Covalently crosslinked polysaccharides and methods of use thereof

Abstract

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 are described herein.

Description

[0001] Priority declaration

[0002] This application claims priority to U.S. Application No. 63 / 357,877, filed on July 1, 2022, and U.S. Application No. 63 / 452,090, filed on March 14, 2023. The disclosure of each of the foregoing applications is incorporated herein by reference in its entirety. Background Art

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

[0004] Described herein are polysaccharide polymers that can be covalently cross-linked with another part, such as another polysaccharide polymer, and hydrogel capsules comprising the polysaccharide polymers, as well as their compositions and uses. In one embodiment, the polysaccharide polymer comprises a clickable cross-linking moiety (such as a compound of formula (IV) or (V)) or a pharmaceutically acceptable salt thereof. In one embodiment, the polysaccharide polymer comprises a clickable cross-linking moiety (e.g., a compound of formula (IV) or (V)) and a compound of formula (I) (e.g., a defibrotic compound) or a pharmaceutically acceptable salt thereof. These polysaccharide polymers can be incorporated into hydrogel capsules that can encapsulate cells. Incorporating the clickable cross-linking moiety into the polysaccharide polymer and then into the hydrogel capsule incorporated with the polysaccharide polymer can allow for the regulation of certain properties of the hydrogel capsule, including capsule diameter, stability, and integrity.

[0005] The details of one or more embodiments of the present invention are set forth herein. Other features, objects, and advantages of the present invention will be apparent from the detailed description, drawings, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is a schematic diagram depicting exemplary architectures of polymers and associated hydrogel capsules described herein. DETAILED DESCRIPTION

[0008] The present disclosure provides a polysaccharide polymer comprising a clickable cross-linking portion and a compound of formula (I), as well as related compositions and hydrogel capsules comprising the polysaccharide polymer, and preparation methods and uses thereof.

[0009] Abbreviations and definitions

[0010] In order to more easily understand 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 ordinary technicians in the field to which the present invention belongs.

[0011] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0012] "About" or "approximately" when used herein means to modify a numerically defined parameter (e.g., a physical description of a hydrogel capsule such as diameter, sphericity, the number of cells encapsulated therein, the number of capsules in a formulation), and means that the listed value is within the acceptable functional range of the defined parameter determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system, including the acceptable error range of the measurement system. For example, "about" can mean a range of 20% above and below the listed 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 may have a diameter of 1.2 to 1.8 mm and may encapsulate 4 M to 6 M 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 may vary by as much as 15%, 10%, or 5% above and below the stated value of the parameter. Alternatively, particularly with respect to certain properties of the devices described herein, such as cell productivity, or defibrotic compounds, the term "about" can mean within an order of magnitude above or below 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) or an image or physical entity (e.g., a sample) by "directly obtaining" or "indirectly obtaining" a value or physical entity. "Directly obtaining" means performing a process (e.g., performing an analytical method or protocol) to obtain a value or physical entity. "Indirectly obtaining" refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly obtains a physical entity or value). Directly obtaining a value or physical entity includes performing a process that includes a physical change of a physical substance or the use of a machine or device. Examples of directly obtaining a value include obtaining a sample from a human subject. Directly obtaining a value includes performing a process that uses a machine or device, such as using a fluorescence microscope to obtain fluorescence microscopy data.

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

[0015] As used herein, "defibrotic" means a compound or material that reduces the foreign body response (FBR). For example, the amount of FBR induced in a biological tissue 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 4) into the tissue is lower than the FBR induced by implanting a reference device without defibrotic (afibrotic-null), i.e., a device lacking any defibrotic compound but having substantially the same composition (e.g., one or more of the same cell types) and structure (e.g., size, shape, number of compartments). In one embodiment, the FBR is induced using an assay known in the art, such as described in WO 2017 / 075630 or using Vegas, A. et al., Nature Biotechnol One or more of the assays / methods described in (above) (e.g., subcutaneous tissue protease measurement of implanted capsules, Masson's trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining and confocal microscopy of macrophages (CD68 or F4 / 80), myofibroblasts (α-muscle actin, SMA) or general cell deposition, quantification of 79 RNA sequences for known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophils on the device (e.g., capsule) retrieved after 14 days in the intraperitoneal space of a suitable test subject (e.g., immunocompetent mouse)) assesses the extent of FBR through an immune response in tissue containing an implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis. In one embodiment, the FBR is assessed by measuring the level of one or more immune response biomarkers (e.g., cathepsins, 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 comprising 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 without FBR, such as a device substantially identical to the device tested or claimed except for the lack of a device for reducing FBR (e.g., a hydrogel capsule that does not comprise a defibrotic compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of one or more biomarkers) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or more.

[0016] As used herein, "cell" refers to an engineered cell or a 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 of a ligand derived from a cell adhesion molecule (CAM) (e.g., a cell adhesion molecule that mediates cell-matrix attachment or cell-cell attachment). The length of CBP is less than 50, 40, 30, 25, 20, 15 or 10 amino acids. In one embodiment, the length of CBP is between 3 and 12 amino acids, the length is between 4 and 10 amino acids, or the length is 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. The CBP amino acid sequence may be identical to the naturally occurring binding domain sequence, or may 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, CBP comprises a cell binding sequence listed in Table 1 below or a conservatively substituted variant thereof. In one embodiment, CBP comprises at least one of the cell binding sequences listed in Table 1 below. In one embodiment, CBP consists essentially of the cell binding sequences listed in Table 1 below. In one embodiment, CBP is an RGD peptide, which means that the peptide comprises the amino acid sequence RGD (SEQ ID NO: 43) and optionally comprises one or more additional amino acids located at one or both of the N-terminus and / or the C-terminus. In one embodiment, CBP is a cyclic peptide comprising RGD (SEQ ID NO: 43), such as one of the cyclic RGD peptides described in Vilaca, H. et al., Tetrahedron 70(35): 5420-5427 (2014). In one embodiment, CBP is a linear peptide comprising RGD (SEQID NO: 43) and is less than 6 amino acids in length. In one embodiment, CBP is a linear peptide consisting essentially of RGD (SEQID NO: 43) or RGDSP (SEQ ID NO: 59).

[0018] Table 1: Exemplary CAM ligand proteins and cell binding sequences

[0019]

[0020] As used herein, "CBP-polymer" means a polymer comprising at least one cell-binding peptide molecule covalently attached to a 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 an alginate, such as sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., essentially consisting of a single amino acid, or a peptide of several identical or different amino acids), which is connected to the N-terminus or C-terminus of CBP via a peptide bond. In one embodiment, the C-terminus of the amino acid linker is connected to the N-terminus of CBP, and the N-terminus of the amino acid linker is connected to at least one side 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 CBP-polysaccharide (e.g., CBP-alginate) are unmodified by CBP, e.g., the unmodified moieties have free carboxyl groups or lack modifiable pendant carboxyl groups. In one embodiment, the number of polysaccharide moieties with covalently attached CBP is less than any of the following values: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%.

[0021] In one embodiment, the density of CBP modification in a CBP-polymer is estimated by combustion analysis of 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 the density of linker-RGD molecule modification (e.g., conjugation density) is about 0.05% nitrogen (N) to 1.00% N, about 0.10% N to about 0.75% N, about 0.20% N to about 0.50% N, or about 0.30% N to about 0.40% N as determined using an assay described herein. In one embodiment, the conjugation density of the 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 in solution (e.g., saline solution) with a viscosity of 80 to 120 cP as determined by any assay capable of quantifying the amount of peptide conjugated to a polymer, e.g., a quantitative peptide conjugation assay described herein. Unless otherwise expressly stated or apparent from the context, a particular recited numerical concentration, concentration range, density, or density range of CBP in a CBP-polymer refers 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.

[0022] As used herein, "cell binding polypeptide (CBPP)" means a polypeptide of at least 50, at least 75, or at least 100 amino acids in length 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.

[0023] As used herein, "CBP-density" refers to the density of a CBP-polymer (e.g., 1-3 RGD (SEQ ID NO: 63) or G 1-3 The amount or concentration of the linker-CBP moiety in the alginate modified with RGDSP (SEQ ID NO: 64) is not limited to that in the present invention, unless otherwise specifically stated herein.

[0024] As used herein, "cell binding substance (CBS)" means any chemical, biological or other type of substance (e.g., small organic compounds, peptides, polypeptides) that can simulate at least one activity of a ligand of a cell adhesion molecule (CAM) or other cell surface molecule that mediates cell-matrix connection or cell-cell connection or other receptor-mediated signaling. In one embodiment, when present in a polymer composition that encapsulates cells, CBS is able to form a temporary or permanent bond or contact with one or more of the cells. In one embodiment, CBS promotes the interaction between two or more living cells encapsulated in a polymer composition. In one embodiment, the presence of CBS in a polymer composition that encapsulates multiple cells (e.g., living 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 in a test subject (e.g., mouse). In one embodiment, CBS is physically attached to one or more polymer molecules in a polymer composition. In one embodiment, CBS is a cell binding peptide or cell binding polypeptide as defined herein.

[0025] As used herein, "conservatively modified variants" or "conservative substitutions" refer to variants of reference peptides or polypeptides that are identical to a reference molecule except that they have one or more conservative amino acid substitutions in the amino acid sequence. In one embodiment, the conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. Conservative amino acid substitutions refer to substitutions of amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) and that have minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are listed in Table 2 below.

[0026] Table 2. Exemplary conservative amino acid substitution groups.

[0027]

[0028] As used throughout the specification and claims, "consisting essentially of" and variations such as "consisting essentially of" or "consisting essentially of" means including any recited elements or groups of elements, and optionally including other elements of similar or different properties to the recited elements, which other elements do not substantially change 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 include one or more amino acids (including substitutions of one or more amino acid residues in the recited amino acid sequence) with one or more amino acid residues that do not substantially affect the relevant biological activity of the cell-binding peptide or therapeutic protein, respectively. 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 substantially change the relevant biological activity of the cell-binding peptide, for example, its ability to increase the viability or productivity of encapsulated cells as described herein.

[0029] As used herein, "crosslinked" and variations thereof; such as "crosslinking" or "x-linked" refer to at least one chemical bond (e.g., an ionic bond, such as a covalent bond) between two polymers. In some embodiments, when two or more chemical bonds are present, crosslinking refers to a mixture of covalent bonds and ionic bonds. In some embodiments, when two or more chemical bonds are present, crosslinking refers to covalent bonds of different types (e.g., covalent bonds comprising different or orthogonal functional groups). In some embodiments, when two or more chemical bonds are present, crosslinking refers to covalent bonds of the same type (e.g., covalent bonds comprising the same functional groups). In some embodiments, when two or more chemical bonds are present, crosslinking refers to ionic bonds of the same type (e.g., covalent bonds comprising the same ions (e.g., Ba). 2+ ) of ionic bonds).

[0030] As used herein, "derived from" with respect to cells refers to cells obtained from a tissue, cell line, or cell, which are then optionally cultured, passaged, differentiated, induced, etc. to produce derived cells. For example, mesenchymal stem cells can be derived from mesenchymal tissue and then differentiated into a variety of cell types.

[0031] As used herein, "device" refers to any implantable article described herein (e.g., a particle, a hydrogel capsule, an implant, a 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 byproducts and / or therapeutic agents produced by living cells to be released from the device.

[0032] As used herein, "differential volume" refers to the volume of a compartment in the device described herein, excluding the space occupied by one or more other compartments. For example, the differential volume of the second compartment (e.g., outer compartment) in a 2-compartment device with an inner compartment and an outer compartment refers to the volume in the second compartment, excluding the space occupied by the first compartment (inner compartment).

[0033] As used herein, "effective amount" refers to the amount of a component of a biological response, such as to treat a disease, illness or condition, a device composition, or a device or device composition, such as a plurality of hydrogel capsules containing a cell (e.g., engineered cell) or an agent (e.g., therapeutic agent) produced by a cell (e.g., engineered RPE cell). In some embodiments, the term "effective amount" refers to the amount of the parts of the device (e.g., the number of cells in the device, the density of the defibrotic compound arranged on the surface of the device and / or in the barrier compartment). As will be understood by those of ordinary skill in the art, the effective amount can vary depending on factors such as the following: desired biological endpoints; pharmacokinetics of therapeutic agents, compositions or devices (e.g., capsules, particles); the condition treated; mode of administration; and age and health of the subject. The effective amount encompasses therapeutic and preventive treatments. For example, in order to mitigate FBR, an effective amount of the 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. As another example, an effective amount of CBS can enhance the viability (e.g., number of viable cells) of cells in the device and / or increase the level of therapeutic agents expressed and / or secreted by the cells. The effective amount of a device, composition, or component (e.g., a defibrotic compound or CBS) can be determined by any technique known in the art or described herein.

[0034] As used herein, "endogenous nucleic acid" is a nucleic acid that naturally occurs in the cells of a subject.

[0035] As used herein, an "endogenous polypeptide" is a polypeptide that naturally occurs in the cells of a subject.

[0036] As used herein, "engineered cells" are cells with non-natural changes and are generally comprised of nucleic acid sequences (e.g., DNA or RNA) or polypeptides that are not present (or present at different levels) in other similar cells under similar conditions that are not engineered (exogenous nucleic acid sequences). In one embodiment, the engineered cells comprise exogenous nucleic acids (e.g., vectors or altered chromosomal sequences). In one embodiment, the engineered cells comprise exogenous polypeptides. In one embodiment, the engineered cells comprise exogenous nucleic acid sequences, such as sequences that are not present in similar cells that are not engineered, such as DNA or RNA. In one embodiment, the exogenous nucleic acid sequence is chromosomal, for example, the exogenous nucleic acid sequence is an exogenous sequence placed in 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 comprises an RNA sequence, such as an mRNA. In one embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extrachromosomal exogenous nucleic acid sequence, and the chromosomal or extrachromosomal exogenous nucleic acid sequence comprises a sequence expressed as RNA, such as an mRNA or a regulatory RNA. In one embodiment, the exogenous nucleic acid sequence comprises a chromosome or an extrachromosomal nucleic acid sequence, and the chromosome or the extrachromosomal nucleic acid sequence comprises a coded polypeptide or is expressed as a sequence of a polypeptide. In one embodiment, the exogenous nucleic acid sequence comprises a first chromosome or an extrachromosomal exogenous nucleic acid sequence, and the first chromosome or the extrachromosomal exogenous nucleic acid sequence regulates the conformation or expression of the second nucleic acid sequence, wherein the second amino acid sequence can be exogenous or endogenous. For example, the engineered cell may comprise an exogenous nucleic acid controlling the expression of an endogenous sequence. In one embodiment, the engineered cell comprises a polypeptide existing at a level different from that found in unengineered similar cells or a distribution. In one embodiment, the engineered cell includes an RPE engineered to produce RNA or polypeptide. For example, the engineered cell may comprise an exogenous nucleic acid sequence, and the exogenous nucleic acid sequence comprises a chromosome or an extrachromosomal exogenous nucleic acid sequence, and the chromosome or the extrachromosomal exogenous nucleic acid sequence comprises an RNA expressed as, for example, a sequence of mRNA or regulatory RNA. In one embodiment, engineered cell (for example, RPE cell) comprises exogenous nucleic acid sequence, and described exogenous nucleic acid sequence comprises chromosome or extrachromosomal nucleic acid sequence, and described chromosome or extrachromosomal nucleic acid sequence comprise coded polypeptide or are expressed as the sequence of polypeptide.In one embodiment, polypeptide is encoded by codon-optimized sequence, to realize polypeptide expression higher than naturally occurring encoding sequence.Codon-optimized sequences can use commercially available algorithms, such as GeneOptimizer (ThermoFisherScientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA) or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol. 33, Supplement No. 2, pp. W526-W531 (2005) produce. In one embodiment, the engineered cell (e.g., RPE cell) comprises an exogenous nucleic acid sequence that regulates the conformation or expression of an endogenous sequence. In one embodiment, the engineered cell (e.g., RPE cell) is cultured from a colony of a stably transfected cell or from a monoclonal cell line.

[0037] As used herein, "exogenous nucleic acid" is a nucleic acid that does not naturally occur in the cells of a subject.

[0038] As used herein, "exogenous polypeptide" is a polypeptide that does not naturally occur in a subject cell (e.g., an engineered cell). Reference to an amino acid position of a particular sequence means the position of the amino acid in a reference amino acid sequence, e.g., the sequence of a full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise indicated), and does not exclude the presence of variations (e.g., deletions, insertions, and / or substitutions) at other positions in the reference amino acid sequence.

[0039] Unless otherwise indicated, "factor VII protein" or "FVII protein" as used herein refers to a polypeptide comprising an amino acid sequence of a naturally occurring factor VII protein or its variant, as determined by an art-recognized assay, and the polypeptide has a FVII biological activity, such as promoting blood coagulation. Naturally occurring FVII exists as a single-chain zymogen, a zymogen-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 zymogen-like and FVIIa. The FVII protein produced by equipment described herein (such as equipment containing engineered RPE cells) includes wild-type primates (such as humans), porcine animals, canines, and murine proteins, and variants of such wild-type proteins, including fragments, mutants, variants with one or more amino acid substitutions and / or deletions. In some embodiments, variant FVII protein can be activated into a fully activated double-chain form (factor VIIa), and the double-chain form has at least 50%, 75%, 90% or more (including>100%) of the activity of wild factor VIIa. Variants of FVII and FVIIa are known, such as marzeptacog alpha (activated) (MarzAA) and the variants described in European Patent No. 1373493, US Patent No. 7771996, US Patent No. 9476037 and US Published Application No. US20080058255.

[0040] Unless otherwise indicated, factor VII biological activity can be quantitatively determined by art-recognized assays. For example, the FVII biological activity in a sample of a biological fluid (e.g., blood plasma) can be measured in the following manner: (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 the factor X hydrolysis in an aqueous system; (iii) using an instrument based on surface plasmon resonance to measure its physical binding to TF (Persson, FEBS Letts.413:359-363, 1997); or (iv) measuring the hydrolysis of a synthetic substrate; and / or (v) measuring the generation of thrombin in an in vitro system independent of TF. In one embodiment, FVII activity is assessed by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuville sur Oise, France) in which a biological sample containing FVII is mixed with thromboplastin calcium, Factor X, and SXa-11 (a chromogenic substrate specific for Factor Xa).

[0041] Unless otherwise indicated, "factor VIII protein" or "FVIII protein" as used herein refers to a polypeptide comprising an amino acid sequence of a naturally occurring factor VIII polypeptide or a variant thereof, as determined by an art-recognized assay, and the polypeptide has FVIII biological activity, such as coagulation activity. The FVIII protein that can be expressed by equipment described herein (such as equipment containing engineered RPE cells) includes wild-type primates (such as humans), porcine animals, canines and murine proteins, and variants of such wild-type proteins, including fragments, mutants, variants with one or more amino acid substitutions and / or deletions, B domain deletions (BDD) variants, single-chain variants, and any one of the aforementioned wild-type or variants and the fusion of a half-life extension polypeptide. In one embodiment, the cell is engineered to encode a precursor factor VIII polypeptide (for example, with a signal sequence) that is completely or partially missing with a B domain. In one embodiment, the cell is engineered to encode a single-chain factor VIII polypeptide containing a variant FVIII protein, preferably having at least 50%, 75%, 90% or more (including>100%) of the coagulation activity of the corresponding wild-type factor VIII. Assays for measuring the coagulation activity of the FVIII protein include a primary or two-stage coagulation assay (Rizza et al., 1982, Coagulation assay of FVIII: Can and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or a chromogenic substrate FVIII: C assay (Rosen, S. 1984. Scand J Haematol 33: 139-145, Supplement).

[0042] Many FVIII-BDD variants are known, and include, for example, variants with complete or partial B domain deletions 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 2 and Example 1); 5,171,844 (e.g., column 4, line 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 6,060,447; 6,228,620; 6,316,226 (e.g., Col. 4, line 4 to Col. 5, line 28 and Examples 1-5); 6,346,513; 6,458,563 (e.g., Col. 4, line 25-53) and 7,041,635 (e.g., Col. 2, line 1 to Col. 3, line 19, Col. 3, line 40 to Col. 4, line 67, Col. 7, line 43 to Col. 8, line 26, and Col. 11, line 5 to Col. 13, line 39).

[0043] In some embodiments, the FVIII-BDD protein produced by the apparatus described herein (e.g., expressed by an engineered cell contained in the apparatus) 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 required for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 09122); (ii) amino acids 747-1638 (Hoeben RC, 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. USA 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)); deletion of amino acids 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597); including one or more residues in the furin recognition sequence, such as LKRHQR (SEQ ID NO: 65), including any of the specific deletions cited in U.S. Patent No. 9,956,269 at column 10, line 65 to column 11, line 36.

[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, e.g., residues 757, 784, 828, 900, 963, or optionally 943, the first 226 amino acids, or the first 163 amino acids (Miao, HZ, et al., Blood 103(a): 3412-3419 (2004); Kasuda, A., et al., J. Thromb. Haemost. 6: 1352-1359 (2008); and Pipe, SW, 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), wherein the substitution or deletion prevents proteolytic cleavage at this site, including any substitutions at positions R1645 and R1648 described in U.S. Pat. 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: F309S substitution to improve expression of FVIII-BDD protein (Miao, HZ, et al., Blood 103(a):3412-3419 (2004); albumin fusion (WO 2011 / 020866); and Fc fusion (WO 04 / 101740).

