chemistry.

[0085] When a range of values is listed, it is intended to encompass every value and sub-range within that range. For example, "C1-C6 alkyl" is intended to encompass 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 alkyls.

[0086] As used herein, "alkyl" refers to a group of straight-chain or branched saturated hydrocarbon groups having from 1 to 24 carbon atoms ("C1-C 24 alkyl"). In some embodiments, the alkyl has from 1 to 12 carbon atoms ("C1-C 12 alkyl"), from 1 to 10 carbon atoms ("C1-C 12 alkyl"), 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 has from 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyls include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tert-pentyl (C5) and n-hexyl (C6). Additional examples of alkyls include n-heptyl (C7), n-octyl (C8), etc. Each instance of an alkyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents or 1 substituent ("substituted alkyl").

[0087] As used herein, "alkenyl" refers to a group of straight-chain or branched hydrocarbon groups having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds and no triple bonds ("C2-C 24 alkenyl"). In some embodiments, the alkenyl has from 2 to 10 carbon atoms ("C2-C 10"alkenyl"), having from 2 to 8 carbon atoms ("C2-C8 alkenyl"), having from 2 to 6 carbon atoms ("C2-C6 alkenyl"), having from 2 to 5 carbon atoms ("C2-C5 alkenyl"), having from 2 to 4 carbon atoms ("C2-C4 alkenyl"), having from 2 to 3 carbon atoms ("C2-C3 alkenyl") or having 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds can be internal (as in 2-butenyl) or terminal (as in 1-butenyl). Examples of C2-C4 alkenyl include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. Examples of C2-C6 alkenyl include the aforementioned C 2-4 alkenyl and pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each instance of alkenyl can be independently optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted by one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents or 1 substituent ("substituted alkenyl").

[0088] As used herein, the term "alkynyl" refers to a group of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 alkenyl"). In some embodiments, the alkynyl has from 2 to 10 carbon atoms ("C2-C 10 alkynyl"), having from 2 to 8 carbon atoms ("C2-C8 alkynyl"), having from 2 to 6 carbon atoms ("C2-C6 alkynyl"), having from 2 to 5 carbon atoms ("C2-C5 alkynyl"), having from 2 to 4 carbon atoms ("C2-C4 alkynyl"), having from 2 to 3 carbon atoms ("C2-C3 alkynyl") or having 2 carbon atoms ("C2 alkynyl"). One or more carbon-carbon triple bonds can be internal (as in 2-butynyl) or terminal (as in 1-butynyl). Examples of C2-C4 alkynyl include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. Each instance of alkynyl can be independently optionally substituted, i.e., unsubstituted ("unsubstituted alkynyl"), or substituted by one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents or 1 substituent ("substituted alkynyl").

[0089] As used herein, the term "heteroalkyl" refers to a non-cyclic stable straight-chain or branched-chain or combinations thereof that includes at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The one or more heteroatoms O, N, P, S, and Si may be placed at any position of the heteroalkyl. Exemplary heteroalkyls 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 may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. In the recitation of "heteroalkyl" followed by a recitation of a specific heteroalkyl such as -CH2O, -NR C R D etc., it will be understood that the terms heteroalkyl and -CH2O or -NR C R D are not redundant or mutually exclusive. Rather, the recitation of the specific heteroalkyls is for purposes of increased clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyls such as -CH2O, -NR C R D etc. Each instance of a heteroalkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heteroalkyl") or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted heteroalkyl").

[0090] Unless otherwise specified, the terms "alkylene", "alkenylene", "alkynylene", or "heteroalkylene" alone or as part of another substituent each mean a divalent group derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene may be described, for example, as a C1-C6 membered alkylene, C2-C6 membered alkenylene, C2-C6 membered alkynylene, or C1-C6 membered heteroalkylene, where the term "membered" refers to the non-hydrogen atoms within the moiety. In the case of a heteroalkylene, the heteroatom may also occupy one or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkylamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the chemical formula of the linking group is written. For example, the formula -C(O)2R'- may represent both -C(O)2R'- and -R'C(O)2-.

[0091] As used herein, "aryl" refers to a group of monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring systems (e.g., having 6, 10, or 14 π electrons shared in the ring array), having 6-14 ring carbon atoms and zero heteroatoms ("C6-C 14 aryl"). In some embodiments, aryl has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl has 10 ring carbon atoms ("C 10 aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, aryl has 14 ring carbon atoms ("C 14 aryl"; e.g., anthracenyl). Aryl can be described as, for example, C6-C 10 aryl, where the term "aryl" refers to the non-hydrogen ring atoms within the moiety. Aryl includes phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of aryl can be independently optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl").

[0092] As used herein, "heteroaryl" refers to a group of 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring systems (e.g., having 6 or 10 π electrons shared in the ring array), having ring carbon atoms and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl containing one or more nitrogen atoms, when the valence allows, the point of attachment can be a carbon or nitrogen atom. The bicyclic heteroaryl system can include one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems where a heteroaryl ring as defined above is fused to one or more aryl groups, where the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members represents the number of ring members in the fused (aryl / heteroaryl) ring system. For bicyclic heteroaryl where one ring contains no heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring carrying the heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl). Heteroaryl can be described as, for example, 6-10 membered heteroaryl, where the term "membered" refers to the non-hydrogen ring atoms within the moiety.

[0093] In some embodiments, heteroaryl is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of heteroaryl can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents.

[0094] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 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, aza Oxalic acid thia Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0095] As used herein, the terms “arylene” and “heteroarylene” alone or as part of another substituent each refer to a divalent group derived from an aryl and a heteroaryl, respectively.

[0096] As used herein, “cycloalkyl” refers to a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C 10 cycloalkyl”) and 0 heteroatoms in a non-aromatic ring system. In some embodiments, the cycloalkyl 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 can be described, for example, as a C4-C7 cycloalkyl moiety, where the term “moiety” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyls 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 cycloalkyls include, but are not limited to, the foregoing C3-C6 cycloalkyls and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubyl (C8), bicyclo[1.1.1]pentyl (C5), bicyclo[2.2.2]octyl (C8), bicyclo[2.1.1]hexyl (C6), bicyclo[3.1.1]heptyl (C7), etc. Exemplary C3-C 10 cycloalkyls include, but are not limited to, the foregoing C3-C8 cycloalkyls and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), etc. As illustrated by the foregoing examples, in certain embodiments, the cycloalkyl is monocyclic (“monocyclic cycloalkyl”) or contains a fused, bridged, or spiro ring system (such as a bicyclic system (“bicyclic cycloalkyl”)), and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems where a cycloalkyl ring as defined above is fused to one or more aryl groups, where the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl ring system. Each instance of cycloalkyl can be independently optionally substituted, i.e., unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”).

[0097] "Heterocyclic group" as used herein refers to 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 ("3-10 membered heterocyclic group"). In a heterocyclic group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, provided that the valence allows. The heterocyclic group may be monocyclic ("monocyclic heterocyclic group") or a fused, bridged, or spiro ring system, such as a bicyclic system ("bicyclic heterocyclic group"), and may be saturated or may be partially unsaturated. The bicyclic heterocyclic group system may include one or more heteroatoms in one or both rings. "Heterocyclic group" also includes a ring system wherein a heterocyclic group ring as defined above is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl or heterocyclic group ring; or a ring system wherein a heterocyclic group ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic group ring, and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic group ring system. The heterocyclic group may be described, for example, as a 3-7 membered heterocyclic group, wherein the term "membered" refers to non-hydrogen ring atoms within said moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of the heterocyclic group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In certain embodiments, the heterocyclic group is an unsubstituted 3-10 membered heterocyclic group. In certain embodiments, the heterocyclic group is a substituted 3-10 membered heterocyclic group.

[0098] In some embodiments, the heterocyclic group is a 5- 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 (“5- to 10-membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 8-membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 6-membered heterocyclic group”). In some embodiments, the 5- to 6-membered heterocyclic group has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclic group has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclic group has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrol-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, dithiolanyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridyl, and thianyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholin-1,1-dioxide. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocanyl, oxocanyl, and thioocanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, and the like. Exemplary 6-membered heterocyclic groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0099] As used herein, “amino” refers to the group -NR 70 R 71, where R 70 and R 71 are each independently hydrogen, C1-C8 alkyl, C3-C 10 cycloalkyl, C4-C 10 heterocycloalkyl, C6-C 10 aryl, and C5-C 10 heteroaryl. In some embodiments, amino refers to NH2.

[0100] As used herein, "cyano" refers to -CN.

[0101] Unless otherwise specified, as used herein, "halo" or "halogen" independently or as part of another substituent means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0102] As used herein, "hydroxy" refers to the group -OH.

[0103] The alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl as 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" heterocycloalkyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). Generally, the term "substituted", whether or not preceded by the term "optionally", means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced by an admissible substituent, where the admissible substituent is, for example, a substituent that results in a stable compound, such as a compound that does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, or other reactions. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents at each position are the same or different. The term "substituted" is intended to include substitution with all admissible substituents of organic compounds (e.g., any of the substituents described herein that result in the formation of a stable compound). The present disclosure contemplates any and all such combinations to arrive at a stable compound. For the purposes of the present disclosure, a heteroatom (such as nitrogen) can have a hydrogen substituent and / or any suitable substituent that satisfies the valence of the heteroatom and results in the formation of a stable moiety.

[0104] Two or more substituents may optionally be joined to form an aryl, heteroaryl, cycloalkyl, or heterocyclic group. It has been found that such so-called ring-forming substituents are generally but not necessarily attached to the cyclic backbone structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the backbone structure. For example, two ring-forming substituents attached to adjacent members of the cyclic backbone structure form a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the backbone structure. For example, two ring-forming substituents are attached to a single member of the cyclic backbone structure, resulting in a spiro ring structure. In another embodiment, the ring-forming substituents are attached to non-adjacent members of the backbone structure.

[0105] The compounds of formula (I) described herein may contain one or more asymmetric centers and can thus exist in a number of isomeric forms (e.g., enantiomers and / or diastereomers). For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers (including racemic mixtures and mixtures enriched in one or more stereoisomers). Isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be prepared by asymmetric synthesis. See, e.g., 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 at page 268 (E.L. Eliel, editor, Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure further encompasses the compounds described herein in the form of individual isomers substantially free of other isomers, and alternatively in the form of mixtures of different isomers.

[0106] As used herein, an enantiomerically pure compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., is enantiomerically enriched). In other words, the "S" form of the compound is substantially free of the "R" form of the compound and is thus enantiomerically enriched in the "R" form. The term "enantiomerically pure" or "enantiomerically enriched" means 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% by weight of the enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0107] The compounds of formula (I) described herein may also contain one or more isotopic substitutions. For example, H may be in any isotopic form, including 1 H, 2 H (D or deuterium), and 3 H (or tritium); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O, and 18 O, etc.

[0108] The term "pharmaceutically acceptable salts" is intended to include salts of the active compounds prepared with relatively non-toxic acids or bases on the basis of specific 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, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (pure 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 functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfide, hydroiodic acid or phosphorous acid, etc.; and those 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 arginine salts, etc.; and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see, for example, Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds used in the devices of the present disclosure (e.g., particles, hydrogel capsules) contain both basic and acidic functional groups that allow the compound to be converted into a base addition salt or an acid addition salt. These salts can be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable carriers known to those skilled in the art are also suitable for use in the present disclosure.

[0109] As used herein, "polysaccharide" refers to a polymer of monosaccharide or disaccharide carbohydrates linked together by glycosidic bonds. Polysaccharides can 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, aldonic acid, mannuronic acid and guluronic acid. Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, amylopectin, glycogen, gelatin, cellulose, amylose, chitin, chitosan or their derivatives or variants, for example, as described in Laurienzo (2010), Mar Drugs 9:2435-65.

[0110] The devices of the present disclosure may contain compounds of formula (I) in the form of prodrugs. Prodrugs are those compounds that are readily subject to chemical change under physiological conditions to provide the compounds useful in preparing the devices of the present disclosure. Additionally, prodrugs may be converted into the useful compounds of formula (I) by chemical or biochemical means in an ex vivo environment.

[0111] Certain compounds of formula (I) described herein may exist in unsolvated forms as well as solvated forms (including hydrated forms). In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of formula (I) described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses encompassed by the present disclosure and are intended to fall within the scope of the present disclosure.

[0112] The term "solvate" refers to a form of a compound that is usually associated with a solvent through a solvolysis reaction. Such physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. The compounds described herein may be prepared, for example, in crystalline form and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates.

[0113] The term "hydrate" refers to a compound associated with water. Generally, the number of water molecules contained in the hydrate of a compound is in a certain ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound may be represented, for example, by the general formula R·x H2O, where R is the compound and where x is a number greater than 0.

[0114] As used herein, the term "tautomer" refers to a compound structure that is an interchangeable form and varies in terms of hydrogen atom and electron shift. Thus, through the movement of π electrons and atoms (usually H), the two structures may be in equilibrium. For example, enols and ketones are tautomers because they can be rapidly interconverted by treatment with acid or base. Tautomeric forms may be related to the acquisition of superior chemical reactivity and biological activity of related compounds.

[0115] As used herein, the symbol refers to a connection to the surface of an entity, such as a polymer (e.g., a polymer forming a hydrogel, such as alginate) or an implantable device (e.g., a particle, a hydrogel capsule). By The indicated linkage can refer to a direct attachment to an entity (e.g., a polymer or an implantable element), or can refer to a linkage to an entity through an attachment group. An "attachment group" as described herein refers to a moiety for linking a compound of formula (I) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein), and can include any attachment chemistry known in the art. A list of exemplary attachment groups is outlined in Bioconjugate Techniques (3rd Edition, Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated herein by reference in its entirety. In some embodiments, the attachment group includes alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(R D )-, -NCN-, -C(=N(R C )(R 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, where each of R A , R C , R D , R F , R G , x and y is independently as described herein. In some embodiments, the attachment group includes amine, ketone, ester, amide, alkyl, alkenyl, alkynyl or thiol. In some embodiments, the attachment group is a crosslinker. In some embodiments, the attachment group is –C(O)(C1-C6-alkylene)-, where the alkylene is substituted by R 1 , and R 1 is as described herein. In some embodiments, the attachment group is -C(O)(C1-C6-alkylene)-, where the alkylene is substituted by 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is -C(O)C(CH3)2 -. In some embodiments, the attaching group is -C(O)(methylene)-, wherein the alkylene group is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attaching group is -C(O)CH(CH3)-. In some embodiments, the attaching group is -C(O)C(CH3)-.

[0116] As used herein, the terms "covalent", "covalent bond", and "covalently linked" refer to a class of chemical bonds involving the sharing of electrons between two adjacent atoms. Examples of covalent bonds include bonds formed between carbon and hydrogen (C-H bond), carbon atoms (C-C bond), carbon and oxygen atoms (C-O bond), and carbon and nitrogen (C-N bond). Depending on the nature of the atoms, the covalent bond can be a single bond, double bond, or triple bond, i.e., the covalent bond may involve sharing one, two, or three pairs of electrons.

[0117] As used herein, the terms "ionic", "ionic bond", and "ionically linked" refer to a class of chemical bonds involving the Coulombic attraction between adjacent atoms with opposite charges (i.e., ions).

[0118] Modified polysaccharide polymer

[0119] The polysaccharide polymers described herein are covalently modified in part with a covalent crosslinker. In one embodiment, the polysaccharide polymer can be a linear, branched, or crosslinked polysaccharide polymer, or a polysaccharide polymer having 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, dendritic polymers, graft-copolymers, ladder polymers, and dendrimers. The polysaccharide polymer can be a thermosensitive polymer, such as a gel (e.g., becoming solid or liquid upon exposure to heat or a certain temperature) or a photocrosslinkable polymer. In some embodiments, the polysaccharide polymer can be biodegradable, e.g., containing labile bonds, or can be dissociated by an enzyme (e.g., a lyase). In some embodiments, the polysaccharide polymer consists of a single type of repeating monomer unit. In other embodiments, the polysaccharide polymer consists of different types of repeating monomer units (e.g., two types of repeating monomer units, three types of repeating monomer units, e.g., a polymeric blend). In some embodiments, the polysaccharide can consist of mannuronic acid and guluronic acid monomers.

[0120] 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, such as carboxymethyl cellulose. In one embodiment, the polysaccharide polymer is polylactic acid, glycan, or polycaprolactone. In one embodiment, the polysaccharide polymer is a hyaluronate, such as sodium hyaluronate. In one embodiment, the polymer is collagen, elastin, or gelatin. In one embodiment, the polymer is chitin.

[0121] In some embodiments, the polysaccharide polymer is a polymer that forms a hydrogel. The polymer that forms a hydrogel contains hydrophilic structures that enable it to accommodate a large amount of water in a three-dimensional network. The polymer that forms a hydrogel may include polymers that form homopolymer hydrogels, copolymer hydrogels, or multi-polymer interpenetrating polymer hydrogels, and may be amorphous, semi-crystalline, or crystalline in nature, for example, as described in Ahmed (2015) J Adv Res 6:105-121. Exemplary polymers that form hydrogels include proteins (e.g., collagen), gelatin, polysaccharides (e.g., starch, alginate, hyaluronate, agarose), and synthetic polysaccharides.

