gels

Review Thiol-Mediated Chemoselective Strategies for In Situ Formation of Hydrogels

Jing Su

Department of Chemistry, Northeastern Illinois University, Chicago, IL 60625, USA; [email protected]; Tel.: +1-773-442-5773


Received: 31 July 2018; Accepted: 31 August 2018; Published: 2 September 2018


Abstract: Hydrogels are three-dimensional networks composed of hydrated polymer chains and have been a material of choice for many biomedical applications such as drug delivery, biosensing, and tissue engineering due to their unique biocompatibility, tunable physical characteristics, flexible methods of synthesis, and range of constituents. In many cases, methods for crosslinking polymer precursors to form hydrogels would benefit from being highly selective in order to avoid cross-reactivity with components of biological systems leading to adverse effects. Crosslinking reactions involving the thiol group (SH) offer unique opportunities to construct hydrogel materials of diverse properties under mild conditions. This article reviews and comments on thiol-mediated chemoselective and biocompatible strategies for crosslinking natural and synthetic macromolecules to form injectable hydrogels for applications in drug delivery and cell encapsulation.

Keywords: hydrogel; chemical crosslinking; chemoselective reaction; thiols

1. Introduction Hydrogels are a class of highly hydrated materials with three-dimensional (3D) networks composed of hydrophilic polymers, which are either synthetic or natural in origin [1]. The structural integrity of hydrogels depends on the crosslinks formed between polymer chains via various physical interactions and chemical bonds. Because they have mechanical properties similar to the extracellular matrix in native tissues, hydrogels have been widely employed as implantable medical devices such as contact lenses and biosensors [2–5], surgical adhesives [6,7], immunoisolating capsules for tissue transplantations [8,9], scaffolds for tissue regeneration [10–12], and materials for drug delivery [13,14]. In particular, in situ forming hydrogels have been very attractive since they allow the delivery of polymer precursors in combination with cells and soluble drugs in aqueous solutions through injection, resulting in the formation of 3D functional hydrogel networks at desired locations [15,16]. Tremendous natural and synthetic materials have been developed for the in situ formation of physical hydrogels by noncovalent electrostatic attraction, hydrogen bonding, and hydrophobic interactions [15,17]. Many of these, however, need to be initiated by changes in pH, temperature, or ionic concentration, such as pH-sensitive leucine-zipper protein assembly [18], thermosensitive collagen gelation [19], Alginate-Ca2+ crosslinking [20], and peptide amphiphile assembly [21–23]. These environmental triggers are not always physiologically relevant or biocompatible, and can be irreversibly detrimental to encapsulated cells and macromolecule drugs. It is also difficult to reproducibly control these conditions in clinical settings. In addition, physically crosslinked hydrogels do not have sufficient mechanical strength and structural stability against environmental changes or even hydrodynamic shearing. On the other hand, the crosslinking of polymers through covalent chemical bond formation in physiological conditions can produce robust hydrogel networks bearing tunable mechanical strength and stability in a much greater range. Hydrogels formed in situ through chemical crosslinking alone or through a hybrid of physical and chemical crosslinking have been shown

Gels 2018, 4, 72; doi:10.3390/gels4030072 www.mdpi.com/journal/gels Gels 2018, 4, 72 2 of 22 Gels 2018, 4, x FOR PEER REVIEW 2 of 22 tobeen meet shown the needsto meet of the many needs different of many biomedical different applications,biomedical applications, from artificial from load-bearing artificial load-bearing connective tissue,connective to 3D tissue, tissue to scaffolds, 3D tissue to scaffolds, controlled to delivery controlled of therapeuticsdelivery of therapeutics [24,25]. [24,25]. In order to develop chemically crosslinked hydrogels to achieve a desired biomedical function, the right polymer precursors, crosslinking methods, and degradation properties of formed hydrogel are all essential. A good understanding of the biological system of interest is required to evaluate the interactions between the system and the applied polymerpolymer precursors,precursors, crosslinkingcrosslinking catalyst/initiators,catalyst/initiators, any possible possible product product released released from from the crosslinking the crosslinking reaction, reaction, and degradation and degradation products fromproducts hydrogels. from Inhydrogels. many cases, In many methods cases, of methods polymer of crosslinking polymer crosslinking would benefit would from benefit being from highly being selective highly selective to avoid cross-reactivityto avoid cross-reactivity and adverse and effects adverse on effects functional on functional components components of the biological of the system biological (Figure system1). In(Figure physiologically 1). In physiologically relevant environments, relevant environments, as focused on as infocused this review, on in chemoselectivitythis review, chemoselectivity is defined as theis defined preferred as the reactivity preferred of reactivity a chemical of groupa chemical toward group another toward specific another functionality specific functionality in the presence in the ofpresence multiple of potentially multiple potentially reactive functionalities, reactive functionalities, especially thoseespecially existing those in existing biological in complexes. biological Thecomplexes. past two The decades past two have decades witnessed have awitnessed remarkable a remarkable advancement advancement of bio-orthogonal of bio-orthogonal chemical reactionschemical thatreactions covalently that covalently connect unnatural connect chemical unnatural structures chemical [26 structures], for example, [26], for 1,3-dipolar example, click 1,3- cycloadditiondipolar click and cycloaddition Diels–Alder and cycloaddition, Diels–Alder providing cycloaddition, promising providing solutions promising to eliminate solutions interference to witheliminate biological interference systems withduring biological the formation systems of polymeric during the hydrogels, formation as summarized of polymeric in hydrogels, several recent as reviewssummarized[24,25 in,27 several]. However, recent these reviews unnatural, [24,25,27]. usually However, expensive these building unnatural, blocks usually may significantly expensive increasebuilding the blocks cost may of materials, significantly and increase this limits the the cost use of of materials, bio-orthogonal and this reactions limits the for use producing of bio- hydrogelsorthogonal in reactions reality. for producing hydrogels in reality.

Figure 1. In situ crosslinking of hydrogels in the presence of the biological complex including cells, Figure 1. In situ crosslinking of hydrogels in the presence of the biological complex including extracellular components, and therapeutic agents. Hydrogel networks should form upon cells, extracellular components, and therapeutic agents. Hydrogel networks should form upon chemoselective interactions between polymer precursors in order to minimize the disturbance to the chemoselective interactions between polymer precursors in order to minimize the disturbance to biological systems under study. the biological systems under study.

On the other hand, polymers presenting naturally existing functionalities such as the amino groups (NH2) and thiols (SH, sulfhydryl), are still widely used in biomedical research and groups (NH2) and thiols (SH, sulfhydryl), are still widely used in biomedical research and applications becauseapplications of the because relatively of low the costrelatively and great low availability. cost and great For example, availability. the For natural example, polymer the chitosan natural presentspolymer aminochitosan groups; presents polypeptides amino groups; can presentpolypeptides amino can groups present through amino lysine groups residues through and lysine thiol groupsresidues at and cysteine thiol residues;groups at and cysteine synthetic residues; macromolecules and synthetic functionalized macromolecules with amine functionalized or thiol groups with areamine readily or thiol available groups from are manyreadily chemical available suppliers from many at affordable chemical prices. suppliers When at affordable these polymers prices. are When used inthese the polymers presence ofare biological used in the components, presence of a commonlybiological appliedcomponents, strategy a commonly for achieving applied chemoselectivity strategy for duringachieving hydrogel chemoselectivity crosslinking during is by hydrogel kinetic control,crosslinking in which is by exogenouskinetic control, polymer in which precursors exogenous are appliedpolymer at precursors much higher are applied concentrations at much than higher those concentrations biological components than those thatbiological are potentially components reactive, that drivingare potentially crosslinking reactive, reactions driving to crosslinking occur mainly reactions between to externally occur mainly supplied between polymers. externally supplied polymers.Compared to the amino group, the thiol group occurs at lower abundance in naturally existingCompared molecules to the and amino therefore group, bears the relativelythiol group higher occurs chemoselectivity, at lower abundance which in cannaturally be kinetically existing “manipulated”molecules and to therefore direct reactions bears mainly relatively taking higher place betweenchemoselectivity, exogenous which thiol-containing can be kinetically polymers and“manipulated” crosslinkers. to This direct review reactions summarizes mainly taking the strategies place between for chemically exogenous crosslinking thiol-containing polymers polymers through and crosslinkers. This review summarizes the strategies for chemically crosslinking polymers through thiol-mediated reactions to form hydrogels in situ for biomedical applications. The goal of this review is not to provide a complete coverage of works that utilized these strategies, but rather to

Gels 2018, 4, 72 3 of 22 thiol-mediated reactions to form hydrogels in situ for biomedical applications. The goal of this review is not to provide a complete coverage of works that utilized these strategies, but rather to highlight inspiring research that opened new horizons of chemoselective hydrogels. Discussions are aimed at giving guidance on how the crosslinking method shall be chosen based on the type of polymer precursors, the desired gelation kinetics, the need for modification of hydrogel networks with biochemical cues, and the effects of crosslinking on the biological targets and surroundings.