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

[0048] Unless otherwise indicated, "factor IX protein" or "FIX protein" as used herein refers to a polypeptide comprising an amino acid sequence of a naturally occurring factor IX protein or its variant, as determined by an art-recognized assay, and the polypeptide has a FIX biological activity, such as coagulation activity. FIX is produced as an inactive zymogen, which is converted into an active form by excision of the factor XIa of an activating peptide, thereby producing a heavy chain and a light chain linked together by one or more disulfide bonds. The FIX protein that can be produced by equipment described herein (such as equipment containing engineered RPE cells) includes wild-type primates (such as humans), porcine animals, canines and murine proteins, and variants of such wild-type proteins, including fragments, mutants, variants with one or more amino acid substitutions and / or deletions, and any one of the aforementioned wild-type or variant proteins and a fusion of a half-life-extending polypeptide. In one embodiment, the cell is engineered to encode a full-length wild-type human factor IX polypeptide (for example, with a signal sequence) or its functional variant. Variant FIX albumen preferably has at least 50%, 75%, 90% or higher (including>100%) coagulant activity of wild-type factor VIX.The mensuration for measuring the coagulant activity of FIX albumen includes Biophen Factor IX mensuration (Hyphen BioMed) and primary coagulation mensuration (activation of partial thromboplastin time (aPTT) (for example, as described in EP 2 032 607), thrombin generation time mensuration (TGA) and rotational thromboelastometry (for example, as described in WO 2012 / 006624).

[0049] Many functional FIX variants are known and can be expressed by the 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, page 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; WO 2007 / 149406, page 4, lines 1 to page 19, line 11; WO 2007 / 149406 A2, page 19, lines 12 to page 20, line 9; WO 08 / 118507, page 5, lines 14 to page 6, line 5; WO 09 / 051717, page 9, lines 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 / 130198 A2, page 4, line 26 to page 12, line 6; WO 09 / 140015, page 11, paragraph

[0043] to page 13, paragraph

[0053] ; WO 2012 / 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, a PAS sequence, transferrin, CTP (a 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with 4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding polypeptide, an albumin binding small molecule, 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) bound together by two disulfide bonds in the hinge region of the Fc.

[0051] FIX variants also include gain of function and loss of function variants.An example of gain of function variants is " Padua " variant of people FIX, which has L (leucine) instead of R (arginine) (corresponding to SEQ ID NO:2 amino acid position 384) at position 338 of mature protein, and has larger catalysis and coagulation activity (Chang et al., J.Biol.Chem., 273:12089-94 (1998)) compared with wild-type people FIX.An example of loss of function variants is to replace lysine with alanine in the 5th amino acid position starting from mature protein, which produces a protein (e.g., loss of function) reduced in combination with collagen IV.

[0052] Unless otherwise indicated, "interleukin-2 protein" or "IL-2 protein" as used herein means a polypeptide comprising an amino acid sequence of a naturally occurring IL-2 protein or a variant thereof, as determined by an assay recognized in the art, the polypeptide has IL-2 biological activity, such as IL-2 receptor signaling in activated Treg cells. The IL-2 protein that can be produced by the equipment described herein (such as a device containing engineered RPE cells) includes wild-type primates (such as humans), porcine animals, canines and murine proteins, and variants of such wild-type proteins. Variant IL-2 proteins preferably have at least 50%, 75%, 90% or higher (including> 100%) of corresponding wild-type IL-2 biological activity. The bioactivity assay of IL-2 protein is described in U.S. Patent No. 10,035,836, and includes, for example, measuring the phosphorylated STAT5 protein level of Treg cells compared with 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.

[0053] As used herein, "islet cells" are intended to include any naturally occurring or any synthetically produced or modified cells that are intended to partially or completely reproduce, simulate or otherwise express some or all of the functions of Langerhans islet cells. The term "islet cells" includes glucose-responsive insulin-producing cells derived from stem cells (e.g., derived from induced pluripotent stem cell lines).

[0054] Unless specifically stated otherwise, "mannitol" as used herein refers to D-mannitol.

[0055] As used herein, "medium molecular weight alginate" or "MMW-Alg" means alginate having an approximate molecular weight of 75 kDa to 150 kDa.

[0056] As used herein, the term "mesenchymal stem cell" or "MSFC" refers to a cell derived from a mesodermal lineage or a cell having at least one characteristic of a cell of a mesodermal lineage, and wherein the MSFC: i) is not in a terminal state of differentiation; and ii) can be terminally differentiated into one or more cell types. MSFC does not include cells of endoderm origin, such as intestinal cells, or cells of ectoderm origin, such as cells derived from the skin, CNS, or neural cells. In one embodiment, MSFC is multipotent. In one embodiment, MSFC is not fully potent. In one embodiment, MSFC comprises one or more of the following features:

[0057] a) It comprises mesenchymal stem cells (MSC) or cells derived therefrom, including: cells derived from primary cell cultures of MSC; cells directly isolated (without long-term culture, e.g., less than 5 or 10 passages or cell division cycles since isolation) from naturally occurring MSC, e.g., from humans or other mammals; cells derived from transformed, potent, immortalized or long-term (e.g., more than 5 or 10 passages or cell division cycles) MSC cultures.

[0058] 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;

[0059] c) it is pluripotent, e.g. as measured by any assay capable of providing information on the pluripotency of a cell (e.g. microscopy);

[0060] d) It presents a characteristic mononuclear oval, stellate, 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. MSFC (e.g., MSC) may have a small amount of cytoplasm but with many thin processes that appear to extend from the nucleus;

[0061] 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;

[0062] f) it is able to differentiate into mesenchymal cell lineages such as osteoblasts, chondroblasts, adipocytes, or fibroblasts;

[0063] 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;

[0064] h) it does not express significant levels of one, two, three or any of CD34, CD31, VE-cadherin, CD45, HLA-DR, CD11b and glycophorin or leukocyte differentiation antigens (e.g., CD14, CD33, CD3 and CD19);

[0065] 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;

[0066] j) it has an anti-inflammatory or immunosuppressive effect, e.g. as measured by any method capable of providing information about inflammation (e.g. in vivo inhibition of T-cell proliferation);

[0067] k) it is able to adhere, for example plastically, for example as determined by visual inspection; or

[0068] l) can grow in three dimensions, e.g. as determined by visual inspection.

[0069] As used herein, "parathyroid hormone" or "PTH" means a polypeptide or peptide comprising an amino acid sequence of a naturally occurring parathyroid hormone polypeptide or peptide or a variant thereof, e.g., as determined by an assay recognized in the art, the polypeptide or peptide has PTH biological activity. PTH polypeptides and peptides that may be expressed by the encapsulated cells described herein include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. Such PTH polypeptides and peptides may consist essentially of the wild-type human sequence of the preproPTH polypeptide (115 amino acids), the proPTH polypeptide (90 amino acids), the mature 84 amino acid peptide (PTH (1-84)), and biologically active variants thereof, such as the 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. 20130217630. PTH variants preferably have at least 50%, 75%, 90% or more (including> 100%) of the biological activity of the corresponding wild-type PTH. U.S. Patent No. 8383417 describes an assay for detecting certain PTH variants by tandem mass spectrometry. U.S. Patent No. 7410948 describes a biological activity assay for PTH peptide variants - determining the stimulation of adenylate cyclase by measuring cAMP levels.

[0070] As used herein, "poloxamers" are 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.

[0071] As used herein, "Poloxamer 188" or "P 188" refers to a poloxamer having an approximate molecular weight of 1800 g / mol of the polyoxypropylene core and an oxyethylene 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, for example, as determined by size exclusion chromatography, and an oxyethylene 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 process described herein substantially meets the specifications set forth in the poloxamer monograph published by the United States Pharmacopoeia-National Formulary (USP-NF) or the European Pharmacopoeia (Ph. Eur.), which is officially published when the process is performed.

[0072] As used herein, a "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 may be natural and synthetic. Homopolymers contain one type of structural unit or monomer, while copolymers contain more than one type of monomer.

[0073] 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.

[0074] As used herein, "Prevention," "prevent," and "preventing" refer to treatments that include administering or applying a therapy, such as administering a composition of a device that encapsulates cells (e.g., as described herein), prior to the onset of a disease, disorder, or condition to preclude physical manifestations of the disease, disorder, or condition. In some embodiments, "Prevention," "prevent," and "preventing" require that signs or symptoms of a disease, disorder, or condition have not yet developed or have not been observed. In some embodiments, treatment includes prevention, and in other embodiments, it does not include prevention.

[0075] "Alternative therapy" or "alternative protein" is a therapeutic protein or its functional fragment that replaces or enhances the beneficial function of a protein that is reduced, present in insufficient amounts, changed (e.g., mutated) or lacking in a subject suffering from a disease or illness associated with a reduced, altered or lacking protein. Examples are certain blood coagulation factors in certain blood coagulation disorders or certain lysosomal enzymes in certain lysosomal storage diseases. In one embodiment, an alternative therapy or alternative protein provides the function of an endogenous protein. In one embodiment, an alternative therapy or alternative protein has the same amino acid sequence of a naturally occurring variant (e.g., a wild-type allele or an allele unrelated to the disease) of the replaced protein. In one embodiment, an alternative therapy or alternative protein is different from a naturally occurring variant (e.g., a wild-type allele or an allele unrelated to the disease, such as an allele carried by a subject) in an amino acid sequence at no more than about 1%, 2%, 3%, 4%, 5%, 10%, 15% or 20% of the amino acid residues.

[0076] As used herein with respect to a claimed device (e.g., a hydrogel capsule), a "reference device" refers to a device (e.g., a hydrogel capsule) that: (i) lacks a specific feature, e.g., a FBR mitigation means (e.g., a barrier compartment comprising a defibrotizing compound (as defined herein) or a CBS (as defined herein) (e.g., an RGD polymer), (ii) encapsulates approximately the same number of cells of one or more of the same cell types as in the claimed device in the cell-containing compartment, and (iii) has a substantially similar polymer composition and structure as in the claimed device except for the lack of the specific feature (e.g., a defibrotizing compound or CBS). In one embodiment, the number of viable cells in the cell-containing compartment of the reference device is within 80% to 120%, or within 90% to 110% of the number of viable cells in the cell-containing compartment of the claimed device. In one embodiment, the cells in the reference device and the claimed device are obtained from the same cell culture. In one embodiment, substantially similar polymer compositions mean that all polymers in the reference device and the claimed device, including any CBP polymers and polymer components of the defibrotizing polymers, as applicable, are of the same chemical and molecular weight class (e.g., alginate with high G content and the same molecular weight range). For example, in one embodiment, the cell-containing compartment of the CBP-null reference device is formed of an unmodified version of the polymer (e.g., alginate) of the CBP polymer used to form the cell-containing compartment of the claimed device. In some embodiments, wherein the claimed two-compartment hydrogel microcapsules have (i) an inner compartment formed of a CBP polymer encapsulating a plurality of cells and (ii) an outer compartment formed of 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 of the same polymer mixture, while the inner compartment of the reference capsule is formed of 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 (eg, one, two, three, etc.) and approximately the same size and shape.

[0077] As used herein, "RPE cells" refer to 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 (ATCC ®CRL-2302™) or cells derived therefrom (e.g., cells cultured from an ARPE-19 cell line by stable transfection with an exogenous sequence encoding a therapeutic protein, or such cultured ARPE-19 cells are otherwise engineered to express an exogenous protein or other exogenous substance), cells derived from primary cell cultures of RPE cells, cells directly isolated from naturally occurring RPE cells (e.g., from humans or other mammals) (without long-term culture, e.g., less than 5 or 10 generations or rounds of cell division since isolation), derived from transformed, immortalized or long-term (e.g., more than 5 or 10 generations or rounds of cell division) b) cells obtained from less differentiated cells, such as cells developed, programmed or reprogrammed (e.g., in vitro) into RPE cells, or cells substantially similar to one or more of naturally occurring RPE cells or cells from primary or long-term cultures of RPE cells, except for any genetic engineering (e.g., cells may be derived from IPS cells); c) cells having one or more of the following properties: i) they express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) they do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) they occur naturally in the retina and form a monolayer above the choroidal vessels in Bruch's membrane; iv) they are responsible for epithelial transport, light absorption, secretion, and immunomodulation in the retina; or v) they are synthetically produced or modified by naturally occurring cells to have similar properties to immortalized RPE cell lines (e.g., ARPE-19 cell line (ATCC ® In one embodiment, RPE cell described herein is engineered to express therapeutic agent when being encapsulated in the polymer composition that comprises CBP or CBS.In other embodiments, RPE cell is unengineered.

[0078] As used herein, "saline solution" means normal saline, ie, water containing 0.9% NaCl, unless otherwise specified.

[0079] When used herein to refer to two nucleotide sequences or two amino acid sequences, "sequence identity" or "percentage identity" means that when two sequences are compared and aligned over a comparison window or a specified region to obtain maximum correspondence, the two sequences are identical within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotides or amino acid positions within a specified region. Sequence identity can be determined using standard techniques known in the art, including but not limited to any algorithm described in U.S. Patent Application Publication No. 2017 / 02334455. In one embodiment, the specified percentage of identical nucleotides or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0080] 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 have waves and undulations on the surface, for example. Spheres and sphere-like objects can be mathematically defined by rotating a circle, ellipse, or combination around each of three perpendicular axes a, b, and c. For a sphere, the three axes are the same length. In general, a sphere-like shape is an ellipsoid whose semi-major axes differ from each other by 10%, 5%, or 2.5% (with respect to its average surface). The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-major axes.

[0081] The term "spheroid" when used herein to refer to a device (e.g., a hydrogel capsule or other particle) means that the device has (i) a perfect or classic oblate spheroid or prolate spheroid shape, or (ii) has a surface that is generally spheroidal, e.g., may have corrugations and undulations and / or may be an ellipsoid with semi-major axes within 100% of each other (with respect to its average surface).

[0082] As used herein, "subject" refers to humans or non-human animals. 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, a teenager) 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 be at any stage of development. The non-human animal can be a transgenic animal.

[0083] As used herein, "total volume" refers to the volume in one compartment of a multi-compartment device, including the space occupied by another compartment. For example, the total volume of the second compartment (e.g., outer compartment) of a two-compartment device refers to the volume in the second compartment, including the space occupied by the first compartment.

[0084] As used herein, the terms "treatment", "treat" and "treating" refer to reducing, reversing, alleviating one or more of the symptoms, manifestations or potential causes of a disease, disorder or illness, delaying its onset or inhibiting its progression. In one embodiment, treatment includes reducing, reversing, alleviating the symptoms of a disease, disorder or illness, delaying its onset or inhibiting its progression. In one embodiment, treatment includes reducing, reversing, alleviating the manifestations of a disease, disorder or illness, delaying its onset, or inhibiting its progression. In one embodiment, treatment includes reducing, reversing, alleviating, alleviating the potential causes of a disease, disorder or illness, or delaying its onset. In some embodiments, "treatment" requires that signs or symptoms of the disease, disorder or illness have appeared or have been observed. In other embodiments, treatment may be administered when there are no signs or symptoms of the disease or illness, such as in preventive treatment. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., taking into account symptom history and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, such as to delay or prevent their recurrence. In some embodiments, treatment includes prevention, and in other embodiments, it does not include prevention.

[0085] 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, which polypeptide has VWF biological activity, e.g., FVIII binding activity, as determined by an art-recognized assay. VWF proteins that can be produced by the devices described herein (e.g., expressed by engineered cells contained in the devices) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins. The encapsulated cells can be programmed to encode any of the following VWF polypeptides: 2813 amino acid precursor VWF, VWF lacking a 22 amino acid signal peptide and an optional 741 amino acid propeptide, 2050 amino acid mature VWF protein and truncated variants thereof (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. Pat. No. 9458223). The variant VWF protein preferably has at least 50%, 75%, 90% or more (including >100%) of the biological activity of the corresponding wild-type VWF protein. Art-recognized assays for determining the biological activity of VWF include ristocetin cofactor activity (Federici AB et al., 2004). Haematologica 89:77-85), VWF binding to GP Ibα of platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), and collagen binding (Kallas & Talpsep. 2001. Annals of Hematology 80:466-471).

[0086] In some embodiments, the VWF protein produced by the device 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, a PAS sequence, transferrin, CTP (a 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with 4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding polypeptide, an albumin binding small molecule, or any combination thereof.

[0087] "VLVG" or "VLVG alginate" refers to a very low viscosity sodium alginate having an average molecular weight of less than about 75 kDa and comprising greater than 60% guluronate units (ie, having a guluronate to mannuronic acid ratio greater than or equal to 1.5).

[0088] "SG100" or "SG100 alginate" refers to sodium alginate having an average molecular weight of about 150 to 250 kDa and comprising greater than 60% guluronic acid units (ie, having a guluronic acid to mannuronic acid ratio greater than or equal to 1).

[0089] Selected chemical definitions

[0090] The definition of specific functional groups and chemical terms is described in more detail below. Chemical elements are identified according to the periodic table CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the 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.

[0091] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valence known in the chemical arts.

[0092] When a range of values ​​is listed, it is intended to encompass each value and subrange 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 alkyl.

[0093] As used herein, "alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C 12 alkyl”), 1 to 10 carbon atoms (“C1-C 12 In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), s-propyl (C4), s-propyl (C5), s-propyl (C6), s-propyl (C7), s-propyl (C8), s-propyl (C9), s-propyl (C10), s-propyl (C11), s-propyl (C12), s-propyl (C13), s-propyl (C14), s-propyl (C15), s-propyl (C16), s-propyl (C17), s-propyl (C18), s-propyl (C19), s-propyl (C21), s-propyl (C22), s-propyl (C23), s-propyl (C24), s-propyl (C25), s-propyl (C26), s-propyl (C27), s-propyl (C28), s-propyl (C29), s-propyl (C31), s-propyl (C32), s-propyl (C33), s-propyl (C34), s-propyl (C35), s-propyl (C36), s-propyl (C37), s-propyl (C38), s-propyl (C39). ), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6). Additional examples of alkyl include n-heptyl (C7), n-octyl (C8), etc. Each example of alkyl can 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").

[0094] As used herein, "alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-C 10The carbon-carbon double bond(s) may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C 2-4 alkenyl and pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each instance of alkenyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkenyl").

[0095] As used herein, the term "alkynyl" refers to a straight chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-C 10 The carbon-carbon triple bond(s) may be internal (as in 2-butynyl) or terminal (as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each example of an alkynyl group may independently be optionally substituted, i.e., unsubstituted (“unsubstituted alkynyl”), or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted alkynyl”).

[0096] As used herein, the term "heteroalkyl" refers to a non-cyclic stable straight or branched chain or a combination thereof including 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 atom and the sulfur atom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The one or more heteroatoms O, N, P, S and Si may be placed in 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. When "heteroalkyl" is described, the specific heteroalkyl group is described, such as -CH2O, -NR C R D It will be understood that the term heteroalkyl and -CH2O or -NR C R D The terms "heteroalkyl" and "heteroalkyl" are not redundant or mutually exclusive. Instead, specific heteroalkyl groups are described to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -CH2O, -NR C R D Etc. Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted (“unsubstituted heteroalkyl”) or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted heteroalkyl”).

[0097] Unless otherwise indicated, the term "alkylene", "alkenylene", "alkynylene" or "heteroalkylene" alone or as part of another substituent means a divalent group derived from an alkyl, alkenyl, alkynyl or heteroalkylene, respectively. Alkylene, alkenylene, alkynylene or heteroalkylene may be described, for example, as C1-C6 membered alkylene, C2-C6 membered alkenylene, C2-C6 membered alkynylene or C1-C6 membered heteroalkylene, wherein the term "membered" refers to the non-hydrogen atoms within the moiety. In the case of heteroalkylene, heteroatoms may also occupy one or both chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, 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-.

[0098] As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring array) radical having 6-14 ring carbon atoms and zero heteroatoms ("C6-C 14 In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 Aryl is a "group" such as an anthracenyl group. Aryl groups may be described as, for example, C6-C 10 The term "membered" refers to the non-hydrogen ring atoms within the moiety. Aryl includes phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of aryl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted ("substituted aryl") with one or more substituents.

[0099] As used herein, "heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in the ring array) having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom when valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups, where one ring contains no heteroatoms (e.g., indolyl, quinolyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring bearing the heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl). Heteroaryl groups can be described, for example, as 6-10 membered heteroaryl groups, where the term "membered" refers to non-hydrogen ring atoms within the moiety.

[0100] 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.

[0101] 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-, oxazinyl, and 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.

[0102] As used herein, the terms "arylene" and "heteroarylene" by themselves or as part of another substituent refer to a divalent radical derived from an aryl and heteroaryl group, respectively.

[0103] As used herein, "cycloalkyl" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C8 cycloalkyl"). 10 Cycloalkyl"). Cycloalkyl can be described, for example, as a C4-C7 membered cycloalkyl, where the term "membered" 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), and the like. Exemplary C3-C8 cycloalkyls include The cycloalkyl radicals include, but are not limited to, the aforementioned C3-C6 cycloalkyl radicals and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), etc. Exemplary C3-C6 cycloalkyl radicals include, but are not limited to, cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), etc. 10 Cycloalkyl includes, but is not limited to, the aforementioned C3-C8 cycloalkyl and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9), 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. 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 ring system ("bicyclic cycloalkyl")), and may be saturated or may be partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each example of a cycloalkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl"), or substituted ("substituted cycloalkyl") with one or more substituents.

