Microcapsule crosslinking

Abstract

Compositions and methods for hydrogel formation, such as microcapsule hydrogel shell formation, are disclosed herein. Methods and compositions herein yield microcapsule populations having predictable microcapsule properties such as shell widths and microcapsule diameters, and may comprise bioparticles that may be analyzed using reactions performed in series. Approaches using biocompatible catalysis of hydrogel formation, as well as uncatalyzed hydrogel crosslinking approaches, are disclosed herein.

Description

MICROCAPSULE CROSSLINKINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This document claims the benefit of priority to US Prov Ser No. 63 / 723,706, filed November 22, 2024, the contents of which are hereby incorporated by reference in their entirety. BACKGROUND

[0002] Hydrogels are particularly suitable for bioanalyte analysis. As solids, hydrogels maintain structural integrity so as to be capable of containing bioanalytes or target analytes in the interior of aqueous core microcapsules or solid hydrogel beads. At the same time, their porosity allows sequential delivery of reagents such that iterative reaction steps may be performed on contained bioparticles.

[0003] Hydrogel microcapsules have been developed to allow sequential iterative delivery of reagents to contained bioparticles, facilitating a broad range of chemical manipulations and biological approaches. Examples of this technology are disclosed in, for example, W02023 / 099610, published June 8, 2023, which is hereby incorporated by reference in its entirety. Orthogonal chemistries for hydrogel formation can be used in combination, simultaneously or sequentially, to modulate the hydrogel properties, such as permeability to biomolecules.

[0004] However, there remains a need for a broad range of hydrogel formation chemistries, so as to facilitate a hydrogel formation under a broad range of reaction conditions, reagents and catalysts, so as to control microcapsule population parameters and biomolecule stresses pursuant to microcapsule formation.SUMMARY

[0005] Disclosed herein are compositions and methods related to hydrogel formation, as may be used in the partitioning and iterative manipulation of bioanalytes or target analytes.

[0006] Disclosed herein are methods of synthesizing a hydrogel, such methods comprising one or more of the steps of localizing, in a partition, a population of shell precursor molecules, each comprising a shell structural component and a crosslinking moiety comprising at least one nonsaturated bond, and inducing crosslinking at the non-saturated bond of the crosslinking moieties. The non-saturated bond is in some cases a carbon-carbon bond, while in other cases the nonsaturated bond is a carbon-heteroatom bond or a heteroatom-heteroatom bond. The crosslinking moiety in some cases comprises a linear carbon chain comprising the non-saturated carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bond. Alternatively, in some cases the crosslinking moiety comprises a ring comprising the non-saturated carbon-carbon, carbonheteroatom or heteroatom-heteroatom bond. The crosslinking moiety in some cases comprises a nitrogen atom; alternatively or in combination, the crosslinking moiety comprises a sulfur atom. In some cases the crosslinking moiety comprises an oxygen atom. In some cases the crosslinking moiety comprises a phosphorous atom. The partition is often a droplet in an emulsion. The nonsaturated carbon-carbon bond is often a non-conjugated alkene, though allenes and alkynes are also contemplated and consistent with the disclosure herein. The non-saturated carbon-carbon bond is in some cases conjugated to a carbonyl group. As mentioned above, the non-saturated carbon-carbon bond is in some cases a non-conjugated alkyne. Often, the structural component comprises a carbohydrate, such as dextran. The shell precursor molecules are often localized to the interior of the droplet and may be uniformly distributed throughout the droplet or localized at the perimeter of the droplet. The shell precursor molecules in some cases further comprise a hydrophobic hydrocarbon chain, such as a butyryl chain. The hydrophobic carbon chain in some cases comprises at least 2 carbon atoms. In some cases the hydrophobic carbon chain comprises no more than 6 carbon atoms. Exemplary hydrophobic carbon chains comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbons. The hydrophobic carbon chain is in some cases linear. Alternately or in combination, the hydrophobic carbon chain is in some cases branched, while in some cases the hydrophobic carbon chain is cyclic or aromatic. Some methods further comprise localizing a population of core precursor molecules to the partition, such as core precursors that do not comprise a crosslinking moiety, or do not comprise a hydrophobic carbon chain, or comprise neither a crosslinking moiety nor a hydrophobic group. In some methods, the core precursors each comprise a shell structural component that is not eligible for crosslinking. The shell precursors and the core precursors in some cases each comprise a common structural component, such as a polysaccharide, for example dextran among others. In some methods the shell precursors and the core precursors comprise distinct structural components. In some methods inducing crosslinking comprises irradiation. Alternatively or in combination, inducing crosslinking comprises introducing a free radical in the partition. Alternatively or in combination, inducing crosslinking comprises inducing crosslinking comprises introducing an oxidative stress in the partition. Alternatively or in combination, inducing crosslinking comprises introducing a thiol in the partition. In some methods the droplet comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Some embodiments comprise inducing crosslinking comprises introducing LAP in the partition. In some cases, the methods comprise colocalizing a biological component or other analyte to the partition. In various embodiments of the method herein, the hydrogel forms a microcapsule having an aqueous interior and a shell. Some methods further comprise degrading the structural component to release contents of the microcapsule. The degrading variously comprises contacting to an enzyme, such as a carbohydrate degrading enzyme, for example a dextranase. In some cases the degrading comprises physical disruption of the such as via ball-milling or freeze-thaw treatment. In some methods herein, crosslinking is facilitated because the non-saturated carbon-carbon bond is strained. In some cases the strained non-saturated carbon-carbon bond is a constituent of a cyclic moiety. The cyclic moiety in some cases comprises at least 8 atoms. In some cases the strained non-saturated carbon-carbon bond is an allene. In some cases the strained non-saturated carbon-carbon bond is an alkene. In some cases the strained non-saturated carbon-carbon bond is an alkyne. In some cases the strained non-saturated carbon-carbon bond is a bond in a norbornene moiety. In some cases the strained non-saturated carbon-carbon bond is a bond in a cyclooctyne moiety, such as a dibenzocyclooctyne moiety. In some cases the population of shell precursor molecules comprises a norbornene moiety, such that in some cases the population of shell precursor molecules comprises dextran having a crosslinking moiety comprising norbornene moiety, or in some cases the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising nitrone moiety, or alternatively the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising tetrazine moiety. In some aspects the population of shell precursor molecules comprises molecules comprising an alkyne crosslinking moiety, such as a bond in a cyclooctyne moiety, such as a dibenzocyclooctyne moiety or a pentynoate crosslinking moiety. In some aspects the population of shell precursor molecules further comprises molecules comprising a bis-azido-PEG crosslinking moiety. In some aspects the method comprises localizing, in the partition, a bis-azido-PEG crosslinking moiety. In some cases the population of shell precursor molecules comprises molecules comprising an azide-containing moiety, such as a 4-(azidomethyl)benzoate crosslinking moiety or an azido-PEG-carboxylate crosslinking moiety. In some methods the population of shell precursor molecules comprises dextran modified by butyryl and methacryloyl moieties. In some cases the shell structural component comprises dextran and the crosslinking moiety comprisesmaleimide, such as protected maleimide or deprotected maleimide. In some methods the population of shell precursor molecules directly react with one another to form a hydrogel, while in alternatives the population of shell precursor molecules are linked by bridging molecules to form a hydrogel, such as bridging molecules that comprise dithiol, for example 1,4-dithiothreitol (DTT). In some cases, the bridging molecules comprise diazide, such as bis-azido-PEG.

[0007] Also disclosed herein are emulsions, such as emulsions for which a droplet of the emulsion comprises a shell precursor molecule, the shell precursor molecule comprising a shell structural component, and a crosslinking moiety comprising a non-saturated bond, such as a nonsaturated bond is a carbon-carbon bond, a carbon-heteroatom bond or a heteroatom-heteroatom bond, for example a non-saturated bond comprising a nitrogen atom, an oxygen atom, a sulfur atom, or a phosphorous atom. The non-saturated bond is in some cases strained, so as to render it more reactive. In some aspects the non-saturated bond is an alkene, or an alkyne. In some emulsions, the shell precursor molecule is selected from a molecule of Fig. 1 or another Figure herein.

[0008] In some emulsions, the shell precursor molecule comprises a cyclic moiety, which in some cases is planar. A cyclic moiety may comprise at least 8 atoms at least 9 atoms, or for example 6, 7, 8, 9, 10, 11, 12 or more atoms. The cyclic moiety comprises in some cases fused benzene rings. An exemplary cyclic moiety is cyclooctyne, for example dibenzocyclooctyne. The shell structural component in some cases comprises dextran and the crosslinking moiety comprises a non-saturated carbon-carbon bond comprising a strained alkene or alkyne. In some cases, the crosslinking moiety comprises norbornene moiety. The droplet in some cases further comprises a dithiol, such as 1,4-dithiothreitol (DTT). In some emulsions, the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising a non-saturated carbonheteroatom and heteroatom-heteroatom bonds comprising tetrazine moiety. In some aspects, the shell structural comprises dextran and the crosslinking moiety comprises a non-saturated carboncarbon bond comprises pentynoate. In some aspects the droplet further comprises DTT. In some aspects the droplet further comprises bis-azido-PEG. In some aspects the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising an azide moiety, such as a diazide harboring molecule. In some aspects the droplet further comprises a second shellprecursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising non-saturated carbon-carbon, carbonheteroatom, heteroatom-heteroatom bonds comprising a 4-(azidomethyl)benzoate moiety. In some aspects the shell structural component comprises dextran and a crosslinking moiety comprising non-saturated carbon-carbon and carbon-heteroatom bonds comprises methacryloyl, and wherein the shell precursor molecule further comprises a butyryl moiety. In some aspects the droplet further comprises DTT. In some aspects the dextran and a crosslinking moiety comprising a non-saturated bond comprises maleimide in a protected or deprotected form. In some aspects the droplet further comprises DTT. In some aspects the droplet further comprises LAP. In some aspects the droplet further comprises a radical. In some aspects the droplet further comprises a redox reagent. In some aspects the droplet is heated to at least 30C for at least 15 minutes. In some aspects the droplet is heated to at least 60C for at least 8 hours. In some aspects the droplet is heated to at least 100C for at least 15 minutes. In some aspects the droplet further comprises a biomolecule.

[0009] Also disclosed herein are microcapsule populations, such as those generated by any of the methods disclosed herein or generated using any one of the compositions herein.

[0010] Also disclosed are compositions comprising the constituents of a droplet of an emulsion of any one of the embodiments herein.

[0011] Consistent with the above, disclosed herein are methods of synthesizing hydrogel precursor compositions and the composition consistent therewith. Some such methods comprise modifying a hydrogel precursor, by for example adding butyl moieties to constituents of a polymer such as dextran, such that its hydrophobicity after addition of a crosslinking moiety such as a moiety having an unsaturated carbon bond is sufficient to allow the hydrogel precursor to accumulate at the periphery or perimeter of a water-in-oil microcapsule precursor droplet, without becoming immiscible with the precursor to the microcapsule’s aqueous or liquid interior, and without becoming miscible with the oil carrier of the emulsion.INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 presents a range of dextran shell hydrogel constituent options.

[0014] Fig. 2A presents a hydrogel precursor composition.

[0015] Fig. 2B presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0016] Fig. 3A presents a hydrogel precursor composition.

[0017] Fig. 3B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0018] Fig. 3C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0019] Fig. 4A presents a hydrogel precursor composition.

[0020] Fig. 4B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0021] Fig. 4C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0022] Fig. 5A presents a hydrogel precursor composition.

[0023] Fig. 5B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0024] Fig. 5C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0025] Fig. 6 A presents a hydrogel precursor composition.

[0026] Fig. 6B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0027] Fig. 6C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0028] Fig. 7A presents a hydrogel precursor composition.

[0029] Fig. 7B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0030] Fig. 7C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0031] Fig. 8 A presents a hydrogel precursor composition.

[0032] Fig. 8B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0033] Fig. 8C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0034] Fig. 9 A presents a hydrogel precursor composition.

[0035] Fig. 9B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0036] Fig. 9C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0037] Fig. 10A presents a hydrogel precursor composition.

[0038] Fig. 10B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0039] Fig. 10C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0040] Fig. 11 A presents a hydrogel precursor composition.

[0041] Fig. 1 IB presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0042] Fig. 11C presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0043] Fig. 12A presents a hydrogel precursor composition.

[0044] Fig. 12B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0045] Fig. 12C presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above using a protocol incorporating shaking.

[0046] Fig. 12D presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above, with shaking.

[0047] Fig. 13A presents a hydrogel precursor composition.

[0048] Fig. 13B presents an image of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0049] Fig. 14A presents a hydrogel precursor composition.

[0050] Fig. 14B presents parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.

[0051] Fig. 14C and Fig. 14D present parameters of a hydrogel microcapsule population formed through use of the hydrogel precursor composition above.DETAILED DESCRIPTIONIntroduction and hydrogel technology

[0052] Disclosed herein are compositions and methods related to bead or microcapsule formation, particularly microcapsule formation from compartmentalized reagent mixtures such as emulsion droplets. Through approaches herein, one may form solid hydrogel beads or solid microcapsules comprising hydrogel shells and aqueous or liquid cores, and in some cases further comprising reaction substrates or analytes for downstream analysis. Hydrogel particles may then be removed from the partitions such as those formed by an oil emulsion carrier, and subjected to downstream reactions or analysis, such as counting, imaging, sorting, or the sequential addition of one or more reagents across the hydrogel constituent of the hydrogel particles.

[0053] Disclosed herein are a broad range of chemistries and compositions suitable for hydrogel particle formation such as hydrogel bead or hydrogel microcapsule formation, so as to facilitate downstream manipulation such as that discusses above or otherwise contemplated in the art. Many such chemistries comprise reactions involving an unsaturated carbon bond to facilitate or effect crosslinking or hydrogel formation.