2. Reactivity of the Thiol Group A thiol compound refers to an organic molecule containing a sulfhydryl (SH) group bonded to a carbon atom. The SH group is a naturally existing functionality, as seen on the side chain of the amino acid cysteine in proteins and peptides, and in low-molecular-weight natural molecules [28]. The SH group participates in the formation of disulfide bonds (S–S) that are essential to tertiary structures of proteins. This functionality or its anion form (–S−) is also present in the active sites of many enzymes such as caspase proteases and ubiquitin-conjugating enzymes [29,30], using its nucleophilicity to mediate various transformations of substrates. These natural biochemical reactions inspired the development of thiol-based conjugation methods, mainly utilizing the good nucleophilicity of the sulfur atom toward electron-deficient moieties including α-halo carbonyl-containing compounds and α, β-unsaturated compounds, such as maleimide and vinyl sulfone, as reviewed by Stenzel [31]. At the physiological pH, the nucleophilicity of thiol is 1000 times stronger than the ionized amino group, enabling thiols to react with electrophiles with a much higher selectivity. Therefore, the thiol group has been an attractive functionality used in the chemical modification and crosslinking of polymers [31], despite the strong odors of thiol-containing compounds. Most of the thiol-engaged crosslinking methods can be assigned to two categories according to the mechanisms of reactions: addition reactions and substitution reactions. Addition reactions simply connect the reactive groups present on the polymers (or crosslinkers) without releasing any side products to the solution environment. Some of the most widely used bioconjugation methods including Michael-type addition and photoinitiated thiol-ene addition belong to this category. In substitution reactions each bonding formation is accompanied by the release of by-products, usually low-molecular-weight molecules. Several reactions for the formation of disulfide bonds, native chemical ligation and the thiol-epoxy reaction are examples of thiol-involved substitution reactions. Presumably, addition reactions would be ideal for the crosslinking of biocompatible polymeric molecules because no side products are generated and interfere with the biological targets in contact with the hydrogels formed. On the other hand, the use of substitution reactions for in situ hydrogel formation does require additional examination of how released side products affect a system of study, such as encapsulated drugs and cells/tissues. Methods based on both types of reaction mechanisms have been applied in the development of in situ forming hydrogels. Table1 outlines the pros and cons of each crosslinking reaction that will be discussed in detail in Sections3 and4.

Table 1. Thiol-mediated reactions for hydrogel crosslinking in situ.

Reaction Advantages Limitations

• Produces disulfide links that break • Slow gelation down in a reductive environment • Relatively low selectivity Disulfide Formation Suitable for developing smart gels When the thiol-disulfide exchange responsive to glutathione and other method is used, by-products are reducing agents released.

• Catalyst-free Low-molecular-weight thiol by-product Native Chemical Ligation Generates thiol-presenting networks for released structure modifications Gels 2018, 4, x FOR PEER REVIEW 4 of 22 Gels 2018, 4, 72 4 of 22

• Catalyst-free Table 1. Cont. Relatively slow gelation, high Thiol-Epoxy Reaction concentrations of reactants No organic by-product released Reaction Advantages Limitationsneeded

• Catalyst-free Relatively• Possible slow cross-reactivity gelation, high with Thiol-Epoxy Reaction • Catalyst-free concentrationsamino groups of reactants needed No• Rapid organic gelation by-product and released high • Nonuniform crosslinking at the

Michael-Type crosslinking degree • Possible cross-reactivity with • Catalyst-free microscale Additions amino groups • Rapid gelation and high Michael-Type Additions No by-product released • Nonuniform crosslinking at crosslinking degree Somethe microscalecrosslinks are unstable. No by-product released • Fast gelation and tunable Some crosslinks are unstable. • Require the use of photo- crosslinking density • Fast gelation and tunable initiators that may be cytotoxic Photo-initiated Thiol- • Allow spatial and temporal control crosslinking density • Require the use of photo-initiators Photo-initiated Thiol-Ene Ene and Thiol-Yne • of gelAllow formation spatial and temporal control that may be cytotoxic and Thiol-Yne Additions UV light can cause cellular Additions of gel formation UVdamage. light can cause cellular damage. NoNo by-product by-product released released

3. Substitution Reactions of Thiols for Hydrogel Crosslinking

3.1. Formation of DisulfideDisulfide Bonds Figure2 summarizes 2 summarizes the strategies the strategies for constructing for constructing disulfide-crosslinked disulfide-crosslinked hydrogels. Traditionally hydrogels. disulfideTraditionally formation disulfide results formation from the results oxidation from ofthe thiols oxidation exposed of thiols to molecular exposed oxygen to molecular in ambient oxygen air orin mildambient oxidizing air or mild reagents oxidizing such asreagents Cu(II)SO such4 (Figure as Cu(II)SO2A). Disulfide4 (Figure bonds2A). Disulfide can dissociate bonds incan a reductivedissociate environment,in a reductive forenvironment, example, in for the example, presence in of the free presence thiols such of free as glutathionethiols such as (GSH) glutathione and dithiothreitol (GSH) and (DTT),dithiothreitol which allows(DTT), thewhich design allows of redox-responsivethe design of redox-responsive degradation [32 degradation,33]. [32,33].

Figure 2. Strategies of disulfide bond formation for in situ crosslinking of polymer hydrogels. (A) Figure 2. Strategies of disulfide bond formation for in situ crosslinking of polymer hydrogels. (A) Mild Mild oxidation of thiols. (B) Thiol-disulfide exchange (a substitution reaction). The disulfide- oxidation of thiols. (B) Thiol-disulfide exchange (a substitution reaction). The disulfide-crosslinked crosslinked network can break down when treated with reductive agents (e.g., soluble thiols). network can break down when treated with reductive agents (e.g., soluble thiols).

The formation of disulfide-crosslinkeddisulfide-crosslinked hydrogel nanoparticles was was reported reported by by Zhang Zhang et et al. al. using hyperbranched polyglycerol terminated with reduced dithiolane groups (Figure 33))[ [34].34]. In a stepwise manner,manner, thiol thiol groups groups on on the the polymers polymers were were partially partially used used for for disulfide disulfide crosslinking, crosslinking, while while the remainingthe remaining thiols thiols were were reserved reserved to conjugate to conjugate maleimide-tagged maleimide-tagged doxorubicin doxorubicin (DOX). (DOX). By carrying By carrying out theout gelthe formationgel formation and and DOX DOX conjugation conjugation in different in different orders, orders, the researchersthe researchers were were able able to control to control the presentationthe presentation of the of DOXthe DOX drug drug molecules molecules either either at the at gel the surface gel surface or within or within the gel the particles. gel particles. Bothtypes Both oftypes nanogel of nanogel particles particles were were shown shown to be to redox-degradable be redox-degradable through through disulfide disulfide cleavage cleavage and and tested tested for deliveringfor delivering the the DOX DOX to cancer to cancer cells cells in vitro. in vitro.

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Figure 3. Hydrogel nanoparticles generated by the crosslinking of PEGs terminated with reduced FigureFiguredithiolane 3. HydrogelHydrogel that can nanoparticles form nanoparticles disulfide generated bonds. generated by Maleimide-tagged the by crosslinking the crosslinking of doxorubicin PEGs of terminated PEGs can terminated be with attached reduced with either reduced at the dithiolanedithiolanesurface of that thatthe can cangel form particles form disulfide disulfide (A )bonds. or bonds. in Maleimide-taggedthe Maleimide-taggedinterior (B) depending doxorubicin doxorubicin on can the be orderattached can be of attached eithergel crosslinking at the either at theand surfacesurfacedrug conjugation of of the the gel gel particles particles. Image ( Amodified) ( Aor) in or the in with theinterior interiorpermission (B) depending (B) dependingfrom [34].on the Copyright onorder the of order gel 2014 crosslinking of Elsevier. gel crosslinking and and drugdrug conjugation conjugation.. Image Image modified modified with with permission permission from from[34]. Copyright [34]. Copyright 2014 Elsevier. 2014 Elsevier. Because a strong oxidative environment can damage the function of many bioactive molecules, Because a strong oxidative environment can damage the function of many bioactive molecules, mildBecause oxidative a strong conditions oxidative are environment needed to crosslink can damage thiol-presenting the function of polymers many bioactive to form molecules, disulfide mild oxidative conditions are needed to crosslink thiol-presenting polymers to form disulfide mild oxidative conditions are needed to crosslink thiol-presenting polymers to form disulfide hydrogels hydrogels for for encapsulating encapsulating drugs drugs and and cells. cells. However, However, most of most these of mild these oxidation mild oxidation conditions conditions for encapsulating drugs and cells. However, most of these mild oxidation conditions cannot cannot produceproduce a asufficiently sufficiently crosslinked crosslinked network network in a short in a periodshort periodof time of(usually time (usuallyit needs moreit needs more producethan a a day) day) a sufficiently [35]. [35]. The The slow crosslinked slow reaction reaction network kinetics kinetics allows in a allows short undesiredperiod undesired interactions of time interactions (usually between it between the needs thiol- more the thanthiol- apresenting day) [35]. polymers/crosslinkers polymers/crosslinkers The slow reaction and kinetics and those those allowsnaturally naturally undesired occurring occurring interactionsthiols, disulfidesthiols, betweendisulfides and electrophiles the and thiol-presenting electrophiles to to polymers/crosslinkersbecome competitive, competitive, which which and may thosemay alter alter naturally the biologicalthe biological occurring system system thiols,of study of disulfides [36];study therefore, [36]; and therefore, this electrophiles crosslinking this crosslinking to become competitive,method is is not not which always always may considered alterconsidered the asbiological biocompatible. as biocompatible. system Additionally, of study Additionally, [36 ]; the therefore, slow the crosslinking slow this crosslinking crosslinking prevents method prevents is notdisulfide always hydrogels hydrogels considered from from as applications biocompatible. applications in clinical in Additionally,clinical settings settings where the where the slow rapid crosslinking the gelation rapid gelation of prevents injectable of disulfide injectable hydrogelsmaterials is is fromnecessary. necessary. applications in clinical settings where the rapid gelation of injectable materials Thiol-disulfide exchange is a substitution reaction used by nature to form disulfide bonds is necessary.Thiol-disulfide exchange is a substitution reaction used by nature to form disulfide bonds (Figure 2B); however, it also occurs at a relatively slow rate unless high local concentrations of thiols Thiol-disulfide exchange is a substitution reaction used by nature to form disulfide bonds (Figureand disulfides 2B); however, are available it also [37,38]. occurs Zhang at a andrelatively Waymouth slow recently rate unless reported high a localthiol-triggered concentrations ring- of thiols (Figure2B); however, it also occurs at a relatively slow rate unless high local concentrations of thiols and andopening disulfides of thiolane are available disulfide [37,38]. in polymeric Zhang self-assemblyand Waymouth produced recently free reported thiol groups, a thiol-triggered which ring- disulfidesopeningparticipated are of in availablethiolane the formation [ disulfide37,38 of]. a Zhang dynamic in polymeric and thiol-disulfide Waymouth self-assembly recently exchange reported producedhydrogel a network thiol-triggered free thiolwith a groups, self- ring-opening which ofparticipatedhealing thiolane property disulfide in the(Figure formation in polymeric4) [39]. ofMaleimide a self-assemblydynamic was thiol-disulfide added produced to consume freeexchange thiolfree thiol groups,hydrogel groups which network within participated the with a self- in thehealinghydrogel formation property and of prohibited a dynamic(Figure thiol-disulfide 4) thiol-disulfide [39]. Maleimide exchange, exchange was changing added hydrogel anto consume adaptable network free with network thiol a self-healing to groups a rigid, within property the (Figurehydrogelpermanent4)[ 39 andnetwork.]. Maleimide prohibited was thiol-disulfide added to consume exchange, free thiol changing groups an within adaptable the hydrogel network and prohibitedto a rigid, thiol-disulfidepermanent network. exchange, changing an adaptable network to a rigid, permanent network.