[0104] As used herein, "heterocyclyl" refers to a group of a 3-10 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 heterocyclyl"). In heterocyclyls containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as long as valence permits. The heterocyclyl group may be a monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system, such as a bicyclic system ("bicyclic heterocyclyl"), and may be saturated or may be partially unsaturated. Heterocyclyl bicyclic ring systems may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the heterocyclyl ring as defined above is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl or heterocyclyl ring; or ring systems in which the heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Heterocyclyl can be described as, for example, 3-7 membered heterocyclyl, wherein the term "member" refers to the non-hydrogen ring atoms within the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") by one or more substituents. In certain embodiments, heterocyclyl is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, heterocyclyl is a substituted 3-10 membered heterocyclyl.

[0105] In some embodiments, heterocyclyl is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon (“5-10 membered heterocyclyl”). In some embodiments, heterocyclyl is a 5-8 membered non-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 heterocyclyl”). In some embodiments, heterocyclyl is a 5-6 membered non-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 heterocyclyl”). In some embodiments, 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0106] Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridine, oxirane and thioethane. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetane and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrophenylthio, dihydrophenylthio, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-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, dihydropyridinyl 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 thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepane, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocyclooctanyl, oxoctanyl, and thiepanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic rings) include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, etc. Exemplary 6-membered heterocyclic groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocyclic rings) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0107] As used herein, "amino" refers to the group -NR 70 R 71 , where R 70 and R 71 are independently hydrogen, C1-C8 alkyl, C3-C 10 Cycloalkyl, C4-C 10 Heterocyclic group, C6-C 10 Aryl and C5-C 10 In some embodiments, amino refers to NH2.

[0108] As used herein, "cyano" refers to -CN.

[0109]

[0043] As used herein, "halo" or "halogen," by itself or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0110] As used herein, "hydroxy" refers to the group -OH.

[0111] As defined herein, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). In general, the term "substituted", whether preceded by the term "optionally" or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, such as a substituent that, upon substitution, results in a stable compound, such as a compound that does not spontaneously transform, such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when substitution occurs at more than one position in any given structure, the substituent at each position is the same or different. The term "substituted" is intended to include substitution with all permissible substituents of an organic compound (e.g., any substituent described herein that results in the formation of a stable compound). The present disclosure contemplates any and all such combinations to achieve stable compounds. For purposes of the present disclosure, a heteroatom (such as nitrogen) may have a hydrogen substituent and / or any suitable substituent that satisfies the valence of the heteroatom and produces the formation of a stable portion as described herein.

[0112] Two or more substituents may be optionally connected to form an aryl, heteroaryl, cycloalkyl or heterocyclyl group. It has been found that such so-called ring-forming substituents are usually but not necessarily attached to a cyclic base structure. In one embodiment, the ring-forming substituent is attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of the cyclic base structure form a condensed ring structure. In another embodiment, the ring-forming substituent is attached to a single member of the base structure. For example, two ring-forming substituents are attached to a single member of the cyclic base structure, producing a spiro ring structure. In another embodiment, the ring-forming substituent is attached to non-adjacent members of the base structure.

[0113] The compounds of formula (I) described herein may contain one or more asymmetric centers and thus may exist in a variety 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 stereoisomer mixtures (including racemic mixtures and mixtures enriched in one or more stereoisomers). Isomers may 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 preferred isomers may 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, p. 268 (EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present 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.

[0114] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, the "S" form of the compound is substantially free of the "R" form of the compound and is therefore an enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" means that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight of the enantiomer. In certain embodiments, weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0115] 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 1H. 2 H (D or deuterium) and 3 H (or tritium); C can be in any isotopic form, including 12 C. 13 C and 14 C; O can be in any isotopic form, including 16 O and 18 O etc.

[0116] The term "pharmaceutically acceptable salt" is intended to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular 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 (neat or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds used in the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, neat 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, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, etc.; and salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginine salts, etc.; and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see, e.g., Berge et al., Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds used in the disclosed devices (e.g., particles, hydrogel capsules) contain both basic and acidic functional groups that allow the compound to be converted into base addition salts or acid addition salts. 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.

[0117] As used herein, "polysaccharide" refers to a polymer of monosaccharide or disaccharide carbohydrates that are bound together by glycosidic linkages. 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, uronic acid, mannuronic acid and guluronic acid. Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, amylopectin, glycogen, gelatin, cellulose, straight-chain starch, chitin, chitosan or its derivatives or variants, for example, as described in Laurienzo (2010), Mar Drugs 9:2435-65.

[0118] The devices of the present disclosure may contain compounds of formula (I) in the form of prodrugs. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide compounds that can be used to prepare the devices of the present disclosure. In addition, prodrugs can be converted into useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0119] The compounds of some formula (I) described herein can exist in non-solvated form and solvated form (including hydrated form). In general, the solvated form is equivalent to the non-solvated form and is included in the scope of the present disclosure. The compounds of some formula (I) described herein can exist in multiple crystals or amorphous forms. In general, all physical forms are equivalent for the purposes covered by the present disclosure and are intended to fall within the scope of the present disclosure.

[0120] The term "solvate" refers to a form of a compound associated with a solvent, typically by a solvolysis reaction. Such physical association may include hydrogen bonding. Commonly used solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. 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.

[0121] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is in a certain ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R·x H2O, where R is a compound and where x is a number greater than 0.

[0122] As used herein, the term "tautomer" refers to a compound structure that is an interchangeable form and that varies in hydrogen atom and electronic displacement. Therefore, by the movement of π electrons and atoms (usually H), the two structures can be in equilibrium. For example, enols and ketones are tautomers because they can be rapidly converted to each other by treating with acids or bases. The tautomeric form may be relevant to the optimization of chemical reactivity and biological activity of the related compounds.

[0123] As used herein, the symbol " ” refers to attachment to a surface of an entity, such as a polymer (e.g., a hydrogel-forming polymer such as alginate) or an implantable device (e.g., a particle, a hydrogel capsule). By “ " " may refer to direct attachment to an entity (e.g., a polymer or an implantable element), or may refer to linkage to an entity through an attachment group. As used herein, an "attachment group" refers to a moiety used to link a compound of Formula (I) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein), and may include any attachment chemistry known in the art. A list of exemplary attachment groups is summarized 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, heterocyclyl, 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 metal, where R A , R C , RD , R F , R G Each of x and y is independently as described herein. In some embodiments, the attachment group comprises an amine, a ketone, an ester, an amide, an alkyl, an alkenyl, an alkynyl, or a thiol. In some embodiments, the attachment group is a crosslinker. In some embodiments, the attachment group is -C(O)(C1-C6-alkylene)-, wherein the alkylene is replaced by R 1 Replace, and R 1 As described herein. In some embodiments, the attachment group is -C(O)(C1-C6-alkylene)-, wherein the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is -C(O)C(CH 3)2 -. In some embodiments, the attachment 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 attachment group is -C(O)CH(CH3)-. In some embodiments, the attachment group is -C(O)C(CH3)-.

[0124] As used herein, the terms "covalent", "covalent bond" and "covalent bonding" refer to a type of chemical bond involving the sharing of electrons between two adjacent atoms. Examples of covalent bonds include bonds formed between carbon and hydrogen (CH bonds), carbon atoms (CC bonds), carbon and oxygen atoms (CO bonds), and carbon and nitrogen (CN bonds). Depending on the nature of the atoms, a covalent bond can be a single bond, a double bond, or a triple bond, i.e., a covalent bond may involve the sharing of one, two, or three electron pairs.

[0125] As used herein, the terms "ionic," "ionic bond," and "ionic bonding" refer to a type of chemical bond involving Coulombic attractions between adjacent oppositely charged atoms (ie, ions).

[0126] Modified polysaccharide polymers

[0127] The polysaccharide polymers described herein are covalently modified by covalent crosslinking agents. In one embodiment, the polysaccharide polymer can be a linear, branched or crosslinked polysaccharide polymer, or a polysaccharide polymer with a selected molecular weight range, degree of polymerization, viscosity or melt flow rate. Branched polysaccharide polymers may include one or more of the following types: star polymers, comb polymers, brush polymers, dendritic polymers, graft-copolymers, ladder polymers and dendritic polymers. In some embodiments, the branched polysaccharide polymer is a star polymer. In some embodiments, the branched polysaccharide polymer is a comb polymer. In some embodiments, the branched polysaccharide polymer is a brush polymer. In some embodiments, the branched polysaccharide polymer is a dendritic polymer. In some embodiments, the branched polysaccharide polymer is a graft-(co)polymer. In some embodiments, the branched polysaccharide polymer is a ladder polymer. In some embodiments, the branched polysaccharide polymer is a dendritic polymer. The polysaccharide polymer can be a thermosensitive polymer, such as a gel (e.g., becoming solid or liquid when exposed to heat or a certain temperature) or a photocrosslinkable polymer. In some embodiments, polysaccharide polymer is a cross-linkable polymer, wherein the cross-linking portion is activated by another agent. In some embodiments, polysaccharide polymer can be biodegradable, for example, containing unstable bonds, or can be dissociated by enzymes (such as lyases). In some embodiments, polysaccharide polymer is composed of a single type of repeating monomer units. In other embodiments, polysaccharide polymer is composed of different types of repeating monomer units (for example, two types of repeating monomer units, three types of repeating monomer units, for example, polymer blends). In some embodiments, polysaccharide can be composed of mannuronic acid and guluronic acid monomers.

[0128] 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, polyglycoside 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.

[0129] In some embodiments, polysaccharide polymer is a polymer forming a hydrogel. The polymer forming a hydrogel comprises a hydrophilic structure that enables it to accommodate a large amount of water in a three-dimensional network. The polymer forming a hydrogel may include a polymer forming a homopolymer hydrogel capsule, a copolymer hydrogel capsule or a polypolymer interpenetrating polymer hydrogel capsule, 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 forming a hydrogel include proteins (e.g., collagen), gelatin, polysaccharides (e.g., starch, alginate, hyaluronate, agarose) and synthetic polysaccharides.

[0130] Exemplary polysaccharides include alginate, agar, agarose, carrageenan, hyaluronate, pullulan, glycogen, gelatin, cellulose, amylose, chitin, chitosan or its derivatives or variants, for example, as described in Laurienzo (2010), Mar Drugs 9:2435-65. The polysaccharide polymer may include heparin, chondroitin sulfate, dermatan, dextran or carboxymethyl cellulose. In some embodiments, the polysaccharide polymer is a cross-linked polymer. In some embodiments, the polysaccharide polymer is a cell surface polysaccharide.

[0131] 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, alginate is 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, alginate is 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 ratio of M:G is about 1.In some embodiments, the ratio of M:G is less than 1.In some embodiments, the ratio of M:G is greater than 1.In some embodiments, 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.

[0132] The polysaccharide polymer (for example any polymer described herein, for example any alginate described herein) or its pharmaceutically acceptable salt comprising the sugar part with formula (I) structure can be modified on one or more monomeric units. In some embodiments, at least 0.5% of the sugar monomer in the polysaccharide polymer has the structure of formula (I) (for example, 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 sugar monomer has 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 monomer of the polysaccharide polymer has the structure of formula (I). In some embodiments, 1% to 20% of the sugar monomer of the polysaccharide polymer has the structure of formula (I). In some embodiments, 1% to 10% of the sugar monomer of the polysaccharide polymer has the structure of formula (I). In some embodiments, 1% to 50% of the sugar monomers have the structure of formula (I).

[0133] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having a structure of Formula I) comprises (compared to the unmodified polymer) an increase in N% of at least 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% N by weight, wherein N% is determined by elemental analysis and corresponds to the amount of compound of Formula I in the modified polymer.

[0134] In some embodiments, the polysaccharide polymer (when comprising a sugar monomer having a structure of Formula I) comprises (compared to the unmodified polymer) an increase in N% of 0.1 wt% to 10 wt% N, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0135] In some embodiments, the polysaccharide polymer (when comprising sugar monomers having a structure of Formula (I)) comprises an increase in N% (compared to the unmodified polymer) of 0.1 wt% to 2 wt% N, wherein N% is determined by elemental analysis and corresponds to the amount of compound of Formula I in the modified polymer.

[0136] In some embodiments, the polysaccharide polymer (when comprising a sugar monomer having a structure of Formula (I)) comprises (compared to the unmodified polymer) an increase in N% of 2 wt% to 4 wt% N, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0137] In some embodiments, the polysaccharide polymer (when comprising a sugar monomer having a structure of Formula (I)) comprises (compared to the unmodified polymer) an increase in N% of 4 to 8 wt% N, wherein N% is determined by elemental analysis and corresponds to the amount of the compound of Formula I in the modified polymer.

[0138] In some embodiments, any polysaccharide polymer described herein (e.g., alginate) comprises a sugar monomer having one or more of formula (Ia), (Ib), (Ic), (Id), (Ie), (If) or a pharmaceutically acceptable salt thereof. In some embodiments, the polysaccharide polymer comprises a sugar monomer having a structure of formula (II-a). In some embodiments, the polymer is modified by a compound of formula (II-b). In some embodiments, the polysaccharide polymer comprises a sugar monomer having a structure of formula (II-c). In some embodiments, the polysaccharide polymer comprises a sugar monomer having a structure of formula (II-d). In some embodiments, the polysaccharide polymer comprises a sugar monomer having a structure of formula (II-e). In some embodiments, the polysaccharide polymer comprises a sugar monomer having a structure of formula (II-f).

[0139] In some embodiments, the polymer (eg, alginate) is modified with the compounds shown in Table 3.

[0140] In some embodiments, the polymer modified with a compound of formula (I) (e.g., alginate) is not a modified polymer described in any of WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 187225, WO2016 / 019391, WO2017 / 075630, WO 2017 / 075631, WO 2018 / 067615, WO 2019 / 169333, and US 2016-0030359.

[0141] Defibrotizing compounds

[0142] In some embodiments, the polysaccharide polymers described herein further comprise at least one defibrotizing compound of formula (I):

[0143] (I)

[0144] or a pharmaceutically acceptable salt thereof, wherein:

[0145] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C(O)O–, –C(O)–, –OC(O)–, –N(R C )–、–N(R C )C(O)–,–C(O)N(R C )–、-N(R C )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 linked to an attachment group (such as an attachment group described herein) and is optionally supported by one or more R 1 Replacement; L 1 and L 3 Each of the R is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 Replacement; L 2 is a bond; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 substituted; P is absent, cycloalkyl, heterocyclyl or heteroaryl, each of which is optionally substituted by one or more R 4 Substituted; Z is hydrogen, alkyl, alkenyl, alkynyl, 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, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 5 Replace; 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 Replacement; or R C and R D Together with the nitrogen atom to which it is attached, it forms a 6 substituted ring (eg, 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 Replace; each R A1 , R B1 , R C1 , R D1, R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3 or 4.

[0146] In some embodiments, the compound of formula (I) is a compound of formula (la):

[0147] (Ia),

[0148] or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )–、–N(R C )C(O)–,–C(O)N(R C )–、–N(R C )N(R D )–、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 linked to an attachment group (such as an attachment group described herein) and is optionally supported by one or more R 1 Replacement; L 1 and L 3 Each of the R is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 Replacement; L 2is a bond; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 substituted; P is optionally substituted with one or more R 4 substituted heteroaryl; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 Replace; 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 Replacement; or R C and R D Together with the nitrogen atom to which it is attached, it forms a 6 substituted ring (eg, 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 Replace; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3 or 4.

[0149] In some embodiments, for Formula (I) and (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-、-N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-or-N(R C )-. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C )-. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C )-. In some embodiments, A is alkyl, -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 is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(RC )C(O)(C1-C6-alkylene)-, wherein the alkylene is R 1 In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, and R 1 is alkyl (eg, methyl). In some embodiments, A is -NHC(O)C(CH3)2-. In some embodiments, A is -N(R C )C(O)(methylene)-, and R 1 is alkyl (eg, methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0150] In some embodiments, for Formula (I) and (Ia), L 1 is a bond, alkyl or heteroalkyl. 1 is a bond or an alkyl group. In some embodiments, L 1 is a bond. In some embodiments, L 1 In some embodiments, L 1 is C1-C6 alkyl. 1 is -CH2-, -CH(CH3)-, -CH2CH2CH2 or -CH2CH2-. In some embodiments, L 1 It is -CH2- or -CH2CH2-.

[0151] In some embodiments, for Formula (I) and (Ia), L 3 is a bond, alkyl or heteroalkyl. 3 is a bond. In some embodiments, L 3 In some embodiments, L 3 It is C1-C 12 In some embodiments, L 3 is C1-C6 alkyl. 3 is -CH2-. In some embodiments, L 3 In some embodiments, L 3 is optionally replaced by one or more R 2 (e.g., oxo) substituted C1-C 12 In some embodiments, L 3 is optionally replaced by one or more R 2 In some embodiments, L 3is -C(O)OCH2-, -CH2(OCH2CH2)2-, -CH2(OCH2CH2)3-, CH2CH2O-, or -CH2O-. In some embodiments, L 3 It is -CH2O-.

[0152] In some embodiments, for formula (I) and formula (Ia), 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, wherein 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 replaced by R 7 (For example, 1 R 7 ) substituted phenyl. In some embodiments, M is In some embodiments, R 7 It's CF3.

[0153] In some embodiments, for Formula (I) and Formula (Ia), P is absent, heterocyclic or heteroaryl. In some embodiments, P is absent. In some embodiments, for Formula (I) and (Ia), 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 a tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl or thiazolyl. In some embodiments, P is a tetrazolyl, imidazolyl, pyrazolyl, or triazolyl or pyrrolyl. In some embodiments, P is an 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 .

[0154] In some embodiments, P is a heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl or a 6-membered heterocyclyl. In some embodiments, P is an imidazolidinone. In some embodiments, P is In some embodiments, P is thiomorpholinyl-1,1-dioxide.

[0155] In some embodiments, P is .

[0156] In some embodiments, for Formula (I) and Formula (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is a monocyclic or bicyclic heterocyclyl. In some embodiments, Z is an oxygen-containing heterocyclyl. In some embodiments, Z is a 4-membered heterocyclyl, a 5-membered heterocyclyl, or a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is , or In some embodiments, Z is a 4-membered oxygen-containing heterocyclic group. In some embodiments, Z is .

[0157] 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 thiomorpholinyl-1,1-dioxyl 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 .

[0158] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is optionally substituted with one or more R 5 In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonyl. In some embodiments, Z is In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is .

[0159] In some embodiments, for Formula (I) and (Ia), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is (e.g., with 1 R 5 ) is a monosubstituted phenyl. In some embodiments, Z is a monosubstituted phenyl, wherein 1 R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 is NH2. In some embodiments, Z is a monosubstituted phenyl group wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 Located in the opposite position.

[0160] In some embodiments, for Formula (I) and (Ia), Z is alkyl. In some embodiments, Z is C1-C 12 In some embodiments, Z is C1-C 10 In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is substituted by 1-5 R 5 In some embodiments, Z is replaced by 1 R 5 In some embodiments, Z is replaced by 1 R 5 Substituted C1-C8 alkyl, wherein 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 replaced by 1 R 5 Substituted C1-C8 alkyl, wherein R 5 Yes-OR A1 or-C(O)OR A1 In some embodiments, Z is replaced by 1 R 5 Substituted C1-C8 alkyl, wherein R 5 Yes-ORA1 or -C(O)OH. In some embodiments, Z is -CH3.

[0161] In some embodiments, for Formula (I) and (Ia), Z is heteroalkyl. In some embodiments, Z is C1-C 12 In some embodiments, Z is C1-C 10 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 In some embodiments, Z is substituted with 1-5 R 5 Substituted nitrogen and sulfur containing heteroalkyl. In some embodiments, Z is N-methyl-2-(methylsulfonyl)eth-1-amino free radical (aminyl).

[0162] 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).

[0163] In some embodiments, Z is hydrogen.

[0164] In some embodiments, L 2 is a key and P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0165] In some embodiments, the compound of formula (I) is a compound of formula (Ib):

[0166] (Ib),

[0167] or a pharmaceutically acceptable salt thereof, wherein ring M 1 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 3 Substitution; Ring Z 1 is optionally replaced by 1-5 R 5 Substituted cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2a , R 2b , R2c and R 2d Each of is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl, or R 2a and R 2b or R 2c and R 2d Each of the groups together form an oxo group; X is absent, N(R 10 )(R 11 ), O or S; R C is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by 1-6 R 6 Replace; 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, heterocyclyl, aryl or heteroaryl; R 10 and R 11 Each of the following 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, 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; each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; each m and n is independently 1, 2, 3, 4, 5 or 6; and “ " refers to a connection to an attachment group or polymer described herein. In some embodiments, for each R 3 and R 5 , each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl or heterocyclyl.

[0168] In some embodiments, the compound of formula (Ib) is a compound of formula (Ibi):

[0169] (Ibi),

[0170] or a pharmaceutically acceptable salt thereof, wherein ring M 2 is optionally replaced by one or more R 3 Substituted aryl or heteroaryl; Ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of the groups together form an oxo group; X is absent, O or S; each R 3 and R 5 are 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 Together with ring Z 2 A fused 5-6 membered ring; 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 a connection to an attachment group or polymer described herein.