[0054] Hydrogels may be formed as particles or as shells for containing an interior such as an aqueous interior. Generally, particles may be formed by mixing reagents in a partition under reaction conditions or in some cases in combination with a reaction catalyst or trigger such as a free radical generated by exposure of light to lithium phenyl-2,4,6-trimethylbenzoylphosphinate (“LAP,” CAS: 85073-19-4), a thiol, a copper ion or other triggering reagent or catalyst. A more extensive list of additional exemplary triggers comprises 1) copper-(I) as a trigger for copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition, 2) UV or blue light as a trigger for radical polymerization via LAP, 3) higher temperature to initiate deprotection of mal eimide group for example, 4) addition of small molecule crosslinking agents with two reactive ends (dithiol, diazide, dialkyne, dialkene, bis-NHS esters, dihydrazide, bis-epoxide, diamine, among others) or5) uncatalyzed reaction between an azide group and an alkyne such as an alkyne under strain as may occur when the alkyne is a constituent of a cyclic molecule or moiety.

[0055] To form a microcapsule, unlike a solid hydrogel bead, the hydrogel reagents are combined with an inert component configured to accumulate in the center of the partition such as an emulsion droplet, so as to sequester or occupy an interior volume from which the hydrogel is excluded.

[0056] Hydrogels may be formed by localizing, in a partition, a population of shell precursor molecules, such as shell precursors comprising a shell structural component and a crosslinking moiety comprising a non-saturated carbon-carbon bond. The crosslinking moiety may comprise a non-saturated chain or a ring or a combination of two, comprising carbon, nitrogen, oxygen, phosphorous or sulfur atoms in various combinations. The non-saturated carbon-carbon bond is in some cases a ‘strained’ double bond that is held in a high energy state, as one sees in the norbornene moiety. A hydrogel is formed by inducing crosslinking at the non-saturated carboncarbon bond of the crosslinking moieties.

[0057] In some cases the inert component differs from the hydrogel precursors in its relative hydrophilicity or hydrophobicity, such that the hydrogel precursors phase-separate from the inert component on that basis. For example, some hydrogel precursors are conjugated to or modified by a butyl moiety or other hydrophobic moiety that renders the precursors relatively hydrophobic, which may promote their accumulation at the perimeter of the partition in proximity to the hydrophobic carrier, while unconjugated or non-functionalized inert components are relatively hydrophilic, encouraging their phase or other separation and accumulation in the center of the microcapsule precursor.

[0058] In some microcapsule formation precursors, the hydrogel precursor or precursors and the inert component share a common structural component, while in other cases the hydrogel precursors and the inert component are structurally unrelated.

[0059] Exemplary hydrogel constituents include carbohydrates such as dextrans or other carbohydrates such as disaccharides or polysaccharides. Accordingly, in some cases both the hydrogel precursors and the inert component comprise structural carbohydrates, in some cases identical or common structural components. An example of a structural carbohydrate shared between hydrogel components and an inert component is dextran, which in the case of thehydrogel component or components is modified by butyl, other alkyl, aryl, or other hydrophobic group so as to render it relatively hydrophobic.

[0060] Hydrogel precursor compositions may further comprise an analyte or a population of analytes, such that upon hydrogel formation the analytes are contained within the hydrogel beads or microcapsules. When microcapsules having hydrophilic cores are being generated, the analyte or analytes often colocalize with the inert component such that upon microcapsule formation an analyte is contained within, or as part of the aqueous interior of, a microcapsule.

[0061] A broad range of chemistries are consistent with hydrogel formation. Some of these chemistries are discussed below, often in the context of a dextran structural component. Exemplary constituents are presented in Fig. 1. It is understood, however, that a broad range of structural components, such as other carbohydrates or non-carbohydrate structural components may be used with the crosslinking chemistries disclosed below.

[0062] Also disclosed herein are hydrogel populations, such as those arising from practice of the disclosure herein, having particular parameters as to microparticle size, size distribution, or hydrogel shell thickness, among other parameters.

[0063] Some microcapsule technologies have been disclosed, for example in PCT Publication W02023 / 099610, published June 8, 2023, which is hereby incorporated by reference in its entirety.Methods and Compositions for hydrogel formation

[0064] Disclosed herein and consistent with the examples below are methods of microcapsule or bead formation. Methods variously comprise partitioning a shell solution and in some cases a core solution into an emulsion droplet or other partition, and inducing crosslinking among shell solution constituents, for example crosslinking involving an unsaturated carbon bond.

[0065] Shell solutions variously comprise a population of at least one shell precursor molecule comprising a shell hydrogel constituent or shell structural component, a crosslinking moiety and in some cases a hydrophobicity moiety. The hydrogel constituent has very few general limitations, though some constituents are of course quite specific. Hydrogel constituents are generally hydrophilic and biocompatible. Exemplary hydrogel constituents are carbohydrates, such as polysaccharides. In all of the examples below and in Fig. 1, the shell constituent is the polysaccharide dextran, such as 500 kDa molecular weight dextran, though alternatives todextran having similar chemical properties and comparable or equivalent conjugated functional moieties are also contemplated.

[0066] A partial descriptive summary of shell constituents is provided in Table 1, below. In this table, “Dextran” refers to branched poly-a-D-glucosides, as may be of microbial origin or synthesized, having glycosidic bonds predominantly C1->C6. “B50” refers to synthesis conditions whereby reagents are present at a stoichiometric ratio such that no more than 50% of glucose units of the Dextran may react with a butyl moiety to form butanoic acid esters. “B90” refers to synthesis conditions whereby reagents are present at a stoichiometric ratio such that no more than 90% of glucose units of the Dextran may react with a butyl moiety to form butanoic acid esters.Table 1. Hydrogel precursor molecules

[0067] A shell precursor molecule may comprise a crosslinking moiety, such as a moiety comprising at least one non-saturated bond. Some exemplary non-saturated bonds include a double or triple carbon-carbon bond, a double or triple carbon-heteroatom bond, which is in some cases under strain of up to 20 kcal / mol or more. A number of heteroatoms are suitable for the present disclosure, such as oxygen, nitrogen or other heteroatom. Carbon-oxygen bonds suitable for the present disclosure include carbonyl bonds, such as a ketone or aldehyde, for example, while other suitable bonds may comprise carbon and nitrogen atoms, or carbon and another heteroatom, or a double or triple heteroatom-heteroatom bond, such as between two nitrogen atoms, among nitrogen atoms in a resonant azide moiety, between nitrogen and oxygen or other unsaturated non-carbon bond.

[0068] The bond is in some cases strained, so as to be held in a high energy state or is otherwise held in a configuration that is distorted from an ideal or energetically more favorable state. An example of a strained double bond is found in the moiety norbornene, though other strained bonds are also suitable. An example of a stable triple bond is found in cyclic moieties consisting of 8 or more atoms, such as cyclooctynes and their derivatives.

[0069] The crosslinking moiety in various cases comprises a non-saturated chain or a ring or a combination of two, comprising carbon, nitrogen, oxygen or sulfur or phosphorus atoms in various combinations. The non-saturated bond such as a carbon-heteroatom or carbon-carbon bond, or an azide moiety, is variously conjugated or non-conjugated.

[0070] A shell solution in some cases further comprises a bridging molecule that may react with a shell precursor molecule, such as at a non-saturated bond, so as to tether one shell precursor to at least one other in a hydrogel. A bridging molecule generally comprises at least two reactive moiety positions, such that it may react with at least two shell precursor molecules to tether them together in a hydrogel. Exemplary bridging molecules include dithiol such as dithiothreitol (DTT), for example 1 ,4-dithiothreitol, bis-azido-PEG, or cystamine dihydrochloride, among other suitable bridging molecules.

[0071] Alternately or in combination, a shell solution may comprise a molecule having a shell hydrogel constituent or shell structural component having an alternate moiety, such as by being tethered to a ‘half bridging moiety, such as the PEG-azide moiety tethered to a shell hydrogel constituent as seen in Fig. 8 A. Alternate ‘half bridging moieties sharing chemical properties of active components of the bridging moieties above are also contemplated. Molecules having a shell hydrogel constituent or shell structural component and a moiety other than a half bridging moiety are also contemplated herein.

[0072] A shell solution is in some cases mixed with a core solution so as to create a hydrophilic core that does not crosslink to form a hydrogel, such that a microcapsule comprising a solid crosslinked hydrogel shell and an aqueous core, that is not crosslinked, is formed pursuant to hydrogel crosslinking. The core solution may comprise or may be mixed with a target analyte such as at least one nucleic acid, protein, bioparticle, viral particle or cell, such that upon hydrogel formation the target analyte is encased within the microcapsule.

[0073] Hydrogel formation may be uncatalyzed or may require no external factors, or may be triggered by application of a chemical, light, thermal energy, pH change or other source.

[0074] Exemplary chemical catalysts or hydrogel crosslinking triggers comprise radicals, as may be induced through photoexcitation of a radical forming moiety such as LAP upon excitation with light, such as at least one 405 nm wavelength pulse of, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 45, 60 or greater than 60 seconds, or a duration spanned by, less than or exceeding the previous range. Another well-known chemical initiation of a radical crosslinking reaction isaccomplished using ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). TEMED may be delivered through the continuous phase of an emulsion in droplet generation, while APS is dissolved in the aqueous phase containing the hydrogel precursors. Their interaction at the droplet border is sufficient to initiate radical crosslinking.

[0075] Additional catalyst or triggering moiety examples comprise copper (I) ions, as may be generated from a solution of copper (II) species such as CuSO4 upon treatment with ascorbate, for example, a strained molecule, such as a trans-cyclooctene or norbornene or a cyclooctyne, covalently attached to the backbone of a polysaccharide, tetrazine moiety, covalently attached to the backbone of a polysaccharide, azide moiety, covalently attached to the backbone of a polysaccharide, alkyne moiety, covalently attached to the backbone of a polysaccharide, maleimide moiety, in a protected or deprotected form, covalently attached to the backbone of a polysaccharide, diazides such as found in azido-PEG5-azide (CAS: 356046-26-9), dialkynes, such as found in 1,7-octadiyne (CAS: 871-84-1), ditetrazines, such as are found in 3,3'-[(l ,1 '- biphenyl)-2,2'-diyl]-6,6'-bis[phenyl]-l,2,4,5-tetrazine, bis-maleimides, such as found in 1,4- bis(maleimido)butane (CAS: 28537-70-4) or dithiols or redox moieties such as are found in dithiothreitol or beta-mercaptoethanol. In some reactions, a CuSO4 initiator is mixed with shell precursors to avoid premature crosslinking prior to droplet formation because Cu2+does not catalyze the reaction such that copper (II) species can be included in the shell solution together with reactive groups: alkyne and azide. Cu+(or copper (I) species) rather than Cu2+initiate the reaction. Cu+2reduction to Cu+happens only in the droplets. A suitable reducing agent is sodium ascorbate and is delivered via the core solution. Therefore, reduction and subsequent click reaction catalysis can occur upon droplet formation when the core and shell polymer solutions meet.

[0076] Alternate chemical triggers may comprise pH change, such as an acidic or basic pH change. Shell precursors are often delivered at a neutral or close to neutral pH, such as 7.4 or about 7.4, or a number spanned by the range of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. pH changes to induce hydrogel formation may comprise introduction of basic conditions, such as a pH of 8.0, about 8.0, or greater than 8.0, such as at least 8.1, 8.2, 8.3, 8.4, 8.5, or greater.

[0077] A catalyst or trigger may be included with inert core reagents such that it comes into contact with the shell reagents upon mixture of core and shell solutions. Such contacting may occur prior to or concurrent with partitioning such as partitioning of a shell and core solutionmixtures into an emulsion. Alternately, in cases where no core reagents or constituents are to be added, a catalyst or trigger may nonetheless be added prior to or concurrent with partitioning as may occur in solid hydrogel bead formation.

[0078] Alternate triggers may be provided by application of thermal energy, such as heat to above room temperature, for example 25C, 30C, 30C, 35C, 40C, 45C, 50C, 55C, 60C, 65C, 70C, 75C, 80C, 85C, 90C, 95C, or greater, or a number spanned by the range of values given herein.

[0079] Some hydrogel formation may occur in the absence of an external catalyst, such as a catalyst which is not covalently attached to a shell precursor molecule (such as a copper ion, among others), or in the absence of externally added variations in temperature or energy such that incubation of shell precursor molecules at room temperature over time is sufficient to lead to hydrogel formation, such as no more than 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3, hours, 4 hours, 8 hours, 16 hours, overnight, 24 hours, or greater than 24 hours, or a time spanned by the range of times presented herein. In some cases reaction of shell precursors is substantially or effectively immediate. Such formation approaches allow biomolecule encapsulation in the absence of chemical or other external catalysis.

[0080] A common feature of many hydrogel formation catalysts or triggers disclosed herein or consistent with the present disclosure is biocompatibility. Many of the triggers herein are delivered with or mixed with a composition comprising an analyte such as a biological analyte, anything ranging from a biomaterial comprising an active enzyme or functionally folded protein or protein complex, to a viral particle or viable cell or cell population. Some hydrogel formation is effected rapidly, so as to minimize or reduce the time during which bioanalytes or target molecules may be harmed. Alternate approaches are effected so as to minimize or reduce the involvement or dependence upon a chemical catalyst or trigger, so as to reduce a likelihood or impact of a chemical perturbation to which a biomolecule or target analyte may be exposed pursuant to hydrogel formation. Some reactions or hydrogel formations are accomplished using reagents that, although reactive to one another, are bioinert or relatively unreactive with target analytes such as cells, cell contents such as proteins, lipids, nucleic acids or other biomaterials. Some such examples are alkyne-azide click-reaction-based hydrogel formation compositions, as neither of these reactant moieties are highly reactive with many of the target analytes such as cells, cell contents such as proteins, lipids, nucleic acids or other biomaterials that may beintended to be analyzed or encapsulated in beads or aqueous core microcapsules formed through mixture of hydrogel reagents such as those disclosed herein.

[0081] Thus, hydrogel crosslinking is effected without substantial negative impact on activity or viability of eventual microcapsule contents.Microcapsule populations

[0082] Consistent with the chemical compositions and methods disclosed above, disclosed herein are microcapsule populations having specified microcapsule parameters. Microcapsule parameters are specified, for example, by selecting the chemistry, the reagents, and in some cases the parameters employed to generate the populations. Microcapsule populations are in some cases formed through use of reagents that are not substantially reactive with biomolecules, such as azides and alkynes, such that the population is formed with little impact upon microcapsule contents such as biomolecules.