Figure 4. Crosslinking of dithiolane-containing polymeric micelles by thiol-triggered disulfide-thiol exchange to form self-healing hydrogels. The addition of a thiol-capturing compound, maleimide, decreased the amount of free thiols necessary for the disulfide-thiol exchange, changing the dynamic FiguregelFigure to a 4.permanent 4.Crosslinking Crosslinking network. of of dithiolane-containing Imagedithiolane-containing modified with permission polymeric polymeric from micelles micelles [39]. Copyright by by thiol-triggered thiol-triggered 2017 ACS. disulfide-thiol disulfide-thiol exchangeexchange toto formform self-healing self-healing hydrogels.hydrogels. TheThe additionaddition ofof aa thiol-capturingthiol-capturing compound,compound, maleimide,maleimide, decreaseddecreased the the amount amount of of free free thiols thiols necessary necessary for for the the disulfide-thiol disulfide-thiol exchange, exchange, changing changing the the dynamic dynamic gelgel to to a a permanent permanent network. network. Image Image modified modified with with permission permission from from [ 39[39].]. Copyright Copyright 2017 2017 ACS. ACS.

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A photo-initiated, radical-mediated thiol-disulfide exchange reaction was demonstrated by A photo-initiated, radical-mediated thiol-disulfide exchange reaction was demonstrated by Wang et al. to construct hydrogels using a comb-like polymer of polyethylene glycol grafted with Wang et al. to construct hydrogels using a comb-like polymer of polyethylene glycol grafted with disulfide-linkedGels 2018, 4, x FOR poly(ethyl PEER REVIEW methacrylate) derivative P(EMA-SS-PEG) [40]. UV irritation 6 of 22 in the disulfide-linked poly(ethyl methacrylate) derivative P(EMA-SS-PEG) [40]. UV irritation in the presence presence of a radical initiator triggered the breakage of some disulfides between PEG and grafted of a radicalA photo-initiated, initiator triggered radical-mediated the breakage thiol-disulfide of some disulfides exchange between reaction PEG was and demonstrated grafted EMA by and EMA and generated free thiols that formed new disulfides among PEG polymer chains to produce generatedWang et free al. thiolsto construct that formed hydrogels new using disulfides a comb-like among polymer PEG polymerof polyethylene chains glycol to produce grafted crosslinked with crosslinked network through a mechanism suggested in Figure 5. networkdisulfide-linked through a mechanism poly(ethyl methacrylate) suggested in derivative Figure5. P(EMA-SS-PEG) [40]. UV irritation in the presence of a radical initiator triggered the breakage of some disulfides between PEG and grafted EMA and generated free thiols that formed new disulfides among PEG polymer chains to produce crosslinked network through a mechanism suggested in Figure 5.

FigureFigure 5. 5. TheThe mechanism mechanism of of UV-triggered UV-triggered thiol-disulfide thiol-disulfide exchange exchange for for the the formation formation of of hydrogels. hydrogels. ImageImageFigure reproduced reproduced 5. The mechanism with with permission permission of UV-triggered from from [40]. [40 thiol-disulfide]. Copyright Copyright 2016exchange 2016 RSC. RSC. for the formation of hydrogels. Image reproduced with permission from [40]. Copyright 2016 RSC. AA modified modified version version of of thiol-disulfide thiol-disulfide exchange exchange between between thiols thiols and and activated activated disulfides disulfides (e.g., (e.g., pyridylpyridyl disulfide) disulfide)A modified proceeds proceeds version offaster faster thiol-disulfide than than thiol thiol exchange oxidation oxidation between to to form form thiols disulfides, disulfides, and activated and and has hasdisulfides been been applied applied(e.g., to to polymerpolymerpyridyl crosslinking crosslinking disulfide) proceedsto to form form nanogelsfaster nanogels than within thiol within oxidation hours hours [41]. to [41 formRecent]. Recentdisulfides, examples examples and include has includebeen the applied work the by workto Peng by etPeng al.polymer that et al. demonstrated crosslinking that demonstrated to theform crosslinking nanogels the crosslinking within of hours pyridyl of [41]. pyridyl disulfide-presenting Recent disulfide-presenting examples include copolymers the work copolymers by with Peng four- with et al. that demonstrated the crosslinking of pyridyl disulfide-presenting copolymers with four- armed,four-armed, thiol-terminated thiol-terminated linkers linkers [42]. [42 The]. The disulfide-crosslinked disulfide-crosslinked nanogels nanogels were were used used for for the the armed, thiol-terminated linkers [42]. The disulfide-crosslinked nanogels were used for the encapsulationencapsulation and and deactivation deactivation of of a a cellulase. cellulase. The The enzyme enzyme activity activity of of the the cellulase cellulase was was recovered recovered as as encapsulation and deactivation of a cellulase. The enzyme activity of the cellulase was recovered as thethe protein proteinthe protein was was was released released released from from from the the the hydrogel hydrogelhydrogel traptrap trap uponupon upon DTT-induced DTT-induced DTT-induced disulfide disulfide disulfide bond bond bond breaking breaking breaking and gel and and gel gel degradationdegradationdegradation (Figure (Figure (Figure 66).). 6).

Figure 6. Crosslinking of pyridyl disulfide-presenting polymer self-assembly with thiol-terminated Figure 6. Crosslinking of pyridyl disulfide-presenting polymer self-assembly with thiol-terminated linkerslinkers for thefor the encapsulation encapsulation and and deactivation deactivation of of a a cellulase. cellulase. ImageImage modifiedmodified with with permission permission from from [42]. FigureCopyright[42]. 6. Copyright Crosslinking 2016 Wiley. 2016 ofWiley. pyridyl disulfide-presenting polymer self-assembly with thiol-terminated linkers for the encapsulation and deactivation of a cellulase. Image modified with permission from [42]. Copyright 2016 Wiley.

Gels 2018, 4, x FOR PEER REVIEW 7 of 22 Gels 2018, 4, 72 7 of 22 A rapid exchange between thiols and thiosulfonates was reported by Schäfer and co-workers [43]. PolyamidesGels 2018, 4, x FOR presenting PEER REVIEW S-ethylsulfonyl- L-cysteine units were used to react with free thiols 7 of 22 and generateA rapid disulfide exchange tags between along the thiols polymer and thiosulfonates chains within was a reported minute by (Figure Schäfer 7). and This co-workers substitution [43]. A rapid exchange between thiols and thiosulfonates was reported by Schäfer and co-workers reactionPolyamides was presentingshown to Sbe-ethylsulfonyl- quantitative Land-cysteine highly units selective, were used and toreleased react with small free alkylsulfonate thiols and generate as a [43]. Polyamides presenting S-ethylsulfonyl-L-cysteine units were used to react with free thiols and sidedisulfide product tags with along low the toxicity. polymer This chains method within has a minutebeen used (Figure by the7). Thissame substitution research group reaction to react was generate disulfide tags along the polymer chains within a minute (Figure 7). This substitution shown to be quantitative and highly selective, and released small alkylsulfonate as a side product with thiolsulfonate-containingreaction was shown to block be quantitative copolymers and with highly dithiol selective, crosslinkers and released to interconnect small alkylsulfonate self-assembled as a low toxicity. This method has been used by the same research group to react thiolsulfonate-containing micellesside (Figure product 7B)with [44], low andtoxicity. may This serve method as an has effective been used strategy by the forsame the research production group ofto disulfidereact hydrogelblockthiolsulfonate-containing copolymers networks within situ dithiol for block other crosslinkers copolymers applications. to with interconnect dithiol crosslinkers self-assembled to interconnect micelles self-assembled (Figure7B) [ 44], and maymicelles serve (Figure as an 7B) effective [44], and strategy may serve for the as production an effective strategyof disulfide for the hydrogel production networks of disulfide in situ for otherhydrogel applications. networks in situ for other applications.