[0171] In some embodiments, the compound of formula (Ibi) is a compound of formula (Ib-ii):

[0172] (Ib-ii),

[0173] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2c and R2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2c and R 2d together to form an oxo group; each R 3 and R 5 are 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; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and “ ” refers to a connection to an attachment group or polymer as described herein.

[0174] In some embodiments, the compound of formula (I) is a compound of formula (Ic):

[0175] (Ic),

[0176] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2c and R 2d together to form an oxo group; each R 3 and R 5 are 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 a connection to an attachment group or polymer described herein.

[0177] In some embodiments, the compound of formula (I) is a compound of formula (Id):

[0178] (Id),

[0179] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O or S; R 2a , R2b , R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of the R 5 are 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; 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 connection to an attachment group or polymer described herein.

[0180] In some embodiments, the compound of formula (I) is a compound of formula (Ie):

[0181] (Ie),

[0182] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O or S; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of the R 5 are 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; 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 connection to an attachment group or polymer described herein.

[0183] In some embodiments, the compound of formula (I) is a compound of formula (If):

[0184] (If),

[0185] or a pharmaceutically acceptable salt thereof, wherein M is optionally replaced by one or more R 3 substituted alkyl; ring P is optionally substituted with one or more R 4 Substituted heteroaryl; L 3 is optionally replaced by one or more R 2 substituted alkyl or heteroalkyl; Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 Replacement; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together to form an oxo group; each R 2 , R 3 , R 4 and R 5 are 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 a connection to an attachment group or polymer described herein.

[0186] In some embodiments, the compound of formula (I) is a compound of formula (II):

[0187] (II)

[0188] or a pharmaceutically acceptable salt thereof, wherein M is a bond, an alkyl group or an aryl group, wherein the alkyl group and the aryl group are optionally replaced by one or more R 3 Replacement; L 3 is optionally replaced by one or more R 2 substituted alkyl or heteroalkyl; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or -OR A , wherein alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally replaced by one or more R 5 Replacement; R A is hydrogen; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together to form an oxo group; each R 2 , R 3 and R 5are 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 a connection to an attachment group or polymer described herein.

[0189] In some embodiments, the compound of formula (II) is a compound of formula (II-a):

[0190] (II-a),

[0191] 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 substituted; Z is hydrogen, alkyl, heteroalkyl or -OR A , wherein alkyl and heteroalkyl are optionally replaced by one or more R 5 Replacement; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together to form an oxo group; each R 2 , R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; R A is hydrogen; 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 a connection to an attachment group or polymer described herein.

[0192] In some embodiments, the compound of formula (I) is a compound of formula (III):

[0193] (III)

[0194] or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R 5 Replacement; R 2a , R 2b , R2c and R 2d Each of R is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R 2a and R 2b or R 2c and R 2d Together they 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 by 1-6 R 6 Replacement; R 3 , R 5 and R 6 Each of the following 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; q is an integer from 0 to 25; and “ ” refers to a connection to an attachment group or polymer described herein.

[0195] In some embodiments, the compound of formula (III) is a compound of formula (III-a):

[0196] (III-a),

[0197] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl, halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1is 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 connection to an attachment group or polymer described herein.

[0198] In some embodiments, the compound of formula (III-a) is a compound of formula (III-b):

[0199] (III-b),

[0200] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl, halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following 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 a connection to an attachment group or polymer described herein.

[0201] In some embodiments, the compound of formula (III-a) is a compound of formula (III-c):

[0202] (III-c),

[0203] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R') or S(O) x ; R' and R" are each independently hydrogen, alkyl, halogen or cycloalkyl; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl or halo; or2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following 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 a connection to an attachment group or polymer described herein.

[0204] In some embodiments, the compound of formula (III-c) is a compound of formula (III-d):

[0205] (III-d),

[0206] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R') or S(O) x ; R' and R" are each independently hydrogen, alkyl, halogen or cycloalkyl; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl or halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following 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 a connection to an attachment group or polymer described herein.

[0207] In some embodiments, the compound of formula (I) is a compound of formula (III-e):

[0208] (III-e),

[0209] or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R 2a and R 2b or R 2c and R 2d Each of the R C is hydrogen, alkyl, alkenyl, alkynyl or heteroalkyl, wherein each of the alkyl, alkenyl, alkynyl or heteroalkyl is optionally substituted by 1-6 R 6 Replacement; R 3 , R 5 and R 6 Each of the following is 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 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; w is 0 or 1; and “ ” refers to a connection to an attachment group or polymer described herein.

[0210] In some embodiments, the compound of formula (I) is a compound of formula (III-f):

[0211] (III-f),

[0212] or a pharmaceutically acceptable salt thereof, wherein ring Z 1 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl, halo; or 2a and R 2b or R2c and R 2d Together they 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 by 1-6 R 6 Replacement; R 3 , R 5 and R 6 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R 12 are 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.

[0213] In some embodiments, the compound of formula (I) is a compound of formula (III-g):

[0214] (III-g),

[0215] or a pharmaceutically acceptable salt thereof, wherein ring Z 1 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 5 Replacement; 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 groups is optionally substituted with 1-6 R 6 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen or alkyl; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 , R 5 and R 6 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -ORA1 , –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 as described herein.

[0216] In some embodiments, the compound of formula (I) is a compound of formula (III-h):

[0217] (III-h),

[0218] 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 groups is optionally substituted with 1-6 R 6 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen or alkyl; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 , R 5 and R 6 Each of the following 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 a connection to an attachment group or polymer as described herein.

[0219] In some embodiments, the compound of formula (I) is a compound of formula (III-i):

[0220] (III-i),

[0221] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R') or S(O) x ; R' and R" are each 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 groups is optionally substituted with 1-6 R 6 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen or alkyl; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 , R 5 and R 6 Each of the following 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 a connection to an attachment group or polymer as described herein.

[0222] In some embodiments, the compound is a compound of formula (I). In some embodiments, L 2 is a key and P and L 3 Does not exist independently.

[0223] In some embodiments, the compound is a compound of formula (Ia). In some embodiments of formula (II-a), L 2 is a bond, P is a heteroaryl, L 3is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl. 2 is a key and P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0224] In some embodiments, the compound is a compound of formula (Ib). In some embodiments, P is absent, L 1 Yes -NHCH2, L 2 is a bond, M is an aryl group (e.g., phenyl), L 3 is -CH2O, and Z is a heterocyclic group (eg, a nitrogen-containing heterocyclic group such as thiomorpholinyl-1,1-dioxide).

[0225] In some embodiments of Formula (Ib), P is absent, L 1 Formula -NHCH2, L 2 is a key, M does not exist, L 3 is a bond, and Z is a heterocyclic group (eg, an oxygen-containing heterocyclic group such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl or oxiranyl).

[0226] In some embodiments, the compound is a compound of Formula (Ibi). In some embodiments of Formula (Ibi), R 2a and R 2b Each of R is independently hydrogen or CH3, 2c and R 2d Each of the above is independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M 2 is optionally replaced by one or more R 3 Substituted phenyl, R 3 is -CF3, and Z 2 is a heterocyclic group (eg, an oxygen-containing heterocyclic group such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl or oxiranyl).

[0227] In some embodiments, the compound is a compound of Formula (Ib-ii). In some embodiments of Formula (Ib-ii), R 2a , R 2b , R 2c and R 2d Each of is independently hydrogen, q is 0, p is 0, m is 1, and Z 2is a heterocyclic group (eg, an oxygen-containing heterocyclic group such as tetrahydropyranyl).

[0228] In some embodiments, the compound is a compound of Formula (Ic). In some embodiments of Formula (Ic), R 2c and R 2d Each of the above is independently hydrogen, m is 1, p is 1, q is 0, R 5 is -CH3, and Z is a heterocyclic group (eg, a nitrogen-containing heterocyclic group such as piperazinyl).

[0229] In some embodiments, the compound is a compound of Formula (Id). In some embodiments of Formula (Id), R 2a , R 2b , R 2c and R 2d Each of is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is a heterocyclyl (eg, an oxygen-containing heterocyclyl such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxirane).

[0230] In some embodiments, the compound is of formula (If). In some embodiments of formula (If), R 2a and R 2b Each of the above is independently hydrogen, n is 1, M is -CH2-, P is a nitrogen-containing heteroaryl group (eg, imidazolyl), L 3 is -C(O)OCH2-, and Z is CH3.

[0231] 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 of the above is independently hydrogen, n is 1, q is 0, L 3 is -CH2(OCH2CH2)2, and Z is -OCH3.

[0232] In some embodiments of Formula (II-a), R 2a and R 2b Each of the above is independently hydrogen, n is 1, L 3 is a bond or –CH 2， and Z is hydrogen or –OH 。

[0233] 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 of the above is independently hydrogen, m is 1, n is 2, q is 3, p is 0, RC is hydrogen, and Z 1 is optionally replaced by R 5 Substituted heteroalkyl (e.g., -N(CH3)(CH2CH2)S(O)2CH3).

[0234] 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 of is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 It is 1 R 5 (e.g., -NH2) substituted aryl (e.g., phenyl).

[0235] 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 of the above is independently hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 2 is a heterocyclic group (eg, a nitrogen-containing heterocyclic group, such as a nitrogen-containing spiroheterocyclic group, such as 2-oxa-7-azaspiro[3.5]nonyl).

[0236] 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 of is independently hydrogen, m is 1, n is 2, q is 1, 2, 3 or 4, p is 0, and X is S(O)2. In some embodiments of Formula (III-d), R 2a and R 2b Each of the above is independently hydrogen, m is 1, n is 2, q is 1, 2, 3 or 4, p is 0, and X is S(O)2.

[0237] In some embodiments, the compound is a compound of formula (Ib), (Id) or (Ie). In some embodiments, the compound is a compound of formula (Ib), (Id) or (II). In some embodiments, the compound is a compound of formula (Ib), (Id) or (If). In some embodiments, the compound is a compound of formula (Ib), (Id) or (III).

[0238] 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, US 2016-0030359, or US 2016-0030360.

[0239] In some embodiments, the compound of formula (I) comprises a compound shown in Table 3, or a pharmaceutically acceptable salt thereof. In some embodiments, the external surface and / or one or more compartments within the devices described herein comprise a small molecule compound shown in Table 3, or a pharmaceutically acceptable salt thereof.

[0240] Table 3: Exemplary compounds of formula (I)

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Conjugation of any of the compounds in Table 3 to a polymer (e.g., alginate) can be performed as described in Example 2 of WO 2019 / 195055 or any other suitable chemical reaction.

[0248] In some embodiments, the compound is a compound of Formula (I) (e.g., (Ia), (Ib), (Ic), (Id), (Ie), (If), (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:

[0249] , and , or a pharmaceutically acceptable salt thereof.

[0250] In some embodiments, a polysaccharide polymer or device (e.g., a hydrogel capsule) described herein comprises a compound , , or a pharmaceutically acceptable salt of either compound.

[0251] In some embodiments, the compound of Formula (I) (e.g., compound 101 in Table 3) is covalently attached to alginate (e.g., alginate with an approximate MW <75 kDa, a G:M ratio ≥ 1.5) with 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, as determined by combustion analysis for nitrogen percentage as described in WO 2020 / 069429.

[0252] Clickable crosslinkers

[0253] Described herein are polysaccharides covalently bound to a cross-linking agent (i.e., a clickable cross-linking moiety) capable of performing a click chemistry reaction. In some embodiments, the clickable cross-linking moiety is capable of performing a [2+3] Huisgen cycloaddition reaction of an azide and an alkyne. The clickable cross-linking moiety may comprise a pair of agents, such as an azide and an alkyne capable of reacting with another. In some embodiments, the click reagent is modified by a suitable spacer to be attached to the polysaccharide. In some embodiments, the spacer comprises an alkyl, alkenyl, heteroalkyl, amine, ether, ester, or other functional group. In some embodiments, the spacer comprises a heteroalkyl. In some embodiments, the spacer comprises an alkylamine. In some embodiments, the spacer comprises an ethylene glycol group. In some embodiments, the click reagent comprises two or more functional groups. In some embodiments, the click reagent comprises two, three, four, five, or more functional groups.

[0254] The clickable cross-linking moiety may or may not require a catalyst (such as a copper catalyst) to react. In some embodiments, the click reaction requires a copper-free click chemistry reaction.

[0255] In some embodiments, the click chemistry reagent comprises an azide. In some embodiments, the azide is selected from the group consisting of alkyl azides, aryl azides, and acyl azides. In some embodiments, the azide is an alkyl azide.

[0256] In some embodiments, the click crosslinker is a compound of formula (IV):

[0257] (IV), or a pharmaceutically acceptable salt or tautomer thereof, wherein A is optionally replaced by one or more R 1 substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; T is absent, O, S, NR 53 , or C(R 54a )(R54b );R 52 , R 53 , R 54a and R 54b Each of 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; R 1 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 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 Replace; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, hydroxy, cycloalkyl, or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

[0258] In some embodiments of Formula (IV), A is heteroalkyl (eg, ethylene glycol or polyethylene glycol), T is C(R 54a )(R 54b );R 52 N(R C1 )(R D1 ); and R 54a , R 54b , R C1 and R D1 Each of is hydrogen.

[0259] In some embodiments, the click cross-linker is a compound of formula (IV-a):

[0260] (IV-a), or a pharmaceutically acceptable salt or tautomer thereof, wherein T is absent, O, S, NR 53 , or C(R 54a )(R 54b );R 50 , R 51a , R 51b and R 52 Each of 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; or, R 51a and R 51b together to form an oxo group; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R7 m and n are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0261] In some embodiments, T is NR 53 And R 53 is hydrogen. In some embodiments, R 52 is hydrogen. In some embodiments, n is 1, 2, 3, or 4. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, the click crosslinker is compound 201, 202, or 203.

[0262] In some embodiments, the cross-linking agent is a compound of formula (IV-b):

[0263] (IV-b), or a pharmaceutically acceptable salt or tautomer thereof, wherein R 50 , R 51a and R 51b Each of 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; or, R 51a and R 51b Together they form an oxo group; R 52a and R 52b Each of the following is independently alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R A1 , R B1 , R C1 , R D1 , R E1 and R F1are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 m and n are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and each of m and n is 0, 1, 2, 3, 4, 5 or 6.

[0264] In some embodiments, the click cross-linker is a compound of formula (IV-c):

[0265] (IV-c), or a pharmaceutically acceptable salt or tautomer thereof, wherein R 50 , R 51a and R 51b Each of 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; or R 51a and R 51b Together they form an oxo group; R 52 is alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 m and n are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and each of m and n is 0, 1, 2, 3, 4, 5 or 6.

[0266] In some embodiments, the compound of formula (IV) is a compound selected from Table 4 or a pharmaceutically acceptable salt thereof.

[0267] Table 4: Exemplary compounds of formula (IV)

[0268]

[0269] In some embodiments, the clickable cross-linking moiety is , or .

[0270] In some embodiments, the click crosslinker comprises an alkyne. In some embodiments, the click crosslinker comprises a cycloalkyne. In some embodiments, the click crosslinker comprises a cyclooctyne. In some embodiments, the click crosslinker comprises a fluorinated cyclooctyne. In some embodiments, the click crosslinker comprises an aryl cyclooctyne. In some embodiments, the crosslinker is a compound of formula (V):

[0271] (V), or a pharmaceutically acceptable salt or tautomer thereof, wherein ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1 to 6 R 7 Replacement; R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R 53 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, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1are 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, hydroxyl, cycloalkyl or heterocyclyl; or two R 7 They may be joined together to form a cycloalkyl, heterocyclyl, aryl or heteroaryl ring.

[0272] In some embodiments, the click cross-linker of formula (V) is a compound of formula (Va):

[0273] , Formula (Va), or a pharmaceutically acceptable salt or tautomer thereof, wherein R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h and R 53i Each of 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, heteroaryl; R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, –C(O)R B1 , –N(R C1 )(R D1 ), cycloalkyl, heterocyclyl, aryl, 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 7are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0274] In some embodiments, the compound of formula (V) is a compound of formula (Vb):

[0275] Formula (Vb), or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T and Y is independently C(R 40 )(R 41 ), O, or N(R 42 );R 40 , R 41 , R 42 , R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h , R 53i , R 53 and R 54 Each of 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, heteroaryl; or R 53a and R 53b , R 53b and R 53c , R 53c and R 53d , R 53f and R 53g , R 53g and R 53h , R 53h and R 53i Any of the following, taken together with the atoms to which they are attached, form an aryl or heteroaryl ring, each of which may be replaced by 1 to 6 R 7 Replace; each R A1 , R B1 , R C1 , R D1 and R E1are 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.

[0276] In some embodiments, R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h and R 53i Each of is independently hydrogen. In some embodiments, Y is O. In some embodiments, T is C(R 40 )(R 41 ) and R 40 and R 41 In some embodiments, each of R 54 In some embodiments, R 53 In some embodiments, the click cross-linker is compound 220.

[0277] Additional clickable cross-linking moieties can be found in U.S. Patent No. USRE47539E1 and U.S. Patent No. 8,133,515, which are incorporated herein by reference in their entirety.

[0278] In some embodiments, the compound of Formula (V) is a compound selected from Table 5 or a pharmaceutically acceptable salt thereof.

[0279] Table 5. Exemplary compounds of formula (V)

[0280]

[0281] In some embodiments, the polysaccharide polymer comprises a first clickable cross-linking portion and a second clickable cross-linking portion. In one embodiment, the first clickable cross-linking portion is a compound of formula (IV) or a pharmaceutically acceptable salt thereof. In one embodiment, the first clickable cross-linking portion is an azide, such as a compound provided in Table 4, or a pharmaceutically acceptable salt thereof. In one embodiment, the second clickable cross-linking portion is a compound of formula (V) or a pharmaceutically acceptable salt thereof. In one embodiment, the first clickable cross-linking portion is an alkyne, such as a compound provided in Table 5, or a pharmaceutically acceptable salt thereof. In some embodiments, the first clickable cross-linking portion is a compound of formula (IV) or a pharmaceutically acceptable salt thereof (e.g., polyethylene glycol azide), and the second clickable cross-linking portion is a compound of formula (V) or a pharmaceutically acceptable salt thereof (e.g., dibenzocyclooctyne (DBCO)).

[0282] Modified polymers with clickable cross-linking moieties

[0283] The clickable cross-linking moiety can be covalently bound to a polysaccharide polymer (e.g., alginate). In one embodiment, the polysaccharide comprises a structure of formula (VI):

[0284] (VI), or a pharmaceutically acceptable salt or tautomer thereof, wherein each of U and Y is independently C(R 40 )(R 41 ), O, or N(R 42 );R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 Each of 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, heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1are 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; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl or heterocyclyl; and the cross-linking agent has a structure of formula (IV), (IV-a), (IV-b), (IV-c), (V), (Va), or (Vb).

[0285] In one embodiment, the polysaccharide of formula (VI) is of formula (VI-a):

[0286] The polysaccharide of (VI-a), or a pharmaceutically acceptable salt or tautomer thereof, wherein each of U and Y is independently C(R 40 )(R 41 ), O, or N(R 42 );R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 Each of 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, heteroaryl; A is optionally replaced by one or more R 1 Substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, or heterocyclyl; R 1 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 ), SRE1 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 Replace; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; T is absent, O, S, NR 53 , or C(R 54a )(R 54b ); x is 1 or 2; and y is 2, 3, or 4; and R 52 , R 53 , R 54a and R 54b Each of 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.

[0287] In one embodiment, the polysaccharide of formula (VI) is of formula (VI-b):

[0288] The polysaccharide of (VI-b), or a pharmaceutically acceptable salt or tautomer thereof, wherein U is C(R 40 )(R 41 ), O, or N(R 42 );R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 , R 50 , R 51a , R 51b , R 52 and R 53 Each of 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, heteroaryl; or, R 51a and R 51b together to form an oxo group; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7m and n are independently 1, 2, 3, 4, 5, or 6.

[0289] In one embodiment, the polysaccharide of formula (VI) is of formula (VI-d):

[0290] The polysaccharide of (VI-d), or a pharmaceutically acceptable salt or tautomer thereof, wherein U is C(R 40 )(R 41 ), O, or N(R 42 );Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1 to 6 R 7 Replacement; R 38a , R 38b , R 39a , R 39b , R 50 , R 51a , R 51b , R 52 and R 53 Each of 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, heteroaryl; each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1 to 6 R 7Replace; 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, hydroxyl, cycloalkyl or heterocyclyl; or two R 7 They may be joined together to form an aryl or heteroaryl ring.

[0291] In one embodiment, the polysaccharide of formula (VI) is of formula (VI-e):

[0292] (VI-e), or a pharmaceutically acceptable salt or tautomer thereof, wherein each of U and Y is independently C(R 40 )(R 41 ), O, or N(R 42 );R 38a , R 38b , R 39a , R 39b , R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h and R 53i Each of 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, heteroaryl; or R 53a and R 53b , R 53b and R 53c , R 53c and R 53d , R 53f and R 53g, R 53g and R 53h , R 53h and R 53i Any of the following, taken together with the atoms to which they are attached, form an aryl or heteroaryl ring, each of which may be replaced by 1 to 6 R 7 Replace, 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.