[0122] Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, amylopectin, glycogen, gelatin, cellulose, amylose, chitin, chitosan, or derivatives or variants thereof, for example, as described in Laurienzo (2010), Mar Drugs 9:2435-65. The polysaccharide polymer may contain heparin, chondroitin sulfate, dermatan sulfate, 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.

[0123] 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 a high-guluronic acid (G) alginate and contains greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more guluronic acid (G). In some embodiments, the alginate is a high-mannuronic acid (M) alginate and contains greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 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 an optional 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.

[0124] A polysaccharide polymer (such as any polymer described herein, such as any alginate described herein) containing a sugar moiety having the structure of formula (I) or a pharmaceutically acceptable salt thereof can be modified on one or more monomer units. In some embodiments, at least 0.5% of the sugar monomers in 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% 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).

[0125] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having the structure of Formula I) comprises an N% increase of at least 0.1 wt%, 0.2 wt%, 0.5 wt%, 1.0 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt% or 10 wt% N compared to the unmodified polymer, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0126] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having the structure of Formula I) comprises an N% increase of 0.1 wt% to 10 wt% N compared to the unmodified polymer, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0127] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having the structure of Formula (I)) comprises an N% increase of 0.1 wt% to 2 wt% N compared to the unmodified polymer, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0128] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having the structure of Formula (I)) comprises an N% increase of 2 wt% to 4 wt% N compared to the unmodified polymer, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0129] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having the structure of Formula (I)) comprises an N% increase of 4 wt% to 8 wt% N compared to the unmodified polymer, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0130] In some embodiments, any polysaccharide polymer described herein (e.g., alginate) comprises 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 comprises 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 comprises sugar monomers having the structure of formula (II-c). In some embodiments, the polysaccharide polymer comprises sugar monomers having the structure of formula (II-d). In some embodiments, the polysaccharide polymer comprises sugar monomers having the structure of formula (II-e). In some embodiments, the polysaccharide polymer comprises sugar monomers having the structure of formula (II-f).

[0131] In some embodiments, the polymer (e.g., alginate) is modified with the compounds shown in Table 3.

[0132] In some embodiments, the polymer (e.g., alginate) modified with a compound of formula (I) is not the modified polymer described in any one of WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 187225, WO2016 / 019391, WO2017 / 075630, WO 2017 / 075631, WO 2018 / 067615, WO 2019 / 169333, and US2016-0030359.

[0133] Defibrillation compound

[0134] In some embodiments, the polymers described herein further comprise at least one defibrillation compound of formula (I):

[0135]

[0136] or a pharmaceutically acceptable salt thereof, wherein:

[0137] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, 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 )C(O)(C1-C6-alkylene)–, -N(R C )C(O)(C1-C6-alkenylene)–, –N(R C )N(R D)–, –NCN–, –C(=N(R C )(R 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 attached to an attachment group (such as the attachment groups described herein) and is optionally substituted by one or more R 1 ; L 1 and L 3 are each independently a bond, an alkyl or a heteroalkyl, where each alkyl and heteroalkyl is optionally substituted by one or more R 2 ; L 2 is a bond; M is absent, an alkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, each of which is optionally substituted by one or more R 3 ; P is absent, a cycloalkyl, a heterocycloalkyl or a heteroaryl, each of which is optionally substituted by one or more R 4 ; Z is hydrogen, an alkyl, an alkenyl, an alkynyl, a heteroalkyl, –OR A , –C(O)R A , –C(O)OR A , –C(O)N(R C )(R D ), –N(R C )C(O)R A , a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by one or more R 5 ; each R A , R B , R C , R D , R E , R F and R G are independently hydrogen, an alkyl, an alkenyl, an alkynyl, a heteroalkyl, a halogen, an azide, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by one or more R 6 ; or R C and RD Together with the nitrogen atom to which it is attached, form an optionally substituted by one or more R 6 substituted ring (e.g., 5- to 7-membered ring); each R 1 , R 2 , R 3 , R 4 , R 5 and R 6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 , S(O) x R E1 , –OS(O) x R E1 , –N(R C1 )S(O) x R E1 , –S(O) x N(R C1 )(R D1 ), –P(R F1 ) y , cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is optionally substituted by one or more R 7 ; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by one or more R 7 ; each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group; x is 1 or 2; and y is 2, 3 or 4.

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

[0139]

[0140] or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, 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 ), N(R C )C(O)(C1-C6-alkylene)–, -N(R C )C(O)(C1-C6-alkenylene)–, –NCN–, –C(=N(R C )(R 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 attached to an attachment group (such as an attachment group described herein) and is optionally substituted by one or more R 1 ; Each of L 1 and L 3 is independently a bond, alkyl or heteroalkyl, each alkyl and heteroalkyl being optionally substituted by one or more R 2 ; L 2 is a bond; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 3 ; P is a heteroaryl optionally substituted by one or more R 4 ; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 ; Each R A , R B , R C , R D , R E , R F and R GIndependently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with one or more R 6 ; or R C and R D together with the nitrogen atom to which it is attached form a ring (e.g., 5- to 7-membered ring) optionally substituted with one or more R 6 ; each R 1 , R 2 , R 3 , R 4 , R 5 and R 6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 , S(O) x R E1 , –OS(O) x R E1 , –N(R C1 )S(O) x R E1 , –S(O) x N(R C1 )(R D1 ), –P(R F1 ) y , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with one or more R 7 ; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with one or more R 7 ; each R 7independently an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, halogen, cyano group, oxo group, hydroxyl group, cycloalkyl group or heterocyclic group; x is 1 or 2; and y is 2, 3 or 4.

[0141] In some embodiments, for formula (I) and (I-a), A is an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group, -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 )-. In some embodiments, A is an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group, -O-, -C(O)O-, -C(O)-, -OC(O)- or -N(R C ). In some embodiments, A is an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, -O-, -C(O)O-, -C(O)-, -OC(O)- or -N(R C ). In some embodiments, A is an alkyl group, -O-, -C(O)O-, -C(O)-, -OC(O) or -N(R C ). 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 ). In some embodiments, A is -N(R C ), and R C and R D are independently hydrogen or an alkyl group. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, wherein the alkylene is substituted by R 1 . In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, and R 1 is an alkyl group (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 an alkyl group (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0142] In some embodiments, for Formulas (I) and (I-a), L 1 is a bond, an alkyl, or a heteroalkyl. In some embodiments, L 1 is a bond or an alkyl. In some embodiments, L 1 is a bond. In some embodiments, L 1 is an alkyl. In some embodiments, L 1 is a C1-C6 alkyl. In some embodiments, L 1 is -CH2-, -CH(CH3)-, -CH2CH2CH2 or -CH2CH2-. In some embodiments, L 1 is -CH2- or -CH2CH2-.

[0143] In some embodiments, for Formulas (I) and (I-a), L 3 is a bond, an alkyl, or a heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is an alkyl. In some embodiments, L 3 is a C1-C 12 alkyl. In some embodiments, L 3 is a C1-C6 alkyl. In some embodiments, L 3 is -CH2-. In some embodiments, L 3 is a heteroalkyl. In some embodiments, L 3 is a C1-C 2 heteroalkyl optionally substituted with one or more R 12 (e.g., an oxo group). In some embodiments, L 3 is a C1-C6 heteroalkyl optionally substituted with one or more R 2 (e.g., an oxo group). 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-.

[0144] 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 In some embodiments, M is substituted with R 7 (e.g., 1R 7 ) substituted phenyl. In some embodiments, M is In some embodiments, R 7 is CF3.

[0145] In some embodiments, for Formulas (I) and (I-a), P is absent, heterocyclic, or heteroaryl. In some embodiments, P is absent. In some embodiments, for Formulas (I) and (I-a), P is a tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is a monocyclic heteroaryl. In some embodiments, P is a nitrogen-containing heteroaryl. In some embodiments, P is a monocyclic nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl. In some embodiments, P is a 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 In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is

[0146] In some embodiments, P is a heterocyclic group. In some embodiments, P is a 5-membered heterocyclic group or a 6-membered heterocyclic group. In some embodiments, P is an imidazolidinone group. In some embodiments, P is In some embodiments, P is a thiomorpholino-1,1-dioxide group.

[0147] In some embodiments, P is

[0148] In some embodiments, for formula (I) and formula (I-a), Z is an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group. In some embodiments, Z is a heterocyclic group. In some embodiments, Z is a monocyclic or bicyclic heterocyclic group. In some embodiments, Z is an oxygen-containing heterocyclic group. In some embodiments, Z is a 4-membered heterocyclic group, a 5-membered heterocyclic group or a 6-membered heterocyclic group. In some embodiments, Z is a 6-membered heterocyclic group. In some embodiments, Z is a 6-membered oxygen-containing heterocyclic group. In some embodiments, Z is a tetrahydropyranyl group. In some embodiments, Z is In some embodiments, Z is a 4-membered oxygen-containing heterocyclic group. In some embodiments, Z is

[0149] In some embodiments, Z is a bicyclic oxygen-containing heterocyclic group. In some embodiments, Z is a phthalic anhydride group. In some embodiments, Z is a sulfur-containing heterocyclic group. In some embodiments, Z is a 6-membered sulfur-containing heterocyclic group. In some embodiments, Z is a 6-membered heterocyclic group containing a nitrogen atom and a sulfur atom. In some embodiments, Z is a thiomorpholino-1,1-dioxide group. In some embodiments, Z is In some embodiments, Z is a nitrogen-containing heterocyclic group. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclic group. In some embodiments, Z is

[0150] In some embodiments, Z is a bicyclic heterocyclic group. In some embodiments, Z is an optionally one or more R 5 substituted bicyclic nitrogen-containing heterocyclic group. In some embodiments, Z is a 2-oxa-7-azaspiro[3.5]nonyl group. In some embodiments, Z is In some embodiments, Z is a 1-oxa-3,8-diazaspiro[4.5]dec-2-one. In some embodiments, Z is

[0151] In some embodiments, for formula (I) and (I-a), Z is an aryl group. In some embodiments, Z is a monocyclic aryl group. In some embodiments, Z is a phenyl group. In some embodiments, Z is (for example, substituted by one R 5)Monosubstituted phenyl. 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 5 is an oxygen-containing heteroalkyl. In some embodiments, Z is monosubstituted phenyl, where one R 5 is OCH3. In some embodiments, Z is monosubstituted phenyl, where one R 5 is in the ortho position. In some embodiments, Z is monosubstituted phenyl, where one R 5 is in the meta position. In some embodiments, Z is monosubstituted phenyl, where one R 5 is in the para position.

[0152] 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 1-5 R 5 groups. In some embodiments, Z is C1-C8 alkyl substituted by one R 5 group. In some embodiments, Z is C1-C8 alkyl substituted by one R 5 group, where 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 group, where R 5 is -OR A1 or -C(O)OR A1 . In some embodiments, Z is C1-C8 alkyl substituted by one R 5 group, where R 5 is -OR A1 or -C(O)OH. In some embodiments, Z is -CH3.

[0153] In some embodiments, for formulas (I) and (I-a), Z is heteroalkyl. In some embodiments, Z is C1-C12 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 optionally substituted with one or more R 5 substituted nitrogen-containing heteroalkyl. In some embodiments, Z is substituted with 1-5 R 5 substituted nitrogen- and sulfur-containing heteroalkyl. In some embodiments, Z is the N-methyl-2-(methylsulfonyl)eth-1-aminyl radical.

[0154] 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).

[0155] In some embodiments, Z is hydrogen.

[0156] In some embodiments, L 2 is a bond and P and L 3 independently do not exist. 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.

[0157] In some embodiments, the compound of formula (I) is a compound of formula (I-b):

[0158]

[0159] or a pharmaceutically acceptable salt thereof, wherein ring M 1 is cycloalkyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with 1-5 R 3 substituents; ring Z 1 is cycloalkyl, heterocyclic, aryl or heteroaryl optionally substituted with 1-5 R 5 substituents; R 2a , R 2b , R 2c and R 2d each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclic, aryl or heteroaryl, or R 2a and R 2b or R2c and R 2d each 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, heterocycloalkyl, aryl or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted with 1-6 R 6 ; each R 3 , R 5 and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl; each of R 10 and R 11 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocycloalkyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl; each m and n is independently 1, 2, 3, 4, 5 or 6; and refers to the attachment to an attachment group or polymer described herein. In some embodiments, for each R 3 and R 5 , each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl or heterocycloalkyl.

[0160] In some embodiments, the compound of formula (I-b) is a compound of formula (I-b-i):

[0161]

[0162] or a pharmaceutically acceptable salt thereof, wherein ring M 2 is an aryl or heteroaryl optionally substituted with one or more R 3 ; ring Z 2 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen, alkyl or heteroalkyl, or each of R 2a and R 2b or each of R 2c and R 2d forms an oxo group together; X is absent, O or S; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; or two R 5 form a 5- to 6-membered ring fused to ring Z 2 ; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and refers to the attachment to an attachment group or polymer described herein.

[0163] In some embodiments, the compound of formula (I-b-i) is a compound of formula (I-b-ii):

[0164]

[0165] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl; each of R 2c and R 2d is independently hydrogen, alkyl or heteroalkyl, or R 2c and R 2d form an oxo group together; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)ORA1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and refers to the attachment to an attachment group or polymer described herein.

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

[0167]

[0168] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl; each of R 2c and R 2d is independently hydrogen, alkyl or heteroalkyl, or R 2c and R 2d together form an oxo group; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m is 1, 2, 3, 4, 5 or 6; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and refers to the attachment to an attachment group or polymer described herein.

[0169] In some embodiments, the compound of formula (I) is a compound of formula (I-d):

[0170]

[0171] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl; X is absent, O or S; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d together form an oxo group; each R 5Independently an alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and refers to a linkage to an attachment group or polymer described herein.

[0172] In some embodiments, the compound of formula (I) is a compound of formula (I-e):

[0173]

[0174] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl; X is absent, O or S; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen, alkyl or heteroalkyl, or each of R 2a and R 2b or R 2c and R 2d together form an oxo group; each R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and refers to a linkage to an attachment group or polymer described herein.

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

[0176]

[0177] or a pharmaceutically acceptable salt thereof, wherein M is an alkyl optionally substituted with one or more R 3 ; ring P is a heteroaryl optionally substituted with one or more R 4 ; L 3 is optionally substituted with one or more R 2Substituted alkyl or heteroalkyl; Z is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted with one or more R 5 substituents; R 2a and R 2b are each independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together form an oxo group; each R 2 , R 3 , R 4 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and refers to the attachment to the attachment group or polymer described herein.

[0178] In some embodiments, the compound of formula (I) is a compound of formula (II):

[0179]

[0180] or a pharmaceutically acceptable salt thereof, wherein M is a bond, alkyl or aryl, wherein the alkyl and aryl are optionally substituted with one or more R 3 substituents; L 3 is alkyl or heteroalkyl optionally substituted with one or more R 2 substituents; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -OR A , wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more R 5 substituents; R A is hydrogen; R 2a and R 2b are each independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together form an oxo group; each R 2 , R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and Refers to the attachment to an attachment group or polymer as described herein.

[0181] In some embodiments, the compound of formula (II) is a compound of formula (II-a):

[0182]

[0183] or a pharmaceutically acceptable salt thereof, wherein L 3 is alkyl or heteroalkyl, each of which is optionally substituted by one or more R 2 ; Z is hydrogen, alkyl, heteroalkyl or -OR A , wherein the alkyl and heteroalkyl are optionally substituted by one or more R 5 ; each of R 2a and R 2b is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together form an oxo group; each R 2 , R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; R A is hydrogen; each of R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and Refers to the attachment to an attachment group or polymer as described herein.

[0184] In some embodiments, the compound of formula (I) is a compound of formula (III):

[0185]

[0186] or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is 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, heterocycloalkyl, aryl or heteroaryl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R Cis hydrogen, alkyl, alkenyl, alkynyl or heteroalkyl, wherein each of the alkyl, alkenyl, alkynyl or heteroalkyl is optionally substituted with 1-6 R 6 ; R 3 , R 5 and R 6 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; and refers to a linkage to an attachment group or polymer described herein.

[0187] In some embodiments, the compound of formula (III) is a compound of formula (III-a):

[0188]

[0189] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with 1-5 R 5 ; R 2a , R 2b , R 2c and R 2d are each independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; o and p are each independently 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; and refers to a linkage to an attachment group or polymer described herein.

[0190] In some embodiments, the compound of formula (III-a) is a compound of formula (III-b):

[0191]

[0192] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with 1-5 R 5 substituents; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen, alkyl, heteroalkyl, halogen; or R 2a and R 2b or R 2c and R 2d together form an oxo group; each of R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; o and p are each independently 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; and refers to the attachment to an attachment group or polymer described herein.

[0193] In some embodiments, the compound of formula (III-a) is a compound of formula (III-c):

[0194]

[0195] or a pharmaceutically acceptable salt thereof, wherein X is C(R’)(R”), N(R’) or S(O) x ; each of R’ and R” is independently hydrogen, alkyl, halogen or cycloalkyl; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen, alkyl, heteroalkyl or halogen; or R 2a and R 2b or R 2c and R 2d together form an oxo group; each of R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R A1 and R B1Independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; and refers to the attachment to an attachment group or polymer described herein.

[0196] In some embodiments, the compound of formula (III-c) is the compound of formula (III-d):

[0197]

[0198] or a pharmaceutically acceptable salt thereof, wherein X is C(R’)(R”), N(R’), or S(O) x ; each of R’ and R” is independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d are each independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; each of R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 ; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; and refers to the attachment to an attachment group or polymer described herein.