[0083] A parameter such as microcapsule shell thickness may be varied so as to affect microcapsule stability, diffusion time for delivery of a reagent such as an aqueous reagent across the shell, or degradation time for execution of a shell degradation protocol.

[0084] A number of shell degradation approaches are consistent with the disclosure herein, such as enzymatic degradation, mechanical shearing as may comprise vortexing alone or in combination with solid particles, ball-milling, or a thermal degradation such as heating or in the alternative at least one freeze-thaw cycle.

[0085] Microcapsule shell thickness may be selected in various chemistries to have a minimum value in um of no greater than, no less than or about 4, 5, 6, 7, or greater than 7, or may be selected to have a minimum value of no less than, no greater than or about 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.5, 5, 6, 7, or greater than 7, or a number spanned by the range herein Some microcapsule populations may have, or methods may selected to yield, microcapsule shell thicknesses of 1 - 20um, 2-15 um, 3-10 um, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a value spanned by or near this range, or a range having a lower limit listed above, or a range having an upper limit listed above, or a range having upper and lower limit listed above.Similarly, microcapsule shell thickness may be selected so as to have a median thickness in um of about 5, about 5.5, about 6, about 6.5, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or greater than 13. Similarly, microcapsules may be selected to have a maximum shellthickness of in some cases no greater than, no less than or about 30, no greater than 8.5, no greater than 8, no greater than 10, or no greater than 7, among other shell thicknesses or widths. Similarly, microcapsule populations may be selected to have a shell thickness range spanning at least 70%, 80%, 90% or a percentage spanned by the listed range of particles of no greater than 15, no greater than 4, no greater than 3.5, no greater than 3, no greater than 2.5, or no greater than 2.

[0086] Similarly, microcapsule diameter for a microcapsule population may be selected to have a minimum diameter of no greater than, no less than or about 70, 52, 72, 75, 65, 68, 83, 74, 70, or a number spanned by the range of values listed. Microcapsule diameter for a microcapsule population may be selected to have a maximum diameter of no greater than, no less than or about 115, 92, 103, 94, 96, 100, 155, or a number spanned by the range of values listed. In some cases a broader range of microcapsule diameters is achieved, such as 100-300 um, 50-400 um, or 20- 500 um, 25-150 um, 50-100 um or a value spanned by any of these ranges. Similarly, a microcapsule may have a diameter of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 250, 300, 350, 400 or other value in um spanned by or adjacent to this set, or a range having a lower limit spanned by this set, or a range having an upper limit spanned by this set, or a range having a lower and upper limit spanned by this set, in um.

[0087] Similarly, microcapsule populations may be selected to have a median diameter of, for example, a bimodal diameter distribution, with a major peak at about 97 ± 5 and a minor peak at 80 ± 2; or a median diameter of 70 ± 2.5; 92.5 ± 2.5; 86 ± 2.5; 78 ± 5; 82.5 ± 2.5; 94 ± 1; 83 ± 2; or 110 ± 10.

[0088] Similarly, microcapsule populations may be selected to have a diameter range for 70%, 80%, or 90%, or a percentage spanned by the listed values of constituents spanning no more than 40, 35, 30, 20, 15, 12, 10, 8, or 7.

[0089] Microcapsule populations in some cases further comprise members harboring target analytes or bioparticles in their aqueous cores, such that a first microcapsule comprises a first bioparticle and a second microcapsule comprises a second bioparticle. Because of the flexibilities of the chemistries disclosed herein, one may generate microcapsule populations harboring active or viable bioparticles even when the bioparticles are vulnerable to, for example, radicals, or copper ions such as found in CuSO4, temperatures above room temperature, or other hydrogel formation triggers. A microcapsule or hydrogel bead population may exhibit a targetanalyte or bioparticle occupancy of, for example, at least or at most 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% occupancy. The activity of contained bioparticles may be, for example, at least or at most 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% relative to activity prior to encapsulation or hydrogel formation. The viability of contained cells may be, for example, at least or at most 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% relative to activity prior to encapsulation or hydrogel formation.

[0090] Microcapsule shell thickness and microcapsule diameter, as well as comparable dimensions in droplets prior to hydrogel formation or otherwise not subjected to crosslinking or in which crosslinking or hydrogel formation do not occur, may be measured by any of a broad range of techniques known in the art. Measurement is in some cases effected through microscopy, such that an image of a microcapsule or microcapsule population is captured and analyzed via computer to identify shell thickness, microcapsule diameter, or both microcapsule shell thickness and microcapsule diameter. Microcapsule images are in some cases compared directly to a scale bar, or a scale bar is superimposed on one or more microcapsule images, or the microcapsule diameter or microcapsule shell thickness is measured in the image and the measurement compared to a scale bar. Suitable microscopy approaches include bright field microscopy, dark field microscopy, phase contrast microscopy, though other approaches such as fluorescence microscopy or confocal microscopy may also be used, as may any approach that generates an image of either entire microcapsules or that depicts microcapsules transparently such that microcapsule shell thickness may be measured.Hydrogel precursor synthesis

[0091] Disclosed herein are methods of synthesizing a hydrogel precursor composition, and hydrogel precursor compositions arising therefrom. Through practice of such methods or use of such compositions, one generates water-in-oil emulsions comprising droplets having hydrogel precursor molecules at their perimeter that are readily crosslinked to form a microcapsule hydrogel shell, as well as aqueous moieties interior to the droplets that are resistant to crosslinking, so as to form an aqueous or liquid microcapsule core. For such methods and compositions to be effective, the hydrogel precursor must be sufficiently hydrophobic toaccumulate at the emulsion droplet exterior or perimeter, but not so hydrophobic as to be miscible with the oil carrier or to be immiscible with the aqueous structural moieties.

[0092] Accumulation or localization to an emulsion droplet perimeter may be understood a number of ways. Hydrogel precursors may be demonstrated to accumulate at a droplet perimeter if, upon contacting to a crosslinking agent such as an oxidative stress, the droplet forms a microcapsule having a hydrogel shell as disclosed elsewhere herein, surrounding an aqueous, liquid or un-crosslinked core. Hydrogel formation as a shell in those cases indicates localization of the hydrogel precursor in the region of the droplet that forms the hydrogel shell.

[0093] A droplet perimeter may be understood as a particular thickness of or region of the droplet adjacent to the carrier or farthest from the droplet center. This thickness may in some cases be understood to be the distal-most segment of the radius of the droplet. This thickness is variously l-20um, 2-15 um, 3-10 um, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a value spanned by or near this range, or a range having a lower limit listed above, or a range having an upper limit listed above, or a range having upper and lower limit listed above, in um.

[0094] Similarly, a droplet perimeter may be understood as the portion of the droplet that comes into direct contact with the carrier, or as the portion of the droplet that is exposed to sufficient oxidative stress to trigger hydrogel formation, or the portion of the droplet that comes into contact with the nonaqueous carrier so as to draw relatively hydrophobic moieties in the aqueous droplet to accumulate therein.

[0095] A perimeter is contrasted to a core or to an aqueous core precursor, referring to the portion of the droplet that does not form a hydrogel in response to a signal or stress such as oxidative stress, or to the portion of the microcapsule internal to the hydrogel shell. This region does not substantially crosslink, in some cases due to the hydrogel precursors accumulating at the perimeter rather than in the aqueous core, while constituent molecules lacking crosslinking moieties such as unsaturated carbon bonds and in some cases lacking hydrophobic moieties such as butyl moieties that may draw them to the perimeter or to the carrier interface. The core or aqueous cor is in some cases the volume defined by the the radius of droplet, minus the distal or eternal most segment, variously l-20um, 2-15 um, 3-10 um, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a value spanned by or near this range. The perimeter and core are often understood in terms of the radius of droplets. However, nonspherical droplets suchas elliptical droplets or lozenge shaped droplets, or droplets assuming a cross-section of a channel through which they flow or are retained, may also have a core and perimeter analogous to those as described herein, which the perimeter being understood as the outermost portion or portion adjacent to a carrier or channel wall and first being exposed to a signal such as an oxidative stress that may triger perimeter hydrogel formation. Thicknesses of perimeters in droplets of these nonspherical shapes are often comparable to those of sphetical droplets discussed above.

[0096] Hydrogel precursor hydrophobicity is in part impacted by chemical properties of hydrogel precursor crosslinking moieties that are added to structural moieties, for example carbohydrates such as dextran or other carbohydrate polymer. Crosslinking moieties are often hydrophobic but may no convey sufficient hydrophobicity to lead to sufficient accumulation at a droplet perimeter. Accordingly, hydrogel precursor structural moieties are additionally modified by addition of hydrophobic moieties, such as butyl moieties or other moieties described herein or otherwise sufficient to modulate or impact hydrogel precursor hydrophobicity. Using such as approach, one may control hydrogel precursor hydrophobicity so as to achieve perimeter localization in emulsion droplets without losing miscibility with the aqueous structural moieties.

[0097] Some such methods comprise one or more of the steps of synthesizing a hydrogel precursor, the method comprising: selecting a structural moiety; selecting a crosslinking moiety; modifying the structural component using a hydrophobic moiety such that, upon addition of the crosslinking moiety, the hydrogel precursor exhibits a hydrophobicity sufficient to localize it to a perimeter of an aqueous droplet in a water-in-oil emulsion; and adding the crosslinking moiety. The aqueous droplet in many cases comprises hydrogel precursor to form a microcapsule shell and unmodified structural moiety to form a microcapsule aqueous core upon crosslinking of the hydrogel precursor. Exemplary structural moieties comprise carbohydrates, such as glucose polymers. An exemplary structural moiety is dextran. The crosslinking moiety in many cases comprises an unsaturated carbon bond, such as an alkene or alkyne. The crosslinking moiety may be cyclic, such that if the unsaturated carbon bond is an alkyne it is held in a strained configuration. The crosslinking moiety in some cases further comprises one or more benzyl moieties. A number of hydrophobic moieties are consistent with the methods herein, such as aliphatic moieties, for example butyl moieties, though other length carbon moieties are alsoconsistent with the disclosure herein, such as moieties comprising 2, 3, 4, 5, 6, 7, 8 ,9 or more carbons.Microcapsule formation approaches

[0098] Disclosed herein are a plurality of approaches for microcapsule formation consistent with the methods, compositions, microcapsule populations disclosed herein . One understands that the approaches are indicative of broad ranges of approaches, wherein the hydrogel structural component dextran is exchanged for a functionally comparable disaccharide, oligosaccharide or polysaccharide, hydrophobicity modifiers are exchanged, or crosslinking moieties are substituted with either functionally or structurally comparable moieties. Similarly, a catalyst or reaction conditions may be exchanged so as to come to a comparable outcome relating to microcapsule formation or population parameters. Some approaches use reagents that are not substantially reactive with biomolecules, such as azides and alkynes, such that the population is formed with little impact upon microcapsule contents such as biomolecules.

[0099] Capsules via 1,3-Dipolar Cycloaddition Between Nitrone and Norbornene. Some approaches comprise cycloaddition involving a dipole moiety and an exposed non-conjugated carbon-carbon double bond. For example, DexB50-Norbornene (LG-049) and DexB50-Nitrone (LG-056A), as shown in Fig. 2A, may react to form a crosslinked hydrogel.

[0100] Reactions may be supported by heating, for example to a temperature of at least, no more than about or exactly lower than 30C, 30C, 35C, 40C, 45C, 50C, 55C, 60C, 65C, 70C, 75C, 80C, 85C, 90C, 95C, or greater, or a number spanned by the range of values given herein. Incubation is performed for at least, at most, about or exactly 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, overnight, or for 1, 2, or more than 2 days. Some reactions occur in no more than 20, 15, 10, 50, or 2 minutes, or 30 seconds or less, while some reactions are substantially immediate. Exemplary embodiments comprise overnight incubation at 60C.

[0101] Shell solution and core solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions are performed under physiological conditions, such as IX PBS at a pH of 7.4, though other buffer conditions and pH levels are also consistent with the technology herein.

[0102] Reactions are in some cases uncatalyzed.

[0103] Single component capsule shells (Radical Crosslinking). Some approaches comprise free-radical-triggered crosslinking of members of a uniform single component population, such as a component comprising an non-conjugated carbon-carbon double or triple bond. For example, DexB50-Norbornene (LG-049), as shown in Fig. 3A, or DexB90-Pentynoate (LG-048), as shown in Fig. 5A. may autoreact to form a hydrogel.

[0104] Reactions are in some cases catalyzed, for example by a free radical. The radical may be introduced through a source separate from the shell solution, such as an inert core solution or a catalyst carrying solution.

[0105] A number of catalysts are consistent with this approach. Exemplary catalysts are induced to generate radicals, such as through photoinduction, as is the case with the photoinducible radical producing LAP.

[0106] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions are induced through the activation by a catalyst or a free radical, such as through photoexcitation at 405 nm or another appropriate excitation wavelength for a given photo-excitable radical generator, such as at least, at most, about or exactly 365, 375, 385, 395, 405, 415, 425, 535, 445, 455, 475, 500 or greater than 500 nm, or a value spanned by or outside of the listed wavelengths.

[0107] Reactions are allowed to proceed at any of a range of temperatures, such as room temperature, at least, no more than about or exactly lower than room temperature, 4C, 10C, 20C, 30C, 35C, 37C, 40C, 45C, 50C, 55C, 60C, 65C, 70C, 75C, 80C, 85C, 90C, 95C, or greater, or a number spanned by the range of values given herein.

[0108] Bridging component Capsules (Radical Crosslinking with / without Thiol-Ene / Yne Reaction). Some approaches comprise radical triggered crosslinking of members of a shell constituent population such as a component comprising a non-conjugated carbon-carbon double bond or triple bond, using bridging or linking molecules. For example, DexB50-Norbornene (LG-049) or DexB90-Pentynoate (LG-048) may react with a dithiol such as dithiothreitol, as shown in Fig. 4A or Fig. 6A, to form a hydrogel.

[0109] Reactions are in some cases catalyzed, for example by a free radical. The radical may be introduced through a source separate from the shell solution, such as an inert core solution or a catalyst carrying solution.