[0293] In other embodiments, polysaccharide (e.g., alginate) can be modified through two or more cross-linking agents. In some embodiments, the two or more cross-linking agents have cross-reactivity. In some embodiments, two or more cross-linking agents from different clickable pairs do not have cross-reactivity.

[0294] Cross-linking agent-modified polysaccharide polymers and defibrotic small molecule compounds

[0295] In some embodiments, polysaccharides (e.g., alginate) can be modified through clickable cross-linking moieties and defibrotic small molecule compounds. In some embodiments, the defibrotic compound is one of the compounds shown in Table 3. In some embodiments, polysaccharides can be modified through a type of clickable cross-linking moieties and a type of defibrotic compound. In some embodiments, polysaccharides can be modified through more than one type of clickable cross-linking moieties and more than one type of defibrotic compound.

[0296] In some embodiments, the polysaccharide is a polymer of formula (VI):

[0297] (VI), or a pharmaceutically acceptable salt or tautomer thereof, wherein W, T 1 、T 2 , U 1 and U 2 Each of which is independently C(R 40 )(R 41 ), O, or N(R 42 );R 38a , R 38b , R 39a and R39b Each of 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, 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; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl or heterocyclyl; and the clickable cross-linking portion is absent or has a structure of formula (IV-a), (IV-b), (IV-c), (V) or (Va); and the defibrotizing compound is absent or has a structure of formula (I), (Ia), (Ib), (Ibi), (Ib-ii), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), (III-d), (III-e), (III-f), (III-g), (III-h) or (III-i).

[0298] In some embodiments, the polysaccharide (eg, alginate) comprising a clickable cross-linking moiety and optionally a compound of formula (I) is a compound shown in Table 6. In Table 6, n is an integer greater than 1.

[0299] In some embodiments, polysaccharide polymers (e.g., alginate polymers) are modified with clickable cross-linking moieties, which can then be covalently cross-linked to form clickable cross-linked hydrogels. In some embodiments, clickable cross-linked hydrogels can encapsulate therapeutic products. In some embodiments, clickable cross-linked hydrogels can encapsulate mammalian cells. In some embodiments, clickable cross-linked hydrogels can encapsulate engineered mammalian cells.

[0300] In one embodiment, the modified polysaccharide polymer of formula (VI) is a compound selected from Table 6.

[0301] Table 6. Exemplary compounds of formula (VI)

[0302]

[0303]

[0304] In some embodiments, the cross-linking agent reaction may further comprise an additive, such as a catalyst. In some embodiments, the click chemistry reaction is carried out in the presence or absence of a catalyst. In some embodiments, the click chemistry reaction is carried out in the presence of a copper catalyst. In some embodiments, the click chemistry reaction is carried out in the presence of a copper (I) catalyst. In some embodiments, the click chemistry reaction is carried out in the absence of a catalyst.

[0305] In some embodiments, the cross-linking reaction is carried out in an aqueous solvent or an organic solvent. In some embodiments, the cross-linking reaction is carried out in a 1:1 solution of PBS and acetonitrile. In some embodiments, the cross-linking reaction is carried out in a 1:1 solution of water and acetonitrile.

[0306] In some embodiments, the click reaction is carried out at 20-40°C.

[0307] The purity of polysaccharide polymer (for example, alginate polymer) can be measured by any method known in the art. In some embodiments, modified polysaccharide polymer (for example, alginate polymer) has a purity greater than about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 99% or higher. In some embodiments, modified polysaccharide polymer has a purity between 50-100%, 75-100%. In one embodiment, modified polysaccharide polymer has a purity of 50%. In one embodiment, modified polysaccharide polymer has a purity greater than 75%. In one embodiment, modified polysaccharide polymer has a purity greater than 99.9%.

[0308] The polysaccharide polymers described herein can be modified on any suitable functional group (e.g., carboxyl or hydroxyl). In one embodiment, the polysaccharide polymer is modified on a single type of functional group. In one embodiment, the polysaccharide polymer is modified on more than one type of functional group.

[0309] In one embodiment, the degree of modification (i.e., the percentage of functional groups of the polymer modified by the clickable cross-linking portion) 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%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 50%. In one embodiment, the degree of modification of the polysaccharide polymer is between about 1% and about 45%. In preferred embodiments, not all functional groups (eg, carboxyl groups) of the modified polysaccharide are substituted, which allows for ionic and covalent cross-linking.

[0310] In one embodiment, the degree of modification (i.e., the percentage of functional groups of the polymer modified by the clickable cross-linking moiety) 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 by the clickable cross-linking moiety) 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 cross-linking moieties) 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%.

[0311] In some embodiments, for example, when the polysaccharide polymers described herein are used to prepare hydrogel capsules with dual crosslinking, the polymer retains sufficient unreacted carboxylic acid groups to allow ionic crosslinking. In some embodiments, the polysaccharide polymers described herein do not contain more than 10% modified carboxylic acid groups. In some embodiments, the polysaccharide polymers described herein do not contain more than 5% modified carboxylic acid groups. In some embodiments, the polysaccharide polymers described herein do not contain more than 5%, 6%, 7%, 8%, 9% or 10% modified carboxylic acid groups.

[0312] 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 clickable cross-linking moieties (e.g., compounds of formula (IV) and / or formula (V), compounds of formula (I) and cell adhesion molecules (e.g., RGD)). In one embodiment, the polysaccharide polymers described herein are modified with clickable cross-linking moieties. In one embodiment, the polysaccharide polymers described herein are modified with compounds of formula (I). In one embodiment, the polysaccharide polymers described herein are modified with cell adhesion molecules. In one embodiment, the polysaccharide polymers described herein are modified with clickable cross-linking moieties and cell adhesion molecules. In a preferred embodiment, the polysaccharide polymers described herein are modified with clickable cross-linking moieties and compounds of formula (I). In one embodiment, the polysaccharide polymers described herein are modified with cell adhesion molecules and compounds of formula (I).

[0313] Characteristics of hydrogels and hydrogel capsules

[0314] The present disclosure further features a hydrogel capsule comprising a polysaccharide polymer as described herein. The hydrogel capsule can be cross-linked (i.e., covalently cross-linked) by clickable cross-linking groups or by, for example, in the presence of a divalent cation (e.g., Ba 2+ ) is produced by ionic crosslinking in the presence of ionomer. In one embodiment, the hydrogel capsules described herein are produced by reacting clickable crosslinking moieties. 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 gamma radiation in the presence of an appropriate initiator.

[0315] Modified polymers with clickable reagents may be able to undergo further polymerization, such as reactions with compatible functional groups on the same or different polymers. In one embodiment, a polymer modified to include a thiol group and a second polymer modified to include an olefin group make it possible to form a cross-linked polymer by reacting the first polymer and the second polymer. In some embodiments, the hydrogel is formed by cross-linking unsaturated functional groups via a chain growth polymerization process. In other embodiments, the hydrogel is formed by cross-linking unsaturated functional groups via a step-growth polymerization process. The step-growth polymerization process preferably comprises a reaction between one or more unsaturated functional groups (e.g., alkenyl) of a polysaccharide chain and the thiolation functional group of another polymer chain. In some embodiments, the step-growth polymerization is a thiol-ene photo-click reaction. In one embodiment, a hydrogel capsule is formed by cross-linking an unsaturated functional group on the first polymer with a functional group on the second polymer. In a preferred embodiment, a hydrogel capsule is formed by cross-linking an alkynyl functional group on the first polymer with an azide functional group on the second polymer.

[0316] The hydrogel capsules described herein are formed by cross-linking of one or more types of polysaccharide polymers. In one embodiment, the hydrogel capsules only comprise polysaccharide polymers. In one embodiment, the hydrogel capsules comprise polysaccharide polymers of the same type, such as alginate polymers. In one embodiment, the hydrogel capsules are formed by the polymerization of two identical polysaccharides. In one embodiment, the hydrogel capsules are formed by the polymerization of two different polysaccharides. In one embodiment, the hydrogel capsules comprise a plurality of polymers, such as a plurality of polysaccharide polymers. In one embodiment, the hydrogel capsules comprise a polysaccharide polymer and a non-polysaccharide polymer.

[0317] The hydrogel capsules described herein may be homogeneous, i.e., may not contain non-polysaccharide polymers. In one embodiment, the hydrogel capsules described herein do not contain a polymer selected from polyacrylamide, poly(vinyl alcohol), poly(ethylene oxide), polyethylene glycol (PEG), and polyphosphazene. In one embodiment, the hydrogel capsule does not contain poly(vinyl alcohol). In one embodiment, the hydrogel capsule does not contain poly(ethylene oxide). In one embodiment, the hydrogel capsule does not contain polyethylene glycol (PEG). In one embodiment, the hydrogel capsule does not contain polyphosphazene.

[0318] 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 by the same type of modified polysaccharide. In one embodiment, the two compartments are formed by different types of modified polysaccharides.

[0319] In some embodiments, the first and second compartments comprise a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment comprises a blend of polymers. In some embodiments, the second (outer) compartment comprises a blend of polymers. In some embodiments, the first and second compartments comprise a blend of the same polymer. In some embodiments, the first and second compartments comprise a blend of different polymers.

[0320] In some embodiments, the first compartment comprises a blend of polymers and the second compartment does not comprise a blend of polymers. In some embodiments, the first compartment comprises a blend of polymers and the second compartment comprises a single type of polymer.

[0321] 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.

[0322] In some embodiments, the first and second compartments comprise a blend of alginate polymers. In some embodiments, the first compartment comprises a blend of alginate polymers. In some embodiments, the second compartment comprises a blend of alginate polymers.

[0323] In some embodiments of the invention, the first and second compartments comprise a blend of VLVG alginate and SLG100 alginate. In some embodiments of the invention, the first compartment comprises a blend of VLVG alginate and SLG100 alginate. In some embodiments of the invention, the second compartment comprises a blend of VLVG alginate and SLG100 alginate.

[0324] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment comprising VLVG alginate, wherein the VLVG alginate comprises a compound of formula (IV) and the SLG100 alginate comprises a compound of formula (V); and (ii) an outer compartment comprising 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).

[0325] In some embodiments of the invention, the first and second compartments comprise a blend of VLVG alginate and SLG100 alginate. In some embodiments of the invention, the first compartment comprises a blend of VLVG alginate and SLG100 alginate. In some embodiments of the invention, the second compartment comprises a blend of VLVG alginate and SLG100 alginate.

[0326] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment comprising 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 comprising 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).

[0327] In some embodiments, the hydrogel capsule comprises: (i) an inner compartment comprising 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 comprising 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).

[0328] The present disclosure features a dual cross-linked polysaccharide for encapsulating mammalian cells. The dual cross-linked polysaccharide hydrogel capsule contains at least one cell binding substance (CBS) (as defined herein). When the hydrogel is implanted in a subject (e.g., a human or other mammalian subject), the cells are capable of expressing a therapeutic agent. In addition, the device comprises at least one means for mitigating FBR (as defined herein).

[0329] In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, 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, 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.

[0330] In some embodiments, defibration polymer comprises alginate chemically modified with the compound of formula (I).The alginate in defibration polymer can be identical or different with any unmodified alginate present in equipment.In one embodiment, the density (for example, conjugated amount) of the compound of formula (I) in defibration 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 (for example, as determined by the combustion analysis of nitrogen percentage).In one embodiment, the amount of compound 101 produces about 0.5% to 2%, 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N %N increase (compared with unmodified alginate), wherein %N is determined by combustion analysis and corresponds to the amount of the compound 101 in modified alginate.

[0331] Hydrogel capsules described herein may be porous or non-porous. The pores in polysaccharide hydrogels (e.g., alginate hydrogels) serve as selective permeation membranes for small proteins and molecules, while preventing larger unwanted molecules (such as immunoglobulins) from entering the cells encapsulated. In a preferred embodiment, 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.

[0332] In some embodiments, the average pore size of the first compartment and the second compartment of the particle is substantially the same. In some embodiments, the average pore size of the first compartment and the second compartment of the particle differs 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 (for example, the average pore size of the first compartment and / or the average pore size of the second compartment) depends on various factors, such as the presence and density of one or more materials in each compartment and the compound of formula (I).

[0333] Hydrogel capsules described herein should not have holes of a diameter sufficient to allow cells (e.g., immune cells, e.g., dendritic cells) to move through the hydrogel. In some embodiments, hydrogel capsules described herein do not have a pore size greater than 75 μm. In some embodiments, 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.

[0334] 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 a dextran permeability assay). 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 preferred embodiments, the average molecular weight permeability is from about 125 kDa to about 175 kDa.

[0335] In some embodiments, the hydrogel capsules described herein can be characterized by their absolute rupture strength (eg, 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 a volume 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 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or an absolute strength of 800 g.In some embodiments, the hydrogel capsules described herein have a volume of 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, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or an absolute strength of 800 g. In some embodiments, the hydrogel capsules described herein have a mass 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, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, or an absolute strength of 800 g.

[0336] 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 described herein (e.g., hydrogel capsules) 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 In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of between about 1 mm and about 8 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of between about 1 mm and about 4 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of 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.

[0337] 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.

[0338] The present disclosure features particles (e.g., hydrogel capsules) comprising a first compartment, a second compartment, a clickable cross-linking moiety (e.g., a compound of formula (IV) or (V)), and optionally a compound of formula (I). The clickable cross-linking moiety is partially covalently bound to a polysaccharide polymer present in the first and / or second compartment. The particles (e.g., hydrogel capsules) may be spherical or have any other shape. The particles (e.g., hydrogel capsules) may comprise materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The particles (e.g., hydrogel capsules) may be made entirely of one type of material, or may comprise a number of other materials in the second (outer) compartment and the first (inner) compartment.

[0339] 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).

[0340] 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 described herein (e.g., hydrogel capsules) 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 In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of between about 1 mm and about 8 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of between about 1 mm and about 4 mm. In some embodiments, the particles (e.g., hydrogel capsules) have an average diameter or size of 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.

[0341] 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.

[0342] 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 larger, and can be as large as 1 mm. For example, the thickness of the outer compartment in the particle described herein can be 10 nanometers to 1 millimeter, 100 nanometers to 1 millimeter, 500 nanometers to 1 millimeter, 1 micrometer (µm) to 1 millimeter, 1 µm to 1 mm, 1 µm to 500 µm, 1 µm to 250 µm, 1 µm to 1 mm, 5 µm to 500 µm, 5 µm to 250 µm, 10 µm to 1mm, 10 µm to 500 µm, or 10 µm to 250 µm. In some embodiments, the thickness of the outer compartment is between 100 nanometers and 1 millimeter, between 1 μm and 1 mm, between 1 μm and 500 μm, or between 5 μm and 1 mm.

[0343] In some embodiments, the first compartment and the second compartment both comprise the same polymer. In some embodiments, the first compartment and the second compartment comprise different polymers. In some embodiments, the first compartment comprises alginate. In some embodiments, the second compartment comprises alginate. In some embodiments, the first compartment and the second compartment both comprise 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 comprises alginate and the second compartment comprises different polymers (e.g., polysaccharides, such as hyaluronate or chitosan). In some embodiments, the second compartment comprises alginate and the first compartment comprises different polymers (e.g., polysaccharides, such as hyaluronate or chitosan).

[0344] The first compartment and the second compartment can both include a single component (e.g., a polymer) or more than one component (e.g., a blend of polymers). In some embodiments, the first compartment contains only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate). In some embodiments, the second compartment contains only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate). In some embodiments, the first and second compartments each independently contain only alginate (e.g., a chemically modified alginate, or a blend of unmodified alginate and chemically modified alginate).

[0345] In some embodiments, the first and second compartments comprise a blend of polymers (i.e., a mixture of polymers). In some embodiments, the first (inner) compartment comprises a blend of polymers. In some embodiments, the second (outer) compartment comprises a blend of polymers. In some embodiments, the first and second compartments comprise a blend of identical polymers. In some embodiments, the first and second compartments comprise a blend of different polymers. In some embodiments, at least one polymer in the blend comprising the outer compartment is covalently modified through a clickable cross-linking moiety (e.g., a compound of formula (IV), (V) or (VI)) described herein. In some embodiments, at least one polymer in the blend comprising the second (outer) compartment is covalently modified through a defibrotization compound (e.g., a compound of formula (I)) described herein. In some embodiments, at least one polymer in the blend comprising the second (outer) compartment is covalently modified through both clickable cross-linking moieties and defibrotization compounds.

[0346] In some embodiments, the first compartment comprises a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers. In some embodiments, the first compartment comprises a blend of 2 polymers. In some embodiments, the first compartment comprises a blend of 3 polymers. In some embodiments, the first compartment comprises a blend of 4 polymers. In some embodiments, the first compartment comprises a blend of 5 polymers. In some embodiments, the first compartment comprises a blend of 6 polymers. In some embodiments, the first (inner) compartment comprises a blend of 7 polymers. In some embodiments, the first (inner) compartment comprises a blend of 8 polymers. In some embodiments, the first (inner) compartment comprises a blend of 9 polymers. In some embodiments, the first (inner) compartment comprises a blend of 10 polymers.

[0347] In some embodiments, the second (outer) compartment comprises a blend of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers. In some embodiments, the second (outer) compartment comprises a blend of 2 polymers. In some embodiments, the second (outer) compartment comprises a blend of 3 polymers. In some embodiments, the second (outer) compartment comprises a blend of 4 polymers. In some embodiments, the second (outer) compartment comprises a blend of 5 polymers. In some embodiments, the second (outer) compartment comprises a blend of 6 polymers. In some embodiments, the second (outer) compartment comprises a blend of 7 polymers. In some embodiments, the second (outer) compartment comprises a blend of 8 polymers. In some embodiments, the second (outer) compartment comprises a blend of 9 polymers. In some embodiments, the second (outer) compartment comprises a blend of 10 polymers.

[0348] In some embodiments, the first compartment comprises a blend of polymers and the second compartment does not comprise a blend of polymers. In some embodiments, the first compartment comprises a blend of polymers and the second compartment comprises a single type of polymer.

[0349] 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.

[0350] In some embodiments, the first and second compartments comprise a blend of polymers, and the polymers of the blend are any two miscible polymers.

[0351] In some embodiments, the first and second compartments comprise a blend of polymers, and the polymer is selected from the group consisting of alginate, hyaluronate, and chitosan.

[0352] In some embodiments, the first and second compartments comprise a blend of polymers, and the polymer is selected from the group consisting of: alginate, hyaluronate, and chitosan. In some embodiments, the first compartment comprises a blend of polymers, and the polymer is selected from the group consisting of: alginate, hyaluronate, and chitosan. In some embodiments, the second compartment comprises a blend of polymers, and the polymer is selected from the group consisting of: alginate, hyaluronate, and chitosan.

[0353] In some embodiments, the first and second compartments comprise a blend of alginate polymers. In some embodiments, the first compartment comprises a blend of alginate polymers. In some embodiments, the second compartment comprises a blend of alginate polymers.

[0354] In some embodiments, the first and second compartments comprise a blend of alginate polymers, and the alginate polymer is selected from high guluronic acid alginate and high mannuronic acid alginate. In some embodiments, the first compartment comprises a blend of alginate polymers, and the alginate polymer is selected from high guluronic acid alginate and high mannuronic acid alginate. In some embodiments, the second compartment comprises a blend of alginate polymers, and the alginate polymer is selected from high guluronic acid alginate and high mannuronic acid alginate.

[0355] In some embodiments, the first and second compartments include a blend of alginate polymers, and the alginate polymer is 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 includes a blend of alginate polymers, and the alginate polymer is 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 includes a blend of alginate polymers, and the alginate polymer is selected from low molecular weight alginate, medium molecular weight alginate, high molecular weight alginate and ultra-high molecular weight alginate.

[0356] In some embodiments, the first and second compartments comprise a blend of alginate polymers, and the alginate polymer is 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. In some embodiments, the first compartment contains a blend of alginate polymers, and the alginate polymer is 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.In some embodiments, the second compartment contains a blend of alginate polymers, and the alginate polymer is 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.

[0357] In some embodiments, the first and second compartments include 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.

[0358] In some embodiments, the first and second compartments include 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.

[0359] In some embodiments, the polymer of the first compartment of the particle (e.g., hydrogel capsule) is modified with one compound of formula (I), and the polymer of the second compartment of the particle (e.g., hydrogel capsule) is modified with a different compound of formula (I). In some embodiments, the particle (e.g., hydrogel capsule) comprises a mixture of a polymer modified with a compound of formula (I) and an unmodified polymer (e.g., a polymer not modified with a compound of formula (I)). In some embodiments, the first compartment comprises a mixture (i.e., 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 comprises a mixture of a polymer modified with a compound of formula (I) and an unmodified polymer (e.g., a polymer not modified with a compound of formula (I)).

[0360] The polymer of the particles (e.g., hydrogel capsules) described herein may be modified with a compound of formula (I) or a pharmaceutically acceptable salt thereof on one or more monomers of the polymer. The modified polymer of the particle (e.g., hydrogel capsule) may be present in the first (inner) compartment of the particle, the second (outer) compartment of the particle, or both the first (inner) compartment and the second (outer) compartment 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).