Figure 7. Thiol-disulfide exchange between thiols and thiolsulfonates for the functionalization (A) FigureFigure 7. Thiol-disulfide 7. Thiol-disulfide exchange exchange between between thiols thiols and and thiolsulfonates thiolsulfonatesfor for thethe functionalizationfunctionalization ( (AA) ) and and chemical crosslinking (B) of thiol-functionalized polymers. In both cases the formed disulfide chemicaland chemical crosslinking crosslinking (B) of thiol-functionalized(B) of thiol-functionalized polymers. polymers. In bothIn both cases cases the the formed formed disulfide disulfide links linksare redox-responsive. links are redox-responsive. are redox-responsive. This exchange This This exchange exchange reaction reaction reaction in (B) was in in (B shown)) was was shown to shown be orthogonal to to be be orthogonal orthogonal to ester to formation ester to ester formationand 1,3-dipolarformation and 1,3-dipolarand cyclic 1,3-dipolar addition cyclic cyclic used addition addition for labeling used used for thefor labeling gellabeling particles thethe gel withgel particles particles dyes. Imageswith with dyes. dyes. are Images modified Images are withare modified with permission from [44]. Copyright 2017 ACS. modifiedpermission with from permission [44]. Copyright from [44]. 2017 Copyright ACS. 2017 ACS.

3.2.3.2. Native Native3.2. Native Chemical Chemical Chemical Ligation Ligation Ligation Native chemical ligation (NCL) is the substitution reaction between a thioester and a 2- NativeNative chemical chemical ligation ligation (NCL) (NCL) is the is substitution the substitution reaction between reaction a thioesterbetween and a thioester a 2-aminoethanethiol and a 2- aminoethanethiol moiety, for example, an N-terminal cysteine residue of a protein or peptide. The aminoethanethiol moiety, for example, an N-terminal cysteine residue of a protein or peptide. The moiety,reaction for example, yields an aninitialN-terminal thioester exchange cysteine residueproduct that of a spontaneously protein or peptide. undergoes The an reaction S- to N-acyl yields an reactioninitialmigration thioester yields toan exchange form initial a new thioester product amide bondexchange that spontaneously[45], as product shown inthat undergoesFigure spontaneously 8A. an S- to undergoesN-acyl migration an S- to to N form-acyl a migrationnew amide to bondform [a45 new], as amide shown bond in Figure [45],8 asA. shown in Figure 8A. A B

A B

+ +

pH7~8

SH HS pH7~8 H O + RSH H N O N C NH HN C O O SH HS H O + RSH H N O N C NH HN C O O O HN C O NH C O N O H N H HS SH

O O HN C +NH C O N O H N H HS SH Figure 8. (A) A general scheme of NCL. (B) Catalyst-free in situ+ hydrogel crosslinking by native chemical ligation. Four-armed PEG macromonomers terminated with N-terminal cysteine and Figurethioester 8. (A ) groups A general crosslink scheme to form of NCL. gels ( inB) aqueous Catalyst-free media in atsitu pH hydrogel 7~8. Image crosslinking reproduced by with native Figure 8. (A) A general scheme of NCL. (B) Catalyst-free in situ hydrogel crosslinking by native chemicalpermission ligation. from Four-armed [35]. Copyright PEG 2009 macromonomers ACS. terminated with N-terminal cysteine and thioester chemical ligation. Four-armed PEG macromonomers terminated with N-terminal cysteine and groups crosslink to form gels in aqueous media at pH 7~8. Image reproduced with permission from [35]. thioester groups crosslink to form gels in aqueous media at pH 7~8. Image reproduced with Copyright 2009 ACS. permission from [35]. Copyright 2009 ACS.

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The chemoselectivity of NCL lies in that only the 2-aminoethanethiol moiety is reactive with a thioester to complete this transthioesterification-rearrangement process and form a stable amide bond.Gels A2018 thiol, 4, 72 group alone, as on the side chain of a cysteine residue in the middle of polypeptide8 of 22 chains, or an amino group only, does not interfere with the NCL reaction. This mild ligation method has provenThe useful chemoselectivity in the chemical of NCL synthesis lies in that of only large the peptides 2-aminoethanethiol and proteins moiety [46] is and reactive peptide-based with a blockthioester polymers to complete and dendrimers this transthioesterification-rearrangement [47,48]. In an early application process andof NCL form a to stable covalently amide bond. connect polymers,A thiol Colliergroup alone, and as co-workers on the side reportedchain of a cysteinethe cross-linking residue in the of middle pre-assembled of polypeptide β-sheet-forming chains, peptidesor an with amino terminal group only, thioester does not and interfere cysteine with groups the NCL to reaction.increase Thisthe stiffness mild ligation of physical method haspeptide hydrogelsproven [49]. useful in the chemical synthesis of large peptides and proteins [46] and peptide-based block polymersHu et al. andapplied dendrimers NCL to [47 form,48]. InPEG an hydrogels early application in situ of in NCL an toaqueous covalently environment connect polymers, (Figure 8B) [35]. CollierMixing and solutions co-workers of four-armed reported the PEG cross-linking macromonomers of pre-assembled terminatedβ-sheet-forming with thioester peptides and withcysteine terminal thioester and cysteine groups to increase the stiffness of physical peptide hydrogels [49]. groups at pH 7~8 resulted in the formation of robust hydrogels rapidly within minutes. These Hu et al. applied NCL to form PEG hydrogels in situ in an aqueous environment (Figure8B) [ 35]. hydrogels remained stable after treatment with excess reducing agents such as tris(2- Mixing solutions of four-armed PEG macromonomers terminated with thioester and cysteine groups carboxyethyl)phosphineat pH 7~8 resulted in the (TCEP) formation and of 2-mercaptoethanol, robust hydrogels rapidly implying within the minutes. polymeric These hydrogels network was crosslinkedremained by stable amide after bonds treatment formed with through excess reducing the NCL agents mechanism, such as tris(2-carboxyethyl)phosphine rather than by intermolecular disulfide(TCEP) bonds and 2-mercaptoethanol, between Cys groups. implying This the hydrogel polymeric formation network was strategy crosslinked was later by amide applied bonds to the encapsulationformed through of extracellular the NCL mechanism, matrix rather proteins than to by intermolecular engineer the disulfide microenvironment bonds between of Cys human mesenchymalgroups. This stem hydrogel cells formationin 3D culture strategy by wasJung later and applied co-workers to the encapsulation [50]. In a more of extracellular recent application matrix of the strategy,proteins to cysteine-functionalized engineer the microenvironment hyaluronic of human acids mesenchymal were crosslinked stem cells with in 3D multi-armed culture by Jung PEG- thioesterand co-workers macromonomers [50]. In a to more form recent hydrogels application in situ of, the expanding strategy, cysteine-functionalized the choices of biomedical hyaluronic polymers for NCLacids crosslinking were crosslinked [51]. with multi-armed PEG-thioester macromonomers to form hydrogels in situ, expanding the choices of biomedical polymers for NCL crosslinking [51]. Early studies of NCL-mediated hydrogel crosslinking revealed some drawbacks of the method Early studies of NCL-mediated hydrogel crosslinking revealed some drawbacks of the method including the instability of thioesters toward hydrolysis and especially a potential concern about the including the instability of thioesters toward hydrolysis and especially a potential concern about adversethe adverse biological biological effects effects of low-molecular-weight of low-molecular-weight thiol thiol side side products released released from from NCL NCL and and hydrolysishydrolysis of thioesters, of thioesters, which which may may vary vary in in different different applications. applications. The useuse ofof thiolactonethiolactone in in place place of a thioesterof a thioester to react to with react withcysteine cysteine can generate can generate an anamide amide bond bond without without releasing releasing a a soluble soluble thiolthiol by- product,by-product, as shown as shown in Figure in Figure 9. Fan9. Fan and and co-workers co-workers usedused thiolactone-graftedthiolactone-grafted and and cysteine-grafted cysteine-grafted poly(glutamicpoly(glutamic acid) acid) precursors precursors to to form form NCL-crosslinked NCL-crosslinked hydrogels hydrogels compatible compatible with with cultured cultured mouse mouse fibroblastfibroblast cells cells [52]. [52 ].

FigureFigure 9. The 9. Thenative native chemical chemical ligation ligation (NCL) (NCL) reaction reaction between between polymers polymers presentingpresenting thiolactone thiolactone (a cyclic(a thioester) cyclic thioester) and a and 2-aminoethanethiol a 2-aminoethanethiol group group does does not not release release any any soluble soluble thiolthiol by-product by-product as as in the traditionalin the traditional NCL NCL reactions. reactions. All All thiols thiols groups groups are are immobilized immobilized onto onto the the covalent covalent network network of of crosslinkedcrosslinked polymers. polymers.