[0110] A number of catalysts are consistent with this approach. Exemplary catalysts are the ones which generates free radicals upon photoinduction, as is the case with the photoinducible radical producing LAP.

[0111] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions are induced through catalyst activation or radical formation, such as through photoexcitation at 405 nm or another appropriate excitation wavelength for a given photo-excitable radical generator, such as at least, at most, about or exactly 365, 375, 385, 395, 405, 415, 425, 535, 445, 455, 475, 500 or greater than 500 nm, or a value spanned by or outside of the listed wavelengths.

[0112] Reactions are allowed to proceed at any of a range of temperatures, such as room temperature, at least, no more than about or exactly lower than room temperature, 4C, 10C, 20C, 30C, 35C, 37C, 40C, 45C, 50C, 55C, 60C, 65C, 70C, 75C, 80C, 85C, 90C, 95C, or greater, or a number spanned by the range of values given herein.

[0113] Capsules via Copper-Catalyzed Alkyne-Azide 1,3-Cycloaddition (CuAAC). Some approaches comprise copper-catalyzed crosslinking of members of a shell constituent population such as a component comprising a non-conjugated azide or carbon-carbon triple bond, using bridging or linking molecules. For example, DexB90-Pentynoate (LG-048) may react with a bridging moiety or crosslinking agent such as bis-azido-PEG (CAS: 356046-26-9), as shown in Fig. 7A to form a hydrogel.

[0114] Some approaches comprise copper-catalyzed crosslinking of members of a combination of shell constituent populations such as a component comprising an non-conjugated carboncarbon double bond or triple bond, and an azide-moiety-containing shell constituent such as DexB90-PEG- Azide, as shown in Fig. 8 A, to form a hydrogel.

[0115] Some approaches comprise copper-catalyzed crosslinking of members of a combination of shell constituent populations such as a component comprising an non-conjugated carboncarbon double bond or triple bond, and a azide-moiety-containing shell constituent such as DexB90-Azidomethylbenzoate (LG-066), as shown in Fig. 9A, to form a hydrogel.

[0116] Reactions are in some cases catalyzed, for example by copper ions. The catalyst may be introduced through a source separate from the shell solution, such as an inert core solution or a catalyst carrying solution or may be introduced as part of the shell solution.

[0117] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly at room temperature, such that emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 5, 10, 15, 30, or 60 minutes, or a value spanned by or outside of the listed durations.

[0118] Capsules via Inverse Electron Demand Diels-Alder (IEDDA) Between Tetrazine and Norbornene). Some approaches comprise crosslinking via inverse electron demand Diels- Alder reaction of members of a combination of shell constituent populations such as a component comprising a tetrazine moiety or strained alkene or strained alkyne moieties, such as DexB50- Norbornene (LG-049), and a shell component comprising a cyclic nitrogen-containing ring such as DexB90-Tetrazine (LG-068), as shown in Fig. 10A.

[0119] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly at room temperature, such that emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 5, 10, 15, 30, or 60 minutes, or a value spanned by or outside of the listed wavelengths.

[0120] Capsules via Base-Catalyzed Thiol-Ene Reaction. Some approaches comprise basecatalyzed crosslinking of members of a shell constituent population such as a component comprising a carbon-carbon double bond or triple bond, using bridging or linking molecules. For example, DexMAB1090 (dextran modified by methacryloyl and butyryl moieties) may react with a bridging moiety such as 1 ,4-dithiothreitol (DTT), as shown in Fig. 11A, to form a hydrogel.

[0121] Reactions are in some cases catalyzed, for example by a basic environment, such as a buffer with pH of 8. The catalyst may be introduced through a source separate from the shell solution, such as an inert core solution or a catalyst carrying solution.

[0122] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly at room temperature, such that emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 5, 10, 15, 30, 60 or 90 minutes, 2 hours, 3 hours, or a value spanned by or outside of the listed durations.

[0123] Capsules via Retro Diels- Alder and Subsequent Base-Catalyzed Thiol-Ene Reaction.Some approaches comprise base-catalyzed crosslinking of members of a shell constituent population such as a component comprising a conjugated carbon-carbon double or triple bond (specifically an alpha-beta unsaturated carbonyl moiety), using bridging or linking molecules such as dithiols, via a retro Diels-Alder reaction and subsequent Michael addition (1,4-conjugate addition). For example, DexB90-Protected-Maleimide (LG-061) may react with a bridging moiety such as DTT, as shown in Fig. 12A, to form a hydrogel.

[0124] Reactions are in some cases catalyzed, for example by a basic environment, such as a buffer with pH of 8. The catalyst may be introduced through a source separate from the shell solution, such as an inert core solution or a catalyst carrying solution.

[0125] Shell solution and core solution or catalyst solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly upon application of heat, such as at least, at most, about or exactly 80C, 85C, 90C, 95C, 100C, 105C, 110C or greater, or a value spanned by the list of temperatures provided herein.

[0126] Emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 1, 2, 5, 10, 15, 30, or 60 minutes after emulsion formation, or by a value spanned by the list of times presented.

[0127] Capsules via the Formation of Imines. Some approaches comprise crosslinking of members of a shell constituent population such as a component comprising a carbonyl (carbonoxygen double bond), such as an aldehyde, using a cystamine bridging or linking molecules such as a diamine. For example, DexB50-Formylbenzoate (LG-056B) may react with a bridging moiety such as cystamine dihydrochlorate 2HC1, as shown in Fig. 13A to form a hydrogel.

[0128] Shell solution and core solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly.

[0129] Emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 1, 2, 5, 10, 15, 30, or 60 minutes after emulsion formation, or by a value spanned by or outside of the list of times presented.

[0130] Capsules via SPAAC. Some approaches comprise crosslinking of members of a shell constituent population such as a constituent comprising a strained C-C double bond or a C-C triple bond, using bridging or linking molecules such as azide comprising molecules, to reactwith the unsaturated bond in a manner facilitated by the strain. For example, DexB50-DBCO (LG-073) may react with bis-azido-PEG, as shown in Fig. 14A, to form a hydrogel.

[0131] The C-C double bond or a C-C triple bond may be made reactive by introducing strain into the bond. Strain is introduced into C-C double or C-C triple bonds by including them as constituents of cyclic moieties. Cycloalkynes are in some cases too unstable and reactive in cyclic structures of 7 or below, but rings of 8 or more constituents may be stable enough to incorporate an alkyne with a strain energy of about 10 to about 20 kcal / mol.

[0132] For example, benzannulated cyclooctynes (dibenzocyclooctynes) exhibit a strain energy of typically 15-25 kcal / mol. Reactivity is modulated by both the angle strain and electronic effects arising from ring modifying moieties, such as the dibenzocyclooctyne shown in Fig. 14 A.

[0133] Strain is introduced into an alkene or allene C-C double bonds via steric effects, such as tert-butyl groups or even sterically larger groups so as to increase strain energy.

[0134] Strain facilitates bond attack and cyclization via an interaction with an azide moiety,.

[0135] Shell solution and core solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly.

[0136] Emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 1, 2, 5, 10, 15, 30, or 60 minutes after emulsion formation, or by a value spanned by or outside of the list of times presented.

[0137] Notably, the reagents used in these approaches to hydrogel formation, strained unsaturated C-C bonds and azide moieties, are generally poorly reactive with biomolecules and bioanalytes such as cells, cell contents, proteins, lipids, nucleic acids such as RNA and DNA, so as to facilitate hydrogel and microcapsule formation with a reduced impact on bioanalytes to be included in the microcapsules.

[0138] Shell solution and core solution are comingled pursuant to partitioning, for example, into droplets of a water-in-oil emulsion. Reactions generally proceed rapidly.

[0139] Emulsion breaking may be performed soon after emulsion formation, such as at least, at most, about or exactly 1, 2, 5, 10, 15, 30, or 60 minutes after emulsion formation, or by a value spanned by or outside of the list of times presented.

[0140] Turning to the Figures, one sees the following.

[0141] At Fig. 1, one sees a variety of shell constituent precursors consistent with the disclosure herein. Each of these examples comprises a carbohydrate foundation and a modification that renders the molecule relatively hydrophobic such that it localizes to the perimeter of an emulsion droplet such as that in a water-in-oil emulsion. Each molecule further comprises an active group that facilitates crosslinking for hydrogel formation, such as a conjugated or non-conjugated alkene or alkyne, nitrone, thiol, azide or azido group, tetrazine or maleimide in a protected or deprotected form. All listed functionalized dextran polymers were formulated as 20% (w / w) solutions in lx PBS, pH 7.4.

[0142] In these examples the carbohydrate foundation is dextran, but other carbohydrates and non-carbohydrate foundations are also consistent with the disclosure herein. Similarly, in each case in this Figure, the modification that renders the molecule relatively hydrophobic, such that it promotes the localization of the molecule to the perimeter of an emulsion droplet in a hydrophobic carrier, is the modification with butyryl groups. However, other hydrophobic moieties are also consistent with this role in the hydrogel precursor shell, such as pentyl or other length hydrophobic carbon chain. It can be saturated or unsaturated, linear, cyclic or branched alkyl chains (acetyl, propyl, butyl, cyclohexyl, pivaloyl, crotonyl) or aryl groups (unsaturated rings like benzene or pyridine) or a combination of both alkyl and aryl chains. In some cases a hydrophobic moiety comprises at least 2, at least 3, at least 4, at least 5, at least 6, or no more than 2, no more than 3, no more than 4, no more than 5, or no more than 6 carbons, alone or in combination with at least one other heteroatom.

[0143] Fig. 2 A presents a composition suitable for hydrogel formation comprising two of the molecules presented in Fig. 1. These molecules present an example of a non-conjugated alkene reacting with a nitrone group to form a crosslinked hydrogel in the absence of a trigger such as a pH change or a free radical.

[0144] Fig. 2B presents the microcapsules generated from the composition in Fig. 2A. Scale bar is 100 um.

[0145] Fig. 3A shows an exemplary molecule capable of catalyzing self-crosslinking. Upon induction of radical formation by exposure of LAP to 405 nm light, free radicals are generated so as to catalyze crosslinking among the non-conjugated alkenes such as those in a population of the molecule presented herein.

[0146] Fig. 3B presents an analysis of microcapsules generated from the composition in Fig. 3A.

[0147] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 60 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0148] At right, one sees microcapsule number on the y-axis, ranging from 0 to 15 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 65 to 120 in intervals of 5 um.

[0149] Fig. 3C presents the microcapsules generated from the composition in Fig. 3A. Scale bar is 100 um.

[0150] Fig. 4A shows an exemplary composition suitable for hydrogel formation. Upon induction of radical formation by exposure of LAP to 405 nm light, free radicals are generated so as to catalyze crosslinking among the non-conjugated alkenes such as those in a population of the molecule presented herein and between non-conjugated alkenes and dithiols such as of the 1 ,4-dithiothreitol (DTT) in a radical -promoted thiol-ene reaction provided therewith.

[0151] Fig. 4B presents an analysis of microcapsules generated from the composition in Fig. 4A.

[0152] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 80 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0153] At right, one sees microcapsule number on the y-axis, ranging from 0 to 20 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 50 to 100 in intervals of 2.5 um.

[0154] Fig. 4C presents the microcapsules generated from the composition in Fig. 4A. Scale bar is 100 um.

[0155] Fig. 5A shows an exemplary molecule capable of catalyzed self-crosslinking. Upon induction of radical formation by exposure of LAP to 405 nm light, radicals are generated so as to catalyze crosslinking among the non-conjugated alkynes such as those in a population of the molecule presented herein.

[0156] Fig. 5B presents an analysis of microcapsules generated from the composition in Fig. 5A.

[0157] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 80 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0158] At right, one sees microcapsule number on the y-axis, ranging from 0 to 25 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 60 to 110 in intervals of 2.5 um.

[0159] Fig. 5C presents the microcapsules generated from the composition in Fig. 5A. Scale bar is 100 um.

[0160] Fig. 6 A shows an exemplary composition suitable for hydrogel formation. Upon induction of radical formation by exposure of LAP to 405 nm light, radicals are generated so as to catalyze crosslinking among the non-conjugated alkynes such as those in a population of the molecule presented herein and between non-conjugated alkynes and dithiols such as of the 1,4- dithiothreitol (DTT) in a radical-promoted thiol-yne reaction provided therewith.

[0161] Fig. 6B presents an analysis of microcapsules generated from the composition in Fig. 6A.

[0162] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 110 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0163] At right, one sees microcapsule number on the y-axis, ranging from 0 to 30 in labeled intervals of 5, with the x-axis presenting microcapsule diameter in um ranging from 60 to 110 in intervals of 2.5 um.

[0164] Fig. 6C presents the microcapsules generated from the composition in Fig. 6A. Scale bar is 100 um.

[0165] Fig. 7A shows an exemplary composition suitable for hydrogel formation. Upon reduction of copper (II) species to copper (I) species, which are the catalyst in this reaction, by the employment of reducing agent such as sodium ascorbate so as to catalyze crosslinking among the alkyne-containing moiety in a form of pentynoate and bis-azides as crosslinking agents such as bis-azido-PEG molecules in copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition reaction provided therewith.

[0166] Fig. 7B presents an analysis of microcapsules generated from the composition in Fig. 7A.

[0167] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 60 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0168] At right, one sees microcapsule number on the y-axis, ranging from 0 to 15 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 50 to 100 in intervals of 2.5 um.

[0169] Fig. 7C presents the microcapsules generated from the composition in Fig. 7A. Scale bar is 100 um.

[0170] Fig. 8A shows an exemplary composition suitable for hydrogel formation. Upon reduction of copper (II) species to copper (I) species, which are the catalyst in this reaction, by the employment of reducing agent such as sodium ascorbate, crosslinking is catalyzed between alkyne-containing moiety in a form of pentynoate and azide-containing moieties in a form of azido-PEG-carboxylate in copper-catalyzed alkyne-azide 1,3 -dipolar cycloaddition reaction provided therewith.

[0171] Fig. 8B presents an analysis of microcapsules generated from the composition in Fig. 8A.

[0172] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 70 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0173] At right, one sees microcapsule number on the y-axis, ranging from 0 to 20 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 50 to 100 in intervals of 2.5 um.