[0199] In some embodiments, the compound of formula (I) is the compound of formula (III-e):

[0200]

[0201] or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with 1 - 5 R 5 ; each of R 2a , R 2b , R 2c , and R 2dEach of them is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R C is hydrogen, alkyl, alkenyl, alkynyl or heteroalkyl, wherein each of the alkyl, alkenyl, alkynyl or heteroalkyl is optionally substituted with 1-6 R 6 ; R 3 , R 5 and R 6 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; each R 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro or amino; each R A1 and R B1 are each independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; w is 0 or 1; and refers to the attachment to the attachment group or polymer described herein.

[0202] In some embodiments, the compound of formula (I) is a compound of formula (III-f):

[0203]

[0204] or a pharmaceutically acceptable salt thereof, wherein ring Z 1 is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted with 1-5 R 5 ; R 2a , R 2b , R 2c and R 2d are each independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R C is hydrogen, alkyl, alkenyl, alkynyl or heteroalkyl, wherein each of the alkyl, alkenyl, alkynyl or heteroalkyl is optionally substituted with 1-6 R 6 ; R 3 , R 5 and R 6 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)ORA1 or -C(O)R B1 ; each R 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halogen, cyano, nitro or amino; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; o and p are each independently 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; w is 0 or 1; and refers to a connection to an attachment group or polymer described herein.

[0205] In some embodiments, the compound of formula (I) is a compound of formula (III-g):

[0206]

[0207] or a pharmaceutically acceptable salt thereof, wherein ring Z 1 is cycloalkyl, heterocyclic, aryl or heteroaryl, each of which is optionally substituted with 1-5 R 5 ; R C is hydrogen, alkyl, –N(R C )C(O)R B , –N(R C )C(O)(C1-C6-alkyl) or –N(R C )C(O)(C1-C6-alkenyl), wherein each of the alkyl and alkenyl is optionally substituted with 1-6 R 6 ; R 2a , R 2b , R 2c and R 2d is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 , R 5 and R 6 is independently alkyl, heteroalkyl, halogen, oxo, –OR A1 , –C(O)OR A1 or –C(O)R B1 ; R 12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halogen, cyano, nitro or amino; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; x is 0, 1 or 2; and refers to a connection to an attachment group or polymer described herein.

[0208] In some embodiments, a compound of formula (I) is a compound of formula (III-h):

[0209]

[0210] or a pharmaceutically acceptable salt thereof, wherein R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C1-C6-alkyl) or -N(R C )C(O)(C1-C6-alkenyl), wherein each of the alkyl and alkenyl is optionally substituted with 1-6 R 6 ; each of R 2a , R 2b , R 2c and R 2d is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; each of R 3 , R 5 and R 6 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 ; R 12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro or amino; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; x is 0, 1 or 2; z is 0, 1, 2, 3, 4, 5 or 6, and refers to the attachment to an attachment group or polymer as described herein.

[0211] In some embodiments, a compound of formula (I) is a compound of formula (III-i):

[0212]

[0213] or a pharmaceutically acceptable salt thereof, wherein X is C(R’)(R”), N(R’) or S(O) x ; each of R’ and R” is independently hydrogen, alkyl or halogen; R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C)C(O)(C1-C6-alkyl) or -N(R C )C(O)(C1-C6-alkenyl), wherein each of the alkyl and alkenyl is optionally substituted with 1-6 R 6 ; R 2a 、R 2b 、R 2c and R 2d are each independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 、R 5 and R 6 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; R 12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro or amino; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; x is 0, 1 or 2; z is 0, 1, 2, 3, 4, 5 or 6, and refers to the attachment to the attachment group or polymer described herein.

[0214] 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.

[0215] 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.

[0216] In some embodiments, the compound is a compound of formula (I-b). In some embodiments, P is absent, L1 is -NHCH2, L 2 is a bond, M is an aryl group (e.g., phenyl), L 3 is -CH2O, and Z is a heterocyclic group (e.g., a nitrogen-containing heterocyclic group, such as thiomorpholinyl-1,1-dioxide).

[0217] In some embodiments of formula (I-b), P is absent, L 1 the formula -NHCH2, L 2 is a bond, M is absent, L 3 is a bond, and Z is a heterocyclic group (e.g., an oxygen-containing heterocyclic group, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl or oxiranyl).

[0218] 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 independently is hydrogen or CH3, R 2c and R 2d each independently is hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M 2 is a phenyl group optionally substituted by one or more R 3 substituents, R 3 is -CF3, and Z 2 is a heterocyclic group (e.g., an oxygen-containing heterocyclic group, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl or oxiranyl).

[0219] 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 independently is hydrogen, q is 0, p is 0, m is 1, and Z 2 is a heterocyclic group (e.g., an oxygen-containing heterocyclic group, such as tetrahydropyranyl).

[0220] 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 independently is hydrogen, m is 1, p is 1, q is 0, R 5 is -CH3, and Z is a heterocyclic group (e.g., a nitrogen-containing heterocyclic group, such as piperazinyl).

[0221] 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 independently is hydrogen, m is 1, n is 3, X is O, p is 0, and Z is a heterocyclic group (e.g., an oxygen-containing heterocyclic group such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).

[0222] 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 independently is hydrogen, n is 1, M is -CH2-, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), L 3 is -C(O)OCH2-, and Z is CH3.

[0223] 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 independently is hydrogen, n is 1, q is 0, L 3 is -CH2(OCH2CH2)2, and Z is -OCH3.

[0224] In some embodiments of formula (II-a), R 2a and R 2b each independently is hydrogen, n is 1, L 3 is a bond or –CH2, and Z is hydrogen or –OH.

[0225] 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 each independently is hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 1 is a heteroalkyl optionally substituted by R 5 (e.g., -N(CH3)(CH2CH2)S(O)2CH3).

[0226] 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 each independently is hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is an aryl (e.g., phenyl) substituted by 1 R 5 (e.g., -NH2).

[0227] 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 each independently is hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 2 is a heterocyclic group (e.g., a nitrogen-containing heterocyclic group, e.g., a nitrogen-containing spiro heterocyclic group, e.g., 2-oxa-7-azaspiro[3.5]nonyl).

[0228] 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 each independently is 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 each independently is hydrogen, m is 1, n is 2, q is 1, 2, 3 or 4, p is 0, and X is S(O)2.

[0229] 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).

[0230] In some embodiments, the compound of formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO 2017 / 075630, US2012-0213708, US2016-0030359 or US2016-0030360.

[0231] In some embodiments, the compound of formula (I) comprises the compounds shown in Table 3, or a pharmaceutically acceptable salt thereof. In some embodiments, the outer surface and / or one or more compartments within the devices described herein comprise the small molecule compounds shown in Table 3, or a pharmaceutically acceptable salt thereof.

[0232] Table 3: Exemplary Compounds of Formula (I)

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] Conjugation of any of the compounds in Table 3 with a polymer (e.g., alginate) can be carried out as described in Example 2 of WO 2019 / 195055 or any other suitable chemical reaction.

[0241] In some embodiments, the compound is a compound of formula (I) (e.g., (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, and is selected from:

[0242]

[0243] or a pharmaceutically acceptable salt thereof.

[0244] In some embodiments, the polysaccharide polymer or device (e.g., hydrogel capsule) described herein comprises the compound or a pharmaceutically acceptable salt of any one of the two compounds.

[0245] In some embodiments, as determined by combustion analysis for nitrogen percentage as described in WO 2020 / 069429, the compound of formula (I) (e.g., compound 101 in Table 3) is covalently attached to alginate (e.g., alginate having an approximate MW < 75 kDa, G:M ratio ≥ 1.5) at a conjugation density of at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0 - 7.0, 5.0 to 7.0, or 6.0 - 7.0 or about 6.8.

[0246] Crosslinking moiety

[0247] In some embodiments, the crosslinking moiety can undergo a thiol-ene click reaction. In some embodiments, the crosslinking moiety contains a thiol. In some embodiments, the thiol includes an alkyl thiol or an aryl thiol. In some embodiments, the click crosslinker can contain more than one thiol group. In some embodiments, the click crosslinker can have two, three, four, five, or six thiol groups. In some embodiments, the thiol is a compound of formula (IV):

[0248] or a pharmaceutically acceptable salt or tautomer thereof, wherein Q is O, NR 33 or C(R 34a )(R 34b );R 33 , R 34a , R 34b , R 60a , R 60b , R 61a , R 61b and R 62 each independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic.

[0249] In some embodiments, the compound of formula (IV) is a compound of formula (IV-a):

[0250] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 60a , R 60b, R 61a , R 61b , R 63a and R 63b each independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1 - 6 R 7 ; and each R 7 independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

[0251] In some embodiments, the compound of formula (IV) is a compound of formula (IV - b):

[0252] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of Q and Y is O, NR 33 or C(R 34a )(R 34b ); R 33 , R 34a , R 34b , R 60a , R 60b , R 61b , R 62 and R 64 each independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

[0253] In some embodiments, the compound of formula (IV) is a compound of formula (IV-c):

[0254] or a pharmaceutically acceptable salt or tautomer thereof, wherein Y is O, NR 33 or C(R 34a )(R 34b ); R 33 , R 34a , R 34b , R 60a , R 60b , R 61b , R 63a , R 63b and R 64 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, heterocyclic group, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1-6 R 7is substituted; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0255] In some embodiments, the crosslinker moiety comprises an alkenyl group. In some embodiments, the crosslinker comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the crosslinker comprises a norbornenyl moiety. In some embodiments, the crosslinker is a compound of formula (V):

[0256] 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 ); R 33 , R 34a , R 34b , R 65a , R 65b , R 65c , R 65d , R 65e , R 65f , R 65g and R 66 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0257] In some embodiments, the crosslinker of formula (V) is a compound of formula (V-a):

[0258] or a pharmaceutically acceptable salt or tautomer thereof, wherein U is O, NR 33 , or C(R 34a )(R 34b ); 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 independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic.

[0259] In some embodiments, the crosslinker of formula (V) is a compound of formula (V-b):

[0260] or a pharmaceutically acceptable salt or tautomer thereof, wherein U is O, NR 33 , or C(R 34a )(R 34b ); 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 , R 68a and R 68b Each of the following is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic.

[0261] In some embodiments, the click crosslinker is a compound of formula (VI):

[0262] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T, Y 1 and Y 2 is independently O, NR 33 , or C(R 34a )(R 34b ); R 33 , R 34a , R 34b , R 69 and R 70 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, –OR A1 , –C(O)OR A1 , –C(O)R B1 , –OC(O)R B1 , –N(RC1 )(R D1 )、 –N(R C1 )C(O)R B1 、 –C(O)N(R C1 )、 SR E1 、 cycloalkyl, heterocyclic group, aryl or heteroaryl; each R A1 、 R B1 、 R C1 、 R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1 - 6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

[0263] In some embodiments, the crosslinker of formula (VI) is a compound of formula (VI - a):

[0264] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of R 69 、 R 70 and R 71 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, heterocyclic group, aryl or heteroaryl; each R A1 、 R B1 、 R C1 、 R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1 - 6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

[0265] In some embodiments, the crosslinker of formula (VI) is a compound of formula (VI-b):

[0266] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 69 、R 70 、R 72a 、R 72b 、R 73a and R 73b each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; each R A1 、R B1 、R C1 、R D1 and R E1 independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7 independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

[0267] In some embodiments, the crosslinker comprises maleimide.

[0268] In some embodiments, the crosslinker comprises aryl or heteroaryl. In some embodiments, the click crosslinker is a compound of formula (VII):

[0269] or a pharmaceutically acceptable salt or tautomer thereof, wherein T is O, NR 33 , or C(R 34a )(R 34b ); ring M is cycloalkyl, heterocyclic group, aryl, heteroaryl, each of which is optionally substituted by 1-6 R 7 ; R 33 、R 34a 、R 34b and R 74Each of the above is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0270] In some embodiments, the cross-linking agent of formula (VII) is a compound of formula (VII-a):

[0271] or a pharmaceutically acceptable salt or tautomer thereof, wherein T is O, NR 33 , or C(R 34a )(R 34b ); Ring M is cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which is optionally substituted by 1-6 R 7 Replacement; R 33 , R 34a , R 34b and R 74 Each of the above is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 RA1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1 - 6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0272] In some embodiments, the crosslinker of formula (VII) is a compound of formula (VII - b):

[0273] or a pharmaceutically acceptable salt or tautomer thereof, wherein ring M is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted with 1 - 6 R 7 ; R 74 , R 75a , R 75b , R 76a and R 76b each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1 - 6 R 7 ; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0274] In some embodiments, the crosslinker of formula (VII) is a compound of formula (VII - c):

[0275] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 74 , R 75a , R 75b , R 76a , R 76b and R 77 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and each R 7 independently is alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic.

[0276] In some embodiments, the crosslinker moiety comprises a tetrazine moiety.

[0277] In some embodiments, the compound of any one of formulas (IV), (V), (VI) and (VII) is selected from the compounds in Table 4:

[0278] Table 4: Exemplary crosslinking moieties

[0279]

[0280] Polymer modified with a crosslinker

[0281] The crosslinker can be covalently bound to a polysaccharide (such as alginate). The modified polysaccharide polymer (such as a modified alginate polymer) may be capable of crosslinking with another polymer. In one embodiment, the polysaccharide polymer is modified with more than one type of crosslinker.

[0282] In one embodiment, the modified polysaccharide is a compound of formula (VIII):

[0283] 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 ); each of R 38a , R 38b , R 39a , R 39b , R 40 , R 41 and R 42 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each of R 32 and R 35 is hydrogen, alkyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic; and the click crosslinker has the structure 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).

[0284] Polysaccharide polymers modified with a click crosslinker and defibrinated small molecule compounds

[0285] In one embodiment, the polysaccharide polymer comprises the structure of formula (IX):

[0286] or a pharmaceutically acceptable salt or tautomer thereof, wherein W, T 1 , T 2 , U 1 and U 2 each independently is C(R 40 )(R 41 ), O, or N(R 42 ); R 38a , R 38b , R 38c , R 38d R 39a , R 39b , R 39a , R 39b , R 40 , R 41 and R 42 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and each R 7independently an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group; p is an integer from 1 to 100; the defibrillation compound has the structure of formula (I), (I-a), (I-b), (I-b-i), (I-b-ii), (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); and the click crosslinker has the structure of formula (IV), (IV-a), (IV-b), (IV-c), (V), (V-a), (V-b), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b) or (VII-c).

[0287] In one embodiment, the modified polysaccharide polymer is a compound selected from Table 5.

[0288] Table 5. Exemplary modified polysaccharide polymers of formulas (VIII and IX)

[0289]

[0290]

[0291]

[0292] or a pharmaceutically acceptable salt thereof.

[0293] The polysaccharide polymers described herein can be modified at any suitable functional group (such as a carboxyl or hydroxyl group). In one embodiment, the polysaccharide polymer is modified at a single type of functional group. In one embodiment, the polysaccharide polymer is modified at more than one type of functional group. In one embodiment, the polysaccharide polymers described herein can be modified at one or more functional groups with a compound of formula (I) and / or a compound of formulas (IV)-(VII).

[0294] In one embodiment, the degree of modification (i.e., the percentage of functional groups of the polymer modified with the photoactive 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 degree of modification of the polysaccharide polymer is between about 1% and about 99%. In one embodiment, the degree of modification is between about 1% and about 80%. In one embodiment, the degree of modification is between about 1% and about 75%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 70%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 65%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 60%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 55%.

[0295] In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 50%.

[0296] In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 45%.

[0297] In a preferred embodiment, not all functional groups (such as carboxyl groups) of the modified polysaccharide are substituted, which allows for ionic and covalent crosslinking.

[0298] 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 crosslinker (e.g., a compound of formula (IV), a compound of formula (I), and a cell adhesion molecule (such as RGD)). In one embodiment, the polysaccharide polymers described herein are modified with a photoactive crosslinker. 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 crosslinker and a cell adhesion molecule. In a preferred embodiment, the polysaccharide polymers described herein are modified with both a photoactive crosslinker and a compound of formula (I). In one embodiment, the polysaccharide polymers described herein are modified with a cell adhesion molecule and a compound of formula (I).

[0299] In one embodiment, the degree of modification (i.e., the percentage of functional groups of the polymer modified with clickable crosslinkers) 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 degree of modification of the polysaccharide polymer is between about 1% and about 99%. In one embodiment, the degree of modification is between about 1% and about 80%. In one embodiment, the degree of modification is between about 1% and about 75%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 70%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 65%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 60%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 55%.

[0300] In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 50%.

[0301] In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 45%.

[0302] In a preferred embodiment, not all functional groups (such as carboxyl groups) of the modified polysaccharide are substituted, which allows for ionic and covalent crosslinking.

[0303] In one embodiment, the degree of modification (i.e., the percentage of functional groups of the polymer modified with clickable crosslinkers) 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 percentage of functional groups of the polymer modified with clickable crosslinkers) 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 percentage of functional groups of the polymer modified with clickable crosslinkers) 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%.