A modifiedA modified version version of the of theNCL NCL method, method, oxo-ester-mediated oxo-ester-mediated NCL NCL (OMNCL, (OMNCL, Figure Figure 10),10), was was used by Strehin and co-workers to crosslink eight-armed PEG macromonomers terminated with used by Strehin and co-workers to crosslink eight-armed PEG macromonomers terminated with N- N-hydroxysuccinimide (NHS) active ester and cysteine [53]. The gelation of these macromonomers hydroxysuccinimide (NHS) active ester and cysteine [53]. The gelation of these macromonomers occurred rapidly within 20 s after mixing, while the crosslinking of macromonomers presenting NHS occurredand amino rapidly groups within was 20 showns after mixing, to be 10 while times slower,the crosslinking confirming of the macromonomers initial, rapid ester presenting exchange NHS and betweenamino groups NHS and was the shown thiol group to be in 10 cysteine, times slower, followed confirming by S→N rearrangement the initial, rapid to form ester an amide exchange between NHS and the thiol group in cysteine, followed by S→N rearrangement to form an amide bond. Although NHS ester is hydrolytically unstable, the hydrolysis product, NHS, also as the side product released from the OMNCL, has much less interference with biological systems than thiols

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Gels 2018, 4, x FOR PEER REVIEW 9 of 22 bond. Although NHS ester is hydrolytically unstable, the hydrolysis product, NHS, also as the side product released from the OMNCL, has much less interference with biological systems than thiols produced in NCL. In a recent report by Boere et al., NCL and OMNCL methods were compared in produced in NCL. In a recent report by Boere et al., NCL and OMNCL methods were compared in connectingconnecting cysteine-grafted cysteine-grafted poly( poly(NN-isopropylacrylamide)-isopropylacrylamide) with with linkers linkers containing containing thioester thioester or NHS or [54 NHS]. [54]. ChemicalChemicalcrosslinking crosslinking by by OMNCL OMNCL produced produced hydrogels hydrogels with higher with stiffnesshigher stiffness and lower andin vitro lowertoxicity in vitro toxicityto mouseto mouse endothelial endothelial cells thancells gelsthan formed gels formed by NCL. by NCL.

FigureFigure 10. A 10. generalizedA generalized reaction reaction scheme scheme for for oxo-ester-mediated oxo-ester-mediated nativenative chemical chemical ligation ligation (OMNCL) (OMNCL) used forused crosslinking for crosslinking eight-armed eight-armed PEG PEG macromonomers macromonomers to form aa chemicalchemical hydrogel hydrogel network. network. Image Image reproducedreproduced with with permission permission from from [53]. [53 ].Copyright Copyright 2013 2013 RSC. RSC.

Hydrogel crosslinking by NCL and OMNCL reactions generates free thiol moieties bound to Hydrogel crosslinking by NCL and OMNCL reactions generates free thiol moieties bound to the the polymeric network, which may introduce anti-oxidative properties to hydrogels and therefore polymericcan increase network, the which survival may of cells introduce cultured anti-oxidative in the hydrogel properties or at the gelto hydrogels surface [55 ].and Furthermore, therefore can increasethese the immobilized survival of thiol cells groups cultured can be in used the tohydrogel conjugate or molecules at the gel containing surface thiol-reactive[55]. Furthermore, structures these immobilizedincluding thiol maleimide groups and canα -halobe used carbonyl to conjugate groups, tomolecules install desired containing functions thiol-reactive in formed hydrogel structures includingnetworks maleimide (Figure 11andA) α-halo [35]. A secondcarbonyl strategy groups, of hydrogel to install functionalization desired functions is to in sacrifice formed a certain hydrogel networksamount (Figure of thiol 11A) groups [35]. inA second the polymer strategy precursors of hydrogel to attach functionalization thiol-reactive molecules is to sacrifice prior a to certain in amountsitu of gelation. thiol groups Su et in al. the utilized polymer a rapid precursors and quantitative to attach thiol-reactive Michael-type additionmolecules to prior conjugate to in a situ gelation.maleimide-terminated, Su et al. utilized a anti-inflammatory rapid and quantitative peptide Michael-type to a low percentage addition of Cys to moieties conjugate of four-armed a maleimide- terminated,PEG-Cys anti-inflammatory macromonomers (Figurepeptide 11 toB) a [56 low]. Subsequent percentage mixing of Cys of moieties the modified of four-armed macromonomers PEG-Cys with thioester-terminated four-armed PEGs resulted in the formation of peptide-conjugated NCL macromonomers (Figure 11B) [56]. Subsequent mixing of the modified macromonomers with hydrogels that promoted the survival of encapsulated pancreatic islet β-cells. However, a limitation of thioester-terminated four-armed PEGs resulted in the formation of peptide-conjugated NCL this functionalization strategy is that bioactive molecules can only be incorporated at low densities hydrogelssince thethat majority promoted of Cysthe groupssurvival need of encapsulated to be retained pancreatic to react with islet thioester β-cells. groups However, for productive a limitation of thiscrosslinking. functionalization Alternatively, strategy the is incorporation that bioactive of themolecules functional can molecules only be (e.g.,incorporated cell survival-promoting at low densities since peptides)the majority into theof Cys initial groups macromonomer need to be structures retained for to NCLreact or with OMNCL thioester crosslinking groups (Figurefor productive 11C), crosslinking.may offer Alternatively, a better way to present the incorporation these molecules of at the more functional variable densities molecules in a hydrogel (e.g., cell system. survival- promoting peptides) into the initial macromonomer structures for NCL or OMNCL crosslinking (Figure 11C), may offer a better way to present these molecules at more variable densities in a hydrogel system.

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FigureFigureFigure 11. Functionalization11.Functionalization Functionalization of of of NCL NCL NCL hydrogels. hydrogels.hydrogels. ((A (AA)) )The The The hydrogel hydrogel hydrogel network network network crosslinked crosslinked crosslinked by the by by NCL the the NCL NCL reactionreactionreaction presents presents presents thiols thiols thiols that that that can can can react react react with withwith maleimide-attachedmaleimide-attached maleimide-attached peptides peptides peptides (or (orother (or other other bioactive bioactive bioactive molecules) molecules) molecules) post-gelation.post-gelation. ( (BB) )( BMaleimide-attached Maleimide-attached) Maleimide-attached moleculesmolecules cancan can be be be conjugated conjugated conjugated to tothiol-polymers to thiol-polymers thiol-polymers prior prior priorto NCL to to NCL NCL crosslinking.crosslinking.crosslinking. ( (CC) ) (MoleculesC Molecules) Molecules of of of desired desired desired properties propertiesproperties cancan can be be be synthetically synthetically synthetically inserted inserted inserted into into intothe thiol-polymersthe the thiol-polymers thiol-polymers oror thioester-polymers thioester-polymersor thioester-polymers to toform form functional functional NCL NCL hydrogels. hydrogels.

An interesting application of the NCL strategy in the dissolution of thioester-linked hydrogels AnAn interesting applicationapplication of of the the NCL NCL strategy strategy in in the the dissolution dissolution of thioester-linkedof thioester-linked hydrogels hydrogels was was demonstrated by Ghobril and co-workers [57]. Dendritic macromers presenting multiple thiol wasdemonstrated demonstrated by Ghobril by Ghobril and co-workers and co-workers [57]. Dendritic[57]. Dendritic macromers macromers presenting presenting multiple multiple thiol termini thiol termini were used to react with a PEG crosslinker containing the NHS active esters to form thiolester were used to react with a PEG crosslinker containing the NHS active esters to form thiolester networks termininetworks were (Figureused to 12). react A small with thiola PEG molecule, crosslinker methyl containing ester of L-cysteine, the NHS added active at estershigh concentrations to form thiolester (Figure 12). A small thiol molecule, methyl ester of L-cysteine, added at high concentrations to the networksto the (Figurehydrogel, 12). was A ablesmall to thioltrigger molecule, the dissolution methyl of ester the gel of byL-cysteine, the NCL mechanismadded at high that concentrationsbroke the tohydrogel, thethioester hydrogel, was links able was in to theable trigger macromolecular to trigger the dissolution the dissolution network. of the This gel of bythe gel the dissolutiongel NCL by the mechanism NCL strategy mechanism was that applied broke that the to broke thethioester the thioesterlinksdevelopment in the links macromolecular in of the dissolvable macromolecular network. sealant This for network. wound gel dissolution closure This gel [57,58], strategy dissolution as wasexamples applied strategy of smart to was the biomaterial development applied to the of developmentdissolvabledesign inspired sealant of dissolvable by for good wound understanding sealant closure for [ 57of wound, chemical58], as closure examples reaction [57,58], mechanisms. of smart as examples biomaterial of design smart biomaterial inspired by designgood understanding inspired by good of chemical understanding reaction of mechanisms. chemical reaction mechanisms.

Figure 12. The use of NCL to dissolve a thioester-linked hydrogel network by the addition of a cysteine derivative. Image modified with permission from [57]. Copyright 2013 Wiley.

Figure 12. The use of NCL to dissolve a thioester-linked hydrogel network by the addition of a Figure 12. The use of NCL to dissolve a thioester-linked hydrogel network by the addition of a cysteine cysteine derivative. Image modified with permission from [57]. Copyright 2013 Wiley. derivative. Image modified with permission from [57]. Copyright 2013 Wiley.