[0174] Fig. 8C presents the microcapsules generated from the composition in Fig. 8A. Scale bar is 100 um.

[0175] Fig. 9A shows an exemplary composition suitable for hydrogel formation. Upon reduction of copper (II) species to copper (I) species, which are the catalyst in this reaction, by the employment of reducing agent such as sodium ascorbate, crosslinking is catalyzed between alkyne-containing moiety in a form of pentynoate and the azide-containing moiety in a form of 4-(azidomethyl)benzoate in copper-catalyzed alkyne-azide 1,3 -dipolar cycloaddition reaction provided therewith.

[0176] Fig. 9B presents an analysis of microcapsules generated from the composition in Fig. 9A.

[0177] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 60 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0178] At right, one sees microcapsule number on the y-axis, ranging from 0 to 20 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 50 to 100 in intervals of 2.5 um.

[0179] Fig. 9C presents the microcapsules generated from the composition in Fig. 9A. Scale bar is 100 um.

[0180] Fig. 10A shows an exemplary composition suitable for hydrogel formation. Crosslinking occurs between non-conjugated strained alkenes and tetrazine moieties in inverse electron demand Diels-Alder reaction provided therewith.

[0181] Fig. 10B presents an analysis of microcapsules generated from the composition in Fig.10 A.

[0182] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 60 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0183] At right, one sees microcapsule number on the y-axis, ranging from 0 to 40 in labeled intervals of 10, with the x-axis presenting microcapsule diameter in um ranging from 70 to 110 in intervals of 2.5 um.

[0184] Fig. 8C presents the microcapsules generated from the composition in Fig. 8A. Scale bar is 100 um.

[0185] Fig. 11A shows an exemplary composition suitable for hydrogel formation. Crosslinking occurs between alpha-beta-unsaturated esters and dithiols, such as DTT, of the composition provided therewith.

[0186] Fig. 1 IB presents an analysis of microcapsules generated from the composition in Fig.11 A.

[0187] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 90 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0188] At right, one sees microcapsule number on the y-axis, ranging from 0 to 30 in labeled intervals of 5, with the x-axis presenting microcapsule diameter in um ranging from 60 to 100 in intervals of 2.5 um.

[0189] Fig. 11C presents the microcapsules generated from the composition in Fig. 11 A. Scale bar is 100 um.

[0190] Fig. 12A shows an exemplary composition suitable for hydrogel formation. Crosslinking occurs between the deprotected maleimide groups and dithiols, such as DTT, under basic conditions of the composition.

[0191] Fig. 12B presents an analysis of microcapsules generated from the composition in Fig. 12A when not subjected to shaking. At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 30 in labeled intervals of 5, with the x-axis presenting shell thickness in um, ranging from 0 to 30 in labeled intervals of 2.5 um.

[0192] At right, one sees microcapsule number on the y-axis, ranging from 0 to 15 in labeled intervals of 2.5, with the x-axis presenting microcapsule diameter in um ranging from 50 to 170 in intervals of 10 um.

[0193] Fig. 12C presents an analysis of microcapsules generated from the composition in Fig. 12A when subjected to shaking.

[0194] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 60 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 13 in labeled intervals of 1 um.

[0195] At right, one sees microcapsule number on the y-axis, ranging from 0 to 40 in labeled intervals of 10, with the x-axis presenting microcapsule diameter in um ranging from 90 to 120 in intervals of 2.5 um.

[0196] Fig. 12D presents the microcapsules generated from the composition in Fig. 12A when subjected to shaking. Scale bar is 100 um.

[0197] One sees in Fig.s 2B-12D that a broad range of crosslinking chemistries and starting moieties may be used to generate microcapsules. Furthermore, by selecting the crosslinking chemistry, one may specify or modulate microcapsule properties such as microcapsule shell thickness and diameter.

[0198] Most chemistries yield microcapsules having a median shell thickness ranging from about 5.5 ±1 um. However, one sees at Fig. 10B, Fig. 12B and Fig. 12C that greater median shell thickness can be achieved through a selection of the appropriate chemistries. At Fig. 10B, for example, one sees a median shell thickness of 7 ± 1 um, while Fig. 12B shows a shell thickness of about 12.5 ± 2.5 um. At Fig. 12C, using the chemistry of Fig. 12A and Fig. 12B with the addition of shaking, one sees a median shell thickness of 8.75 ± 1.5 um.

[0199] Similarly, selection of chemistry has a large impact on microcapsule or semipermeable capsule (SPC) diameter. At Fig. 3B, one sees a bimodal diameter distribution, with a major peak at about 97 ± 5 um and a minor peak at 80 ± 2 um. At Fig. 4B, one sees a median diameter of 70 ± 2.5 um. At Fig. 5B the median is 92.5 ± 2.5 um. At Fig. 6B the median is about 86 ± 2.5 um. Fig. 7B shows a broad median of 78 ± 5 um. Fig.s 8B and 9B show a median of 82.5 ± 2.5 um. Fig. 10B shows a median of 94 ± 1 um. Fig. 1 IB shows a median of 83 ± 2 um. Fig. 12B shows a broad range having a peak at about 110 ± 10 um. Fig. 12C shows a median of 109 ± 3 um.

[0200] Thus, by selecting among the chemistries or protocols disclosed herein, one may specify from among a range of microcapsule thicknesses and microcapsule diameters.

[0201] Fig. 13A shows an exemplary composition suitable for hydrogel formation. Crosslinking occurs between the C-0 double bond (in this case an aldehyde) and cystamine (such as a diamine), under basic conditions of the composition. This composition demonstrates that C-0 double bonds, as well as C-C unsaturated bonds as demonstrated elsewhere, are suitable for hydrogel formation.

[0202] Fig. 13B shows a microcapsule population formed using the composition above.

[0203] Fig. 14A shows an exemplary composition suitable for hydrogel formation via strain- promoted azide-alkyne cycloaddition. Crosslinking occurs between the C-C triple bond (alkyne) and an azide, under neutral conditions and without requiring a catalyst such as a copper (I) catalyst. The alkyne is held under strain, which facilitates the reaction with the azide. This example demonstrates that reactions can occur in the absence of copper, and that reactions can occur between strained alkynes and azide moieties.

[0204] Fig. 14B presents an analysis of microcapsules generated from the composition in Fig. 14 A.

[0205] At left, one sees the number of microcapsules on the y-axis, ranging from 0 to 50 in labeled intervals of 10, with the x-axis presenting shell thickness in um, ranging from 0 to 10 in labeled intervals of 1 um.

[0206] At right, one sees microcapsule number on the y-axis, ranging from 0 to 12 in labeled intervals of 1, with the x-axis presenting microcapsule diameter in um ranging from 50 to 100 in intervals of 10 um.

[0207] Fig. 14C and Fig. 14D present the microcapsules generated from the composition in Fig. 14A. Scale bars are 100 um.

[0208] The disclosure is further understood in light of the following partial list of numbered embodiments.

[0209] 1. A method of synthesizing a hydrogel, comprising localizing, in a partition, a population of shell precursor molecules, each comprising a shell structural component and a crosslinking moiety comprising at least one non-saturated bond, and inducing crosslinking at the non-saturated bond of the crosslinking moieties. 2. The method of embodiment 1 or any previous embodiment, wherein the non-saturated bond is a carbon-carbon bond. 3. The method of embodiment 1 or any previous embodiment, wherein the non-saturated bond is a carbonheteroatom bond. 4. The method of embodiment 1 or any previous embodiment, wherein the non-saturated bond is a heteroatom-heteroatom bond. 5. The method of embodiment 2 or any previous embodiment, wherein the crosslinking moiety comprises a linear carbon chain comprising the non-saturated carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bond. 6. The method of embodiment 2 or any previous embodiment, wherein the crosslinking moiety comprises a ring comprising the non-saturated carbon-carbon, carbon-heteroatom or heteroatom- heteroatom bond. 7. The method of embodiment 1 or any previous embodiment, wherein the crosslinking moiety comprises a nitrogen atom. 8. The method of embodiment 1 or any previous embodiment, wherein the crosslinking moiety comprises a sulfur atom. 9. The method of embodiment 1 or any previous embodiment, wherein the crosslinking moiety comprises an oxygen atom. 10. The method of embodiment 1 or any previous embodiment, wherein the crosslinking moiety comprises a phosphorous atom. 11. The method of embodiment 1 or any previous embodiment, wherein the partition is a droplet in an emulsion. 12. The method of embodiment 2 or any previous embodiment, wherein the non-saturated carbon-carbon bond is a non-conjugated alkene. 13. The method of embodiment 2 or any previous embodiment, wherein the non-saturated carbon-carbon bond is conjugated to a carbonyl group. 14. The method of embodiment 2 or any previous embodiment, wherein the non-saturated carbon-carbon bond is a non-conjugated alkyne. 15. The method of embodiment 1 or any previous embodiment, wherein the structural component comprises a carbohydrate. 16. The method of embodiment 1 or any previous embodiment, wherein the structural component comprises dextran. 17. The method ofembodiment 1 or any previous embodiment, wherein the shell precursor molecules are localized to the interior of the droplet. 18. The method of embodiment 1 or any previous embodiment, wherein the shell precursor molecules are uniformly distributed throughout the droplet. 19. The method of embodiment 1 or any previous embodiment, wherein the shell precursor molecules are localized at the perimeter of the droplet. 20. The method of embodiment 1 or any previous embodiment, wherein the shell precursor molecules each further comprise a hydrophobic hydrocarbon chain. 21. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain is a butyryl chain. 22. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain comprises at least 2 carbon atoms. 23. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain comprises no more than 6 carbon atoms. 24. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain is linear. 25. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain is branched. 26. The method of embodiment 20 or any previous embodiment, wherein the hydrophobic carbon chain is cyclic. 27. The method of embodiment 1 or any previous embodiment, further comprising localizing a population of core precursor molecules to the partition. 28. The method of embodiment 27 or any previous embodiment, wherein the core precursors each do not comprise a crosslinking moiety. 29. The method of embodiment 27 or any previous embodiment, wherein the core precursors each do not comprise a hydrophobic carbon chain. 30. The method of embodiment 27 or any previous embodiment, wherein the core precursors each comprise a shell structural component that is not eligible for crosslinking. 31. The method of embodiment 30 or any previous embodiment, wherein the shell precursors and the core precursors each comprise a common structural component. 32. The method of embodiment 31 or any previous embodiment, wherein the common structural component comprises a polysaccharide. 33. The method of embodiment 32 or any previous embodiment, wherein the polysaccharide is dextran. 34. The method of embodiment 30 or any previous embodiment, wherein the shell precursors and the core precursors comprise distinct structural components. 35. The method of embodiment 1 or any previous embodiment, wherein inducing crosslinking comprises irradiation. 36. The method of embodiment 1 or any previous embodiment, wherein inducing crosslinking comprises introducing a free radical in the partition. 37. The method of embodiment 1 or any previous embodiment, wherein inducing crosslinking comprises introducing an oxidative stress in thepartition. 38. The method of embodiment 1 or any previous embodiment, wherein inducing crosslinking comprises introducing a thiol in the partition. 39. The method of embodiment 1 or any previous embodiment, wherein the droplet comprises lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP). 40. The method of embodiment 1 or any previous embodiment, wherein inducing crosslinking comprises introducing LAP in the partition. 41. The method of embodiment 1 or any previous embodiment, comprising colocalizing a biological component to the partition. 42. The method of embodiment 1 or any previous embodiment, wherein the hydrogel forms a microcapsule having an aqueous interior and a shell. 43. The method of embodiment 1 or any previous embodiment, comprising degrading the structural component to release contents of the microcapsule. 44. The method of embodiment 43 or any previous embodiment, wherein the degrading comprises contacting to an enzyme. 45. The method of embodiment 44 or any previous embodiment, wherein the enzyme comprises a carbohydrate degrading enzyme. 46. The method of embodiment 44 or any previous embodiment, wherein the enzyme comprises a dextranase. 47. The method of embodiment 43 or any previous embodiment, wherein the degrading comprises physical disruption of the microcapsule. 48. The method of embodiment 47 or any previous embodiment, wherein the physical disruption comprises ball-milling. 49. he method of embodiment 47 or any previous embodiment, wherein the physical disruption comprises freeze thaw treatment. 50. The method of embodiment 1 or any previous embodiment, wherein the non-saturated carbon-carbon bond is strained. 51. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is a constituent of a cyclic moiety. 52. The method of embodiment 51 or any previous embodiment, wherein the cyclic moiety comprises at least 8 atoms. 53. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is an allene. 54. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is an alkene. 55. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is an alkyne. 56. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is a bond in a norbornene moiety. 57. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbon bond is a bond in a cyclooctyne moiety. 58. The method of embodiment 50 or any previous embodiment, wherein the strained non-saturated carbon-carbonbond is a bond in a dibenzocyclooctyne moiety. 59. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules comprises a norbornene moiety. 60. The method of embodiment 56 or any previous embodiment, wherein the population of shell precursor molecules comprises dextran having a crosslinking moiety comprising norbornene. 61. The method of embodiment 57 or any previous embodiment, wherein the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising nitrone. 62. The method of embodiment 57 or any previous embodiment, wherein the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising tetrazine. 63. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules comprises molecules comprising an alkyne crosslinking moiety. 64. The method of embodiment 60 or any previous embodiment, wherein the alkyne crosslinking moiety is a pentynoate crosslinking moiety. 65. The method of embodiment 60 or any previous embodiment, wherein the population of shell precursor molecules further comprises molecules comprising a bis-azido-PEG crosslinking moiety. 66. The method of embodiment 60 or any previous embodiment, comprising localizing, in the partition, a bis-azido-PEG crosslinking moiety. 67. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules comprises molecules comprising an azide-containing moiety. 68. The method of embodiment 64 or any previous embodiment, wherein the azide-containing moiety is a 4-(azidomethyl)benzoate crosslinking moiety. 69. The method of embodiment 64 or any previous embodiment, wherein the azide- containing moiety is an azido-PEG-carboxylate crosslinking moiety. 70. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules comprises dextran modified by butyryl and methacryloyl moieties. 71. The method of embodiment 1 or any previous embodiment, wherein the shell structural component comprises dextran and the crosslinking moiety comprises maleimide. 72. The method of embodiment 68 or any previous embodiment, wherein the maleimide is protected. 73. The method of embodiment 68 or any previous embodiment, wherein the maleimide is deprotected. 74. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules directly react with one another to form a hydrogel. 75. The method of embodiment 1 or any previous embodiment, wherein the population of shell precursor molecules are linked by bridging molecules to form a hydrogel. 76. The method of embodiment 72 or any previousembodiment, wherein the bridging molecules comprise dithiol. 77. The method of embodiment 73 or any previous embodiment, wherein the dithiol is 1 ,4-dithiothreitol (DTT). 78. The method of embodiment 72 or any previous embodiment, wherein the bridging molecules comprise diazide. 79. The method of embodiment 75 or any previous embodiment, wherein the diazide comprises bis-azido-PEG. 80. An emulsion, a droplet of the emulsion comprising a shell precursor molecule, the shell precursor molecule comprising a shell structural component, and a crosslinking moiety comprising a non-saturated bond. 81. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is a carbon-carbon bond. 82. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is a carbon-heteroatom bond. 83. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is a heteroatom-heteroatom bond. 84. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond comprises a nitrogen atom. 85. The emulsion of embodiment 80or any previous embodiment, wherein the non-saturated bond comprises an oxygen atom. 86. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond comprises a sulfur atom. 87. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond comprises a phosphorous atom. 88. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is strained. 89. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is an allene. 90. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is an alkene. 91. The emulsion of embodiment 80 or any previous embodiment, wherein the non-saturated bond is an alkyne. 92. The emulsion of embodiment 80 or any previous embodiment, wherein the shell precursor molecule is selected from a molecule of Fig. 1. 93. The emulsion of embodiment 80 or any previous embodiment, wherein the shell precursor molecule comprises a cyclic moiety. 94. The emulsion of embodiment 90 or any previous embodiment, wherein the cyclic moiety is planar. 95. The emulsion of embodiment 90 or any previous embodiment, wherein the cyclic moiety comprises at least 8 atoms. 96. The emulsion of embodiment 90 or any previous embodiment, wherein the cyclic moiety comprises at least 9 atoms. 97. The emulsion of embodiment 90 or any previous embodiment, wherein the cyclic moiety comprises a benzo moiety. 98. The emulsion of embodiment 90 or any previous embodiment, wherein the cyclic moiety is cyclooctyne 99. The emulsion of embodiment 90 or any previous embodiment,wherein the cyclic moiety is dibenzocyclooctyne. 100. The emulsion of embodiment 78 or any previous embodiment, wherein the shell structural component comprises dextran and the crosslinking moiety comprising a non-saturated carbon-carbon bond comprises a strained alkene. 101. The emulsion of embodiment 97 or any previous embodiment, wherein the crosslinking moiety comprises norbornene. 102. The emulsion of embodiment 97 or any previous embodiment, wherein the droplet further comprises dithiol. 103. The emulsion of embodiment 99 or any previous embodiment, wherein the dithiol comprises 1 ,4-dithiothreitol (DTT). 104. The emulsion of embodiment 80 or any previous embodiment, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising a non-saturated carbonheteroatom and heteroatom-heteroatom bonds comprising tetrazine. 105. The emulsion of embodiment 80 or any previous embodiment, wherein the shell structural comprises dextran and the crosslinking moiety comprising a non-saturated carbon-carbon bond comprises pentynoate. 106. The emulsion of embodiment 102 or any previous embodiment, wherein the droplet further comprises DTT. 107. The emulsion of embodiment 102 or any previous embodiment, wherein the droplet further comprises bis-azido-PEG. 108. The emulsion of embodiment 102 or any previous embodiment, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising an azide moiety. 109. The emulsion of embodiment 102 or any previous embodiment, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising non-saturated carbon-carbon, carbon-heteroatom, heteroatom- heteroatom bonds comprising an 4-(azidomethyl)benzoate moiety. 110. The emulsion of embodiment 80 or any previous embodiment, wherein the shell structural component comprises dextran and a crosslinking moiety comprising non-saturated carbon-carbon and carbonheteroatom bonds comprises methacryloyl, and wherein the shell precursor molecule further comprises a butyryl moiety. 111. The emulsion of embodiment 106 or any previous embodiment, wherein the droplet further comprises DTT. 112. The emulsion of embodiment 80 or any previous embodiment, wherein the shell structural component comprises dextran and a crosslinking moiety comprising a non-saturated bond comprises maleimide in a protected or deprotected form. 113. The emulsion of embodiment 108 or any previous embodiment, whereinthe droplet further comprises DTT. 114. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet further comprises LAP. 115. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet further comprises a radical. 116. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet further comprises a redox reagent. 117. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet is heated to at least 30C for at least 15 minutes. 118. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet is heated to at least 60C for at least 8 hours. 119. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet is heated to at least 100C for at least 15 minutes. 120. The emulsion of any one of embodiments 77 - 110 or any previous embodiment, wherein the droplet further comprises a biomolecule. 121. A microcapsule population generated by the method of any one of embodiments 1 - 71. 122. A microcapsule population generated using a composition of any one of embodiments 77 - 117. 123. A composition comprising the constituents of a droplet of an emulsion of any one of embodiments 77 - 117. 123. A method of synthesizing a hydrogel precursor, the method comprising one or more of: selecting a structural moiety; selecting a crosslinking moiety; modifying the structural component using a hydrophobic moiety such that, upon addition of the crosslinking moiety, the hydrogel precursor exhibits a hydrophobicity sufficient to localize it to a perimeter of an aqueous droplet in a water-in-oil emulsion; and adding the crosslinking moiety. The method of any previous embodiment such as 123, wherein the aqueous droplet comprises hydrogel precursor to form a microcapsule shell and unmodified structural moiety to form a microcapsule aqueous core upon crosslinking of the hydrogel precursor. The method of any previous embodiment such as 123, wherein the structural moiety comprises glucose. The method of any previous embodiment such as 123, wherein the structural moiety comprises dextran. The method of any previous embodiment such as 123, wherein the crosslinking moiety comprises an unsaturated carbon bond. The method of any previous embodiment such as 123, wherein the crosslinking moiety is cyclic. The method of any previous embodiment such as 128, wherein the crosslinking moiety comprises a strained alkyne. The method of any previous embodiment such as 123, wherein the hydrophobic moiety comprises a butyl moiety.