[0304] In some embodiments, such as when the polysaccharide polymers described herein are used to prepare double-crosslinked hydrogel capsules, the polymer retains sufficient unreacted carboxyl groups to permit ionic crosslinking. In some embodiments, the polysaccharide polymers described herein do not contain more than 10% carboxyl groups with a degree of modification. In some embodiments, the polysaccharide polymers described herein do not contain more than 5% carboxyl groups with a degree of modification. In some embodiments, the polysaccharide polymers described herein do not contain more than 5%, 6%, 7%, 8%, 9% or 10% carboxyl groups with a degree of modification.

[0305] Characteristics of the Hydrogel Capsules

[0306] The present disclosure further features hydrogel capsules comprising the polysaccharide polymers described herein. The hydrogel capsules can be produced by crosslinking crosslinking groups (i.e., covalently crosslinking) or by ionic crosslinking, for example, in the presence of a divalent cation (such as 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 other methods of initiating polymerization are also possible, including applying heat, ultrasound, and γ-radiation in the presence of a suitable initiator.

[0307] The hydrogel capsules described herein are formed by crosslinking one or more types of polysaccharide polymers. In one embodiment, the hydrogel capsules contain only polysaccharide polymers. In one embodiment, the hydrogel capsules contain the same type of polysaccharide polymer, such as an alginate polymer. In one embodiment, the hydrogel capsules are formed by polymerization of two identical polysaccharides. In one embodiment, the hydrogel capsules are formed by polymerization of two different polysaccharides. In one embodiment, the hydrogel capsules contain a plurality of polymers, such as a plurality of polysaccharide polymers. In one embodiment, the hydrogel capsules contain one polysaccharide polymer and a non-polysaccharide polymer.

[0308] The hydrogel capsules described herein can be homogeneous, i.e., they may not contain non-polysaccharide polymers. In one embodiment, the hydrogel capsules described herein do not contain polymers selected from polyacrylamide, poly(vinyl alcohol), poly(ethylene oxide), polyethylene glycol (PEG), and polyphosphazene. In one embodiment, the hydrogel capsules do not contain poly(vinyl alcohol). In one embodiment, the hydrogel capsules do not contain poly(ethylene oxide). In one embodiment, the hydrogel capsules do not contain polyethylene glycol (PEG). In one embodiment, the hydrogel capsules do not contain polyphosphazene.

[0309] In one embodiment of the present invention, the hydrogel capsule is a two-compartment hydrogel capsule. In a preferred embodiment of the present disclosure, 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.

[0310] In some embodiments, the first and second compartments contain a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment contains a blend of polymers. In some embodiments, the second (outer) compartment contains a blend of polymers. In some embodiments, the first and second compartments contain the same blend of polymers. In some embodiments, the first and second compartments contain different blends of polymers.

[0311] In some embodiments, the first compartment contains a blend of polymers and the second compartment does not contain a blend of polymers. In some embodiments, the first compartment contains a blend of polymers and the second compartment contains a single type of polymer.

[0312] In some embodiments, the first compartment does not contain a blend of polymers and the second compartment contains a blend of polymers. In some embodiments, the first compartment contains a single type of polymer and the second compartment contains a blend of polymers.

[0313] In some embodiments, the first and second compartments contain a blend of alginate polymers. In some embodiments, the first compartment contains a blend of alginate polymers. In some embodiments, the second compartment contains a blend of alginate polymers.

[0314] In some embodiments of the present invention, the first and second compartments contain a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the first compartment contains a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the second compartment contains a blend of VLVG alginate and SLG100 alginate.

[0315] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment that contains VLVG alginate, wherein the VLVG alginate contains a compound of formula (IV) and the SLG100 alginate contains a compound of formula (V); and (ii) an outer compartment that contains a blend of VLVG alginate and SLG100 alginate, wherein the VLVG alginate contains a compound of formula (IV) and the SLG100 alginate contains a compound of formula (V).

[0316] In some embodiments of the present invention, the first and second compartments contain a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the first compartment contains a blend of VLVG alginate and SLG100 alginate. In some embodiments of the present invention, the second compartment contains a blend of VLVG alginate and SLG100 alginate.

[0317] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment containing a blend of VLVG alginate and SLG100 alginate, 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 containing a blend of VLVG alginate and SLG100 alginate, wherein the VLVG alginate comprises a compound of formula (IV) and the SLG100 alginate comprises a compound of formula (V).

[0318] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment containing a blend of VLVG alginate and SLG100 alginate, wherein the VLVG alginate comprises compounds of formula (I) and formula (IV) and the SLG100 alginate comprises a compound of formula (V); and (ii) an outer compartment containing a blend of VLVG alginate and SLG100 alginate, wherein the VLVG alginate comprises compounds of formula (I) and formula (IV) and the SLG100 alginate comprises a compound of formula (V).

[0319] A modified polymer containing a crosslinking moiety may be capable of undergoing further polymerization, for example reacting with compatible functional groups on the same or different polymers. In one embodiment, a polymer modified to contain a thiol group and a second polymer modified to contain an olefin group are such that a crosslinked polymer can be formed by reacting the first polymer and the second polymer. In some embodiments, the hydrogel is formed by crosslinking unsaturated functional groups via a chain-growth polymerization process. In other embodiments, the hydrogel is formed by crosslinking unsaturated functional groups via a step-growth polymerization process. The step-growth polymerization process preferably involves the reaction between one or more unsaturated functional groups (e.g., alkenyl) of one polysaccharide chain and the thiolated functional groups of another polymer chain. In some embodiments, the step-growth polymerization is a thiol-ene photo-click reaction.

[0320] The present disclosure features a dual-crosslinked polysaccharide for encapsulating mammalian cells. The dual-crosslinked polysaccharide hydrogel contains at least one cell-binding substance (CBS) (as defined herein). When the hydrogel is implanted into a subject (e.g., a human or other mammalian subject), the cells are capable of expressing a therapeutic agent. Additionally, the device comprises at least one means for reducing FBR (as defined herein).

[0321] In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, the alginate is a high guluronic acid (G) alginate and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more guluronic acid (G). In some embodiments, the alginate is a high mannuronic acid (M) alginate and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 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–250 kDa and a G:M ratio of ≥1.5.

[0322] In some embodiments, the defibrinated polymer comprises an alginate chemically modified with a compound of formula (I). The alginate in the defibrinated polymer can be the same as or different from any unmodified alginate present in the device. In one embodiment, the density (e.g., conjugation amount) of the compound of formula (I) in the defibrinated alginate is between about 4.0% and about 8.0% nitrogen, between about 5.0% and about 7.0% nitrogen, or between about 6.0% and about 7.0% nitrogen (e.g., as determined by combustion analysis of the nitrogen percentage). In one embodiment, the amount of compound 101 produces a %N increase of about 0.5% to 2%, 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N (compared to the unmodified alginate), where %N is determined by combustion analysis and corresponds to the amount of compound 101 in the modified alginate.

[0323] The hydrogel capsules described herein can be porous or non-porous. The pores in the polysaccharide hydrogel (e.g., alginate hydrogel) act as a selectively permeable membrane for small proteins and molecules while preventing larger unwanted molecules (such as immunoglobulins) from entering the encapsulated cells. In a preferred embodiment, the hydrogels and hydrogel capsules described herein are porous. In one embodiment, the average pore size of the hydrogel capsule is between about 10 nm and about 50 nm. In some embodiments, the average pore size is between about 10 nm and 40 nm. In some embodiments, the average pore size is between about 10 nm and 30 nm. In some embodiments, the average pore size is between about 10 nm and 20 nm.

[0324] 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 dextran permeability measurements). In some embodiments, the average molecular weight permeability is from about 50 kDa to about 400 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 400 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 350 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 300 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 250 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 200 kDa. In some embodiments, the average molecular weight permeability is from about 100 kDa to about 150 kDa. In a preferred embodiment, the average molecular weight permeability is from about 125 kDa to about 175 kDa.

[0325] The hydrogel capsules described herein can be porous or non-porous. The pores in polysaccharide hydrogel capsules (e.g., polysaccharide hydrogel capsules formed from alginate hydrogels) act as a selective permeation membrane for small proteins and molecules while preventing larger unwanted molecules, such as immunoglobulins, from entering the encapsulated cells. In a preferred embodiment, the hydrogels and hydrogel capsules described herein are porous. In some embodiments, the average pore size 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 size 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 size 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.

[0326] In some embodiments, the average pore sizes of the first and second compartments of the particle (e.g., the hydrogel capsule) are substantially the same. In some embodiments, the average pore sizes of the first and second compartments 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 size of the particle (e.g., the average pore size of the first compartment and / or the average pore size of the second compartment) depends on various factors, such as one or more materials within each compartment, the presence and density of the photoactive crosslinker, and the presence and density of the compound of formula (I).

[0327] The hydrogel capsules described herein should not have pores with a diameter sufficient to allow cells (e.g., immune cells, such as dendritic cells) to move through the hydrogel. In some embodiments, the diameter of the pores is small enough to prevent antibodies from moving through the hydrogel. In some embodiments, the hydrogel capsules described herein do not have a pore size greater than 75 μm. In some embodiments, the hydrogel capsules described herein do not have a pore size greater than 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 75 μm.

[0328] In some embodiments, the hydrogel capsules described herein can be characterized by their absolute rupture strength (e.g., crush strength) as determined by using a texture analyzer. In some embodiments, the absolute rupture strength is between 50 and 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.

[0329] The present disclosure features particles (such as hydrogel capsules) that include a first compartment, a second compartment, a crosslinked moiety (such as a compound of formula (IV) or (V)) described herein, and optionally a compound of formula (I).

[0330] The photoactive crosslinked moiety is covalently bonded to a polysaccharide polymer present in the first and / or second compartment. The particles (such as hydrogel capsules) can be spherical or have any other shape. The particles (such as hydrogel capsules) can comprise materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The particles (such as hydrogel capsules) can be made entirely of one type of material or can contain many other materials in the second (outer) and first (inner) compartments.

[0331] 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).

[0332] In some embodiments, the particles (e.g., hydrogel capsules) have a maximum linear dimension (LLD) (e.g., average diameter) or size greater than 1 millimeter (mm), preferably 1.5 mm or greater. In some embodiments, the diameter or size of the particles (e.g., hydrogel capsules) can be as large as 10 mm. For example, the particles (e.g., hydrogel capsules) described herein have a size 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.

[0333] In some embodiments, the particles (e.g., hydrogel capsules) have a maximum linear dimension (LLD) (e.g., average diameter) or size of 1 millimeter (mm) or less. In some embodiments, the particles (e.g., hydrogel capsules) have a size 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.

[0334] In some embodiments, 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 100 nm or greater, and can be as large as 1 mm. For example, the thickness of the outer compartment in the particles described herein can be from 10 nanometers to 1 millimeter, from 100 nanometers to 1 millimeter, from 500 nanometers to 1 millimeter, from 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 from 100 nanometers to 1 millimeter, between 1 μm and 1 mm, between 1 μm and 500 μm, or between 5 μm and 1 mm.

[0335] In some embodiments, both the first and second compartments contain the same polymer. In some embodiments, the first and second compartments 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 and second compartments 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, such as hyaluronate or chitosan). In some embodiments, the second compartment contains alginate and the first compartment contains a different polymer (e.g., a polysaccharide, such as hyaluronate or chitosan).

[0336] Both the first and second compartments can include a single component (e.g., one polymer) or more than one component (e.g., a blend of polymers). In some embodiments, the first compartment contains only alginate (e.g., chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate). In some embodiments, the second compartment contains only alginate (e.g., chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate). In some embodiments, both the first and second compartments independently contain only alginate (e.g., chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate).

[0337] In some embodiments, the first and second compartments contain a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment contains a blend of polymers. In some embodiments, the second (outer) compartment contains a blend of polymers. In some embodiments, the first and second compartments contain the same blend of polymers. In some embodiments, the first and second compartments contain different blends of polymers. In some embodiments, at least one polymer in the blend contained in the outer compartment is covalently modified with a photoactive crosslinker (e.g., a compound of formula (IV), (V), or (VI)) described herein. In some embodiments, at least one polymer in the blend contained in the second (outer) compartment is covalently modified with a defibrillation compound (e.g., a compound of formula (I)) described herein. In some embodiments, at least one polymer in the blend contained in the second (outer) compartment is covalently modified with both a photoactive crosslinker and a defibrillation compound.

[0338] In some embodiments, the first compartment contains a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers. In some embodiments, the first compartment contains a blend of 2 polymers. In some embodiments, the first compartment contains a blend of 3 polymers. In some embodiments, the first compartment contains a blend of 4 polymers. In some embodiments, the first compartment contains a blend of 5 polymers. In some embodiments, the first compartment contains a blend of 6 polymers. In some embodiments, the first (inner) compartment contains a blend of 7 polymers. In some embodiments, the first (inner) compartment contains a blend of 8 polymers. In some embodiments, the first (inner) compartment contains a blend of 9 polymers. In some embodiments, the first (inner) compartment contains a blend of 10 polymers.

[0339] In some embodiments, the second (outer) compartment contains a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers. In some embodiments, the second (outer) compartment contains a blend of 2 polymers. In some embodiments, the second (outer) compartment contains a blend of 3 polymers. In some embodiments, the second (outer) compartment contains a blend of 4 polymers. In some embodiments, the second (outer) compartment contains a blend of 5 polymers. In some embodiments, the second (outer) compartment contains a blend of 6 polymers. In some embodiments, the second (outer) compartment contains a blend of 7 polymers. In some embodiments, the second (outer) compartment contains a blend of 8 polymers. In some embodiments, the second (outer) compartment contains a blend of 9 polymers. In some embodiments, the second (outer) compartment contains a blend of 10 polymers.

[0340] In some embodiments, the first compartment contains a blend of polymers and the second compartment does not contain a blend of polymers. In some embodiments, the first compartment contains a blend of polymers and the second compartment contains a single type of polymer.

[0341] In some embodiments, the first compartment does not contain a blend of polymers and the second compartment contains a blend of polymers. In some embodiments, the first compartment contains a single type of polymer and the second compartment contains a blend of polymers.

[0342] In some embodiments, the first and second compartments contain a blend of polymers, and the polymers of the blend are any two miscible polymers.

[0343] In some embodiments, the first and second compartments contain a blend of polymers, and the polymers are selected from the group consisting of alginate, hyaluronate, and chitosan.

[0344] In some embodiments, the first and second compartments contain a blend of polymers, and the polymers are selected from the group consisting of alginate, hyaluronate, and chitosan. In some embodiments, the first compartment contains a blend of polymers, and the polymers are selected from the group consisting of alginate, hyaluronate, and chitosan. In some embodiments, the second compartment contains a blend of polymers, and the polymers are selected from the group consisting of alginate, hyaluronate, and chitosan.

[0345] In some embodiments, the first and second compartments contain a blend of alginate polymers. In some embodiments, the first compartment contains a blend of alginate polymers. In some embodiments, the second compartment contains a blend of alginate polymers.

[0346] In some embodiments, the first and second compartments contain a blend of alginate polymers, and the alginate polymers are selected from guluronic acid-rich alginate and mannuronic acid-rich alginate. In some embodiments, the first compartment contains a blend of alginate polymers, and the alginate polymers are selected from guluronic acid-rich alginate and mannuronic acid-rich alginate. In some embodiments, the second compartment contains a blend of alginate polymers, and the alginate polymers are selected from guluronic acid-rich alginate and mannuronic acid-rich alginate.

[0347] In some embodiments, the first and second compartments contain a blend of alginate polymers, and the alginate polymers are 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 contains a blend of alginate polymers, and the alginate polymers are 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 contains a blend of alginate polymers, and the alginate polymers are selected from low molecular weight alginate, medium molecular weight alginate, high molecular weight alginate, and ultra-high molecular weight alginate.

[0348] In some embodiments, the first and second compartments contain 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 ULVIL-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 contains 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 ULVIL-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 contains 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 ULVIL-6G, Pronova UP VLVM, Pronova UP LVM, Pronova UP MVM, Pronova UP VLVG, PronovaUP MVG, Pronova UP LVG, Pronova SLM20, Pronova SLM100, Pronova SLG20, and PronovaSLG100.

[0349] 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.

[0350] 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 greater than 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 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.

[0351] In some embodiments, the polymer in the first compartment of the particle (e.g., hydrogel capsule) is modified with a compound of formula (I), and the polymer in 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) contains 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 contains a mixture (i.e., a 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 contains 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)).

[0352] The polymers of the particles (e.g., hydrogel capsules) described herein can be modified on one or more monomers of the polymer with a compound of formula (I) or a pharmaceutically acceptable salt thereof. 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 in the second compartment (which includes the outer particle surface). 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 formula (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).

[0353] In some embodiments, the polymer (e.g., alginate) (when modified with a compound of formula (I), such as compound 101 of Table 3) has an increase in %N of any of the following values (compared to the unmodified polymer (e.g., alginate)): (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% to 10% by weight; (iii) 0.1% to 2% N by weight; (iv) 2% to 4% N by weight; (v) 4% to 8% N by weight; (vi) 5% to 9% N by weight; (vii) 6% to 9% N by weight; (viii) 6% to 8% N by weight; (ix) 7% to 9% N by weight; and (x) 8% to 9% N by weight, where 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.

[0354] Particles (such as hydrogel capsules) (such as the first or second compartment therein) may contain a compound of formula (I) in an amount conferring specific characteristics to the particle. For example, the particle surface (such as the exterior of the outer compartment) may contain a concentration or density of the compound of formula (I) such that the particle has a defibrosing effect (i.e., reduces the foreign body response) in a subject. In one embodiment, the particle surface contains alginate chemically modified with a defibrosing effective amount of Compound 101. In one embodiment, the defibrosing effective amount of Compound 101 produces an increase in %N of about 0.5% to 2%, 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N (compared to unmodified alginate), where %N is determined by combustion analysis (such as as described in Example 2 herein) and corresponds to the amount of Compound 101 in the modified alginate.