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3.3.3.3. Thiol-Epoxy Thiol-Epoxy Reaction Reaction (Ring-Opening (Ring-Opening Reaction Reaction of of Epoxides) Epoxides) TheThe thiol-epoxy thiol-epoxy reaction reaction involves involves a a nucleophilic nucleophilic substitution substitution between between the the thiol/thiolate nucleophilenucleophile and and an an electrophilic electrophilic carbon carbon on on the the epoxy epoxy ring, ring, leading leading to the to thering ring opening opening followed followed by protonby proton transfer transfer to generate to generate a thioether-alcohol a thioether-alcohol product product (Figure (Figure 13A). 13A). Although Although initiated initiated by by the the substitutionsubstitution mechanism, mechanism, the the overall overall outcome outcome of of the the reaction reaction is is similar similar to to an an addition addition reaction reaction in in which which therethere are are no no organic organic by-products. by-products. TheThe thiol-epoxy thiol-epoxy reaction reaction has been has beenused for used the polymerization for the polymerization of monomer of building monomer blocks building [59], theblocks functionalization [59], the functionalization of polymers of [60], polymers and was [60 shown], and wasby Gao shown et al. by for Gao crosslinking et al. for crosslinking poly(N,N- dimethylacrylamide-co-glycidylpoly(N,N-dimethylacrylamide-co-glycidyl methacrylate) methacrylate) to form to hydrogels form hydrogels in situ in situ under under physiological physiological ◦ conditionsconditions (pH (pH 7~8, 7~8, 37 37 °C,C, Figure Figure 13B) 13B) [61]. [61]. The The polymer polymer precursors precursors started started gelation gelation within within seconds seconds whenwhen present present at 10~20% 10~20% weightweight percent percent in in aqueous aqueous solutions, solutions, and and the resultant the resultant hydrogels hydrogels were shown were shownto be nontoxic to be nontoxic to Hela to cells Hela incubated cells incubated together. together. The fast The reaction fast reaction kinetics andkinetics relatively and relatively high selectivity high selectivityshown in thisshown study in suggest this study a strong suggest potential a strong of the potential thiol-epoxy of the reaction thiol-epoxy for the reaction development for the of developmentinjectable hydrogel of injectable materials. hydrogel materials.

FigureFigure 13. 13. (A(A) ) Thiol-triggered Thiol-triggered ring ring opening opening of of epoxides epoxides undergoes undergoes a a nucleophilic nucleophilic substitution substitution mechanismmechanism but but does does not not produce produce byproducts. byproducts. (B (B) )The The thiol-epoxy thiol-epoxy reaction reaction for for the the crosslinking crosslinking of of polymerspolymers to to form injectableinjectable hydrogel. hydrogel. Image Image modified modified with with permission permission from from [61]. [61] Copyright. Copyright 2016 Wiley.2016 Wiley. 4. Addition Reactions Involving Thiols for Hydrogel Crosslinking 4. Addition Reactions Involving Thiols for Hydrogel Crosslinking 4.1. Michael-Type Additions 4.1. Michael-TypeA Michael-type Additions addition reaction refers to the addition of a nucleophile to an electron-deficient carbon–carbonA Michael-type double addition bond reaction (olefin). refers This to type the ofaddition reaction of betweena nucleophile thiols to and an electron-deficient various activated carbon–carbondouble bonds, doublesuch as bondthose in (olefin). acrylates, This maleimide type of reaction and vinyl between sulfones thiols (Figure and 14 various), has been activated widely doubleapplied bonds, in bioconjugation such as those and in acrylates, biomaterials maleimide due to mild and conditionsvinyl sulfones and (Figure high yields. 14), has One been of thewidely early appliedexamples in bioconjugation of thiol-acrylate and Michael biomaterials addition due for to hydrogel mild conditions crosslinking and was high reported yields. One by Hubbell of the early et al., exampleswhere PEGs of thiol-acrylate multi-functionalized Michael withaddition thiols for and hydrogel acrylate crosslinking groups were was crosslinked reported by to formHubbell hydrogels et al., wherefor the PEGs encapsulation multi-functionalized and controlled with thiols release and of acrylate albumin groups [62]. The were same crosslinked strategy to was form used hydrogels by Elia forand the co-workers encapsulation to produce and controlled hydrogels release by connecting of albumin thiol-modified [62]. The same heparin strategy and was hyaluronic used by acidElia and with co-workersPEG-diacrylate to produce for the localhydrogels delivery by ofconnecting growth factors thiol-modified by injection heparin to achieve and stimulated hyaluronic angiogenesis acid with PEG-diacrylatein a mouse model for the [63 local]. Indelivery a very of recentgrowth application factors by injection of this reaction,to achieve four-armed stimulated angiogenesis PEG-acrylate inmacromonomers a mouse model were [63]. crosslinked In a very by recent thiolgroups application of cysteines of this in reaction,heparin-binding four-armed peptides PEG-acrylate to generate macromonomersa 3D environment were for culturing crosslinked encapsulated by thiol mouse groups cardiac of cysteines stem cells in [64 heparin-binding]. The study showed peptides that theto generatecrosslinking a 3D kinetics environment and the for mechanical culturing and encapsulated biodegradation mouse properties cardiac of stem the hydrogelcells [64]. can The be study tuned showedby varying that the the concentrations crosslinking kinetics of the acrylate-macromonomers and the mechanical and and biodegradation the cysteine-bearing properties peptides of the to hydrogelresemble can the natural be tuned mouse by varying heart tissue. the concentrations of the acrylate-macromonomers and the cysteine-bearing peptides to resemble the natural mouse heart tissue.

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Figure 14. Michael-type additions for in situ crosslinking of polymers through reactions of thiols with Figure 14. Michael-type additions for in situ crosslinking of polymers through reactions of thiols with maleimide (A), acrylate (B), or vinyl sulfone (C) groups. maleimide (A), acrylate (B), or vinyl sulfone (C) groups.

While maleimidemaleimide is is an an electron-deficient electron-deficient olefin olefin widely widely used used for thiol-mediated for thiol-mediated bioconjugation bioconjugation[31,65], [31,65],its utilization its utilization in chemical in chemical crosslinking crosslinking of hydrogel of hydrogel has not has been not as been common as common as acrylates as acrylates and vinyl and vinylsulfones. sulfones. Phelps Phelps et al., et demonstrated al., demonstrated that inthat triethanolamine-buffered in triethanolamine-buffered solutions, solutions, four-armed four-armed PEG PEGmacromonomers macromonomers presenting presenting maleimide maleimide groups groups were were able able to to crosslink crosslink by by reacting reacting withwith peptides presenting two two terminal terminal cysteines cysteines with with faster faster kinetics kinetics compared compared to acrylate- to acrylate- or vinyl sulfone-presenting or vinyl sulfone- presentingfour-armed four-armed PEG macromere PEG macromere [66]. However, [66]. vinyl However, sulfone vinyl moieties sulfone have moieties been shown have inbeen other shown reports in otheras the reports more reactive as the inmore Michael-type reactive in additions Michael-type for the additions crosslinking for the of PEGcrosslinking hydrogel of networks PEG hydrogel [36,67]. networksVinyl sulfones [36,67]. are Vinyl expected sulfones to merelyare expected undergo to merely nucleophilic undergo addition nucleophilic at the addition olefin double at the olefin bond, doublewhile the bond, addition while to the maleimide addition is to often maleimide accompanied is often by accompanied a ring opening by reaction a ring opening that generate reaction charged that generateproducts charged that may products alter the that properties may alter of polymers the properties after crosslinkingof polymers [after68]. crosslinking [68]. A drawback of the Michael-type addition for hydrogel crosslinking manifests where nucleophilic molecules molecules other other than than thiol-containing thiol-containing polymers/linkers polymers/linkers are present are duringpresent gel during formation. gel formation.Hammer et Hammer al. investigated et al. investigated the nonspecific the nonspecific chemical interaction chemical betweeninteraction lysosome between and lysosome linear PEGs and linearterminated PEGs withterminated maleimide, with acrylamidemaleimide, oracrylamide vinyl sulfone or vinyl [36]. sulfone Elevating [36]. the Elevating pH of the the environment pH of the environment(inorganic buffers) (inorganic from buffers) 4 to 9 from resulted 4 to in9 resulted increased in PEGincreased conjugation PEG conjugation to lysozyme to lysozyme by each by of eachthe three of the groups three groups tested, tested, with vinyl with sulfonevinyl sulfone giving giving the highest the highest modification modification and and acrylamide acrylamide the thelowest lowest modification modification of theof the protein. protein. Even Even when when high high concentrations concentrations ofof thiol-functionalizedthiol-functionalized PEGs were introduced to compete with lysozyme to to react with these electron-deficientelectron-deficient olefin olefin groups, groups, protein modificationmodification through the vinyl sulfone was still significant,significant, implying a relatively low chemoselectivity ofof vinylvinyl sulfone.sulfone. Additionally,Additionally, these these fast fast Michael-type Michael-type additions additions may may not not be idealbe ideal for forapplications applications that need that needconsistent consistent hydrogel hydrogel crosslinking. crosslinking. For example, For example, nonuniformity nonuniformity at the microscale at the microscaledue to different due degreesto different of crosslinking degrees of werecrosslinking observed were in PEG observed hydrogels in PEG formed hydrogels by the thiol-maleimide formed by the thiol-maleimideaddition, resulting addition, in varied resulting cellular in responses varied cellular to hydrogels, responses as reportedto hydrogels, by Darling as reported et al. [by69 ].Darling et al. The[69]. dynamic character of Michael-type addition reactions has received increasing attention becauseThe of dynamic its potential character use in of the Michael-type development addition of degradable reactions and has malleable received increasingpolymeric hydrogelattention becausematerials of (Figure its potential 15A). Baldwin use in the and development Kiick reported of maleimide-thioldegradable and crosslinked malleable polymeric hydrogels degradedhydrogel materialsthrough a (Figure glutathione-triggered 15A). Baldwin and retro Kiick Michael-type reported maleimide-thiol addition reaction, crosslinked which took hydrogels place at degraded a slower throughrate than agel glutathione-triggered breakdown by reductive retro Michael-type cleavage of disulfides addition [reaction,70]. A similar which strategy took place of crosslinking at a slower rateand than dissolution gel breakdown of hydrogels by reductive through cleavage the reversible of disulfides maleimide-thiol [70]. A similar addition strategy was later of crosslinking utilized by andKharkar dissolution and co-workers of hydrogels to develop through multifunctional the reversible PEG maleimide-thiol hydrogels that addition degraded was in responselater utilized to three by Kharkar and co-workers to develop multifunctional PEG hydrogels that degraded in response to three orthogonal stimuli, one of which is reductive GSH that triggers the retromaleimide-thiol addition by, with tunable degradation kinetics (Figure 15B) [71]. Kuhl et al. and Chakma et al.