[0210] As used herein, the term “about” in the context of a value refers to arrange spanning 10% above to 10% below that value, or in some cases 1 unit above to one unit below the value when the percentages lead to fractional values and fractional values do not convey meaning and cannot be meaningfully rounded. In the context of a range, the term refers to an extended range spanning from 10% below the lower stated limit to 10% above the higher stated limit.

[0211] The term “at least one selected from the list of A, B, and C” refers to a list comprising A, or A and B, or A and C, or B, or B and C, or C, or A, B, and C, alone or in combination with additional members”.

[0212] As used herein, the prefix “u” refers to the metric modifier “micro”.EXAMPLES

[0213] The following examples relate to specific reactions supporting the general concept of using crosslinking involving unsaturated carbon bonds to induce hydrogel formation.

[0214] Example 1. Nitrone-Norbornene 1,3-Dipolar Cycloaddition. DexB50-Norbornene (LG-049) and DexB50-Nitrone (LG-056), as shown in Fig. 2A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 25 uL of 20% (w / w) DexB50-Norbornene solution in lx PBS, 25 uL, 20% (w / w) DexB50-Nitrone solution in lx PBS and 50 uL lx PBS, pH 7.4. This was combined with an inert core solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS and 50 uL lx PBS. The shell and core solutions were mixed and introduced into a water-in-oil emulsion without an additional catalyst, and incubated overnight at 60C. The emulsion was broken and microcapsules were observed.

[0215] Microcapsules were imaged, and the results are shown in Fig. 2B.

[0216] Example 2. Norbornene Capsules (Radical Crosslinking). DexB50-Norbornene (LG- 049) as shown in Fig. 3A, was reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB50-Norbornene solution in lx PBS and 50 uL lx PBS.

[0217] This was combined with an inert core / photoinducer solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 37.5 uL lx PBS and 12.5 uL 4% (w / w) LAP solution. The shell and core solutions were mixed and introduced into a water-in-oil emulsion, and exposed to an excitation light of 405 nm for 30 seconds. The emulsion was broken and microcapsules were observed.

[0218] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 3B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 93.6 um (CV=9%), SPC Shell Radius Mean: 5.73 um (CV=31%), SPC Volume Mean: 438 pL (CV=25%). Population size used for measurements - -1000.

[0219] Example 3. Pentynoate Capsules (Radical Crosslinking). DexB90-Pentynoate (LG- 048) as shown in Fig. 5A, was reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB90-Pentynoate solution in lx PBS and 50 uL lx PBS.

[0220] This was combined with an inert core / photoinducer solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 37.5 uL lx PBS and 12.5 uL 4% LAP. The shell and core solutions were mixed and introduced into a water-in-oil emulsion, and exposed to an excitation light of 405 nm for 30 seconds. The emulsion was broken and microcapsules were observed.

[0221] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 5B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 92.2 um (CV=4%), SPC Shell Radius Mean: 5.58 um (CV=13%), SPC Volume Mean: 413 pL (CV=12%). Population size used for measurements - -1000.

[0222] Example 4. Norbornene Capsules combined with Dithiol (Radical Crosslinking + Thiol-Ene). DexB50-Norbornene (LG-049) as shown in Fig. 4A, was reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB50-Norbornene solution in lx PBS and 50 uL lx PBS.

[0223] This was combined with an inert core / photoinducer solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 33.5 uL lx PBS, 4 uL of 0.1 M solution of DTT in H2O, and 12.5 uL 4% LAP. The shell and core solutions were mixed and introduced into a water-in-oil emulsion, and exposed to an excitation light of 405 nm for 30 seconds. The emulsion was broken and microcapsules were observed.

[0224] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 4B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 83.7 um (CV=6%), SPC Shell Radius Mean: 5.43 um (CV=13%), SPC Volume Mean: 310 pL (CV=18%). Population size used for measurements - -1000.

[0225] Example 5. Pentynoate Capsules combined with Dithiol (Radical Crosslinking + Thiol-Yne). DexB90-Pentynoate (LG-049) as shown in Fig. 6A, was reacted to form a hydrogelmicrocapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB90-Penytnoate solution in lx PBS and 50 uL lx PBS.

[0226] This was combined with an inert core / photoinducer solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 33.5 uL lx PBS, 4 uL 0.1 M DTT solution in H2O, and 12.5 uL 4% LAP. The shell and core solutions were mixed and introduced into a water-in-oil emulsion, and exposed to an excitation light of 405 nm for 30 seconds. The emulsion was broken and microcapsules were observed.

[0227] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 6B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 92.2 um (CV=4%), SPC Shell Radius Mean: 5.58 um (CV=13%), SPC Volume Mean: 413 pL (CV=12%). Population size used for measurements - -1000.

[0228] Example 6. Alkyne-Azide 1,3-Cycloaddition Capsules.

[0229] DexB90-Pentynoate (LG-048) and Bis-azido-PEG, as shown in Fig. 7A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell / pre-catalyst solution of 50 uL 20% (w / w) DexB90-Penytnoate solution in lx PBS, 36 uL lx PBS, 10 uL 20 mM CuSCL solution in H2O and 4 uL 0.1 M bis-azido-PEG in H2O.

[0230] This was combined with an inert core / reductant solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 40 uL lx PBS, and 10 uL 200 mM sodium ascorbate solution in lx PBS. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was broken and microcapsules were observed.

[0231] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 7B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 79.4 um (CV=6%), SPC Shell Radius Mean: 5.50 um (CV=12%), SPC Volume Mean: 265 pL (CV=19%). Population size used for measurements - -1000.

[0232] Example 7. Alkyne-Azide 1,3-Cycloaddition Capsules.

[0233] DexB90-Pentynoate (LG-048) and DexB90-PEG-azide, as shown in Fig. 8A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 25 uL 20% (w / w) DexB90-Penytnoate solution in lx PBS, 25 uL 20% (w / w) DexB90-PEG-azide solution in IxPBS, 40 uL lx PBS, and 10 uL 20 mM CuSCL solution in H2O.

[0234] This was combined with an inert core / reductant solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 40 uL lx PBS, and 10 uL 200 mM sodium ascorbate solution in lx PBS. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was broken and microcapsules were observed.

[0235] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 8B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 83.3 um (CV=5%), SPC Shell Radius Mean: 5.51 um (CV=13%), SPC Volume Mean: 305 pL (CV=15%). Population size used for measurements - -1000.

[0236] Example 8. Alkyne-Azide 1,3-Cycloaddition Capsules.

[0237] DexB90-Pentynoate (LG-048) and DexB90-azidomethylbenzoate, as shown in Fig. 9A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 25 uL 20% (w / w) DexB90-Penytnoate solution in lx PBS, 25 uL 20% (w / w) 2x DexB90-azidomethylbenzoate solution in lx PBS, 40 uL lx PBS, and 10 uL 20mM CuSO4 solution in H2O.

[0238] This was combined with an inert core / inducer solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 40 uL lx PBS, and 10 uL 200 mM sodium ascorbate solution in lx PBS. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was broken and microcapsules were observed.

[0239] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 9B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 80.9 um (CV=5%), SPC Shell Radius Mean: 5.66 um (CV=13%), SPC Volume Mean: 279 pL (CV=17%). Population size used for measurements - -1000.

[0240] Example 9. Tetrazine-Norbornene Capsules (IEDDA).

[0241] DexB50-Norbornene (LG-049) and DexB90-Tetrazine (LG-068), as shown in Fig. 10A were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 25 uL 20% (w / w) DexB90-Tetrazine solution in lx PBS, 25 uL 20% (w / w) DexB50-Norbornene solution in lx PBS, and 50 uL lx PBS.

[0242] This was combined with an inert core solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS and 50 uL lx PBS. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was incubated overnight at room temperature, after which the emulsion was broken and microcapsules were observed.

[0243] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 10B. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 93.3 urn (CV=2%), SPC Shell Radius Mean: 6.95 urn (CV=12%), SPC Volume Mean: 426 pL (CV=6%). Population size used for measurements - -1000.