[0355] In one embodiment, mechanical testing of the hydrogel capsules is performed on a TA.XT plus texture analyzer (Stable Micro Systems, Surrey, United Kingdom) using a 5 mm probe attached to a 5 kg load cell. A single capsule is placed on the platform and compressed from above by the probe at a fixed rate of 0.5 mm / second. Contact between the probe and the capsule is detected when a repulsive force of 1 g is measured. The probe continues to travel 90% of the distance between the contact height of the probe and the platform, thereby compressing the capsule to the burst point. The resistance of the probe to the compression force is measured and plotted as a function of the probe travel (force versus displacement curve). Typically, the capsule will rupture slightly before it bursts completely, and the force applied to the probe will decrease slightly. The analysis macro can be programmed to detect the first occurrence of a decrease of 0.25 - 0.5 g in the force versus displacement curve. The force applied by the probe when this occurs is referred to as the initial rupture force. In one embodiment, the desired mechanical strength of the particles (such as two-compartment hydrogel capsules) described herein has an initial rupture force greater than 1, 1.5, 2, 2.5, or 3 grams or at least 2 grams.

[0356] In one embodiment, the desired mechanical strength of the particles (such as hydrogel capsules) is the ability to remain intact at a desired time point after implantation in a subject. For example, when observed by optical microscopy, such as by bright field imaging as described in the examples herein, the outer and inner compartments of the hydrogel capsule retrieved from the subject are visibly intact after retrieval from an immunocompetent mouse.

[0357] In one embodiment, the particle surface comprises alginate chemically modified with Compound 101 in an amount that provides the particle with defibrillation properties and the desired mechanical strength, e.g., the concentration or density of Compound 101 in the modified alginate results in a %N increase of any of the following values (compared to unmodified alginate): (i) 1% to 3% by weight; (ii) 2% to 4% N by weight; (iii) 4% to 8% N by weight; (iv) 5% to 9% N by weight; (v) 6% to 9% N by weight; (vi) 6% to 8% N by weight; (vii) 7% to 9% N by weight; and (ix) 8% to 9% N by weight; wherein 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.

[0358] When the particle (e.g., the first or second compartment therein) comprises alginate, the alginate can be chemically modified with a compound of formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid moiety can be activated to couple with one or more amine-functionalized compounds to obtain alginate modified with a compound of formula (I). The alginate polymer can be dissolved in water (30 mL / g polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). A solution (0.3 M) of the compound of formula (I) dissolved in a buffer or solvent such as acetonitrile can be added to this mixture. The reactants can be heated to, for example, 55 °C and held for 16 h, then cooled to room temperature and concentrated via rotary evaporation. The residue can then be dissolved in a buffer or solvent such as water. The mixture can then be filtered, for example, through a cyano-modified silica gel bed (Silicycle), and the filter cake washed with water. The resulting solution can then be dialyzed (10,000 MWCO membrane) against a buffer or water for 24 hours, e.g., replacing the buffer or water at least once, at least twice, at least three times or more. The resulting solution can be concentrated, for example, via lyophilization to obtain the desired chemically modified alginate.

[0359] 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 in the first compartment. In some embodiments, the cells are disposed in the second compartment. In some embodiments, the cells are disposed in the first compartment and the second compartment does not contain cells. The particle (e.g., a hydrogel capsule) can comprise an active or inactive fragment of a protein or polypeptide, such as glucose oxidase (e.g., for a glucose sensor), kinase, phosphatase, oxygenase, hydrogenase, reductase.

[0360] The particles described herein (such as hydrogel capsules) can be configured to release a therapeutic agent, such as an exogenous substance, such as the therapeutic agents 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 biomaterial. In some embodiments, the therapeutic agent is a nucleic acid (such as RNA or DNA), a protein (such as a hormone, an enzyme, an antibody, an antibody fragment, an antigen, or an epitope), a small molecule, a lipid, a drug, a vaccine, or any derivative thereof.

[0361] Particles (such as hydrogel capsules) (such as as described herein) can be provided in a formulation 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 (such as hydrogel capsules) in the formulation or composition have the characteristics described herein, such as an average diameter or an average pore size.

[0362] Cells and therapeutic agents

[0363] The hydrogel capsules of the present invention can contain a variety of different cell types (such as 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 any of the foregoing. Exemplary cell types include the cell types described in WO 2017 / 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 before being encapsulated within the hydrogel capsules described herein. The cells in the suspension can be in the form of single cells (such as from a monolayer cell culture), or provided in another form (such as placed on a microcarrier (such as a bead or a matrix)) or as a three-dimensional aggregate of cells (such as a cell cluster or a spheroid). The cell suspension can contain a plurality of cell clusters (such 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 a glucose-responsive manner.

[0364] Compared with the untreated control, the hydrogel capsules of the present disclosure reduce immune cell adhesion. In one embodiment, compared with the untreated control, the hydrogel capsules reduce macrophage adhesion. In one embodiment, compared with the untreated control, macrophage adhesion is reduced by about 1-fold to 10-fold. In one embodiment, compared with the untreated control, macrophage adhesion is reduced by about 1-fold to 8-fold. In one embodiment, compared with the untreated control, macrophage adhesion is reduced by about 1-fold to 7-fold. In one embodiment, compared with the untreated control, macrophage adhesion is reduced by about 1-fold to 6-fold. In one embodiment, compared with the untreated control, macrophage adhesion is reduced by about 1-fold to 5-fold.

[0365] The hydrogels or hydrogel capsules of the present disclosure allow encapsulated cells (e.g., engineered cells) to retain viability (e.g., as determined by a cell viability assay). In some embodiments, the hydrogels or hydrogel capsules allow encapsulated cells to retain viability for at least seven days, at least one month, or at least one year.

[0366] The present disclosure features a cell that produces or is capable of producing a therapeutic agent for preventing or treating a disease, disorder, or condition described herein. In one embodiment, the cell is an engineered cell. In one embodiment, the cell is engineered to sense a stimulant (e.g., a chemical signal) and express a therapeutic agent in response to the stimulant. The therapeutic agent can be any biological substance, such as a nucleic acid (e.g., a nucleotide, DNA, or RNA), polypeptide, lipid, sugar (e.g., a monosaccharide, disaccharide, oligosaccharide, or polysaccharide), or small molecule, each of which is described in further detail below. Exemplary therapeutic agents include those listed in WO 2017 / 075631 and WO2019 / 195055.

[0367] In some embodiments, the cell (e.g., an engineered cell) produces a nucleic acid. The size of the nucleic acid produced by the cells described herein can vary and contains one or more nucleosides or nucleotides, such as greater than 2, 3, 4, 5, 10, 25, 50, or more nucleosides or nucleotides. In some embodiments, the nucleic acid is a short fragment of, for example, RNA or DNA and can be used as a reporter gene 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., a vector, 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 greater.

[0368] 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, such as a hormone, a growth factor, a coagulation factor or clotting factor, an antibody molecule, an enzyme, a cytokine, a cytokine receptor, or a chimeric protein comprising a cytokine or a cytokine receptor) produced by a cell in an implantable element may have a naturally occurring amino acid sequence or may contain variants of a naturally occurring sequence. The variants may be naturally occurring or non-naturally occurring amino acid substitutions, mutations, deletions, or additions relative to a reference naturally occurring sequence. The naturally occurring amino acid sequence may be a polymorphic variant. The naturally occurring amino acid sequence may 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. Additionally, the peptide or polypeptide (e.g., a protein) used in the present invention may be modified in some manner, such as 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 greater.

[0369] 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 growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionyl-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).

[0370] 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).

[0371] In some embodiments, the protein is a clotting factor or coagulation factor, such as a blood clotting factor or blood coagulation factor. In some embodiments, the protein is a protein involved in coagulation, i.e., the process by which blood changes from a liquid to a solid or gel. Exemplary clotting 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-Prower 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, such as protein C.

[0372] 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 comprises 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 comprises a full-length antibody or a full-length immunoglobulin chain. In one embodiment, the antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. In one embodiment, the antibody molecule is a monospecific antibody molecule and binds a single epitope, such as a monospecific antibody molecule having plural immunoglobulin variable domain sequences, each of which binds the same epitope. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it comprises plural immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plural immunoglobulin variable domain sequences has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plural immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, such as 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.

[0373] Antibody molecules of various types can be produced by the cells in the implantable elements described herein, including intact immunoglobulins of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domains of antibodies. The antibody molecule can be an antibody, such as an IgG antibody, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody molecule can be in the form of an antigen-binding fragment, including Fab fragments, F(ab')2 fragments, single-chain variable regions, etc. The antibody can be polyclonal or monoclonal (mAb). Monoclonal antibodies can include "chimeric" antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided that 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 antibody can also be modified recombinantly, e.g., by deletion, addition, or substitution of amino acids, to increase the efficacy of the antibody in mediating the desired function. Exemplary antibodies include anti-β-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, fasimab, tanezumab).

[0374] In some embodiments, the protein is a cytokine or cytokine receptor, or a chimeric protein comprising a cytokine or its receptor, including, for example, tumor necrosis factor α and β, their receptors and their derivatives, renin; lipoprotein; colchicine; adrenocorticotropic hormone; vasopressin; somatostatin; lysine vasopressin; pancreozymin; leuprorelin; α-1-antitrypsin; atrial natriuretic factor; pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; neprilysin; RANTES (regulated upon activation, normal T cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin; Mullerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; murine gonadotropin-associated peptide; chorionic gonadotropin; microbial proteins, such as β-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), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; 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 protein; CD proteins, such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factor; immunotoxin; interferons, such as interferon-α (e.g., interferon-α2A), interferon-β, interferon-γ, interferon-λ and composite interferon; colony-stimulating factor (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (IL), such as IL-1 to IL-10; superoxide dismutase; T cell receptor; surface membrane protein; decay-accelerating factor; transporter protein; homing receptor; addressin; fertility inhibitors, such as prostaglandins; fertility promoters; regulatory 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. Suitable proteins or peptides can be natural or recombinant and include, for example, fusion proteins.

[0375] Examples of polypeptides (such as 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, IFNW1, 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, adrenaline, melatonin, triiodothyronine, prostaglandin, leukotriene, prostacyclin, thromboxane, islet amyloid polypeptide, Müllerian inhibitory factor or hormone, adiponectin, adrenocorticotropic hormone, angiotensin, vasopressin, arginine vasopressin, atrial natriuretic peptide, 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, thyroid-stimulating hormone, thyrotropin-releasing hormone, vasoactive intestinal peptide, androgen, α-glucosidase (also known as acid maltase),Glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine palmitoyl transferase, carnitine, and myoadenylate deaminase.

[0376] 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, such as factor VIII (e.g., comprising a naturally occurring human factor VIII amino acid sequence or a variant thereof) or factor IX (e.g., comprising a naturally occurring human factor IX amino acid sequence or a variant thereof).

[0377] In some embodiments, the cell is engineered to express factor VIII, such as recombinant factor VIII. In some embodiments, the cell is derived from human tissue and is engineered to express factor VIII, such as recombinant factor VIII. In some embodiments, the recombinant factor VIII is a B-domain deleted recombinant factor VIII (FVIII-BDD).

[0378] In some embodiments, the cell is derived from human tissue and is engineered to express factor IX, such as recombinant factor IX. In some embodiments, the cell is engineered to express factor IX, such as wild-type human factor IX (FIX) or a polymorphic variant thereof. In some embodiments, the cell is engineered to express a gain-of-function (GIF) variant of wild-type FIX protein (FIX-GIF), wherein the GIF variant has a higher specific activity than the corresponding wild-type FIX.

[0379] In some embodiments, the replacement therapy or replacement protein is an enzyme, such as alpha-galactosidase, alpha-L-iduronidase (IDUA), or N-sulfo-glucosamine sulfohydrolase (SGSH). In some embodiments, the replacement therapy or replacement protein is an enzyme, such as alpha-galactosidase A (e.g., comprising a 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.

[0380] In some embodiments, the therapeutic agent is a sugar, such as a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. In some embodiments, the sugar includes a triose, tetrose, pentose, hexose, or heptose moiety. In some embodiments, the sugar includes a linear monosaccharide or a cyclized monosaccharide. In some embodiments, the sugar includes 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 attached 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.

[0381] 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 insert into a liposome, vesicle, or membrane. Lipids can include fatty acids, glycerides, glycerophospholipids, sterol lipids, prenol lipids, sphingolipids, glycolipids, polyketides, or sphingoids. Examples of lipids produced by the cells described herein include anandamide, docosahexaenoic acid, prostaglandins, leukotrienes, thromboxanes, eicosanoids, triglycerides, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, ceramides, sphingomyelin, cerebrosides, gangliosides, estrogens, androsterone, testosterone, cholesterol, carotenoids, quinones, hydroquinones, or ubiquinones.

[0382] In some embodiments, the therapeutic agent is a small molecule. Small molecules can include natural products produced by cells. In some embodiments, small molecules have poor availability or do not conform to Lipinski's Rule of Five (a set of guidelines for estimating whether a small molecule is likely to 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., carumonam, daptomycin, fidaxomicin, fosfomycin, ipamycin, micronomicin sulfate, mikamycin, mupirocin, netilmicin sulfate, teicoplanin, thienamycin, rifamycin, erythromycin, vancomycin), antiparasitic agents (e.g., artemisinin, ivermectin), anticancer agents (e.g., doxorubicin, aclarubicin, aminolevulinic acid, arglabin, homoharringtonine, paclitaxel, pentostatin, peplomycin, romidepsin, trabectedin, actinomycin D, bleomycin, chromomycin A, daunorubicin, folinic acid, neocarzinostatin, streptozotocin, trabectedin, vinblastine, vincristine), antidiabetic agents (e.g., voglibose), central nervous system agents (e.g., levodopa, galantamine, ziconotide), statins (e.g., mevastatin), antifungal agents (e.g., fumagillin, cyclosporine), 1-deoxynojirimycin, and theophylline, steroids (cholesterol, estrogen, testosterone). Other small molecule natural products are described in Newman, D.J. And Cragg, M. (2016) J NatProd79:629-661 and Butler, M.S. et al. (2014) Nat Prod Rep 31:1612-1661.

[0383] In some embodiments, the cells are engineered to synthesize non-protein or non-peptide small molecules. For example, in one embodiment, the cells can produce statins (e.g., taurastatin, pravastatin, fluvastatin, or atorvastatin).

[0384] In some embodiments, the therapeutic agent is an antigen (e.g., viral antigen, bacterial antigen, fungal antigen, plant antigen, environmental antigen, or tumor antigen). Those skilled in the art recognize that antigens have immunostimulatory effects, i.e., they are capable of stimulating an immune response or providing effective immunity for the organism or molecule from which they are derived. Antigens can be nucleic acids, peptides, proteins, sugars, lipids, or combinations thereof.

[0385] Cells (e.g., engineered cells such as those 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%, or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% 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, such as 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%, or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the cluster produce multiple therapeutic agents (e.g., a therapeutic agent described herein).

[0386] The therapeutic agents may be related or may form a complex. 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 (e.g., 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 intracellularly. For example, the therapeutic agent can be an enzyme involved in the detoxification or metabolism of an unwanted substance, and the detoxification or metabolism of the unwanted substance occurs intracellularly.

[0387] 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 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 1 M mL -1 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 2 M mL -1 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 3 M mL -1 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 4 M mL -1 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 5 M mL -1 In some embodiments, the hydrogel capsules described herein contain mammalian cells at a concentration of about 6 M mL -1mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 7M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 8M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 9M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 10M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 15M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 20M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 25M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 30M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 35M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 40M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 45M mL -1 mammalian cells. In some embodiments, the hydrogel capsules described herein contain a concentration of about 50M mL -1 mammalian cells.

[0388] In some embodiments, the hydrogel capsules described herein contain a concentration of about 1 - 50M mL -1 、 1 - 45MmL -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 - 30M mL -1 , 5 - 25M mL -1 , 5 - 20M mL -1 , 5 - 15M mL -1 , 5 - 10M mL -1 , 10 - 50M mL -1 , 10 - 45M mL -1 , 10 - 40M mL -1 , 10 - 35M mL -1 , 10 - 30M mL -1 , 10 - 25M mL -1 , 10 - 20M mL -1 , 10 - 15M mL -1 , 15 - 50M mL -1 , 15 - 45M mL -1 , 15 - 40M mL -1 , 15 - 35M mL -1 , 15 - 30M mL -1 , 15 - 25M mL -1 , 15 - 20M mL -1 , 20 - 50M mL -1 , 20 - 45M mL -1 , 20 - 40M mL -1 , 20 - 35M mL -1 , 20 - 30M mL -1 , or 20 - 25M mL -1 mammalian cells between

[0389] Therapeutic method

[0390] Described herein is a method for preventing or treating a disease, disorder, or condition in a subject by administering or implanting 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 alleviate or mitigate at least one symptom of a disease, disorder, or condition (such as type 1 diabetes). In some embodiments, the methods described herein prevent or slow the onset of a disease, disorder, or condition (such as type 1 diabetes). In some embodiments, the subject is a human.