Gels 2018, 4, 72 13 of 22 orthogonal stimuli, one of which is reductive GSH that triggers the retromaleimide-thiol addition by, Gels 2018, 4, x FOR PEER REVIEW 13 of 22 with tunable degradation kinetics (Figure 15B) [71]. Kuhl et al. and Chakma et al. reported that the reversiblereported Michael-addition that the reversible crosslinking Michael-addition couldbe crosslinking used to improve could be the used self-healing to improve properties the self-healing of gels at ◦ elevatedproperties temperature of gels (≥at 60 elevatedC) and temperature increased pH(≥60 (≥ °C)9) and [72 ,73increased]; however, pH (≥9) these [72,73]; conditions however, may these not find immediateconditions application may not infind current immediate biomedical application studies. in current biomedical studies.

FigureFigure 15. Retro 15. Retro Michael-type Michael-type addition addition ( A(A)) can can bebe utilizedutilized to to break break down down networks networks of crosslinked of crosslinked polymerspolymers on demand on demand (B) ( inB) thein the presence presence of of soluble soluble thiolsthiols (e.g., (e.g., glutathione, glutathione, GSH), GSH), as an as orthogonal an orthogonal methodmethod to the to degradationthe degradation by by light light (the (the reactive reactive groupgroup shown in in green) green) and and hydrolysis hydrolysis (the (the reactive reactive group shown in orange). Image B modified with permission from [71]. Copyright 2015 RSC. group shown in orange). Image B modified with permission from [71]. Copyright 2015 RSC. 4.2. Photo-Initiated Thiol-Ene and Thiol-Yne Additions 4.2. Photo-Initiated Thiol-Ene and Thiol-Yne Additions Photo-initiated crosslinking of hydrophilic polymers is one of the most widely used methods for Photo-initiated crosslinking of hydrophilic polymers is one of the most widely used methods for in in situ hydrogel formation. Traditionally, a solution containing polymeric precursors with photo- situ hydrogelreactive groups formation. (e.g., Traditionally,acrylate derivatives) a solution and low containing concentrations polymeric of radical-generating precursors with initiators photo-reactive is groupsdelivered (e.g., acrylate to a desired derivatives) location, andand lowsubsequent concentrations irradiation of of radical-generating UV light triggers the initiators crosslinking is delivered of to a desiredpolymers location, to form andgels subsequent[74–77]. Although, irradiation in principle, of UV lightphotocrosslinking triggers the can crosslinking take place ofpreferably polymers to form gelsamong [74 reactive–77]. Although, polymers inthrough principle, kinetic photocrosslinking control, the reaction can lacks take high place chemoselectivity preferably among because reactive polymersmany through bioactive kinetic molecules control, contain the UV-sensitive reaction lacks groups high and chemoselectivity can be chemically transformed because many upon bioactive UV moleculesirradiation. contain There UV-sensitive are inherent groupslimitations and associated can be chemicallywith the photo-curing transformed process upon such UV as the irradiation. use Thereof are potentially inherent toxic limitations photoinitiators associated [78,79], with the thegeneration photo-curing of highly process reactive such radicals, as the and use the of eventual potentially exothermic effect of photo-reactions, which can be detrimental to functions of biological systems in toxic photoinitiators [78,79], the generation of highly reactive radicals, and the eventual exothermic contact with hydrogels [80]. Limited penetration of light into deeper tissues further restricts the use effectof of photocrosslinked photo-reactions, hydrogels which as can carriers be detrimental of therapeutics to in functions many applications of biological [74]. Nevertheless, systems in the contact with hydrogelsgreat availability [80]. of Limited various polymers penetration with of photo-reactive light into deeper groups tissuesand the furtherconvenience restricts of applying the use of photocrosslinkedlight on demand hydrogels to control as gelation carriers and of thus therapeutics the mechanic in properties many applications of the hydrogels [74]. render Nevertheless, this the greatmethod availability still attractive. of various polymers with photo-reactive groups and the convenience of applying light on demandThe application to control of thiol gelation addition and across thus unsaturated the mechanic carbon-carbon properties bonds, of the hydrogelssuch as the renderdouble this methodbonds still in attractive. alkenes and triple bonds in alkynes, has flourished in the field of biomaterials in the last Thedecade application [81–84]. The of photochemically thiol addition acrossinduced unsaturated thiol-ene reaction carbon-carbon proceeds by bonds, a radical such mechanism as the double to bondsgive in alkenesan anti-Markovnikov-type and triple bonds thioether, in alkynes, which has is flourished compatible inwith the water field and of biomaterialsoxygen and usually in the last accompanied by Michael-type addition (Figure 16). An early study by Roydholm et al. described that decade [81–84]. The photochemically induced thiol-ene reaction proceeds by a radical mechanism to varying the ratios of thiol- and acrylate-containing polymer precursors and reaction conditions could give answitch anti-Markovnikov-type the main hydrogel crosslinking thioether, which mechanism is compatible between the with different water and modes, oxygen Michael-type and usually accompaniedaddition, byradical-initiated Michael-type thiol-ene, addition or (Figure the mixed 16). mechanism, An early study resulting by Roydholmin hydrogels et with al. describedadjustable that varyingmechanical the ratios and of thiol- degradation and acrylate-containing properties [85]. The polymer photochemical precursors thiol-ene and reaction addition conditions has been could switchconsidered the main “clickable” hydrogel crosslinking because of its mechanism high efficiency between and orthogonality the different to modes, a wide Michael-typerange of functional addition, radical-initiatedgroups [86,87]. thiol-ene, Beside orits theuse mixed in bioconjugation mechanism, [88–90], resulting this in reaction hydrogels has with been adjustablewidely applied mechanical to and degradation properties [85]. The photochemical thiol-ene addition has been considered “clickable” Gels 2018, 4, 72 14 of 22

Gels 2018, 4, x FOR PEER REVIEW 14 of 22 because of its high efficiency and orthogonality to a wide range of functional groups [86,87]. Beside its crosslinking of polymeric hydrogels for the delivery and controlled release of therapeutics as use in bioconjugation [88–90], this reaction has been widely applied to crosslinking of polymeric discussed in a recent review [91]. hydrogels for the delivery and controlled release of therapeutics as discussed in a recent review [91].

A B

Figure 16. 16. Photoinitiated, radical-mediated thiol-ene thiol-ene addition generates a covalent bond rapidly between aa thiol-presentingthiol-presenting compound compound and and an an alkene alkene (A ().A When). When electron-deficient electron-deficient alkenes alkenes are are used, used, the photoinitiatedthe photoinitiated thiol-ene thiol-ene reaction reaction is often is accompanied often accompanied by the base-catalyzed by the base-catalyzed Michael-type Michael-type addition reactionaddition (reactionB). (B).