[0244] Example 10. Thiol-Ene Reaction (Base-Catalyzed 1,4-Conjugate Addition).DexMAB1090 and dithiol (DTT) as shown in Fig. 11 A were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) functionalized dextran (modified with 10% of methacryloyl and 90% of butyryl groups based on a repeating unit of dextran) solution in lx PBS, 48 uL lx PBS, 2 uL 1 M DTT solution in H2O.

[0245] This was combined with an inert core / inducer solution of 50 uL 20% (w / w) 500 kDa dextran in lx PBS and 50 uL lx Tris HC1, pH 8.0. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was incubated at room temperature for 1 hour, and then was broken and microcapsules were observed.

[0246] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 1 IB. The microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 83.8 urn (CV=4%), SPC Shell Radius Mean: 5.70 urn (CV=12%), SPC Volume Mean: 310 pL (CV=12%). Population size used for measurements - -1000.

[0247] Example 11. Retro Diels- Alder + Thiol-Ene (Base-Catalyzed).

[0248] DexB90-protected-Maleimide and DTT, as shown in Fig. 12A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB90-Protected-Maleimidesolution in lx PBS, 50 uL lx PBS.

[0249] This was combined with an inert core / crosslinking agent solution of 50 uL 20% (w / w) 500 kDa dextran solution in 100 mM Tris-HCl, pH 8.0, 44 uL 100 mM Tris-HCl, pH 8.0, and 6 uL 0.1 M DTT solution in H2O. The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was incubated for 15 minutes at 100C without (Fig. 12B) and with (Fig. 12C) shaking the emulsion on a thermomixer (500 rpm). The emulsion was cooled to room temperature, broken and microcapsules were observed.

[0250] Microcapsule shell thickness and diameter were quantified, and the results are shown in Fig. 12B and Fig. 12C. At Fig. 12B, without shaking, the microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 108 um (CV=10%), SPC ShellRadius Mean: 13.6 um (CV=28%), SPC Volume Mean: 686 pL (CV=30%). Population size used for measurements - -1000.

[0251] At Fig. 12C, with shaking, the microcapsules were measured and observed to possess the following qualities. SPC Diameter Mean: 109 um (CV=2%), SPC Shell Radius Mean: 8.71 um (CV=11%), SPC Volume Mean: 687 pL (CV=6%). Population size used for measurements - -1000. One observes that shaking during shell formation significantly decreased the coefficient of variation for the resulting microcapsule population.

[0252] Example 12. Imine formation.

[0253] DexB50-formylbenzoate and cystamine dihydrochloride (CAS: 56-17-7), as shown in Fig. 13 A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 20% (w / w) DexB50-formylbenzoate in lx PBS, 50 uL 100 mM CHES, pH 8.9.

[0254] This was combined with an inert core / crosslinking agent solution of 50 uL 20% (w / w) 500 kDa dextran solution in lx PBS, 45 uL 100 mM CHES buffer, pH 8.9, and 5 uL 0.1 M cystamine dihydrochloride in lx PBS.

[0255] The shell and core solutions were mixed and introduced into a water-in-oil emulsion. The emulsion was incubated for 2 hours at room temperature, after which the emulsion was broken and microcapsules were observed. Results are shown in Fig. 13B.

[0256] This example demonstrates hydrogel formation through reaction involving a carbonoxygen double bond.

[0257] Example 13. Strain-Promoted Azide Alkyne Cycloaddition. DexB50- dibenzocyclooctyne (DexB50-DBCO) and bis-azido-PEG, as shown in Fig. 14A, were reacted to form a hydrogel microcapsule population. The reaction comprised a reactive shell solution of 50 uL 2x shell (DexB50-DBCO) in 50 uL lx PBS, pH 7.4.

[0258] This was combined with an crosslinking agent solution of 50 uL 2x core (20% 500 kDa Dextran in IxPBS), 46 uL lx PBS, pH 7.4, and 4 uL 0.1 M bis-azido-PEG in water. Alternately, a crosslinking agent solution of 50 uL 2x shell (DexB50-DBCO, LG-073) 50 uL lx PBS, pH 7.4, and 5 uL of 0.1 M bis-azido-PEG in water can be used.

[0259] The shell and core solutions were mixed for a few seconds at room temperature and introduced into a water-in-oil emulsion. After droplet generation and removal of bottom oil, the emulsion breaking was performed immediately. Results are shown in Fig. 14B.

[0260] SPCs exhibited a mean diameter of 79.1 um (CV=7%), a mean shell radius of 5.49 um (CV=14%), mean volume of 263 pL (CV=21%). Droplets formed with a generation speed of 229 Hz.

[0261] This example demonstrates hydrogel through reactions that do not rely upon a copper ion, instead involving azide reaction with strained alkyne. Both the specificity of the azide for the alkyl rather than biological targets, and the absence of a copper ion both reduced cellular toxicity.

[0262] Example 14. DexB50-DBCO Synthesis. DexB50 is reacted with DMAP and EDC and a modified dibenzocyclooctyne (DBCO-Acid), each at 15 mol%, in DMSO, at 25C for two days under argon atmosphere in the absence of ambient light, to yield DexB50-DBCO, as shown in Fig., 16. Reagents include 500 mg DexB50, 2.336 mmol, 4-dimethylaminopyridine (DMAP) (TCI), CAS No 1122-58-3, at 43 mg or 15 mol %, 0.350 mmol; DBCO-acid >98% (TCI), CAS No 1353016-70-2, at 107 mg, or 15 mol %, 0.350 mmol; EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide) (TCI) 0.877 g / mL at 20 °C (lit), CAS No 1892-57-5, at 62 uL or 15 mol %, 0.350 mmol; Dimethylsulfoxide 99.7 % (DMSO), CAS No 67-68-5, and deionized water.

[0263] To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added in small portions and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, anhydrous DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at RT, to the resulting solution, DBCO-Acid (107 mg, 0.350 mmol) was added which was followed by the addition of EDC (62 pL, 0.350 mmol). The mixture was stirred for 2 days at RT under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 5 days , changing water 6 times (VP / VDIW > 20, VP - volume of product solution in dialysis bags, VDIW - volume of DI water in the dialysis vessel) which was followed by freeze-drying. The product was isolated as crystalline white solid in 86% yield (0.516 g).

[0264] LG-044 Synthesis. To a round-bottomed flask equipped with magnetic stirrer and DMSO (40 mL), dextran (4000 mg, 24.67 mmol [according to repeating unit of the polymer]) was added in small portions and the suspension was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (452 mg, 3.70 mmol) was added. After stirring the mixture for 60 min at RT, to the resulting suspension, R-(-)-glycidyl butyrate (3116 pL, 22.20 mmol) wasadded drop wise at room temperature. The mixture was stirred for 3 days at room temperature. To the homogeneous mixture, 1 M HC1 (3.70 mL, 3.70 mmol) was added dropwise, and the mixture was stirred for 15 min. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline off-white solid (4.228 g, yield = 59%, degree of substitution (DoS): butyrate = 58%).

[0265] LG.045 Synthesis. To a round-bottomed flask equipped with magnetic stirrer and DMSO (40 mL), dextran (4000 mg, 24.67 mmol [according to repeating unit of the polymer]) was added in small portions and the suspension was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (452 mg, 3.70 mmol) was added. After stirring the mixture for 60 min at room temperature, to the resulting suspension, R-(-)-glycidyl butyrate (3116 pL, 22.20 mmol) was added dropwise at room temperature. The mixture was stirred for 3 days at room temperature. To the homogeneous mixture, 1 M HC1 (3.70 mL, 3.70 mmol) was added dropwise, and the mixture was stirred for 15 min. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline off-white solid (4.228 g, yield = 70%, degree of substitution (DoS): butyrate = 36%)

[0266] LG-047 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added in small portions and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, biotin (86 mg, 0.350 mmol) was added which was followed by the addition of EDC (62 pL, 0.350 mmol). The mixture was stirred for 2 days at room temperature under argon. To the homogeneous mixture, 1 M HC1 (0.70 mL, 0.700 mmol) was added dropwise, and the mixture was stirred for 15 min. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 followed by freeze-drying. The product was isolated as crystalline off-white solid (0.518 g, yield = 89%, degree of substitution (DoS): butyrate / biotin = 38 / 6 (%)).

[0267] LG-048 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB90 (500 mg, 2.035 mmol [according to repeating unit of the polymer]) was added and the suspension was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (37 mg, 0.305 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, 4-pentynoic acid (30 mg, 0.305 mmol) was addedwhich was followed by the addition of EDC (54 pL, 0.305 mmol). The mixture was stirred for 2 days at room temperature under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline off-white solid (0.485 g, yield = 93%, degree of substitution (DoS): butyrate / pentynote = 45 / 3%).

[0268] LG-049 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added in small portions and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, 5-norbornene-2-carboxylic acid (predominantly endo isomer) (86 mg, 0.350 mmol) was added which was followed by the addition of EDC (62 pL, 0.350 mmol). The mixture was stirred for 2 days at room temperature under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline off-white solid (0.490 g, yield = 90%, degree of substitution (DoS): butyrate / norbornene = 32 / 2 (%)).

[0269] LG-051 Synthesis. To a round-bottomed flask equipped with magnetic stirrer and EtOH (27 mL), terephthalaldehydic acid (500 mg, 3.33 mmol) was added which was followed by N- methyl hydroxylamine hydrochloride (334 mg, 4.00 mmol) and anhydrous NaOAc (656 mg, 8.00 mmol), and the solution was stirred at room temperature overnight. The reaction mixture was then acidified with 1 M HC1 (10 mL, 10 mmol), and the mixture was extracted with dichloromethane (4x 10 mL). Organic phase was dried with anhydrous MgSO4, and the solvent was removed in vacuo followed by drying the product in high vacuum overnight. The crude product was isolated as a white solid (0.473 g, 85%, the product was isolated as a 1 : 1 mixture of terephthalaldehydic acid and the desired Nitrone.

[0270] LG-052 Synthesis. To a flame-dried round-bottomed flask under argon equipped with magnetic stirrer and degassed deionized water (5 mL), 4-maleimidobutyric acid (229 mg, 1.25 mmol) was added followed by methyl 2-furanoate (400 pL, 3.75 mmol). The suspension was stirred at 50 °C for 3 days under argon. The product was extracted with dichloromethane (3x 10 mL), the organic phase was dried with anhydrous MgSO4, the solvent was removed in vacuo followed by drying the product in a high vacuum overnight. The crude product was isolated as a white solid (312 mg, yield = 66%).

[0271] LG-056A, B Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, Nitrone 1 (111 mg, 0.700 mmol) was added which was followed by the addition of EDC (124 pL, 0.700 mmol). The mixture was stirred for 3 days at room temperature under argon. To the homogeneous mixture, 1 M HC1 (0.35 mL, 0.350 mmol) was added dropwise, and the mixture was stirred for 15 min. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze- drying. The product was isolated as crystalline white solid (0.521 g, yield = 73%, degree of substitution: butyrate / nitrone / formylbenzoate = 36 / 4 / 4 (%)). The product was isolated as a mixture of two products: LG-056A (Dex50-Nitrone) and LG-056B (DexB50-Formylbenzoate).

[0272] LG.057 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, Nitrone 1 (111 mg, 0.700 mmol) was added which was followed by the addition of EDC (124 pL, 0.700 mmol). The mixture was stirred for 3 days at room temperature under argon. To the homogeneous mixture, 1 M HC1 (0.35 mL, 0.350 mmol) was added dropwise, and the mixture was stirred for 15 min. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline white solid (0.521 g, yield = 73%, degree of substitution: butyrate / nitrone / formylbenzoate = 36 / 4 / 4 (%)). The product was isolated as a mixture of two products: LG-056A (Dex50-Nitrone) and LG-056B (DexB50-Formylbenzoate).

[0273] LG-061 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (7 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added in small portions and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, “protected” 4-maleimidobutyric acid (108 mg, 0.350 mmol, 64% molar purity) was added which was followed by the addition of EDC (62 pL, 0.350 mmol). The mixture was stirred for 2 days at room temperature under argon. Themixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline light purple solid (0.514 g, yield = 91%, degree of substitution (DoS): butyrate / ” protected”-maleimide = 28 / 2 (%)).

[0274] LG-064 Synthesis. To a flame-dried round-bottomed flask under argon equipped with magnetic stirrer and anhydrous DMSO (10 mL), 4-(bromomethyl)benzoic acid (1000 mg, 4.65 mmol) was added followed by sodium azide (1810 mg, 27.9 mmol). The suspension was stirred at 80 °C for 2 days under argon. To the reaction mixture deionized water (10 mL) and Et2O (20 mL) were added, and the resulting organic layer was separated and washed with deionized water (4x 10 mL). After drying the organic layer with anhydrous MgSO4, removing the solvent and drying the residue in vacuo overnight, the pure product was collected as a white solid (332 mg, yield = 38%).

[0275] LG-065 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (1 mL), DexB90 (79 mg, 0.322 mmol [according to repeating unit of the polymer]) was added and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (8 mg, 0.0644 mmol) was added. After stirring at room temperature until all solids dissolve, to the resulting solution, azido-PEG4-NHS ester (25 mg, 0.0644 mmol), dissolved in 1 mL of anhydrous DMSO, was added. The mixture was stirred for 2 days at room temperature under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline white solid (0.088 g, yield = 91%, degree of substitution (DoS): butyrate / PEG-azide = 58 / 2 (%)).

[0276] LG-066 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB90 (500 mg, 2.035 mmol [according to repeating unit of the polymer]) was added and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (37 mg, 0.305 mmol) and 4-(azidomethyl)-benzoic acid (54 mg, 0.305 mmol) were added. After stirring at room temperature until all solids dissolve, to the resulting solution, EDC (54 pL, 0.305 mmol) was added. The mixture was stirred for 2 days at room temperature under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as a white solid (0.509 g, yield = 93%, degree of substitution (DoS): butyrate / azidomethylbenzoate = 41 / 2 (%)).