[0391] In some embodiments, the disease, disorder or affliction affects a body system, such as the nervous system (e.g., the peripheral or central nervous system), the vascular system, the skeletal system, the respiratory system, the endocrine system, the lymphatic system, the reproductive system or the gastrointestinal tract. In some embodiments, the disease, disorder or affliction affects a part of the body, such as the blood, the eyes, the brain, the skin, the lungs, the stomach, the mouth, the ears, the legs, the feet, the hands, the liver, the heart, the kidneys, the bones, the pancreas, the spleen, the large intestine, the small intestine, the spinal cord, the muscles, the ovaries, the uterus, the vagina or the penis.

[0392] In some embodiments, the disease, disorder or affliction is an autoimmune disease. In some embodiments, the disease, disorder or affliction is diabetes (type 1 or type 2).

[0393] The present disclosure further includes methods for identifying a subject having or suspected of having a disease, disorder or affliction described herein (such as type 1 diabetes), and, after such identification, administering to the subject a hydrogel capsule comprising (i) a polysaccharide polymer described herein and (ii) islet cells (e.g., 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 criteria associated with diabetes, such as a high blood glucose level (e.g., greater than 300 mg / dL, greater than 400 mg / dL) or a high hemoglobin A1C level (e.g., a hemoglobin A1C level greater than 5.9%, a hemoglobin A1C level greater than 6.5%, a hemoglobin A1C level greater than 7%). In one embodiment, the subject is a human. In one embodiment, the subject is an adult. In one embodiment, the subject is a child (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).

[0394] Method for manufacturing particles

[0395] The present disclosure further includes a method for manufacturing 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 particle is a hydrogel capsule, the method for manufacturing the particle includes contacting a plurality of droplets comprising a first and a second polymer solution (e.g., each comprising a polymer that forms a hydrogel) with an aqueous crosslinking solution. The droplets can be formed using any technique known in the art.

[0396] Each compartment of the particles described herein may contain an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinker, or a blend thereof. Briefly, in a method of preparing particles configured as two-compartment hydrogel capsules, a volume of a first polymer solution (e.g., containing an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinker, or a blend thereof, and optionally containing cells) is loaded into a first syringe connected to the lumen of a coaxial needle. The first syringe can then be connected to an infusion pump vertically oriented above a container containing an aqueous crosslinking solution that includes a crosslinker, a buffer, and an osmolality regulator. A volume of a second polymer solution (e.g., containing an unmodified polymer, a polymer modified with a compound of formula (I), a polymer modified with a crosslinker, or a blend thereof, and optionally containing cells) is loaded into a second syringe connected to the outer lumen of the coaxial needle. The second syringe can then be connected to an infusion pump horizontally oriented relative to the container containing the crosslinking solution. A high voltage electricity generator can then be connected to the top and bottom of the needle. The first and second polymer solutions can then be extruded through the syringes using the syringe pumps and the electricity generator, the settings of which are determined to achieve the desired droplet rate of the polymer solutions into the crosslinking solution. Those skilled in the art can readily determine the various combinations of needle lumen size, voltage range, flow rate, droplet rate, and droplet spacing to produce a two-compartment hydrogel capsule composition in which most (e.g., at least 80%, 85%, 90% or more) of the capsules are within 10% of the target size and have a spheroid-like shape. After depleting the first and second volumes of polymer solution, the droplets can be allowed to crosslink in the crosslinking solution for a period of time, e.g., about five minutes.

[0397] Exemplary process parameters for preparing compositions for millimeter-sized capsules (e.g., millimeter-sized capsules with a 1.5 mm diameter) include the following. The coaxial needle is positioned above the surface of the crosslinking solution at a distance sufficient to provide a drop distance from the tip of the needle to the surface of the solution. In one embodiment, the distance between the tip of the needle and the surface of the solution is between 1 cm and 5 cm. In one embodiment, the first and second polymer solutions are extruded through the needle at a combined 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 combined 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.

[0398] In one embodiment, the voltage of the instrument is between 1 kV and 20 kV, or between 1 kV and 15 kV, or between 1 kV and 10 kV, or between 5 kV and 10 kV. The voltage can be adjusted until the desired droplet rate is achieved. In one embodiment, the droplet rate of the instrument is between 1 drop / 10 seconds and 50 drops / 10 seconds, or between 1 drop / 10 seconds and 25 drops / 10 seconds.

[0399] In one embodiment, the number of non-particulate debris on the surface of the crosslinking solution is determined. The particles that fall to the bottom of the crosslinking container can then be collected, for example, by transferring the crosslinking solution containing the particles to a separate container, leaving any non-particulate debris on the surface of the solution in the original crosslinking container. The removed particles can then be allowed to settle, the crosslinking solution can be removed, and the particles can then be washed one or more times with a buffer (e.g., HEPES buffer). In one embodiment, one or more aliquots of the resulting particle composition (e.g., particle preparation) are examined microscopically to evaluate the quality of the composition, such as the number of particle defects and satellite particles.

[0400] In some embodiments, the crosslinking solution further comprises a process additive (e.g., a hydrophilic nonionic surfactant). The process additive can reduce the surface tension of the crosslinking solution. Reagents that can be used as process 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 nonionic surfactants such as 20, 80, Triton TM X-100, CA-630, poloxamer 188, or poloxamer 407, or a surfactant having substantially the same chemical and physical properties as those listed in the exemplary surfactant table immediately below.

[0401]

[0402]

[0403] a Hydrophilic-lipophilic balance

[0404] b Chemical name and synonyms: polyethylene glycol sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polysorbate 20, polyoxyethylene 20 sorbitan monolaurate

[0405] c Chemical name and synonyms: polyethylene glycol sorbitan monooleate, polyoxyethylene (20) sorbitan monooleate, polysorbate 80, (x)-sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl)

[0406] d Chemical name and synonyms: 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, tert-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether; octylphenol ethoxylate, octylphenol ethylene oxide condensate

[0407] e Chemical name and synonyms: branched octylphenoxypolyethoxyethanol, octylphenoxypoly(ethenyloxy)ethanol

[0408] f Chemical name: poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)

[0409] gChemical Name: Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)

[0410] In some embodiments, the process additive is a non-ionic surfactant. In one embodiment, the process additive comprises more than one surfactant, such as more than one hydrophilic surfactant. In some embodiments, the process additive does not contain 20 (polysorbate 20) or Triton TM X-100. In one embodiment, the process additive is CA-630 (polyethylene glycol sorbitan monooleate). In some embodiments, the process additive is poloxamer 188.

[0411] In some embodiments, the process additive (such as a surfactant) is present in the crosslinking solution at a concentration of at least 0.0001% or higher. In some embodiments, the crosslinking solution comprises at least 0.001%, 0.01% or 0.1% of the process additive. In some embodiments, the process additive is present at a concentration selected from about 0.001% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.01%, and about 0.01% to about 0.5%. In one embodiment, the process additive is a surfactant and is present at a concentration below the critical micelle concentration of the surfactant.

[0412] In some embodiments, the crosslinking agent comprises a single type of divalent cation or a mixture of different types, such as Ba 2+ 、Ca 2+ 、Sr 2+ One or more of. 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).

[0413] In some embodiments, the crosslinking agent is SrCl2 and the process additive is 80 (or a surfactant having substantially the same chemical and physical properties as those listed in the exemplary surfactant table), with a concentration of less than 0.1%, for example, from about 0.005% to 0.05%, from 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 process additive is poloxamer 188 at a concentration of 1%.

[0414] The type and concentration of the buffer in the aqueous crosslinking solution are selected to maintain the solution pH at approximately neutral, such as from about 6.5 to about 7.5, from about 7.0 to about 7.5, or about 7.0. In one embodiment, the buffer is compatible with the biomaterial (such as cells) to be encapsulated in the particles. In some embodiments, the buffer in the aqueous crosslinking solution includes HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0415] The osmotic pressure regulator in the aqueous crosslinking solution is selected to maintain the solution osmotic pressure at a value similar to that of the polymer solution (in some embodiments, which includes a cell suspension), for example, having an osmotic pressure with a higher or lower variation of up to 20%, 10%, or 5%. In some embodiments, the osmotic pressure agent is mannitol at a concentration of 0.1 M to 0.3 M.

[0416] In some embodiments, the crosslinking solution contains 25 mM HEPES buffer, 20 mM BaCl2, 0.2 M mannitol, and 0.01% poloxamer 188.

[0417] In some embodiments, the crosslinking solution contains 50 mM strontium chloride hexahydrate, 0.165 M mannitol, 25 mM HEPES, and 0.01% of a surfactant having substantially the same chemical and physical characteristics as those listed in the exemplary surfactant table of Tween 80.

[0418] In one embodiment, the process additive is poloxamer 188, which is present in the particle composition (such as a particle formulation) in a detectable amount after the washing step. Poloxamer 188 can be detected by any technique known in the art, such as by dissolving the particle partially or completely in an aliquot of the composition via precipitation with sodium sulfate and analyzing the supernatant by LC / MS.

[0419] The reduction of the surface tension of the crosslinking solution can be evaluated by any method known in the art, such as by using a contact angle goniometer or tensiometer, for example, via the du Nouy ring method (see, for example, Davarci et al. (2017) Food Hydrocolloids 62:119 - 127).

[0420] Exemplary Listed Embodiments

[0421] 1. A polysaccharide polymer comprising:

[0422] (i) A crosslinked moiety; and

[0423] (ii) A compound of formula (I):

[0424]

[0425] or a pharmaceutically acceptable salt thereof, wherein:

[0426] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, 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)(C1-C6-alkylene)-, -N(R C )C(O)(C2-C6-alkenylene)-, -C(=N(R C )(R 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 attached to an attachment group (e.g., an attachment group described herein) and is optionally substituted by one or more R 1 ;

[0427] Each of L 1 and L 3 is independently a bond, alkyl or heteroalkyl, each alkyl and heteroalkyl being optionally substituted by one or more R 2 ;

[0428] L 2 is a bond;

[0429] M is absent, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 3 substituted;

[0430] P is heteroaryl optionally substituted by one or more R 4 substituted;

[0431] Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 substituted;

[0432] Each R A 、R B 、R C 、R D 、R E 、R F and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by one or more R 6 substituted;

[0433] Or R C and R D together with the nitrogen atom to which they are attached form a ring (e.g., 5-7 membered ring) optionally substituted by one or more R 6 substituted;

[0434] Each R 1 、R 2 、R 3 、R 4 、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 )、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 )(R D1 )、–P(R F1 ) y 、cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is optionally substituted by one or more R 7 substituents;

[0435] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by one or more R 7 substituents;

[0436] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group;

[0437] x is 1 or 2; and

[0438] y is 2, 3 or 4.

[0439] 2. The polysaccharide polymer according to embodiment 1, wherein the crosslinking moiety is covalently bound to a sugar monomer within the polysaccharide polymer.

[0440] 3. The polysaccharide polymer according to embodiment 2, wherein the crosslinking moiety is bound to a carboxylate moiety within the sugar monomer.

[0441] 4. The polysaccharide polymer according to any one of embodiments 1 to 3, wherein the crosslinking moiety comprises alkyl, alkenyl, alkynyl, ester, ketone, amine, thiol, cycloalkyl, heterocyclic group, aryl or heteroaryl.

[0442] 5. The polysaccharide polymer according to any one of embodiments 1-4, wherein the crosslinking moiety is capable of reacting with a second crosslinking moiety after activation with, for example, heat, acid, base or catalyst.

[0443] 6. The polysaccharide polymer according to any one of embodiments 1-5, wherein the crosslinking moiety is present on the polysaccharide polymer at a density of at least about 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or higher, for example as determined by comparison with a reference standard.

[0444] 7. The polysaccharide polymer according to any one of embodiments 1-6, wherein the crosslinked portion is present on the polysaccharide polymer at a density between 1% and 10%, such as 1%-8%, 1%-6%, or 1%-4%, for example as determined by comparison with a reference standard.

[0445] 8. The polysaccharide polymer according to any one of embodiments 1-7, wherein the polysaccharide polymer is selected from alginate, hyaluronate, and chitosan.

[0446] 9. The polysaccharide polymer according to any one of embodiments 1-8, wherein the polysaccharide polymer is alginate.

[0447] 10. The polysaccharide polymer according to embodiment 9, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

[0448] 11. The polysaccharide polymer according to any one of embodiments 1-10, wherein the crosslinked portion has the structure of formula (IV):

[0449] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0450] Q is O, NR 33 or C(R 34a )(R 34b );

[0451] R 33 , R 34a , R 34b , R 60a , R 60b , R 61a , R 61b and R 62 in each case independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl;

[0452] Each R A1 , R B1 , R C1 , R D1 and RE1 Independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 substituents; and

[0453] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0454] 12. The polysaccharide polymer according to embodiment 11, wherein the crosslinking moiety comprises a thiol moiety.

[0455] 13. The polysaccharide polymer according to any one of embodiments 1-10, wherein the crosslinking moiety has a structure of formula (V):

[0456] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0457] Each of T and U is independently O, NR 33 or C(R 34a )(R 34b );

[0458] R 33 、R 34a 、R 34b 、R 65a 、R 65b 、R 65c 、R 65d 、R 65e 、R 65f 、R 65g and R 66 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl;

[0459] each R A1 、R B1 、R C1 、R D1 and R E1Independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and

[0460] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0461] 14. The polysaccharide polymer according to embodiment 13, wherein the crosslinking moiety comprises a norbornenyl moiety.

[0462] 15. The polysaccharide polymer according to any one of embodiments 1-10, wherein the crosslinking moiety has the structure of formula (VI):

[0463] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0464] T, Y 1 and Y 2 each is independently O, NR 33 , or C(R 34a )(R 34b );

[0465] R 33 , R 34a , R 34b , R 69 and R 70 each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl;

[0466] each R A1 , R B1 , R C1 , R D1 and R E1Independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and

[0467] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0468] 16. The polysaccharide polymer according to embodiment 15, wherein the crosslinking moiety comprises a maleimide moiety.

[0469] 17. The polysaccharide polymer according to any one of embodiments 1-10, wherein the crosslinking moiety has the structure of formula (VII):

[0470] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0471] T is O, NR 33 or C(R 34a )(R 34b );

[0472] Ring M is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted with 1-6 R 7 ;

[0473] R 33 , R 34a , R 34b and R 74 are each independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl;

[0474] each R A1 , R B1 , R C1 , R D1 and R E1Independently being hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 Rs 7 and

[0475] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0476] 18. The polysaccharide polymer according to embodiment 17, wherein the crosslinking moiety comprises a tetrazinyl moiety.

[0477] 19. The polysaccharide polymer according to any one of embodiments 1-18, wherein the crosslinking moiety has a structure selected from Table 4, or a pharmaceutically acceptable salt thereof.

[0478] 20. The polysaccharide polymer according to any one of embodiments 1-19, wherein the polysaccharide polymer comprises one of the compounds of formula (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt thereof.

[0479] 21. The polysaccharide polymer according to any one of embodiments 1-20, wherein the polysaccharide polymer comprises two of the compounds of formula (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt thereof.

[0480] 22. The polysaccharide polymer according to any one of embodiments 1-21, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

[0481] 23. The polysaccharide polymer according to any one of embodiments 1-22, 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.

[0482] 24. The polysaccharide polymer according to any one of embodiments 1-23, wherein the compound of formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

[0483] 25. The polysaccharide polymer according to any one of embodiments 1-24, wherein the polysaccharide polymer is alginate, the crosslinking moiety 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.

[0484] 26. A composition comprising the polysaccharide polymer according to any one of embodiments 1-25.

[0485] 27. A hydrogel capsule comprising the polysaccharide polymer according to any one of embodiments 1-25.

[0486] 28. The hydrogel capsule according to embodiment 27, wherein the hydrogel capsule comprises a single compartment containing the polysaccharide polymer (such as the polysaccharide polymers described herein).

[0487] 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 (such as the polysaccharide polymers described herein).

[0488] 30. The hydrogel capsule according to embodiment 29, wherein the hydrogel capsule comprises an inner compartment and an outer compartment.

[0489] 31. The hydrogel capsule according to embodiment 30, wherein:

[0490] the inner compartment contains a first polysaccharide polymer containing the crosslinked moiety;

[0491] the outer compartment contains a second polysaccharide polymer containing the crosslinked moiety.

[0492] 32. A hydrogel capsule comprising:

[0493] (i) an inner compartment containing a first polysaccharide polymer containing a compound of formula (I):

[0494] or a pharmaceutically acceptable salt thereof, wherein:

[0495] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic, 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)(C1-C6-alkylene)-, -N(R C )C(O)(C2-C6-alkenylene)-, -C(=N(R C )(R 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 attached to an attachment group (e.g., an attachment group described herein) and is optionally substituted with one or more R 1 ;

[0496] L 1 and L 3 are each independently a bond, an alkyl or a heteroalkyl, each alkyl and heteroalkyl being optionally substituted with one or more R 2 ;

[0497] L 2 is a bond;

[0498] M is absent, an alkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, each of which is optionally substituted with one or more R 3 ;

[0499] P is a heteroaryl optionally substituted with one or more R 4 ;

[0500] Z is an alkyl, an alkenyl, an alkynyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, each of which is optionally substituted with one or more R 5 ;

[0501] Each R A , R B , R C , R D , R E , R F and R G are independently hydrogen, an alkyl, an alkenyl, an alkynyl, a heteroalkyl, a halogen, an azide, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl, each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl being optionally substituted with one or more R 6 ;

[0502] Or R C and R D together with the nitrogen atom to which they are attached form a ring (e.g., a 5-7 membered ring) optionally substituted with one or more R 6 ;

[0503] Each R 1 , R 2 , R 3, R 4 , R 5 and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 , S(O) x R E1 , –OS(O) x R E1 , –N(R C1 )S(O) x R E1 , –S(O) x N(R C1 )(R D1 ), –P(R F1 ) y , cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is optionally substituted by one or more R 7 substituents;

[0504] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl, heteroaryl is optionally substituted by one or more R 7 substituents;

[0505] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group;

[0506] x is 1 or 2; and

[0507] y is 2, 3 or 4; and

[0508] (ii) an outer compartment, which contains a second polysaccharide polymer comprising a crosslinked moiety.