[0361] In some embodiments, the polymer (e.g., alginate) (when modified with a compound of Formula (I), e.g., compound 101 of Table 3) comprises an increase in % N of any of the following values ​​(compared to an 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. N; and (x) 8 to 9% by weight N, 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 polymer.

[0362] Particles (such as hydrogel capsules) (such as the first compartment or the second compartment therein) may include a compound of formula (I) in an amount giving the particle specific features. For example, the particle surface (such as the outside of the outer compartment) may include a concentration or density of a compound of formula (I) that allows the particle to have a defibrotic effect (i.e., mitigating foreign body reaction) in a subject. In one embodiment, the particle surface includes alginate chemically modified by compound 101 of a defibrotic effective amount. In one embodiment, the defibrotic 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), wherein % N is determined by combustion analysis (such as described in Example 2 herein) and corresponds to the amount of the compound 101 in the modified alginate.

[0363] In one embodiment, a 5 mm probe attached to a 5 kg load cell is used to perform mechanical testing of hydrogel capsules on a TA.XT plus texture analyzer (Stable Micro Systems, Surrey, United Kingdom). A single capsule is placed on a platform and compressed from above by a probe at a fixed rate of 0.5 mm / sec. When a repulsive force of 1 g is measured, contact between the probe and the capsule is detected. 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 stroke (force versus displacement curve). Typically, before the capsule bursts completely, the capsule will be slightly broken, and the force applied to the probe will be reduced in small amounts. The analysis macro can be programmed to detect the first occurrence of a reduction 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 required mechanical strength of the particles described herein (e.g., two-compartment hydrogel capsules) has an initial rupture force greater than 1, 1.5, 2, 2.5 or 3 grams or at least 2 grams.

[0364] In one embodiment, the desired mechanical strength of a particle (e.g., a hydrogel capsule) is the ability to remain intact at a desired time point after implantation in a subject, e.g., the outer and inner compartments of a hydrogel capsule removed from a subject are visibly intact after retrieval from an immunocompetent mouse when observed by optical microscopy, e.g., by bright field imaging as described in the Examples herein.

[0365] In one embodiment, the particle surface comprises alginate chemically modified with Compound 101 in an amount that provides the particle with defibrotizing properties and desired mechanical strength, for example, the concentration or density of Compound 101 in the modified alginate produces a % N increase (compared to unmodified alginate) of any of the following values: (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 compound of formula (I) in the modified alginate.

[0366] When the particle (e.g., the first compartment or the second compartment thereof) 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 a compound of formula (I) dissolved in a buffer or solvent (such as acetonitrile) can be added to this mixture. The reactant can be heated to, for example, 55°C and kept for 16h, then cooled to room temperature and concentrated via rotary evaporation. The residue can then be dissolved in a buffer or solvent (e.g., water). The mixture can then be filtered, for example, through a cyano-modified silica gel bed (Silicycle), and the filter cake can be washed with water. The resulting solution can then be dialyzed (10,000 MWCO membrane) against buffer or water for 24 hours, e.g., exchanging the buffer or water at least once, at least twice, at least three times or more.The resulting solution can be concentrated, e.g., via lyophilization, to yield the desired chemically modified alginate.

[0367] In some embodiments, the particles described herein include cells. In some embodiments, the cells are engineered to produce therapeutic agents (e.g., proteins or polypeptides, such as antibodies, proteins, enzymes, or growth factors). In some embodiments, the cells are placed in the first compartment. In some embodiments, the cells are placed in the second compartment. In some embodiments, the cells are placed in the first compartment and the second compartment does not include cells. Particles (e.g., hydrogel capsules) may include active or inactive fragments of proteins or polypeptides, such as glucose oxidase (e.g., for glucose sensors), kinases, phosphatases, oxygenases, hydrogenases, reductases.

[0368] The particles described herein (e.g., hydrogel capsules) can be configured to release therapeutic agents, such as exogenous substances, such as 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 (e.g., RNA or DNA), a protein (e.g., 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.

[0369] Particles (e.g., hydrogel capsules) (e.g., as described herein) can be provided in the form of a preparation or composition for implantation or administration to a subject. In some embodiments, at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the particles (e.g., hydrogel capsules) in the preparation or composition have features as described herein, such as average diameter or average pore size.

[0370] Cells and therapeutic agents

[0371] The hydrogel capsule of the present invention may include a variety of different cell types (e.g., human cells), including but not limited to: adipocytes, epidermal cells, epithelial cells, endothelial cells, fibroblasts, pancreatic islet cells, mesenchymal stem cells, pericytes, subtypes of any of the foregoing, cells derived from any of the foregoing, cells derived from induced pluripotent stem cells, and a mixture of one or more of any of the foregoing. Exemplary cell types include cell types described in WO 2017 / 075631 and WO2019 / 195055. In one embodiment, the hydrogel capsule described herein comprises a plurality of cells. In one embodiment, a plurality of cells are in the form of a cell suspension before being encapsulated in a hydrogel capsule described herein. The cells in the suspension may be in the form of a single cell (e.g., from a monolayer cell culture), or in another form (e.g., placed on a microcarrier (e.g., a bead or matrix)) or provided as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension may include multiple cell clusters (e.g., as a spheroid) or a microcarrier. In some embodiments, the hydrogel capsule does not contain any pancreatic islet cells and does not contain any cells capable of producing insulin in a glucose-responsive manner.

[0372] Compared to untreated controls, hydrogel capsules of the present disclosure reduce immune cell adhesion. In one embodiment, compared to untreated controls, hydrogel capsules reduce macrophage adhesion. In one embodiment, compared to untreated controls, macrophage adhesion is reduced by about 1 to 10 times. In one embodiment, compared to untreated controls, macrophage adhesion is reduced by about 1 to 8 times. In one embodiment, compared to untreated controls, macrophage adhesion is reduced by about 1 to 7 times. In one embodiment, compared to untreated controls, macrophage adhesion is reduced by about 1 to 6 times. In one embodiment, compared to untreated controls, macrophage adhesion is reduced by about 1 to 5 times.

[0373] 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.

[0374] The present disclosure is characterized by a cell that produces or can produce a therapeutic agent for preventing or treating a disease, disorder or illness described herein. In one embodiment, the cell is an engineered cell. In one embodiment, the cell is engineered to sense a stimulus (e.g., a chemical signal) and expresses a therapeutic agent in response to the stimulus. The therapeutic agent can be any biological substance, such as nucleic acid (e.g., nucleotides, DNA or RNA), polypeptide, lipid, sugar (e.g., monosaccharide, disaccharide, oligosaccharide or polysaccharide) or small molecule, each of which is further described in detail below. Exemplary therapeutic agents include those listed in WO 2017 / 075631 and WO2019 / 195055.

[0375] In some embodiments, cells (e.g., engineered cells) produce nucleic acids. The size of nucleic acids produced by cells as described herein may vary, and contain one or more nucleosides or nucleotides, for example, greater than 2, 3, 4, 5, 10, 25, 50 or more nucleosides or nucleotides. In some embodiments, nucleic acids are short fragments such as RNA or DNA, and can be used as reporter genes 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., vector, chromosomal DNA). In some embodiments, nucleic acids have an average molecular weight (kD) of about 0.25, 0.5, 1, 1.5, 2, 2.5, 5, 10, 25, 50, 100, 150, 200 or more.

[0376] In some embodiments, the therapeutic agent is a peptide or polypeptide (e.g., protein), such as a hormone, enzyme, cytokine (e.g., proinflammatory cytokine or anti-inflammatory cytokine), growth factor, coagulation factor or lipoprotein. The peptide or polypeptide (e.g., protein, such as a hormone, growth factor, coagulation factor or coagulation factor, antibody molecule, enzyme, cytokine, cytokine receptor, or chimeric protein including cytokine or cytokine receptor) produced by cells in the implantable element may have a naturally occurring amino acid sequence, or may contain a variant of a naturally occurring sequence. The variant may be a naturally occurring or non-natural amino acid substitution, mutation, deletion or addition 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 its naturally occurring variant is a human sequence. In addition, the peptide or polypeptide (e.g., protein) used in the present invention may be modified in some way, 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 more.

[0377] In some embodiments, 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, adrenaline, glucagon, insulin, estrogen, progesterone and testosterone. In some embodiments, protein is insulin (e.g., insulin A chain, insulin B chain or proinsulin). In some embodiments, protein is growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, dephenylalanine human growth hormone and porcine growth hormone. In some embodiments, protein is not insulin (e.g., insulin A chain, insulin B chain or proinsulin).

[0378] 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 factor-I and -II (IGF-I and IGF-II).

[0379] 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 that participates in coagulation, the process by which blood changes from a liquid to a solid or gel. Exemplary clotting factors and coagulation factors include factor I (e.g., fibrinogen), factor II (e.g., prothrombin), factor III (e.g., tissue factor), factor V (e.g., prothrombinogen, unstable 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 precursor), factor XII (e.g., Hagemann 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 anti-clotting factor, such as protein C.

[0380] In some embodiments, 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 comprising at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, a monoclonal antibody (including a full-length antibody with 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 to a single epitope, such as a monospecific antibody molecule with a plurality of immunoglobulin variable domain sequences, and each immunoglobulin variable domain sequence binds to the same epitope.

[0381] In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the plurality of immunoglobulin variable domain sequences has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence in the plurality of immunoglobulin variable domain sequences has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, for example, the same protein (or the subunit of a multimeric protein). In one embodiment, the multispecific antibody molecule comprises the 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 tetraspecific antibody molecule.

[0382] Various types of antibody molecules can be produced by cells in the implantable elements described herein, including complete immunoglobulins of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domains of antibodies. Antibody molecules can be antibodies, such as IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4. Antibody molecules can be in the form of antigen-binding fragments, including Fab fragments, F(ab')2 fragments, single-chain variable regions, etc. Antibodies can be polyclonal or monoclonal (mAb). Monoclonal antibodies can include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and 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, as well as fragments of such antibodies, as long as they specifically bind to the target antigen and / or exhibit the desired biological activity. In some embodiments, the antibody molecule is a single domain antibody (e.g., nanobody). Antibodies can also be modified by recombinant means, such as by the deletion, addition, or substitution of amino acids, to increase the efficacy of antibodies 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., voranumab, fasitumab, tanezumab).

[0383] 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 alpha and beta, their receptors and their derivatives, renin; lipoprotein; colchicine; adrenocorticotropic hormone; vasopressin; somatostatin; lysine vasopressin; pancreatin; leuprolide; alpha-1-antitrypsin; atrial natriuretic factor; pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; enkephalin; RANTES (regulated activation, normal T cell expression and secretion); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin; Müllerian inhibiting substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; chorionic gonadotropin; microbial proteins, such as beta-lactamase; DNA enzymes; inhibins; activins; receptors for hormones or growth factors; integrins; proteins A or D; rheumatoid factor; platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming growth factors (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factors-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 factors; immunotoxins; interferons such as interferon-α (e.g., interferon.α.2A), interferon-β, interferon-γ, interferon-λ and composite interferon; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; transporters; homing receptors; addressins; 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 may be natural or recombinant, and include, for example, fusion proteins.

[0384] Examples of polypeptides (e.g., proteins) produced by cells in the implantable elements described herein also include CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (KC), CXCL2 (SDF1a), CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNFB (LTA), TNFC (LTB), TNFSF4, TNFSF5 (CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL 9. IL21, IL3, IL5, IL6, IL11, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL 16. IL32, IL34, IL10, IL22, IL19, IL20, IL24, IL26, IL29, IFNL1, IFNL2, IFNL3, IL28, IFNA1, IFNA2, IFNA4, I FNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNB1, IFNK, 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, Cer 1. Cklf, Clcf1, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlf1, Ctf2, Ebi3, Edn1, Fam3b, Fasl, Fgf2, Flt3l, Gdf10, Gdf 11. 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, Pglyr p1, 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, prostaglandins, leukotrienes, prostacyclin, thromboxane, islet amylin, 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, thyrotropin, thyrotropin-releasing hormone, vasoactive intestinal polypeptide,Androgens, alpha-glucosidase (also called acid maltase), glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine palmitoyltransferase, carnitine and myoadenylate deaminase.

[0385] In some embodiments, the protein is a replacement therapy or replacement protein. In some embodiments, the replacement therapy or replacement protein is a coagulation 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).

[0386] In some embodiments, the cell is engineered to express factor VIII, e.g., recombinant factor VIII. In some embodiments, the cell is derived from human tissue and is engineered to express factor VIII, e.g., recombinant factor VIII. In some embodiments, the recombinant factor VIII is a recombinant factor VIII with a B domain deletion (FVIII-BDD).

[0387] 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 a wild-type FIX protein (FIX-GIF), wherein the GIF variant has a higher specific activity than the corresponding wild-type FIX.

[0388] In some embodiments, the replacement therapy or replacement protein is an enzyme, such as α-galactosidase, α-L-iduronidase (IDUA) or N-sulfoglucosamine sulfohydrolase (SGSH). In some embodiments, the replacement therapy or replacement protein is an enzyme, such as α-galactosidase A (e.g., comprising a naturally occurring human α-galactosidase A amino acid sequence or a variant thereof). In some embodiments, the replacement therapy or replacement protein is a cytokine or an antibody.

[0389] In some embodiments, the therapeutic agent is a sugar, such as a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide. In some embodiments, sugar includes triose, tetrose, pentose, hexose or heptose moieties. In some embodiments, sugar includes linear monosaccharides or cyclized monosaccharides. In some embodiments, sugar includes glucose, galactose, fructose, rhamnose, mannose, arabinose, glucosamine, galactosamine, sialic acid, mannosamine, glucuronic acid, galacturonic acid, mannuronic acid or guluronic acid moieties. In some embodiments, sugar is attached to protein (e.g., N- connected polysaccharides or O- connected polysaccharides). Exemplary sugars include glucose, galactose, fructose, mannose, rhamnose, sucrose, ribose, xylose, sialic acid, maltose, straight-chain starch, inulin, oligofructose, oligogalactose, mannan, lectin, pectin, starch, cellulose, heparin, hyaluronic acid, chitin, amylopectin or glycogen. In some embodiments, the therapeutic agent is a sugar alcohol.

[0390] In some embodiments, therapeutic agent is lipid.Lipid can be hydrophobic or amphipathic, and can form tertiary structure (such as liposome, vesicle or film) or insert in liposome, vesicle or film.Lipid can include fatty acid, glycerolipid, glycerophospholipid, sterol lipid, isopentenol lipid, sphingolipid, glycolipid, polyketide or sphingolipid.The example of lipid produced by cell described herein includes arachidonic acid, docosahexaenoic acid, prostaglandin, leukotriene, thromboxane, eicosanoid, triglyceride, cannabinoid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, ceramide, sphingomyelin, cerebroside, ganglioside, estrogen, androsterone, testosterone, cholesterol, carotenoid, quinone, hydroquinone or ubiquinone.

[0391] In some embodiments, the therapeutic agent is a small molecule. Small molecules may include natural products produced by cells. In some embodiments, small molecule availability is poor or does not meet the Lipinski five rules (a set of guidelines for estimating whether small molecules may become human oral active drugs; see, for example, Lipinski, CA et al. (2001) Adv Drug Deliv46:2-36). Exemplary small molecule natural products include antibacterial drugs (e.g., calumonan, daptomycin, fidaxomycin, fosfomycin, ipamycin, sulphuric acid, mikamycin, mopicin, netilmicin sulfate, teicoplanin, thiomycin, rifamycin, erythromycin, vancomycin), antiparasitic drugs (e.g., artemisinin, ivermectin), anticancer drugs (e.g., doxorubicin, aclarubicin, aminolevulinic acid, arglabin, homoharringtonine, paclitaxel, pentostatin, peplomycin, romidepsin , trabectin, actinomycin D, bleomycin, chromomycin A, daunorubicin, folinic acid, neocarcinogen, streptozotocin, trabectin, vinblastine, vincristine), antidiabetic drugs (e.g., voglibose), central nervous system drugs (e.g., levodopa, galantamine, zicontide), statins (e.g., mevastatin), antifungal drugs (e.g., fumagillin, cyclosporin), 1-deoxynojirimycin and theophylline, sterols (cholesterol, estrogen, testosterone). Other small molecule natural products are described in Newman, DJ And and Cragg, M. (2016) J Nat Prod 79: 629-661 and Butler, MS et al. (2014) Nat Prod Rep 31: 1612-1661.

[0392] In some embodiments, the cell is engineered to synthesize a non-protein or non-peptide small molecule. For example, in one embodiment, the cell can produce a statin (e.g., taurostatin, pravastatin, fluvastatin, or atorvastatin).

[0393] In some embodiments, the therapeutic agent is an antigen (e.g., a viral antigen, a bacterial antigen, a fungal antigen, a plant antigen, an environmental antigen, or a tumor antigen). Antigens are considered by those skilled in the art to have an immunostimulatory effect, that is, they can stimulate an immune response or provide effective immunity to the organism or molecule from which they are derived. Antigens can be nucleic acids, peptides, proteins, sugars, lipids, or combinations thereof.

[0394] Cells (e.g., engineered cells, such as engineered cells described herein) can produce a single therapeutic agent or multiple therapeutic agents. In some embodiments, cells produce a single therapeutic agent. In some embodiments, cell clusters include 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 cells in a cluster produce a single therapeutic agent (e.g., a therapeutic agent described herein). In some embodiments, cells produce multiple therapeutic agents, such as at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 therapeutic agents. In some embodiments, cell clusters include 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 cells in a cluster produce multiple therapeutic agents (e.g., a therapeutic agent described herein).

[0395] The therapeutic agent 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 (e.g., an enzyme). In some embodiments, the therapeutic agent is not secreted or released from the cell, but is maintained in the cell. For example, the therapeutic agent can be an enzyme that participates in the detoxification or metabolism of an unwanted substance, and the detoxification or metabolism of the unwanted substance occurs in the cell.

[0396] In some embodiments, the hydrogel capsules described herein contain 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 of mammalian cells.

[0397] In some embodiments, the hydrogel capsules described herein contain a concentration of about 1 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 2 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 3 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 4 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 5 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 6 M mL-1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 7 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 8 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 9 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 10 M mL -1 In some embodiments, the hydrogel capsules described herein contain about 15 M mL -1 In some embodiments, the hydrogel capsules described herein contain about 20 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 25 M mL -1 In some embodiments, the hydrogel capsules described herein contain about 30 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 35 M mL -1 In some embodiments, the hydrogel capsules described herein contain about 40 M mL -1 In some embodiments, the hydrogel capsules described herein contain about 45 M mL -1 In some embodiments, the hydrogel capsules described herein contain a concentration of about 50 M mL -1 of mammalian cells.

[0398] In some embodiments, the hydrogel capsules described herein contain a concentration of about 1-50 M mL -1 , 1-45 MmL -1 , 1-40 M mL -1 , 1-35 M mL -1 , 1-30 M mL -1 , 1-25 M mL -1 , 1-20 M mL -1 , 1-15 M mL -1 、1-10M mL -1 , 1-5 M mL -1 , 5-50 M mL -1 , 5-45 M mL -1 , 5-40 M mL-1 , 5-35 M mL -1 , 5-30 M mL -1 、5-25M mL -1 , 5-20 M mL -1 , 5-15 M mL -1 , 5-10 M mL -1 , 10-50 M mL -1 、10-45 M mL -1 , 10-40 M mL -1 、10-35 M mL -1 , 10-30 M mL -1 , 10-25 M mL -1 , 10-20 M mL -1 , 10-15 M mL -1 , 15-50 M mL -1 , 15-45 M mL -1 , 15-40 M mL -1 , 15-35 M mL -1 , 15-30 M mL -1 , 15-25 M mL -1 , 15-20 M mL -1 、20-50M mL -1 , 20-45 M mL -1 , 20-40 M mL -1 , 20-35 M mL -1 , 20-30 M mL -1 , or 20-25 M mL -1 between mammalian cells.

[0399] Treatment

[0400] Described herein are methods of preventing or treating a disease, disorder or condition in a subject via administration or implantation of a hydrogel capsule comprising a polysaccharide polymer described herein. In some embodiments, the methods described herein directly or indirectly reduce or alleviate at least one symptom of a disease, disorder or condition. In some embodiments, the methods described herein prevent or mitigate the occurrence of a disease, disorder or condition. In some embodiments, the subject is a human.

[0401] In some embodiments, the disease, disorder, or condition affects a body system, such as the nervous system (e.g., peripheral or central nervous system), vascular system, skeletal system, respiratory system, endocrine system, lymphatic system, reproductive system, or gastrointestinal tract. In some embodiments, the disease, disorder, or condition affects a part of the body, such as the blood, eyes, brain, skin, lungs, stomach, mouth, ears, legs, feet, hands, liver, heart, kidneys, bones, pancreas, spleen, large intestine, small intestine, spinal cord, muscle, ovaries, uterus, vagina, or penis.

[0402] In some embodiments, the disease, disorder or condition is a neurodegenerative disease, diabetes (type 1 or type 2), heart disease, an autoimmune disease, cancer, liver disease, a lysosomal storage disease, a blood coagulation disorder or a coagulation disorder, an orthopedic disorder, an amino acid metabolism disorder.