[0277] LG-068 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (1 mL), DexB90 (94 mg, 0.382 mmol [according to repeating unit of the polymer])was added and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (9.3 mg, 0.0764 mmol) was added. After stirring at room temperature until all solids dissolve, to the resulting solution, methyltetrazine-NHS ester (25 mg, 0.0764 mmol), dissolved in 2 mL of anhydrous DMSO, was added. The mixture was covered in aluminum foil and was stirred for 4 days at room temperature under argon. The mixture was transferred to a dialysis bag and was dialyzed in deionized water protected from ambient light for 3 days followed by freeze-drying. The product was isolated as crystalline purple solid (0.104 g, yield = 98%, degree of substitution (DoS): B / tetrazine = 62 / 2.5 (%)).

[0278] LG-073 Synthesis. To a round-bottomed flask under argon equipped with magnetic stirrer and DMSO (5 mL), DexB50 (500 mg, 2.336 mmol [according to repeating unit of the polymer]) was added in small portions and the solution was stirred at room temperature until all of dextran dissolved. To the mixture, DMAP (43 mg, 0.350 mmol) was added. After stirring the mixture for 10 min at room temperature, to the resulting solution, DBCO-Acid (107 mg, 0.350 mmol) was added which was followed by the addition of EDC (62 pL, 0.350 mmol). The mixture was stirred for 2 days at room temperature under argon. The mixture was transferred to dialysis bags and was dialyzed in deionized water for 3 days followed by freeze-drying. The product was isolated as crystalline white solid (0.516 g, yield = 86%, degree of substitution (DoS): butyrate / DBCO = 20 / 0.5 (%)).

Claims

CLAIMSWe claim1. A method of synthesizing a hydrogel, comprising localizing, in a partition, a population of shell precursor molecules, each comprising a shell structural component and a crosslinking moiety comprising at least one non-saturated bond, and inducing crosslinking at the nonsaturated bond of the crosslinking moieties.

2. The method of claim 1, wherein the non-saturated bond is a carbon-carbon bond.

3. The method of claim 1, wherein the non-saturated bond is a carbon-heteroatom bond.

4. The method of claim 1, wherein the non-saturated bond is a heteroatom-heteroatom bond.

5. The method of claim 2, wherein the crosslinking moiety comprises a linear carbon chain comprising the non-saturated carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bond.

6. The method of claim 2, wherein the crosslinking moiety comprises a ring comprising the non-saturated carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bond.

7. The method of claim 1 , wherein the crosslinking moiety comprises a nitrogen atom.

8. The method of claim 1, wherein the crosslinking moiety comprises a sulfur atom.

9. The method of claim 1 , wherein the crosslinking moiety comprises an oxygen atom.

10. The method of claim 1, wherein the crosslinking moiety comprises a phosphorous atom.

11. The method of claim 1, wherein the partition is a droplet in an emulsion.

12. The method of claim 2, wherein the non-saturated carbon-carbon bond is a nonconjugated alkene.

13. The method of claim 2, wherein the non-saturated carbon-carbon bond is conjugated to a carbonyl group.

14. The method of claim 2, wherein the non-saturated carbon-carbon bond is a nonconjugated alkyne.

15. The method of claim 1, wherein the structural component comprises a carbohydrate.

16. The method of claim 1, wherein the structural component comprises dextran.

17. The method of claim 1 , wherein the shell precursor molecules are localized to the interior of the droplet.Page 53 of 6118. The method of claim 1, wherein the shell precursor molecules are uniformly distributed throughout the droplet.

19. The method of claim 1, wherein the shell precursor molecules are localized at the perimeter of the droplet.

20. The method of claim 1, wherein the shell precursor molecules each further comprise a hydrophobic hydrocarbon chain.

21. The method of claim 20, wherein the hydrophobic carbon chain is a butyryl chain.

22. The method of claim 20, wherein the hydrophobic carbon chain comprises at least 2 carbon atoms.

23. The method of claim 20, wherein the hydrophobic carbon chain comprises no more than 6 carbon atoms.

24. The method of claim 20, wherein the hydrophobic carbon chain is linear.

25. The method of claim 20, wherein the hydrophobic carbon chain is branched.

26. The method of claim 20, wherein the hydrophobic carbon chain is cyclic.

27. The method of claim 1, further comprising localizing a population of core precursor molecules to the partition.

28. The method of claim 27, wherein the core precursors each do not comprise a crosslinking moiety.

29. The method of claim 27, wherein the core precursors each do not comprise a hydrophobic carbon chain.

30. The method of claim 27, wherein the core precursors each comprise a shell structural component that is not eligible for crosslinking.

31. The method of claim 30, wherein the shell precursors and the core precursors each comprise a common structural component.

32. The method of claim 31, wherein the common structural component comprises a polysaccharide.

33. The method of claim 32, wherein the polysaccharide is dextran.

34. The method of claim 30, wherein the shell precursors and the core precursors comprise distinct structural components.

35. The method of claim 1, wherein inducing crosslinking comprises irradiation.Page 54 of 6136. The method of claim 1, wherein inducing crosslinking comprises introducing a free radical in the partition.

37. The method of claim 1, wherein inducing crosslinking comprises introducing an oxidative stress in the partition.

38. The method of claim 1, wherein inducing crosslinking comprises introducing a thiol in the partition.

39. The method of claim 1, wherein the droplet comprises lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP).

40. The method of claim 1 , wherein inducing crosslinking comprises introducing LAP in the partition.

41. The method of claim 1, comprising colocalizing a biological component to the partition.

42. The method of claim 1, wherein the hydrogel forms a microcapsule having an aqueous interior and a shell.

43. The method of claim 1, comprising degrading the structural component to release contents of the microcapsule.

44. The method of claim 43, wherein the degrading comprises contacting to an enzyme.

45. The method of claim 44, wherein the enzyme comprises a carbohydrate degrading enzyme.

46. The method of claim 44, wherein the enzyme comprises a dextranase.

47. The method of claim 43, wherein the degrading comprises physical disruption of the microcapsule.

48. The method of claim 47, wherein the physical disruption comprises ball milling.

49. he method of claim 47, wherein the physical disruption comprises freeze thaw treatment.

50. The method of claim 1, wherein the non-saturated carbon-carbon bond is strained.

51. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is a constituent of a cyclic moiety.

52. The method of claim 51, wherein the cyclic moiety comprises at least 8 atoms.

53. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is an allene.

54. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is an alkene.Page 55 of 6155. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is an alkyne.

56. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is a bond in a norbornene moiety.

57. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is a bond in a cyclooctyne moiety.

58. The method of claim 50, wherein the strained non-saturated carbon-carbon bond is a bond in a dibenzocyclooctyne moiety.

59. The method of claim 1, wherein the population of shell precursor molecules comprises a norbornene moiety.

60. The method of claim 59, wherein the population of shell precursor molecules comprises dextran having a crosslinking moiety comprising norbornene.

61. The method of claim 60, wherein the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising nitrone.

62. The method of claim 60, wherein the population of shell precursor molecules further comprises dextran having a crosslinking moiety comprising tetrazine.

63. The method of claim 1, wherein the population of shell precursor molecules comprises molecules comprising an alkyne crosslinking moiety.

64. The method of claim 63, wherein the alkyne crosslinking moiety is a pentynoate crosslinking moiety.

65. The method of claim 63, wherein the population of shell precursor molecules further comprises molecules comprising a bis-azido-PEG crosslinking moiety.

66. The method of claim 63, comprising localizing, in the partition, a bis-azido-PEG crosslinking moiety.

67. The method of claim 1, wherein the population of shell precursor molecules comprises molecules comprising an azide-containing moiety.

68. The method of claim 67, wherein the azide-containing moiety is a 4- (azidomethyl)benzoate crosslinking moiety.

69. The method of claim 67, wherein the azide-containing moiety is an azido-PEG- carboxylate crosslinking moiety.Page 56 of 6170. The method of claim 1, wherein the population of shell precursor molecules comprises dextran modified by butyryl and methacryloyl moieties.

71. The method of claim 1, wherein the shell structural component comprises dextran and the crosslinking moiety comprises maleimide.

72. The method of claim 71, wherein the maleimide is protected.

73. The method of claim 71, wherein the maleimide is deprotected.

74. The method of claim 1, wherein the population of shell precursor molecules directly react with one another to form a hydrogel.

75. The method of claim 1, wherein the population of shell precursor molecules are linked by bridging molecules to form a hydrogel.

76. The method of claim 75, wherein the bridging molecules comprise dithiol.

77. The method of claim 76, wherein the dithiol is 1 ,4-dithiothreitol (DTT).

78. The method of claim 75, wherein the bridging molecules comprise diazide.

79. The method of claim 78, wherein the diazide comprises bis-azido-PEG.

80. An emulsion, a droplet of the emulsion comprising a shell precursor molecule, the shell precursor molecule comprising a shell structural component, and a crosslinking moiety comprising a non-saturated bond.

81. The emulsion of claim 80, wherein the non-saturated bond is a carbon-carbon bond.

82. The emulsion of claim 80, wherein the non-saturated bond is a carbon-heteroatom bond.

83. The emulsion of claim 80, wherein the non-saturated bond is a heteroatom-heteroatom bond.

84. The emulsion of claim 80, wherein the non-saturated bond comprises a nitrogen atom.

85. The emulsion of claim 80, wherein the non-saturated bond comprises an oxygen atom.

86. The emulsion of claim 80, wherein the non-saturated bond comprises a sulfur atom.

87. The emulsion of claim 80, wherein the non-saturated bond comprises a phosphorous atom.

88. The emulsion of claim 80, wherein the non-saturated bond is strained.

89. The emulsion of claim 80, wherein the non-saturated bond is an allene.

90. The emulsion of claim 80, wherein the non-saturated bond is an alkene.

91. The emulsion of claim 80, wherein the non-saturated bond is an alkyne.Page 57 of 6192. The emulsion of claim 80, wherein the shell precursor molecule is selected from a molecule of Fig. 1.

93. The emulsion of claim 80, wherein the shell precursor molecule comprises a cyclic moiety.

94. The emulsion of claim 93, wherein the cyclic moiety comprises at least 8 atoms.

95. The emulsion of claim 93, wherein the cyclic moiety comprises at least 9 atoms.

96. The emulsion of claim 93, wherein the cyclic moiety comprises a fused benzene ring.

97. The emulsion of claim 93, wherein the cyclic moiety is cyclooctyne.

98. The emulsion of claim 93, wherein the cyclic moiety is dibenzocyclooctyne.

99. The emulsion of claim 81, wherein the shell structural component comprises dextran and the crosslinking moiety comprising a non-saturated carbon-carbon bond comprises a strained alkene.

100. The emulsion of claim 99, wherein the crosslinking moiety comprises norbornene.

101. The emulsion of claim 99, wherein the droplet further comprises dithiol.

102. The emulsion of claim 101, wherein the dithiol comprises 1 ,4-dithiothreitol (DTT).

103. The emulsion of claim 80, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising a non-saturated carbon-heteroatom and heteroatom- heteroatom bonds comprising tetrazine.

104. The emulsion of claim 80, wherein the shell structural component comprises dextran and the crosslinking moiety comprising a non-saturated carbon-carbon bond comprises pentynoate.

105. The emulsion of claim 104, wherein the droplet further comprises DTT.

106. The emulsion of claim 104, wherein the droplet further comprises bis-azido-PEG.

107. The emulsion of claim 104, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising an azide moiety.

108. The emulsion of claim 104, wherein the droplet further comprises a second shell precursor molecule, the second shell precursor molecule comprising a dextran shell structural component and a crosslinking moiety comprising non-saturated carbon-carbon, carbonheteroatom, heteroatom-heteroatom bonds comprising a 4-(azidomethyl)benzoate moiety.Page 58 of 61109. The emulsion of claim 80, wherein the shell structural component comprises dextran and a crosslinking moiety comprising non-saturated carbon-carbon and carbon-heteroatom bonds comprises methacryloyl, and wherein the shell precursor molecule further comprises a butyryl moiety.

110. The emulsion of claim 108, wherein the droplet further comprises DTT.

111. The emulsion of claim 80, wherein the shell structural component comprises dextran and a crosslinking moiety comprising a non-saturated bond comprises maleimide in a protected or deprotected form.

112. The emulsion of claim 110, wherein the droplet further comprises DTT.

113. The emulsion of any one of claims 80 - 112, wherein the droplet further comprises LAP.

114. The emulsion of any one of claims 80 - 112, wherein the droplet further comprises a radical.

115. The emulsion of any one of claims 80 - 112, wherein the droplet further comprises a redox reagent.

116. The emulsion of any one of claims 80 - 112, wherein the droplet is heated to at least 30C for at least 15 minutes.

117. The emulsion of any one of claims 80 - 112, wherein the droplet is heated to at least 60C for at least 8 hours.

118. The emulsion of any one of claims 80 - 112, wherein the droplet is heated to at least 100C for at least 15 minutes.

119. The emulsion of any one of claims 80 - 112, wherein the droplet further comprises a biomolecule.

120. A microcapsule population generated by the method of any one of claims 1 - 79.

121. A microcapsule population generated using a composition of any one of claims 80 - 119.

122. A composition comprising the constituents of a droplet of an emulsion of any one of claims 80 - 119.

123. A method of synthesizing a hydrogel precursor, the method comprising: selecting a structural moiety; selecting a crosslinking moiety; modifying the structural component using a hydrophobic moiety such that, upon addition of the crosslinking moiety, the hydrogel precursorPage 59 of 61exhibits a hydrophobicity sufficient to localize it to a perimeter of an aqueous droplet in a waterin-oil emulsion; and adding the crosslinking moiety.

124. The method of claim 123, wherein the aqueous droplet comprises hydrogel precursor to form a microcapsule shell and unmodified structural moiety to form a microcapsule aqueous core upon crosslinking of the hydrogel precursor.

125. The method of claim 123, wherein the structural moiety comprises glucose.

126. The method of claim 123, wherein the structural moiety comprises dextran.

127. The method of claim 123, wherein the crosslinking moiety comprises an unsaturated carbon bond.

128. The method of claim 123, wherein the crosslinking moiety is cyclic.

129. The method of claim 128, wherein the crosslinking moiety comprises a strained alkyne.

130. The method of claim 123, wherein the hydrophobic moiety comprises a butyl moiety.Page 60 of 61

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