[0509] 33. The hydrogel capsule according to embodiment 32, wherein the polysaccharide polymer (e.g., the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from alginate, hyaluronate, and chitosan.

[0510] 34. The hydrogel capsule according to any one of embodiments 32-33, wherein the polysaccharide polymer (e.g., the first polysaccharide polymer and / or the second polysaccharide polymer) is alginate.

[0511] 35. The hydrogel capsule according to any one of embodiments 32-34, wherein the first polysaccharide polymer is alginate.

[0512] 36. The hydrogel capsule according to any one of embodiments 32-35, wherein the second polysaccharide polymer is alginate.

[0513] 37. The hydrogel capsule according to any one of embodiments 32-36, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

[0514] 38. The hydrogel capsule according to any one of embodiments 32-37, wherein the crosslinked moiety has the structure of formula (IV):

[0515] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0516] Q is O, NR 33 or C(R 34a )(R 34b );

[0517] R 33 、R 34a 、R 34b 、R 60a 、R 60b 、R 61a 、R 61b and R 62 in each case is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl;

[0518] Each R A1 、R B1 、R C1 、R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with 1-6 R 7 substituents; and

[0519] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl, or heterocycloalkyl.

[0520] 39. The hydrogel capsule according to any one of embodiments 32-37, wherein the crosslinked moiety has the structure of formula (V):

[0521] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0522] Each of T and U is independently O, NR 33 or C(R 34a )(R 34b );

[0523] R 33 、R 34a 、R 34b 、R 65a 、R 65b 、R 65c 、R 65d 、R 65e 、R 65f 、R 65g and R 66 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl, or heteroaryl;

[0524] Each R A1 、R B1 、R C1 、R D1 and RE1 Independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 substituents; and

[0525] each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0526] 40. The hydrogel capsule according to any one of embodiments 32-37, wherein the crosslinked moiety has the structure of formula (VI):

[0527] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0528] T, Y 1 and Y 2 each is independently O, NR 33 , or C(R 34a )(R 34b );

[0529] R 33 , R 34a , R 34b , R 69 and R 70 each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl;

[0530] each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 substituents; and

[0531] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0532] 41. The hydrogel capsule according to any one of embodiments 32 - 37, wherein the crosslinked moiety has the structure of formula (VII):

[0533] or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0534] T is O, NR 33 or C(R 34a )(R 34b );

[0535] Ring M is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted with 1 - 6 R 7 substituents;

[0536] R 33 , R 34a , R 34b and R 74 each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocycloalkyl, aryl or heteroaryl;

[0537] Each R A1 , R B1 , R C1 , R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1 - 6 R 7 substituents; and

[0538] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

[0539] 42. The hydrogel capsule according to any one of embodiments 38-41, wherein the compound of formula (IV), (V), (VI) or (VII) is selected from the compounds in Table 4 or a pharmaceutically acceptable salt thereof.

[0540] 43. The hydrogel capsule according to any one of embodiments 32-42, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

[0541] 44. The hydrogel capsule according to any one of embodiments 32-43, 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.

[0542] 45. The hydrogel capsule according to any one of embodiments 32-44, wherein the compound of formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

[0543] 46. The hydrogel capsule according to any one of embodiments 32-45, wherein the hydrogel capsule has a diameter between 0.1 mm and 5 mm.

[0544] 47. The hydrogel capsule according to any one of embodiments 32-46, wherein the hydrogel capsule has a diameter between 1 mm and 5 mm.

[0545] 48. The hydrogel capsule according to any one of embodiments 32-47, wherein the hydrogel capsule has a diameter between 1 mm and 2.5 mm.

[0546] 49. The hydrogel capsule according to any one of embodiments 32-48, wherein the hydrogel capsule encapsulates cells.

[0547] 50. The hydrogel capsule according to embodiment 49, wherein the cells produce a therapeutic agent.

[0548] 51. The hydrogel capsule according to embodiment 50, wherein the therapeutic agent is a protein, such as a hormone, a clotting factor, an antibody or an enzyme.

[0549] 52. The hydrogel capsule according to any one of embodiments 32-51, wherein the hydrogel capsule is formulated for implantation into a subject (e.g., into the intraperitoneal (IP) space, abdominal cavity, omentum, lesser sac, subcutaneous fat).

[0550] 53. The hydrogel capsule according to any one of embodiments 32-52, wherein the implantable element is formulated for implantation into the IP space of a subject.

[0551] 54. A composition comprising the hydrogel capsule according to any one of embodiments 32 - 53.

[0552] 55. A method of producing a hydrogel capsule comprising the polysaccharide polymer according to any one of embodiments 1 - 24.

[0553] 56. A method of enhancing the stability of a hydrogel capsule comprising a polysaccharide polymer, the method comprising providing means for both ionically cross - linking and covalently cross - linking the polysaccharide polymer.

[0554] 57. The method according to embodiment 56, wherein the means for ionically cross - linking the polysaccharide polymer comprises using a divalent cation (e.g., Ba 2+、 Ca 2+ 、Sr 2+ ).

[0555] 58. The method according to any one of embodiments 56 - 57, wherein the means for covalently cross - linking the polysaccharide polymer comprises using a cross - linking moiety.

[0556] 59. The polysaccharide polymer according to any one of embodiments 1 - 25, wherein after addition of the click cross - linker, the polysaccharide retains additional carboxylic acid groups.

[0557] 60. The polysaccharide polymer according to any one of embodiments 1 - 25 or 59, wherein the polysaccharide is not reduced (e.g., not treated with a reducing agent) before modification with a click cross - linker.

[0558] 61. The polysaccharide polymer according to any one of embodiments 1 - 25 or 59, wherein the polysaccharide polymer is not oxidized (e.g., not treated with an oxidizing agent) before modification with a click cross - linker.

[0559] 62. The polysaccharide polymer according to any one of embodiments 1 - 25 or 59 - 61, wherein the click cross - linker is non - hydrolyzable.

[0560] 63. The polysaccharide polymer according to any one of embodiments 1 - 25 or 59 - 62, wherein the compound of formula (I) is non - hydrolyzable.

[0561] 64. The polysaccharide polymer according to any one of embodiments 1 - 25 or 59 - 63, wherein the click cross - linker is not a thiol or a vinyl sulfone.

[0562] 65. A method of treating a disease, disorder or condition of a subject, the method comprising administering to the subject the hydrogel capsule according to any one of embodiments 32 - 53.

[0563] 66. The method according to embodiment 65, wherein the disease, disorder or condition is diabetes (e.g., type 1 diabetes).

[0564] 67. The method according to embodiment 65, wherein the disease, disorder or condition is not diabetes (e.g., type 1 diabetes).

[0565] 68. The method according to any one of embodiments 65 - 67, wherein the subject is a human.

[0566] Examples

[0567] The following examples are set forth to more fully understand the invention described herein. Synthetic and biological examples described in this application are provided to illustrate the compounds, compositions, devices, and methods provided herein, and the examples should not in any way be construed as limiting their scope.

[0568] The compounds, modified polymers, implantable elements, and their compositions provided herein can be prepared from readily available starting materials using modifications of the specific synthetic schemes set forth below that are well known to those skilled in the art. It should be understood that, given typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), other process conditions can also be used unless otherwise stated. The optimal reaction conditions can vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through routine optimization procedures.

[0569] In addition, 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 protecting groups suitable for specific functional groups and the appropriate conditions for protection and deprotection are well known in the art. For example, many protecting groups and their introduction and removal are described in Greene et al., Protecting Groups in Organic Synthesis, 2nd ed., Wiley, New York, 1991 and the references cited therein.

[0570] Any of the strategies described below can be used to prepare the exemplary compounds, modified polymers, implantable elements, and compositions of the present invention.

[0571] Example 1: Synthesis of Sodium Alginate Modified with Exemplary Thiols and Compounds of Formula (I)

[0572]

[0573] In this example, an alginate polymer comprising a thiol crosslinker and a compound of formula (I) was synthesized. NovaMatrix PRONOVA TM UP LG20 (300 g; 1.25% w / w in water, 3.75 g sodium alginate) was weighed into a 400 mL EasyMax reactor equipped with an overhead stirrer. 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) and endotoxin-free water (20 g) were assembled in a separate 150 mL sterile container and mixed on an orbital shaker at 300 rpm until completely dissolved. Once dissolved, the pH was adjusted to pH 7.0 with 6N and 1N hydrochloric acid and charged into the EasyMax reactor. The stirring speed was set to 300 rpm and the batch temperature was adjusted to 25 °C. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.88 g, 14.02 mmol) and endotoxin-free water (45 g) were assembled in a separate 150 mL container and manually mixed until completely dissolved. The solution was added to the EasyMax reactor over a two-minute period. Once addition was complete, the batch was heated to 35 °C over 1 hour and held at 35 °C for 15 hours, then cooled to 25 °C. Once the reaction was complete, the batch was filtered through a cyanide silica pad and then purified by tangential flow filtration (10 kDa molecular weight cut-off). The solution was purified by performing 10 volume exchanges with physiological saline followed by 10 volume exchanges with endotoxin-free water.

[0574] After purification, the solution was concentrated to a refractive index value of 1.3360 and charged back into the 400 mL EasyMax reactor. The stirring speed was set to 300 rpm and the batch temperature was adjusted to 25 °C. For addition on the thiol crosslinker, exemplary reaction conditions are as follows. In a separate sterile container, methionine sulfoxide hydrochloride (0.91 g, 5.28 mmol) was weighed and dissolved in 1M MES pH 7.0 buffer (7.5 mL), then charged into the reactor. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) was weighed in a sterile container and dissolved in 1M MES pH 7.0 buffer (10 mL), then added to the reactor over two minutes. Once charging was complete, the reaction mixture was heated to 35 °C over 1 hour and held at 35 °C for 15 hours, then cooled to 25 °C. The reaction mixture was purified by performing 10-fold volume exchange with brine via tangential flow filtration (10 kDa molecular weight cut-off). After purification, the solution was concentrated to a refractive index value of 1.3380.

[0575] Example 2: Synthesis of Sodium Alginate Modified with Exemplary Maleimide

[0576]

[0577] In this example, an alginate polymer containing a maleimide crosslinker can be synthesized according to the exemplary procedure outlined below. Weigh Nova Matrix PRONOVA TM UP LG20 (300 g; 1.25% w / w in water, 3.75 g sodium alginate) and place it in a 400 mL EasyMax reactor equipped with an overhead stirrer. In a separate sterile container, weigh 1-(2-aminoethyl)-maleimide hydrochloride (0.93 g, 5.28 mmol) and dissolve it in 1M MES pH 7.0 buffer (7.5 mL), then load it into 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 and dissolve it in 1M MES pH 7.0 buffer (10 mL), then add it to the reactor over two minutes. Once the loading is complete, heat the reaction mixture to 35 °C over 1 hour and hold it at 35 °C for 15 hours, then cool it to 25 °C. Purify the reaction mixture by tangential flow filtration (10 kDa molecular weight cut-off) with a 10-fold volume exchange with brine. After purification, concentrate the solution to a refractive index value of 1.3380.

[0578] Example 3: Synthesis of Dual-Crosslinked Alginate Polymer by Michael Addition Reaction

[0579]

[0580] To form a covalently crosslinked alginate, a thiol crosslinker and a maleimide crosslinker can be coupled together according to the exemplary scheme outlined herein: Dissolve an alginate polymer containing Compound 302 and an alginate polymer containing Compound 304 (molar ratio 1:3) in 1M MES buffer and incubate at 20 - 30 °C for 4 - 12 hours. Purify the reaction mixture by tangential flow filtration (10 kDa molecular weight cut-off) with a 10-fold volume exchange with brine. After purification, concentrate the solution to a refractive index value of 1.3380.

[0581] Example 4: Synthesis of Sodium Alginate Modified with Exemplary Tetrazine and Compound of Formula (I)

[0582]

[0583] In this example, an alginate polymer comprising a thiol crosslinker and a compound of formula (I) was synthesized. NovaMatrix PRONOVA TM UP LG20 (300 g; 1.25% w / w in water, 3.75 g sodium alginate) was weighed into a 400 mL EasyMax reactor equipped with a overhead stirrer. 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) and endotoxin-free water (20 g) were assembled in a separate 150 mL sterile container and mixed on an oscillator at 300 rpm until completely dissolved. Once dissolved, the pH was adjusted to pH 7.0 with 6N and 1N hydrochloric acid and loaded into the EasyMax reactor. The stirring speed was set at 300 rpm and the batch temperature was adjusted to 25 °C. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.88 g, 14.02 mmol) and endotoxin-free water (45 g) were assembled in a separate 150 mL container and manually mixed until completely dissolved. The solution was added to the EasyMax reactor over a two-minute period. Once addition was complete, the batch was heated to 35 °C over 1 hour and held at 35 °C for 15 hours, then cooled to 25 °C. Once the reaction was complete, the batch was filtered through a cyanide silica pad and then purified by tangential flow filtration (10 kDa molecular weight cut-off). The solution was purified by first performing 10 volume exchanges with physiological saline and subsequently 10 volume exchanges with endotoxin-free water.

[0584] After purification, the solution was concentrated to a refractive index value of 1.3360 and transferred back into a 400 mL EasyMax reactor. The stirring speed was set to 300 rpm, and the batch temperature was adjusted to 25 °C. For the addition on the thiol crosslinker, the exemplary reaction conditions were as follows. In a separate sterile container, 1-[4-(1,2,4,5-tetrazin-3-yl)phenyl]methanamine (1.18 g, 5.28 mmol) was weighed and dissolved in 1 M MES pH 7.0 buffer (7.5 mL), and then loaded into the reactor. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) was weighed in a sterile container and dissolved in 1 M MES pH 7.0 buffer (10 mL), and then added to the reactor over two minutes. Once the loading was complete, the reaction mixture was heated to 35 °C over 1 hour and held at 35 °C for 15 hours, then cooled to 25 °C. The reaction mixture was purified by tangential flow filtration (10 kDa molecular weight cut-off) with a 10-fold volume exchange with brine. After purification, the solution was concentrated to a refractive index value of 1.3380.

[0585] Example 5: Synthesis of Alginate Modified with Exemplary Norbornene

[0586]

[0587] In this example, an alginate polymer containing a maleimide crosslinker can be synthesized according to the exemplary procedure outlined below. Nova Matrix PRONOVA TM UP LG20 (300 g; 1.25% w / w in water, 3.75 g of sodium alginate) was weighed into a 400 mL EasyMax reactor equipped with an overhead stirrer. In a separate sterile container, 5-norbornene-2-methanamine (0.84 g, 5.28 mmol) was weighed and dissolved in 1 M MES pH 7.0 buffer (7.5 mL), and then loaded into the reactor. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) was weighed in a sterile container and dissolved in 1 M MES pH 7.0 buffer (10 mL), and then added to the reactor over two minutes. Once the loading was complete, the reaction mixture was heated to 35 °C over 1 hour and held at 35 °C for 15 hours, then cooled to 25 °C. The reaction mixture was purified by tangential flow filtration (10 kDa molecular weight cut-off) with a 10-fold volume exchange with brine. After purification, the solution was concentrated to a refractive index value of 1.3380.

[0588] Example 6: Synthesis of doubly crosslinked alginate polymers by inverse electron demand Diels-Alder reaction

[0589]

[0590] To form covalently crosslinked alginate, a thiol crosslinker and a maleimide crosslinker can 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 1M MES buffer and incubated at 20 - 30 °C for 4 - 12 hours. The reaction mixture is purified by tangential flow filtration (10 kDa molecular weight cut-off) with 10-fold volume exchange with brine. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0591] Example 7: Synthesis of doubly crosslinked alginate polymers by thiol-ene photoclick reaction.

[0592] To form covalently crosslinked alginate, a thiol crosslinker and a norbornene crosslinker can 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) are dissolved in 1M MES buffer together with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator and irradiated with UV light for 0.5 hours with stirring. The reaction mixture is purified by tangential flow filtration (10 kDa molecular weight cut-off) with 10-fold volume exchange with brine. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0593] Example 8: Synthesis of exemplary doubly crosslinked alginate hydrogel capsules

[0594] Before manufacturing a one- or two-compartment alginate hydrogel capsule, the buffer and alginate solutions are sterilized by filtration through a 0.2-μm filter using a sterile process. To prepare particles configured as two-compartment hydrogel capsules with a diameter of approximately 1.5 mm, the electrospray 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 (22G inner lumen, 18G outer lumen, Paragon). The inner lumen was attached to a first 5-ml Luer lock syringe (BD, NJ, USA) which was connected to a vertically oriented syringe pump (Pump11Pico Plus, Harvard Apparatus, Holliston, MA, USA). The outer lumen was connected via a Luer connector to a second 5-ml Luer lock syringe which was connected to a horizontally oriented second syringe pump (Pump11Pico Plus). The two syringe pumps moved the first and second alginate solutions from the syringes through the two inner lumens of the coaxial needle, and individual droplets containing the two alginate solutions were extruded from the needle into a glass dish containing the crosslinking solution. The settings for each Pico Plus syringe pump were for a diameter of 12.06 mm, and the flow rate of each pump was adjusted to achieve the various test flow rates in the examples below, but the total flow rate was kept set at 10 ml / h.