[0403] In some embodiments, the disease, disorder or condition is an autoimmune disease. In some embodiments, the disease, disorder or condition is diabetes (type 1 or type 2 diabetes). In one embodiment, the subject suffers from or is diagnosed with diabetes (e.g., type 1 or type 2 diabetes). The subject may have any biomarker or other diagnostic criteria associated with diabetes, such as high blood sugar levels (e.g., greater than 300 mg / dL, greater than 400 mg / dL) or high hemoglobin A1C levels (e.g., greater than 5.9% hemoglobin A1C levels, greater than 6.5% hemoglobin A1C levels, greater than 7% hemoglobin A1C levels).

[0404] In some embodiments, the disease, disorder, or condition is not type I diabetes and / or is not type II diabetes.

[0405] In some embodiments, the disease, disorder or condition is a neurodegenerative disease. Exemplary neurodegenerative diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and cerebral palsy (CP), dentatorubral nucleus-globus pallidus Lewy body atrophy (DRPLA), neuronal intranuclear hyaline inclusion disease (NIHID), Lewy body dementia, Down syndrome, Hallervorden-Spatz disease, prion disease, argyrophilic grain dementia, corticobasal degeneration, dementia pugilistica, diffuse neurofibrillary tangles, Gerstmann-Strauss-Scheinker disease, Jacob-Kreutzfeldt disease, Niemann-Pick disease type 3, progressive supranuclear palsy, subacute sclerosing panencephalitis, spinocerebellar ataxia, Pick's disease and dentatorubral nucleus-globus pallidus Lewy body atrophy.

[0406] In some embodiments, the disease, disorder, or condition is an autoimmune disease, such as scleroderma, multiple sclerosis, lupus, or allergy.

[0407] In some embodiments, the disease is a liver disease, such as hepatitis B, hepatitis C, cirrhosis, NASH.

[0408] In some embodiments, the disease, disorder or condition is cancer. Exemplary cancers include leukemia, lymphoma, melanoma, lung cancer, brain cancer (e.g., glioblastoma), sarcoma, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, prostate cancer, or uterine cancer.

[0409] In some embodiments, the disease, disorder, or condition is an orthopedic condition. Exemplary orthopedic conditions include osteoporosis, osteonecrosis, Paget's disease, or a bone fracture.

[0410] In some embodiments, the disease, disorder or condition is a lysosomal storage disease. Exemplary lysosomal storage diseases include Gaucher disease (e.g., type I, type II, type III), Tay-Sachs disease, Fabry disease, Farber disease, Hurler syndrome (also known as mucopolysaccharidosis type I (MPS I)), Hunter syndrome, lysosomal acid lipase deficiency, Niemann-Pick disease, sialic acid storage disease, Sanfilippo syndrome (also known as mucopolysaccharidosis type IIIA (MPS3A)), multiple sulfatase deficiency, Mara-La syndrome, metachromatic leukodystrophy, Krabbe disease, Scheher-Ellison syndrome, Hurler-Schwein syndrome, Sly syndrome, hyaluronidase deficiency, Pompe disease, Danon disease, gangliosidosis, or Morquio syndrome.

[0411] In some embodiments, the disease, disorder or condition is a blood coagulation disorder or coagulation disorder. Exemplary blood coagulation disorders or coagulation disorders include hemophilia (e.g., hemophilia A or hemophilia B), von Willebrand's disease, thrombocytopenia, uremia, Bernard-Soulier syndrome, factor XII deficiency, vitamin K deficiency, or afibrinogenemia.

[0412] In some embodiments, the disease, condition, or disorder is an amino acid metabolism disorder, such as phenylketonuria, tyrosinemia (eg, type 1 or type 2), alkaptonuria, homocystinuria, hyperhomocysteinemia, maple syrup urine disease.

[0413] In some embodiments, the disease, disorder or condition is a fatty acid metabolism disorder, such as hyperlipidemia, hypercholesterolemia, galactosemia.

[0414] In some embodiments, the disease, disorder, or condition is a disorder of purine or pyrimidine metabolism, such as Lesch-Nyhan syndrome.

[0415] The present invention further includes methods for identifying a subject suffering from or suspected of having a disease, disorder or condition as described herein, and after such identification, administering to the subject an implantable element comprising cells (e.g., optionally encapsulated by a closure member and optionally modified by a compound of formula (I) as described herein, or a composition thereof). In one embodiment, the subject is a human. 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 of age, less than 18 years of age, less than 15 years of age, less than 12 years of age, less than 10 years of age, or less than 6 years of age).

[0416] Method of making pellets

[0417] The present disclosure further includes methods for making particles described herein, e.g., particles comprising a first compartment, a second compartment, and a compound of formula (I). In some embodiments where the particles are hydrogel capsules, the method of making the particles comprises contacting a plurality of droplets comprising a first and a second polymer solution (e.g., each comprising a hydrogel-forming polymer) with an aqueous crosslinking solution. The droplets can be formed using any technique known in the art.

[0418] Each compartment of the particle described herein may include an unmodified polymer, a polymer modified by a compound of formula (I), a polymer modified by a clickable cross-linking part, or a blend thereof. In short, when preparing a method for a particle configured as a two-compartment hydrogel capsule, a certain volume of the first polymer solution (for example, including an unmodified polymer, a polymer modified by a compound of formula (I), a polymer modified by a clickable cross-linking part, or a blend thereof, and optionally containing cells) is loaded into the first syringe connected to the inner cavity of a coaxial needle. The first syringe can then be connected to a syringe pump vertically oriented above a container containing an aqueous cross-linking solution, and the aqueous cross-linking solution includes a cross-linking agent, a buffer and an osmotic concentration regulator. A certain volume of the second polymer solution (for example, including an unmodified polymer, a polymer modified by a compound of formula (I), a polymer modified by a clickable cross-linking part, or a blend thereof, and optionally containing cells) is loaded into the second syringe connected to the outer cavity of a coaxial needle. The second syringe can then be connected to a syringe pump horizontally oriented relative to the container containing the cross-linking solution. A high-voltage power generator can then be connected to the top and bottom of the needle. Then the first and second polymer solutions can be squeezed through a syringe using a syringe pump and a power generator, the setting of the syringe being determined to reach the desired drop rate of the polymer solution into the cross-linking solution. Various combinations of needle lumen size, voltage range, flow rate, drop rate, and drop distance can be easily determined by those skilled in the art to produce a 2-compartment hydrogel capsule composition, wherein most (e.g., at least 80%, 85%, 90% or more) capsules are within 10% of the target size and have a sphere-like shape. After exhausting the first and second volumes of polymer solution, the droplets can be allowed to crosslink in the cross-linking solution for a certain amount of time, e.g., about five minutes.

[0419] Exemplary process parameters for preparing millimeter-sized capsules (e.g., 1.5 mm diameter millimeter-sized capsules) compositions include the following. A coaxial needle is placed above the surface of the cross-linked solution at a distance sufficient to provide a drop distance from the needle tip to the surface of the solution. In one embodiment, the distance between the needle tip 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 total flow rate of 0.05 mL / min to 5 mL / min, or 0.05 mL / min to 2.5 mL / min, or 0.05 mL / min to about 1 mL / min, or 0.05 mL / min to 0.5 mL / min, or 0.1 mL / min to 0.5 mL / min. In one embodiment, the first and second polymer solutions are extruded through the needle at a total flow rate of about 0.05 mL / min, 0.1 mL / min, 0.15 mL / min, 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, or 0.5 mL / min. In one embodiment, the flow rates of the first and second polymer solutions through the needle are substantially the same. In one embodiment, the flow rates of the first and second polymer solutions through the needle are different.

[0420] 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 drop rate is achieved. In one embodiment, the drop 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.

[0421] In one embodiment, the number of non-particle debris on the surface of the cross-linking solution is determined. The particles that fall to the bottom of the cross-linking container can then be collected, for example, by transferring the cross-linking solution containing the particles to a separate container, leaving any non-particle debris on the surface of the solution in the original cross-linking container. The removed particles can then be allowed to settle, the cross-linking solution can be removed, and the particles can then be washed one or more times with a buffer (e.g., a HEPES buffer). In one embodiment, one or more aliquots of the resulting particle composition (e.g., a particle preparation) are examined by microscopy to assess the quality of the composition, such as particle defects and the number of satellite particles.

[0422] In some embodiments, the cross-linking solution further comprises a process additive (e.g., a hydrophilic nonionic surfactant). The process additive can reduce the surface tension of the cross-linking 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 Tween ® 20. Tween ® 80, Triton™ X-100, IGEPAL® CA-630, poloxamer 188, or poloxamer 407, or a surfactant having substantially the same chemical and physical properties as listed in the exemplary surfactant table immediately below.

[0423]

[0424] a Hydrophile-lipophile balance

[0425] b Chemical name and synonyms: Polyethylene glycol sorbitan monolaurate, Polyoxyethylene (20) sorbitan monolaurate, Polysorbate 20, Polyoxyethylene 20 sorbitan monododecanoate

[0426] 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)

[0427] 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

[0428] e Chemical name and synonyms: Branched octylphenoxypolyethoxyethanol, octylphenoxypoly(ethyleneoxy)ethanol

[0429] f Chemical name: Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)

[0430] g Chemical name: Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)

[0431] In some embodiments, the process additive is a nonionic 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 Tween® 20 (polysorbate 20) or Triton™ X-100. In one embodiment, the process additive is IGEPAL® CA-630 (polyethylene glycol sorbitan monooleate). In some embodiments, the process additive is poloxamer 188.

[0432] In some embodiments, a process additive (e.g., a surfactant) is present in the cross-linking solution at a concentration of at least 0.0001% or more. In some embodiments, the cross-linking solution comprises at least 0.001%, 0.01%, or 0.1% of a process additive. In some embodiments, the process additive is present in 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.

[0433] In some embodiments, the crosslinking agent includes a single type of divalent cation or a mixture of different types, such as Ba 2+ , Ca 2+ , Sr 2+ In some embodiments, the cross-linking 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 cross-linking agent is, for example, CaCl2 at a concentration of 50 mM to 100 mM. In some embodiments, the cross-linking agent is, for example, SrCl2 at a concentration of 37.5 mM to 100 mM. In some embodiments, the cross-linking 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).

[0434] In some embodiments, the crosslinking agent is SrCl2 and the process additive is Tween ®80 (or a surfactant having substantially the same chemical and physical properties as listed in the exemplary surfactant table) at 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%.

[0435] The type and concentration of the buffer in the aqueous cross-linking solution is selected to maintain the solution pH at approximately neutral, e.g., about 6.5 to about 7.5, about 7.0 to about 7.5, or about 7.0. In one embodiment, the buffer is compatible with the biomaterial (e.g., cells) to be encapsulated in the particles. In some embodiments, the buffer in the aqueous cross-linking solution comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0436] The osmotic pressure regulating agent in the aqueous crosslinking solution is selected to maintain the solution osmotic pressure at a value similar to the osmotic pressure of the polymer solution (in some embodiments, which includes a cell suspension), for example, with a higher or lower osmotic pressure of up to 20%, 10% or 5%. In some embodiments, the osmotic pressure regulating agent is mannitol at a concentration of 0.1 M to 0.3 M.

[0437] In some embodiments, the crosslinking solution comprises 25 mM HEPES buffer, 20 mM BaCl 2、 0.2M mannitol and 0.01% poloxamer 188.

[0438] In some embodiments, the cross-linking solution comprises 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 properties as listed in the Exemplary Surfactant Table for Tween 80.

[0439] In one embodiment, the process additive is poloxamer 188, which is present in a detectable amount in the particle composition (e.g., particle formulation) after the washing step. Poloxamer 188 can be detected by any technique known in the art, such as by partially or completely dissolving the particles in an aliquot of the composition via sodium sulfate precipitation and analyzing the supernatant by LC / MS.

[0440] The reduction in the surface tension of the cross-linked solution can be assessed by any method known in the art, such as by using a contact angle goniometer or a tensiometer, for example via the du Nouy ring method (see, e.g., Davarci et al. (2017) Food Hydrocolloids 62: 119-127).

[0441] Exemplary embodiments

[0442] 1. A polysaccharide polymer comprising:

[0443] (i) a clickable cross-linking portion; and

[0444] (ii) a compound of formula (I):

[0445] (I)

[0446] or a pharmaceutically acceptable salt thereof, wherein:

[0447] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-、-N(R C )C(O)-、-C(O)N(R C )-、-N(R C )N(R D )-、-NCN-、-N(R C )C(O)(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 linked to an attachment group (e.g., an attachment group described herein) and is optionally bound by one or more R 1 replace;

[0448] L 1 and L 3 Each of is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace;

[0449] L 2 is the key;

[0450] M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace;

[0451] P is optionally replaced by one or more R 4 substituted heteroaryl;

[0452] Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 replace;

[0453] 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 replace;

[0454] or R C and R D Together with the nitrogen atom to which they are attached, they form a 6 substituted rings (e.g., 5-7 membered rings);

[0455] 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace;

[0456] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace;

[0457] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0458] x is 1 or 2; and

[0459] y is 2, 3, or 4.

[0460] 2. The polysaccharide polymer of embodiment 1, wherein the clickable cross-linking moiety is covalently bound to a sugar monomer within the polysaccharide polymer.

[0461] 3. The polysaccharide polymer of embodiment 2, wherein the clickable cross-linking moiety is bound to the carboxylate moiety within the sugar monomer.

[0462] 4. A polysaccharide polymer as described in any one of embodiments 1 to 3, wherein the clickable cross-linking portion comprises an alkyl, alkenyl, alkynyl, ester, ketone, amine, azide, cycloalkyl, heterocyclic, aryl or heteroaryl group.

[0463] 5. The polysaccharide polymer of any one of embodiments 1-4, wherein the clickable cross-linking moiety is capable of reacting with a second clickable cross-linking moiety when activated with heat and optionally in the absence of a copper catalyst.

[0464] 6. A polysaccharide polymer as described in any of embodiments 1-5, wherein the clickable cross-linking moieties are present on the polysaccharide polymer at a density of at least about 1%, e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, e.g., as determined by comparison with a reference standard.

[0465] 7. A polysaccharide polymer as described in any of embodiments 1-6, wherein the clickable cross-linking moieties are 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.

[0466] 8. The polysaccharide polymer of any one of embodiments 1-7, wherein the polysaccharide polymer is selected from alginate, hyaluronate and chitosan.

[0467] 9. The polysaccharide polymer of any one of embodiments 1-8, wherein the polysaccharide polymer is alginate.

[0468] 10. The polysaccharide polymer of embodiment 9, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

[0469] 11. The polysaccharide polymer of any one of embodiments 1-8, wherein the clickable cross-linking moiety has a structure of formula (IV):

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

[0471] A is optionally replaced by one or more R 1 substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; T is absent, O, S, NR 53 or C(R 54a )(R 54b );

[0472] R 52 , R 53 , R 54a and R 54b Each of 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;

[0473] R 1 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 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace;

[0474] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace;

[0475] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0476] x is 1 or 2; and

[0477] y is 2, 3, or 4.

[0478] 12. The polysaccharide polymer according to embodiment 11, wherein the compound of formula (IV) is a compound of formula (IV-b):

[0479] (IV-b), or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0480] R 50 , R 51a and R 51b Each of 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; or, R 51a and R 51b Together they form an oxo group;

[0481] R 52a and R 52b Each of the following is independently alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace;

[0482] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and

[0483] Each of m and n is 0, 1, 2, 3, 4, 5 or 6.

[0484] 13. A polysaccharide polymer as described in embodiment 12, wherein m is 2, and R 52a, R 52b , R 51a and R 51b Each of is independently hydrogen.

[0485] 14. A polysaccharide polymer as described in any of embodiments 1-13, wherein the clickable cross-linking portion has a structure selected from Table 4, or a pharmaceutically acceptable salt thereof.

[0486] 15. The polysaccharide polymer of any one of embodiments 1-10, wherein the clickable cross-linking moiety has a structure of formula (V):

[0487] (V), or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0488] Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1-6 R 7 replace;

[0489] R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0490] R 53 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, heterocyclyl, aryl or heteroaryl;

[0491] 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

[0492] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; or

[0493] Two R's 7 Together they may form an aryl ring or a heteroaryl ring.

[0494] 16. The polysaccharide polymer of embodiment 15, wherein the clickable cross-linking moiety has a structure of formula (Va):

[0495] , Formula (Va), or a pharmaceutically acceptable salt or tautomer thereof, wherein: R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h and R 53i Each of the above 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 , cycloalkyl, heterocyclyl, aryl, heteroaryl;

[0496] R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, –C(O)R B1 , –N(R C1 )(R D1 ), cycloalkyl, heterocyclyl, aryl, heteroaryl;

[0497] 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

[0498] Each R 7are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0499] 17. The polysaccharide polymer according to embodiment 16, wherein R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h , R 53i Each of is independently hydrogen.

[0500] 18. A polysaccharide polymer as described in any of embodiments 1-17, wherein the clickable cross-linking portion has a structure selected from Table 5, or a pharmaceutically acceptable salt thereof.

[0501] 19. The polysaccharide polymer of any one of embodiments 1-18, wherein the polysaccharide polymer comprises one of the compound of formula (IV) or the compound of formula (V), or a pharmaceutically acceptable salt thereof.

[0502] 20. The polysaccharide polymer of any one of embodiments 1-18, wherein the polysaccharide polymer comprises each of the compound of formula (IV) and the compound of formula (V), or a pharmaceutically acceptable salt thereof.

[0503] 21. The polysaccharide polymer of any one of embodiments 1-20, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

[0504] 22. A polysaccharide polymer as described in any one of embodiments 1-21, 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.

[0505] 23. The polysaccharide polymer of any one of embodiments 1-22, wherein the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

[0506] 24. A polysaccharide polymer as described in any one of embodiments 1-23, wherein the polysaccharide polymer is alginate, the clickable cross-linking portion is selected from the compounds listed in Table 4 or pharmaceutically acceptable salts thereof, and the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

[0507] 25. A composition comprising the polysaccharide polymer of any one of embodiments 1-24.

[0508] 26. A hydrogel capsule comprising the polysaccharide polymer of any one of embodiments 1-24.

[0509] 27. The hydrogel capsule of embodiment 26, wherein the hydrogel capsule comprises a single compartment comprising the polysaccharide polymer (eg, a polysaccharide polymer described herein).

[0510] 28. The hydrogel capsule of embodiment 26, wherein the hydrogel capsule comprises a plurality of compartments, wherein one of the compartments comprises the polysaccharide polymer (eg, a polysaccharide polymer described herein).

[0511] 29. The hydrogel capsule of embodiment 28, wherein the hydrogel capsule comprises an inner compartment and an outer compartment.

[0512] 30. The hydrogel capsule of embodiment 29, wherein:

[0513] The inner compartment comprises a first polysaccharide polymer comprising the clickable cross-linking moiety;

[0514] The outer compartment comprises a second polysaccharide polymer comprising the clickable cross-linking moiety.

[0515] 31. A hydrogel capsule comprising:

[0516] (i) an inner compartment comprising a first polysaccharide polymer comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0517] Formula (I):

[0518] (I)

[0519] in:

[0520] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-、-N(R C )C(O)-、-C(O)N(R C )-、-N(R C )N(R D )-、-NCN-、-N(R C )C(O)(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 linked to an attachment group (e.g., an attachment group described herein) and is optionally bound by one or more R 1 replace;

[0521] L 1 and L 3 Each of is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace;

[0522] L 2 is the key;

[0523] M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace;

[0524] P is optionally replaced by one or more R 4 substituted heteroaryl;

[0525] Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 replace;

[0526] 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 replace;

[0527] or R C and R D Together with the nitrogen atom to which they are attached, they form a 6substituted rings (e.g., 5-7 membered rings);

[0528] 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace;

[0529] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace;

[0530] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0531] x is 1 or 2; and

[0532] y is 2, 3, or 4; and

[0533] (ii) an outer compartment comprising a second polysaccharide polymer comprising clickable cross-linkable moieties.

[0534] 32. The hydrogel capsule of any one of embodiments 26-31, wherein the polysaccharide polymer (eg, the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from the group consisting of alginate, hyaluronate, and chitosan.

[0535] 33. The hydrogel capsule of any one of embodiments 26-32, wherein the polysaccharide polymer (eg, the first polysaccharide polymer and / or the second polysaccharide polymer) is alginate.

[0536] 34. The hydrogel capsule of any one of embodiments 30-33, wherein the first polysaccharide polymer is alginate.

[0537] 35. The hydrogel capsule of any one of embodiments 30-33, wherein the second polysaccharide polymer is alginate.

[0538] 36. The hydrogel capsule of any one of embodiments 33-35, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

[0539] 37. The hydrogel capsule of any one of embodiments 31-36, wherein the clickable cross-linking moiety has a structure of formula (IV):

[0540] (IV), or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0541] A is optionally replaced by one or more R 1 substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; T is absent, O, S, NR 53 or C(R 54a )(R 54b );

[0542] R 52 , R 53 , R 54a and R 54b Each of 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;

[0543] R 1 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 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace;

[0544] Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace;

[0545] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0546] x is 1 or 2; and

[0547] y is 2, 3, or 4;

[0548] And / or the structure of formula (V):

[0549] (V), or a pharmaceutically acceptable salt or tautomer thereof, wherein:

[0550] Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1-6 R 7 replace;

[0551] R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0552] R 53 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, heterocyclyl, aryl or heteroaryl;

[0553] 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

[0554] Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; or

[0555] Two R's 7 Together they may form an aryl ring or a heteroaryl ring.