[0595] For the manufacture of both two-compartment and one-compartment double-crosslinked alginate hydrogel capsules, after extruding the desired volume of 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. Capsules that fell to the bottom of the crosslinking vessel were collected by pipetting into a conical tube. After the capsules had settled in the tube, the crosslinking buffer was removed and the capsules were washed. The capsules were washed four times with HEPES buffer and then suspended in

[0596] In some experiments, the quality of the capsules in the two-compartment or one-compartment capsule compositions was examined. An aliquot containing at least 200 capsules was removed from the composition and transferred to a well plate, and the quality of the entire aliquot was examined by optical microscopy by counting the number of spherical capsules in 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 herein.

[0597] Example 9: Preparation of a two-compartment hydrogel containing a modified polysaccharide polymer

[0598] Using the method of Example 8, a two-compartment hydrogel can be synthesized from the alginate polymers described in Examples 3, 6, and 7. As a non-limiting example, a two-compartment hydrogel can be synthesized according to this method that contains an inner compartment and an outer compartment of the double-crosslinked alginate of Example 3. As another example, a two-compartment hydrogel can be synthesized that contains an inner compartment of the double-crosslinked alginate of Example 6 and an outer compartment that contains the double-crosslinked alginate of Example 7.

[0599] Example 10: Synthesis of Exemplary Double-Crosslinked Alginate Hydrogel Capsules

[0600] The rupture or mechanical strength of a particle (e.g., a hydrogel capsule) can be determined by performing a rupture test using a texture analyzer after fabrication but before implantation. In one embodiment, mechanical testing of the hydrogel capsules is performed on a TA.XT plus texture analyzer (StableMicro Systems, Surrey, United Kingdom) using a 5 mm probe attached to a 5 kg load cell. A single capsule is placed on the platform and compressed from above by the probe at a fixed rate of 0.5 mm / sec. Contact between the probe and the capsule is detected when a repulsive force of 1 g is measured. The probe continues to travel 90% of the distance between the contact height of the probe and the platform, thereby compressing the capsule to the burst point. The resistance of the probe to the compression force is measured and plotted as a function of the probe travel (force vs. displacement curve). Typically, the capsule will rupture slightly before it fully bursts, and the force applied to the probe will decrease slightly. The 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 referred to as the initial rupture force. In one embodiment, the rupture force of a capsule formulation manufactured using the device described herein is the average of the initial rupture forces of at least 10, 20, 30, or 40 capsules.

[0601] In this example, three exemplary alginate hydrogel capsule formulations are prepared according to the protocols outlined in Examples 3, 6, and 7 and their rupture strengths are analyzed. The hydrogel capsule architectures are described in Table 6. As Figures 1A - 1C shown, compared to hydrogels consisting solely of ion (e.g., Ba 2+ -mediated) crosslinking, the double-crosslinked alginate hydrogels exhibit increased average rupture strength. In Figures 1A - 1C , (1), (3), and (5) refer to ionically crosslinked alginate hydrogels, while (2), (4), and (6) refer to hydrogel capsules 1, 2, and 3 as shown in Table 6.

[0602] Table 6. Exemplary Two-Compartment Hydrogel Architectures

[0603]

[0604] Example 11: Encapsulation of mammalian cells in a hydrogel comprising a modified polysaccharide polymer

[0605] Exemplary mammalian cells can be encapsulated in the hydrogel described in Example 10. Cells (e.g., at a density of 5 - 8 x 10 6 cells / mL -1 can be added to the inner and / or outer layer of the two-compartment hydrogel).

[0606] Equivalents and scope

[0607] This application incorporates by reference in their entirety various published patents, published patent applications, journal articles, and other publications. If any incorporated reference conflicts with this specification, this specification shall control. In addition, any particular embodiment of the disclosure within the prior art may be explicitly excluded from any one or more of the claim terms. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly set forth as excluded herein. Any particular embodiment of the disclosure may be excluded from any claim term for any reason, whether or not related to the existence of prior art.

[0608] Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited to the above specification, drawings, or embodiments, but rather as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications may be made to this specification without departing from the spirit or scope of the disclosure as defined in the following claims.

Claims

1. A polysaccharide polymer comprising: (i) a crosslinked portion; and (ii) a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, 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)(C1-C6-alkylene)-, -N(R C )C(O)(C2-C6-alkenylene)-, -C(=N(R C )(R 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 attached to an attachment group (e.g., an attachment group described herein) and is optionally substituted by one or more R 1 ; L 1 and L 3 each independently is a key, alkyl or heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted with one or more R 2 substituents; L 2 is a key; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted with one or more R 3 substituted; P is an optionally substituted heteroaryl group substituted by one or more R 4 groups; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted with one or more R 5 substituted; Each R A 、R B 、R C 、R D 、R E 、R F and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted by one or more R 6 substituents; or R C and R D together with the nitrogen atom to which they are attached form an optionally one or more R 6 substituted ring (e.g., 5-7 membered ring); Each R 1 、R 2 、R 3 、R 4 、R 5 and R 6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 、S(O) x R E1 、–OS(O) x R E1 、–N(R C1 )S(O) x R E1 、–S(O) x N(R C1 )(R D1 )、–P(R F1 ) y 、cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is optionally substituted by one or more R 7 ; Each R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with one or more R 7 substituents; Each R 7 is independently an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group; x is 1 or 2; and y is 2, 3 or 4.

2. The polysaccharide polymer according to claim 1, wherein the crosslinked portion is covalently bound to a sugar monomer within the polysaccharide polymer.

3. The polysaccharide polymer according to claim 2, wherein the crosslinked portion is bound to a carboxylate moiety within the sugar monomer.

4. The polysaccharide polymer according to claim 1, wherein the crosslinked portion comprises an alkyl, alkenyl, alkynyl, ester, ketone, amine, thiol, cycloalkyl, heterocyclic, aryl or heteroaryl group.

5. The polysaccharide polymer according to claim 1, wherein the crosslinked portion is capable of reacting with a second crosslinked portion after activation with, for example, heat, acid, base or a catalyst.

6. The polysaccharide polymer according to claim 1, wherein the crosslinked portion is present on the polysaccharide polymer at a density of at least about 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or higher, for example as determined by comparison with a reference standard.

7. The polysaccharide polymer according to claim 1, wherein the crosslinked portion is present on the polysaccharide polymer at a density between 1% and 10%, such as 1%-8%, 1%-6%, or 1%-4%, for example as determined by comparison with a reference standard.

8. The polysaccharide polymer according to claim 1, wherein the polysaccharide polymer is selected from alginate, hyaluronate and chitosan.

9. The polysaccharide polymer according to claim 1, wherein the polysaccharide polymer is alginate.

10. The polysaccharide polymer according to claim 9, wherein the alginate is guluronic acid (G)-rich alginate or mannuronic acid (M)-rich alginate.

11. The polysaccharide polymer according to claim 1, wherein the crosslinked moiety has the structure of formula (IV): or a pharmaceutically acceptable salt or tautomer thereof, wherein: Q is O, NR 33 or C(R 34a )(R 34b )); 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, azido, 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, heterocyclic, aryl or heteroaryl; Each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and Each R 7 is independently an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a halogen, a cyano group, an oxo group, a hydroxy group, a cycloalkyl group or a heterocyclic group.

12. The polysaccharide polymer according to claim 11, wherein the crosslinked moiety contains a thiol moiety.

13. The polysaccharide polymer according to claim 1, wherein the crosslinked moiety has the structure of formula (V): 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 ); R 33 、R 34a 、R 34b 、R 65a 、R 65b 、R 65c 、R 65d 、R 65e 、R 65f 、R 65g and R 66 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 , R B1 , R C1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1 - 6 R 7 ; and Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

14. The polysaccharide polymer according to claim 13, wherein the crosslinked moiety contains a norbornenyl moiety.

15. The polysaccharide polymer according to claim 1, wherein the crosslinked moiety has the structure of formula (VI): or a pharmaceutically acceptable salt or tautomer thereof, wherein: T, Y 1 and Y 2 Each of which is independently O, NR 33 , or C(R 34a )(R 34b ); R 33 、R 34a 、R 34b 、R 69 and R 70 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

16. The polysaccharide polymer according to claim 15, wherein the crosslinked moiety contains a maleimide moiety.

17. The polysaccharide polymer according to claim 1, wherein the crosslinked moiety has the structure of formula (VII): or a pharmaceutically acceptable salt or tautomer thereof, wherein: T is O, NR 33 or C(R 34a )(R 34b )); Ring M is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which is optionally substituted with 1-6 Rs 7 substituted; R 33 、R 34a 、R 34b and R 74 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 、R B1 、R C1 、R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

18. The polysaccharide polymer according to claim 17, wherein the crosslinked moiety comprises a tetrazine moiety.

19. The polysaccharide polymer according to claim 1, wherein the crosslinked moiety has a structure selected from Table 4, or a pharmaceutically acceptable salt thereof.

20. The polysaccharide polymer according to claim 1, wherein the polysaccharide polymer comprises one of the compounds of formula (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt thereof.

21. The polysaccharide polymer according to claim 1, wherein the polysaccharide polymer comprises two of the compounds of formula (IV), (V), (VI) or (VII), or a pharmaceutically acceptable salt thereof.

22. The polysaccharide polymer according to claim 1, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

23. The polysaccharide polymer according to claim 1, 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.

24. The polysaccharide polymer according to claim 1, wherein the compound of formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

25. The polysaccharide polymer according to claim 1, wherein the polysaccharide polymer is alginate, the crosslinked moiety 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.

26. A composition comprising the polysaccharide polymer according to any one of claims 1-25.

27. A hydrogel capsule comprising the polysaccharide polymer according to any one of claims 1-25.

28. The hydrogel capsule according to claim 27, wherein the hydrogel capsule comprises a single compartment containing the polysaccharide polymer (such as the polysaccharide polymer described herein).

29. The hydrogel capsule according to claim 27, wherein the hydrogel capsule comprises a plurality of compartments, and one of the compartments contains the polysaccharide polymer (such as the polysaccharide polymer described herein).

30. The hydrogel capsule according to claim 29, wherein the hydrogel capsule comprises an inner compartment and an outer compartment.

31. The hydrogel capsule according to claim 30, wherein: The inner compartment contains a first polysaccharide polymer comprising the crosslinked portion; The outer compartment contains a second polysaccharide polymer comprising the crosslinked portion.

32. A hydrogel capsule comprising: (i) An inner compartment containing a first polysaccharide polymer comprising a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, 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)(C1-C6-alkylene)-, -N(R C )C(O)(C2-C6-alkenylene)-, -C(=N(R C )(R 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 attached to an attachment group (e.g., an attachment group described herein) and is optionally substituted by one or more R 1 ; L 1 and L 3 each independently is a key, alkyl or heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted with one or more R 2 substituents; L 2 is a key; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 3 substituted; P is an optionally substituted heteroaryl group which is substituted by one or more R 4 groups; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, each of which is optionally substituted with one or more R 5 substituted; Each R A 、R B 、R C 、R D 、R E 、R F and R G are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with one or more R 6 substituents; or R C and R D together with the nitrogen atom to which they are attached form an optionally one or more R 6 substituted ring (e.g., 5- to 7-membered ring); Each R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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 、S(O) x R E1 、–OS(O) x R E1 、–N(R C1 )S(O) x R E1 、–S(O) x N(R C1 )(R D1 )、–P(R F1 ) y 、cycloalkyl, heterocyclic group, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclic group, aryl and heteroaryl is optionally substituted by one or more R 7 ; Each R A1 、R B1 、R C1 、R D1 、R E1 and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted by one or more R 7 substituents; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group; x is 1 or 2; and y is 2, 3 or 4; and (ii) an outer compartment, which contains a second polysaccharide polymer comprising a crosslinked portion.

33. The hydrogel capsule according to claim 32, wherein the polysaccharide polymer (e.g., the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from alginate, hyaluronate, and chitosan.

34. The hydrogel capsule according to claim 32, wherein the polysaccharide polymer (e.g., the first polysaccharide polymer and / or the second polysaccharide polymer) is alginate.

35. The hydrogel capsule according to claim 32, wherein the first polysaccharide polymer is alginate.

36. The hydrogel capsule according to claim 32, wherein the second polysaccharide polymer is alginate.

37. The hydrogel capsule according to claim 32, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

38. The hydrogel capsule according to claim 32, wherein the crosslinked moiety has a structure of formula (IV): or a pharmaceutically acceptable salt or tautomer thereof, wherein: Q is O, NR 33 or C(R 34a )(R 34b )); R 33 、R 34a 、R 34b 、R 60a 、R 60b 、R 61a 、R 61b and R 62 Each of which, independently, is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 、R B1 、R C1 、R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 substituents; and Each R 7 is independently an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

39. The hydrogel capsule according to claim 32, wherein the crosslinked portion has a structure of formula (V): 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 ); 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 which independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 、R B1 、R C1 、R D1 and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 ; and Each R 7 is independently an alkyl group, alkenyl group, alkynyl group, heteroalkyl group, halogen, cyano group, oxo group, hydroxyl group, cycloalkyl group or heterocyclic group.

40. The hydrogel capsule according to claim 32, wherein the crosslinked portion has a structure of formula (VI): or a pharmaceutically acceptable salt or tautomer thereof, wherein: T, Y 1 and Y 2 Each of which is independently O, NR 33 , or C(R 34a )(R 34b ); R 33 、R 34a 、R 34b 、R 69 and R 70 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 , R B1 , R C1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with 1-6 R 7 ; and Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocycloalkyl.

41. The hydrogel capsule according to claim 32, wherein the crosslinked portion has a structure of formula (VII): or a pharmaceutically acceptable salt or tautomer thereof, wherein: T is O, NR 33 or C(R 34a )(R 34b )); Ring M is cycloalkyl, heterocyclic, aryl, heteroaryl, each of which is optionally substituted by 1-6 Rs 7 substituted; R 33 、R 34a 、R 34b and R 74 each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, 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, heterocyclic group, aryl or heteroaryl; Each R A1 , R B1 , R C1 , R D1 , and R E1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl is optionally substituted with 1-6 R 7 ; and Each R 7 is independently an alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclic group.

42. The hydrogel capsule according to claim 32, wherein the compound of formula (IV), (V), (VI) or (VII) is selected from the compounds in Table 4 or a pharmaceutically acceptable salt thereof.

43. The hydrogel capsule according to claim 32, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

44. The hydrogel capsule according to claim 32, 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.

45. The hydrogel capsule according to claim 32, wherein the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

46. The hydrogel capsule according to claim 32, wherein the hydrogel capsule has a diameter between 0.1 mm and 5 mm.

47. The hydrogel capsule according to claim 32, wherein the hydrogel capsule has a diameter between 1 mm and 5 mm.

48. The hydrogel capsule according to claim 32, wherein the hydrogel capsule has a diameter between 1 mm and 2.5 mm.

49. The hydrogel capsule according to claim 32, wherein the hydrogel capsule encapsulates cells.

50. The hydrogel capsule according to claim 49, wherein the cells produce a therapeutic agent.

51. The hydrogel capsule according to claim 50, wherein the therapeutic agent is a protein, such as a hormone, a clotting factor, an antibody or an enzyme.

52. The hydrogel capsule according to claim 32, wherein the hydrogel capsule is formulated for implantation into a subject (e.g., into the intraperitoneal (IP) space, abdominal cavity, omentum, lesser omental bursa, subcutaneous fat).

53. The hydrogel capsule according to claim 32, wherein the implantable element is formulated for implantation into the IP space of a subject.

54. A composition comprising the hydrogel capsule according to any one of claims 32 - 53.

55. A method of producing a hydrogel capsule comprising the polysaccharide polymer according to any one of claims 1 - 25.

56. A method of enhancing the stability of a hydrogel capsule comprising a polysaccharide polymer, wherein the method comprises providing means for both ionically cross - linking and covalently cross - linking the polysaccharide polymer.

57. The method according to claim 56, wherein the means for ionically cross - linking the polysaccharide polymer comprises using a divalent cation (e.g., Ba 2+、 Ca 2+ , Sr 2+ ).

58. The method according to any one of claims 56 - 57, wherein the means for covalently cross - linking the polysaccharide polymer comprises using a cross - linking moiety.

59. A method for treating a disease, disorder or condition of a subject, which comprises administering to the subject the hydrogel capsule according to any one of claims 32 - 53 or the composition according to claim 54, thereby treating the disease, disorder or condition of the subject.

60. The method according to claim 59, wherein the disease, disorder or medical condition is diabetes (e.g., type 1 diabetes).

61. The method according to claim 59, wherein the disease, disorder or medical condition is not diabetes (e.g., type 1 diabetes).

62. The method according to claim 59, wherein the subject is a human.

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