[0556] 38. The hydrogel capsule of embodiment 37, wherein the compound of formula (IV) is selected from the compounds in Table 4 or a pharmaceutically acceptable salt thereof.

[0557] 39. The hydrogel capsule of embodiment 37, wherein the compound of formula (V) is selected from the compounds in Table 5 or a pharmaceutically acceptable salt thereof.

[0558] 40. The hydrogel capsule of any one of embodiments 31-39, wherein the outer compartment comprises a first polysaccharide polymer comprising a compound of formula (IV) and a second polysaccharide polymer comprising a compound of formula (V).

[0559] 41. The hydrogel capsule of any one of embodiments 31-40, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

[0560] 42. The hydrogel capsule of any one of embodiments 31-41, 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.

[0561] 43. The hydrogel capsule of any one of embodiments 31-42, wherein the compound of formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

[0562] 44. The hydrogel capsule of any one of embodiments 31-43, wherein the hydrogel capsule has a diameter between 0.1 mm and 5 mm.

[0563] 45. The hydrogel capsule of any one of embodiments 31-44, wherein the hydrogel capsule has a diameter between 1 mm and 5 mm.

[0564] 46. ​​The hydrogel capsule of any one of embodiments 31-45, wherein the hydrogel capsule has a diameter between 1 mm and 2.5 mm.

[0565] 47. The hydrogel capsule of any one of embodiments 31-46, wherein the hydrogel capsule encapsulates cells.

[0566] 48. The hydrogel capsule of embodiment 47, wherein the cells produce a therapeutic agent.

[0567] 49. The hydrogel capsule of embodiment 48, wherein the therapeutic agent is a protein, such as a hormone, a coagulation factor, an antibody, or an enzyme.

[0568] 50. The hydrogel capsule of any one of embodiments 31-49, wherein the hydrogel capsule is formulated for implantation into a subject (eg, into the intraperitoneal (IP) space, the abdominal cavity, the omentum, the lesser sac, subcutaneous fat).

[0569] 51. The hydrogel capsule of any one of embodiments 31-50, wherein the implantable element is formulated for implantation into the IP space of a subject.

[0570] 52. A composition comprising the hydrogel capsule of any one of embodiments 31-51.

[0571] 53. A method of producing a hydrogel capsule comprising the polysaccharide polymer of any one of embodiments 1-24.

[0572] 54. A method of improving 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.

[0573] 55. A method as described in embodiment 54, wherein the means for ionically crosslinking the polysaccharide polymer comprises using divalent cations (e.g., Ba 2+、 Ca 2+ , Sr 2+ ).

[0574] 56. A method as described in any of embodiments 54-55, wherein the means for covalently cross-linking the polysaccharide polymer comprises the use of a clickable cross-linking moiety (e.g., a combination of an azide and an alkyne (e.g., DBCO, or a pharmaceutically acceptable salt thereof)).

[0575] 57. The polysaccharide polymer of any one of embodiments 1-24, wherein the polysaccharide retains additional carboxylic acid groups after adding the clickable cross-linking moiety.

[0576] 58. The polysaccharide polymer of any one of embodiments 1-24 or 57, wherein the polysaccharide is not reduced (eg, not treated with a reducing agent) prior to modification with a clickable cross-linking moiety.

[0577] 59. The polysaccharide polymer of any one of embodiments 1-24 or 57, wherein the polysaccharide polymer is not oxidized (eg, not treated with an oxidizing agent) prior to modification with a clickable cross-linking moiety.

[0578] 60. A polysaccharide polymer as described in any of embodiments 1-24 or 57-59, wherein the clickable cross-linking portion is non-hydrolyzable.

[0579] 61. The polysaccharide polymer of any one of embodiments 1-24 or 57-60, wherein the compound of formula (I) is non-hydrolyzable.

[0580] 62. A polysaccharide polymer as described in any of embodiments 1-24 or 57-61, wherein the clickable cross-linking moiety is not a thiol or vinyl sulfone.

[0581] Example

[0582] In order to more fully understand the invention described herein, the following examples are set forth.The 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 be interpreted in any way as limiting their scope.

[0583] The compounds provided herein, modified polymers, implantable elements and compositions thereof can be prepared from readily available starting materials using modifications of the specific synthetic schemes well known to those skilled in the art as described below. 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 may also be used unless otherwise specified. Optimal reaction conditions may vary according to the specific reactants or solvents used, but such conditions may be determined by those skilled in the art through conventional optimization procedures.

[0584] In addition, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent some functional groups from carrying out undesirable reactions. The selection of protecting groups applicable to specific functional groups and the appropriate conditions for protection and deprotection is 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 edition, Wiley, New York, 1991 and in the references cited therein.

[0585] Any of the strategies described below may be used to prepare the exemplary compounds, modified polymers, implantable devices, and compositions of the invention.

[0586] Example 1: Synthesis of Sodium Alginate Modified with Azide Clickable Crosslinking Moieties and Defibrotizing Compounds

[0587]

[0588] In this embodiment, alginate polymers containing azide clickable cross-linking moieties were synthesized. NovaMatrix PRONOVA™ 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-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 to 300 rpm and the batch temperature was adjusted to 25 ° C. 4- (4,6-dimethoxy-1,3,5-triazine-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 the addition was complete, the batch was heated to 35 ° C within 1 hour and kept at 35 ° C for 15 hours, then cooled to 25 ° C. Once the reaction was complete, the batch was filtered through a cyanosilica pad and then purified by tangential flow filtration (10 kDa molecular weight cut-off). First, 10 volume exchanges were performed with normal saline, followed by 10 volume exchanges with endotoxin-free water to purify the solution.

[0589] After purification, the solution is concentrated to a refractive index value of 1.3360 and loaded back into a 400 mL EasyMax reactor. The stirring speed is set to 300 rpm, and the batch temperature is adjusted to 25 ° C. In order to be added on the azide clickable cross-linking part, exemplary reaction conditions are as follows. In a separate sterile container, 1-amino-11-azido-3,6,9-trioxa undecane (1.15 g, 5.28 mmol) is weighed and dissolved in 1M MES pH 7.0 buffer (7.5 mL), then loaded into the reactor. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) is 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 the loading was complete, the reaction mixture was heated to 35°C within 1 hour and maintained at 35°C for 15 hours before being cooled to 25°C. The reaction mixture was purified via tangential flow filtration (10 kDa molecular weight cutoff) with 10-fold volume exchange with brine. After purification, the solution was concentrated to a refractive index value of 1.3380.

[0590] Example 2: Synthesis of sodium alginate modified with DBCO clickable cross-linking moieties

[0591]

[0592] In this embodiment, alginate polymers comprising DBCO clickable cross-linking moieties can be synthesized according to the exemplary procedures outlined below. Nova Matrix PRONOVA™ UP LG20 (300 g; 1.25% w / w in water, 3.75 g sodium alginate) is weighed into a 400 mL EasyMax reactor equipped with an overhead stirrer. In a separate sterile container, azadibenzocyclooctyne-amine (2.06 g, 5.28 mmol) is weighed and dissolved in 1M MES pH 7.0 buffer (7.5 mL) and then loaded into the reactor. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (1.61 g, 5.82 mmol) is weighed in a sterile container and dissolved in 1M 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 within 1 hour and maintained at 35°C for 15 hours before being cooled to 25°C. The reaction mixture was purified via tangential flow filtration (10 kDa molecular weight cutoff) with 10-fold volume exchange with brine. After purification, the solution was concentrated to a refractive index value of 1.3380.

[0593] Example 3: Synthesis of dual cross-linked alginate polymers via copper-free click chemistry

[0594]

[0595] To form covalently cross-linked alginate, an azide cross-linker and a DBCO cross-linker can be coupled together according to the exemplary protocol outlined herein: an alginate polymer comprising compound 301 and an alginate polymer comprising compound 304 (molar ratio 1:3) 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 cutoff) with 10-fold volume exchange with saline. After purification, the solution is concentrated to a refractive index value of 1.3380.

[0596] Alginates comprising other combinations of azide crosslinkers and DBCO crosslinkers (eg, compounds of Tables 4 and 5) are also contemplated.

[0597] Example 4: Synthesis of Exemplary Double Cross-linked Alginate Hydrogel Capsules

[0598] Before manufacturing a compartment or two compartment alginate hydrogel capsule, buffer and alginate solution are sterilized by using aseptic process via 0.2-μm filter filtration. In order to prepare the particle of the two compartment hydrogel capsule constructed as diameter about 1.5 mm, electrostatic droplet generator is arranged as follows: ES series 0-100 kV, 20 watts of high voltage generators (EQ series, Matsusada, NC, USA) are connected to the top and bottom of coaxial needle (the inner chamber of 22G, the outer chamber of 18G, Paragon). The inner chamber is attached to the first 5-ml Luer lock syringe (BD, NJ, USA), and the syringe is connected to the vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA). The outer chamber is connected to the second 5-ml Luer lock syringe via Luer connector, and the syringe is connected to the second syringe pump (Pump 11 PicoPlus) of horizontal orientation. The two syringe pumps moved the first and second alginate solutions from the syringes through the two lumens of the coaxial needle, and a single droplet containing the two alginate solutions was extruded from the needle into a glass dish containing the crosslinking solution. Each PicoPlus syringe pump was set to 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 remained set to 10 ml / h.

[0599] For the fabrication of both two-compartment and one-compartment dual cross-linked alginate hydrogel capsules, after extruding the required volume of alginate solution, the alginate droplets were ionically cross-linked in a cross-linking solution containing 25 mM HEPES buffer, 20 mM BaCl2, and 0.2 M mannitol for 5 minutes. In some experiments, the cross-linking solution also contained 0.01% poloxamer 188. The capsules that fell to the bottom of the cross-linking container were collected by pipetting into a conical tube. After the capsules settled in the tube, the cross-linking buffer was removed and the capsules were washed. The capsules were washed four times with HEPES buffer and then suspended in

[0600] In some experiments, the quality of the capsules in the two-compartment or one-compartment capsule compositions was checked. An aliquot containing at least 200 capsules was taken from the composition and transferred to a well plate, and the quality of the entire aliquot was checked by counting the number of spherical capsules in the total by light microscopy.

[0601] In some experiments, the mechanical strength of capsules in two-compartment capsule compositions was examined using a texture analyzer to determine the initial rupture force as described herein above.

[0602] Example 5: Preparation of two-compartment hydrogel capsules containing modified polysaccharide polymers

[0603] Using the method of Example 4, a two-compartment hydrogel capsule can be synthesized from the alginate polymer described in Example 2. As a non-limiting example, a two-compartment hydrogel comprising an inner compartment and an outer compartment of the doubly cross-linked alginate of Example 2 can be synthesized according to this method.

[0604] Example 6: Encapsulation of mammalian cells in hydrogel capsules comprising modified polysaccharide polymers

[0605] Exemplary mammalian cells can be...

Claims

1. A polysaccharide polymer comprising: (i) a clickable cross-linking portion; and (ii) a compound of formula (I): (I)， or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-、-N(R C )C(O)-、-C(O)N(R C )-、-N(R C )N(R D )-、-NCN-、-N(R C )C(O)(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 linked to an attachment group (e.g., an attachment group described herein) and is optionally bound by one or more R 1 replace; L 1 and L 3 Each of is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace; L 2 is the key; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace; P is optionally replaced by one or more R 4 substituted heteroaryl; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 replace; 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 replace; or R C and R D Together with the nitrogen atom to which they are attached, they form a 6 substituted rings (e.g., 5-7 membered rings); 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace; Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

2. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety is covalently bound to a sugar monomer within the polysaccharide polymer.

3. The polysaccharide polymer of claim 2, wherein the clickable cross-linking moiety is bound to a carboxylate moiety within the sugar monomer.

4. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety comprises an alkyl, alkenyl, alkynyl, ester, ketone, amine, azide, cycloalkyl, heterocyclyl, aryl, or heteroaryl group.

5. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety is capable of reacting with a second clickable cross-linking moiety when activated with heat and optionally in the absence of a copper catalyst.

6. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moieties are present on the polysaccharide polymer at a density of at least about 1%, e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, e.g., as determined by comparison to a reference standard.

7. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moieties are present on the polysaccharide polymer at a density between 1% and 10%, such as 1%-8%, 1%-6%, or 1%-4%, such as determined by comparison with a reference standard.

8. The polysaccharide polymer of claim 1, wherein the polysaccharide polymer is selected from the group consisting of alginate, hyaluronate and chitosan.

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

10. The polysaccharide polymer of claim 9, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

11. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety has a structure of formula (IV): (IV), or a pharmaceutically acceptable salt or tautomer thereof, wherein: A is optionally replaced by one or more R 1 substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; T is absent, O, S, NR 53 or C(R 54a )(R 54b ); R 52 , R 53 , R 54a and R 54b Each of 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; R 1 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 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace; Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

12. The polysaccharide polymer according to claim 11, wherein the compound of formula (IV) is a compound of formula (IV-b): (IV-b), or a pharmaceutically acceptable salt or tautomer thereof, wherein: R 50 , R 51a and R 51b Each of 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; or, R 51a and R 51b Together they form an oxo group; R 52a and R 52b Each of the following is independently alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and Each of m and n is 0, 1, 2, 3, 4, 5 or 6.

13. The polysaccharide polymer of claim 12, wherein m is 2, and R 52a , R 52b , R 51a and R 51b Each of is independently hydrogen.

14. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety has a structure selected from Table 4, or a pharmaceutically acceptable salt thereof.

15. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety has a structure of formula (V): (V), or a pharmaceutically acceptable salt or tautomer thereof, wherein: Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1-6 R 7 replace; R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R 53 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, 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 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; or Two R's 7 Together they may form an aryl ring or a heteroaryl ring.

16. The polysaccharide polymer of claim 15, wherein the clickable cross-linking moiety has a structure of formula (Va): , Formula (Va), or a pharmaceutically acceptable salt or tautomer thereof, wherein: R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h and R 53i Each of the above 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 , cycloalkyl, heterocyclyl, aryl, heteroaryl; R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, –C(O)R B1 , –N(R C1 )(R D1 ), cycloalkyl, heterocyclyl, aryl, 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.

17. The polysaccharide polymer according to claim 16, wherein R 53a , R 53b , R 53c , R 53d , R 53e , R 53f , R 53g , R 53h , R 53i Each of is independently hydrogen.

18. The polysaccharide polymer of claim 1, wherein the clickable cross-linking moiety has a structure selected from Table 5, or a pharmaceutically acceptable salt thereof.

19. The polysaccharide polymer of claim 1, wherein the polysaccharide polymer comprises one of the compound of formula (IV) or the compound of formula (V), or a pharmaceutically acceptable salt thereof.

20. The polysaccharide polymer of any one of claims 1-18, wherein the polysaccharide polymer comprises each of the compound of formula (IV) and the compound of formula (V), or a pharmaceutically acceptable salt thereof.

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

22. 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.

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

24. The polysaccharide polymer of claim 1, wherein the polysaccharide polymer is alginate, the clickable cross-linking moiety is selected from the compounds listed in Table 4 or pharmaceutically acceptable salts thereof, and the compound of formula (I) is compound 101 or a pharmaceutically acceptable salt thereof.

25. A composition comprising the polysaccharide polymer of any one of claims 1-24.

26. A hydrogel capsule comprising the polysaccharide polymer according to any one of claims 1 to 24.

27. The hydrogel capsule of claim 26, wherein the hydrogel capsule comprises a single compartment comprising the polysaccharide polymer (eg, a polysaccharide polymer described herein).

28. The hydrogel capsule of claim 26, wherein the hydrogel capsule comprises a plurality of compartments, wherein one of the compartments comprises the polysaccharide polymer (eg, a polysaccharide polymer described herein).

29. The hydrogel capsule of claim 26, wherein the hydrogel capsule comprises an inner compartment and an outer compartment.

30. The hydrogel capsule of claim 29, wherein: The inner compartment comprises a first polysaccharide polymer comprising the clickable cross-linking moiety; The outer compartment comprises a second polysaccharide polymer comprising the clickable cross-linking moiety.

31. A hydrogel capsule comprising: (i) an inner compartment comprising a first polysaccharide polymer comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof: Formula (I): (I)， in: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-、-N(R C )C(O)-、-C(O)N(R C )-、-N(R C )N(R D )-、-NCN-、-N(R C )C(O)(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 linked to an attachment group (e.g., an attachment group described herein) and is optionally bound by one or more R 1 replace; L 1 and L 3 Each of is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace; L 2 is the key; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace; P is optionally replaced by one or more R 4 substituted heteroaryl; Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 replace; 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, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 6 replace; or R C and R D Together with the nitrogen atom to which they are attached, they form a 6 substituted rings (e.g., 5-7 membered rings); 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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace; Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4; and (ii) an outer compartment comprising a second polysaccharide polymer comprising clickable cross-linkable moieties.

32. The hydrogel capsule of claim 26, wherein the polysaccharide polymer (eg, the first polysaccharide polymer and / or the second polysaccharide polymer) is selected from the group consisting of alginate, hyaluronate, and chitosan.

33. The hydrogel capsule of claim 26, wherein the polysaccharide polymer (eg, the first polysaccharide polymer and / or the second polysaccharide polymer) is alginate.

34. The hydrogel capsule of claim 30, wherein the first polysaccharide polymer is alginate.

35. The hydrogel capsule of claim 30, wherein the second polysaccharide polymer is alginate.

36. The hydrogel capsule of any one of claims 34-35, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

37. The hydrogel capsule of claim 26, wherein the clickable cross-linking moiety has a structure of formula (IV): (IV), or a pharmaceutically acceptable salt or tautomer thereof, wherein: A is optionally replaced by one or more R 1 substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; T is absent, O, S, NR 53 or C(R 54a )(R 54b ); R 52 , R 53 , R 54a and R 54b Each of 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; R 1 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 、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, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R 7 replace; Each R A1 , R B1 , R C1 , R D1 , R E1 and R F1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4; And / or the structure of formula (V): (V), or a pharmaceutically acceptable salt or tautomer thereof, wherein: Ring M 1 and M 2 Each of the groups is independently aryl or heteroaryl, each of which is optionally substituted by 1-6 R 7 replace; R 52 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , –C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl or heteroaryl; R 53 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, 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 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; or Two R's 7 Together they may form an aryl ring or a heteroaryl ring.

38. The hydrogel capsule of claim 36, wherein the compound of formula (IV) is selected from the compounds in Table 4 or a pharmaceutically acceptable salt thereof.

39. The hydrogel capsule of claim 36, wherein the compound of formula (V) is selected from the compounds in Table 5 or a pharmaceutically acceptable salt thereof.

40. The hydrogel capsule of claim 30, wherein the outer compartment comprises a first polysaccharide polymer comprising a compound of formula (IV) and a second polysaccharide polymer comprising a compound of formula (V).

41. The hydrogel capsule of claim 30, wherein the compound of formula (I) has a structure selected from Table 3, or a pharmaceutically acceptable salt thereof.

42. The hydrogel capsule of claim 30, 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.

43. The hydrogel capsule of claim 30, wherein the compound of formula (I) is Compound 101 or a pharmaceutically acceptable salt thereof.

44. The hydrogel capsule of claim 30, wherein the hydrogel capsule has a diameter between 0.1 mm and 5 mm.

45. The hydrogel capsule of any one of claims 30-43, wherein the hydrogel capsule has a diameter between 1 mm and 5 mm.

46. ​​The hydrogel capsule of any one of claims 30-44, wherein the hydrogel capsule has a diameter between 1 mm and 2.5 mm.

47. The hydrogel capsule of any one of claims 30-45, wherein the hydrogel capsule encapsulates cells.

48. The hydrogel capsule of claim 47, wherein the cells produce a therapeutic agent.

49. The hydrogel capsule of claim 48, wherein the therapeutic agent is a protein, such as a hormone, a coagulation factor, an antibody, or an enzyme.

50. The hydrogel capsule of claim 30, wherein the hydrogel capsule is formulated for implantation into a subject (eg, implantation into the intraperitoneal (IP) space, the abdominal cavity, the omentum, the lesser omental bursa, subcutaneous fat).

51. The hydrogel capsule of claim 30, wherein the implantable element is configured for implantation into the IP space of a subject.

52. A composition comprising the hydrogel capsule of any one of claims 26-51.

53. A method of producing a hydrogel capsule comprising the polysaccharide polymer of any one of claims 1-24.

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

55. The method of claim 54, wherein the means for ionically crosslinking the polysaccharide polymer comprises using divalent cations (e.g., Ba 2+ , Ca 2+ , Sr 2+ ).

56. The method of any one of claims 54-55, wherein the means for covalently cross-linking the polysaccharide polymer comprises the use of a clickable cross-linking moiety (e.g., a combination of an azide and an alkyne such as DBCO or a pharmaceutically acceptable salt thereof).

57. A method for treating a disease, disorder or condition in a subject, comprising administering to the subject the hydrogel capsule of any one of claims 26-51 or the composition of claim 52, thereby treating the disease, disorder or condition in the subject.

58. The method of claim 57, wherein the disease, disorder or condition is diabetes (eg, type 1 diabetes).

59. The method of claim 57, wherein the disease, disorder or condition is not diabetes (eg, type 1 diabetes).

60. The method of claim 57, wherein the subject is a human.

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