The use use of of norbornene norbornene as asthe the ene ene moiety moiety enables enables a strain-promoted a strain-promoted thiol-ene thiol-ene reaction reaction and further and furtherimproves improves the gelation the gelationspeed and speed chemoselectivity and chemoselectivity of this method of this (Figure method 17A) (Figure [77,92]. 17 A)Shih [77 et,92 al.]. Shihreported et al. a comparison reported a between comparison hydrogels between formed hydrogels by thiol-ene formed photocrosslinking by thiol-ene photocrosslinking of PEG-tetra- ofnorbornene PEG-tetra-norbornene (PEG4NB) and (PEG4NB)dithiothreitol and (DTT) dithiothreitol and those by (DTT) Michael and addition those by crosslinking Michael addition of PEG- crosslinkingtetra-acrylate of with PEG-tetra-acrylate DTT [93]. The with thiol-ene DTT photocrosslinking[93]. The thiol-ene resultedphotocrosslinking in faster gelation resulted rate in faster and gelationhigher degree rate and of network higher degree formation of network as revealed formation by rheometry. as revealed Fairbanks by rheometry. et al. demonstrated Fairbanks et the al. demonstratedfunctionalization the of functionalization thiol-norbenene of photocrosslinked thiol-norbenene photocrosslinked hydrogels in two hydrogels ways: (1) in dicysteine- two ways: (1)terminated dicysteine-terminated peptides containing peptides a protease containing substrate a protease sequence substrate were used sequence to react were with used norbornene- to react withterminated norbornene-terminated crosslinkers to form crosslinkers degradable to hydrogels; form degradable (2) the conjugation hydrogels; of (2) cysteine-containing the conjugation ofpeptides cysteine-containing to the formed gels peptides were achieved to the formed through gels a second were achievedthiol-ene reaction through between a second the thiol-ene cysteine reactionthiol and betweenunlinked thenorbornene cysteine on thiol the polymer and unlinked network norbornene [92]. Both methods on the were polymer used network to present [92 an]. BothRGD-presenting methods were cell usedadhesion to present peptide an on RGD-presenting hydrogels to support cell adhesion the attachment peptide and on hydrogels spreading toof supporthuman MSC the attachmentcells (Figure and 17B). spreading The majority of human of studies MSC using cells thiol-norbornene (Figure 17B). The linked majority hydrogels of studies for usingthe delivery thiol-norbornene of therapeutics linked have hydrogels been reviewed for the elsewhere delivery [77,91]. of therapeutics Recent advancement have been in reviewed the fast- elsewherebooming regime[77,91 ]of. Recentthese hydrogels advancement includes in thevarying fast-booming the type of regime polymers, of these radical hydrogels initiators includes [94,95], varyingand light the sources type [95,96] of polymers, combined radical with initiatorsmodern material [94,95], processing and light sourcestechnologies [95,96 [97–99],] combined providing with modernbetter control material over processing the mechanical technologies and biological[97–99], providingproperties better of thiol-ene control hydrogels over the mechanical for biomedical and biologicalapplications. properties of thiol-ene hydrogels for biomedical applications. Photo-initiated thiol-alkyne thiol-alkyne addition addition reactions reactions were were reported reported more more than than half half a century a century ago agobut butnot not used used for for materials materials development development until until recently, recently, and and received received a a lot lot less less attention attention than other photocrosslinkingphotocrosslinking reactions [84,100]. [84,100]. Compared Compared to to the the 1:1 1:1 stoichiometry stoichiometry of of the the thiol-ene thiol-ene reaction, reaction, a athiol-yne thiol-yne addition addition utilizes utilizes each each alkyne alkyne group group to to react react with with two two thiols thiols groups, groups, where where a vinylene sulfide sulfide intermediateintermediate is firstfirst formedformed andand bearsbears higherhigher reactivityreactivity thanthan thethe startingstarting alkynealkyne towardtoward thethe additionaddition of thiolsthiols (Figure 1818).). Theoretically, thiol-ynethiol-yne reactions result in higherhigher crosslinkcrosslink densitydensity of polymerpolymer networks than than thiol-ene thiol-ene hydrogels, hydrogels, and and have have been been verified verified by their by high their conversion high conversion rates under rates ambient under humidityambient humidity and atmospheric and atmospheric oxygen conditions oxygen conditions [101]. In early [101]. demonstrations In early demonstrations of thiol-yne of crosslinked thiol-yne hydrogelscrosslinked by hydrogels Fairbanks by et Fairbanks al. and Chan et al. et and al., uniformChan et networksal., uniform were networks produced were from produced multi-armed from macromonomersmulti-armed macromonomers presenting thiol presenting and alkyne thiol groups and [ alkyne101,102 ]. groups The geometry [101,102]. of The macromonomers geometry of exertedmacromonomers an effect exerted on the an reactivity effect on of the thiol reactivity and alkyne of thiol groups and alkyne toward groups crosslinking, toward crosslinking, observed as theobserved decreased as the network decreased formation network with formation increased with branchness increased of macromonomers branchness of macromonomers (multiplicity of arms)(multiplicity [102]. Unreacted of arms) [102]. thiol andUnreacted alkyne thiol groups and on alkyne the hydrogel groups networkon the hydrogel can be used network for incorporation can be used offor desired incorporation chemical of functionalities desired chemical either duringfunctionalities crosslinking either or during post-gelation crosslinking [103,104 or]. post-gelation [103,104].

Gels 2018, 4, 72 15 of 22 Gels 2018, 4, x FOR PEER REVIEW 15 of 22 Gels 2018, 4, x FOR PEER REVIEW 15 of 22

Figure 17. (A) A general mechanism of radical-mediated thiol-norbornene addition. (B) Four-armed FigureFigure 17. 17. (A(A) )A A general general mechanism mechanism of of radical-mediated radical-mediated thiol-norbornene thiol-norbornene addition. addition. (B (B) )Four-armed Four-armed norbornene-presenting PEG macromonomers were used to crosslink with di-cysteine-terminated norbornene-presentingnorbornene-presenting PEG PEG macromonomers macromonomers were were used used to to crosslink crosslink with with di-cysteine-terminated di-cysteine-terminated peptidespeptidespeptides to to to form form form hydrogels. hydrogels. hydrogels. The The The gel gel gel can can can be be be modified modified modified uniformly uniformly uniformly with with with a a acell cell cell adhesion adhesion adhesion peptide peptide peptide CRGDS CRGDS CRGDS duringduringduring crosslinking crosslinking crosslinking ( C( (CC) ))or or or after after after gelation gelation gelation through through through unreacted unreacted unreacted norborenene norborenene norborenene at at at defined defined defined locations locations locations shown shown shown asasas the the the red red sphere sphere in in ( D (D),), both both supporting supporting cell cell attachment attachment and and spreading. spreading. Images Images Images reproduced reproduced reproduced with with with permissionpermissionpermission from from from [92]. [92]. [92]. Copyright Copyright Copyright 2009 2009 Wiley. Wiley.

Figure 18. (A) A general mechanism of photoinitiated, radical-mediated thiol-yne addition. (B) The FigureFigure 18. 18. (A(A) )A A general general mechanism mechanism of of photoinitiated, photoinitiated, radical-mediated radical-mediated thiol-yne thiol-yne addition. addition. (B (B) )The The crosslinking between four-armed C(CH2OOCCH2CH2SH)4 and a dialkyne linker produced network crosslinkingcrosslinking between between four-armed four-armed C(CH C(CH2OOCCH2OOCCH2CH2CH2SH)2SH)4 and4 and a adialkyne dialkyne linker linker produced produced network network (blue) with a higher crosslinking density and higher elastic modulus, compared to the crosslinking (blue)(blue) with with a ahigher higher crosslinking crosslinking density density and and higher higher elastic elastic modulus, modulus, compared compared to to the the crosslinking crosslinking between the same thiol and a dialkene linker (green). Images reproduced with from [101]. Copyright betweenbetween the the same same thiol thiol and and a adialkene dialkene linker linker (green). (green). Images Images reproduced reproduced with with from from [101]. [101]. Copyright Copyright 2010 ACS. 20102010 ACS. ACS.

AsAsAs an an an alternative alternative alternative to to to the the the photo-initiated photo-initiated photo-initiated thiol-yne thiol-yne thiol-yne reaction, reaction, reaction, a a anon-radical non-radical thiol-yne thiol-yne thiol-yne addition addition addition was was was reportedreportedreported by by by Truong Truong Truong and and Dove Dove as as a a base-catalyzed base-catalyzed nucleophilic nucleophilic nucleophilic addition addition addition reaction reaction reaction between between between thiols thiols thiols and and and electrophilicelectrophilicelectrophilic alkynes alkynes alkynes to to to produce produce produce vinylene vinylene vinylene sulfide sulfide sulfide [105]. [105]. [105]. This This This reaction reaction reaction has has has been been been shown shown shown to to to be be be efficient efficient efficient forforfor crosslinking crosslinking crosslinking thiol- thiol- thiol- and and and alkyne-containing alkyne-containing alkyne-containing PEG PEG PEG macromonomers macromonomers macromonomers in in in PBS PBS PBS solutions solutions solutions at at at pH pH pH 7.4 7.4 7.4 [106], [106], [106 ], andandand applied applied applied to to to the the the formation formation formation of of of ECM-mimicking ECM-mimicking ECM-mimicking hydrogels hydrogels hydrogels as as as a a amodel model model system system system for for for supporting supporting supporting cancer cancer cancer cellcellcell growth growth growth (Figure (Figure (Figure 19) 19)19 )[[107]. [107].107]. AAA unique unique unique capacity capacity capacity of of of photo-initiated photo-initiated crosslinking crosslinking crosslinking methods methods methods (as (as (as well well well as as degradation) degradation) mentioned mentioned aboveaboveabove is is is to to to allow allow allow temporal temporal temporal and and and spatial spatial spatial control control control over over over the the the hydrogel hydrogel hydrogel structure structure structure and and and properties properties properties in in order order in order to to generategenerateto generate complexcomplex complex bioactivebioactive bioactive scaffoldsscaffolds scaffolds [108–110].[108–110]. [108–110 For].For For instance,instance, instance, thethe the thiol-enethiol-ene thiol-ene photocrosslinkingphotocrosslinking photocrosslinking waswas was usedusedused to to to fabricate fabricate ultrathin, ultrathin, ultrathin, micro-patterned micro-patterned micro-patterned hydrogel hydrogel hydrogel films films films onon on solidsolid substrates substrates and and 3D-patterned 3D-patterned peptide-presentingpeptide-presentingpeptide-presenting hydrogels hydrogels hydrogels with with with tunable tunable tunable crosslinking crosslinking crosslinking density density density and and and biochemical biochemical biochemical cues cues cues for for for cell cell cell encapsulationencapsulationencapsulation [92,111–114]. [92,111–114]. [92,111–114 ].Again, Again, Again, as as new asnew new methods methods methods for for photo forphoto photo crosslinking crosslinking crosslinking advance advance advance toward toward toward the the use use the ofofuse light light of inlightin thethe in visible visible the visible and and nearnear and IRIR near rangerange IR range[78,115][78,115] [78 andand,115 initiators]initiators and initiators with with low low with toxicity toxicity low toxicity [95,116,117],[95,116,117], [95,116 these these,117 ], hydrogelshydrogels will will find find even even wider wider applications applications in in developing developing responsive responsive biomaterials biomaterials and and creating creating complexcomplex 3D 3D microenvironments microenvironments for for tissue tissue engineering. engineering.

Gels 2018, 4, 72 16 of 22 these hydrogels will find even wider applications in developing responsive biomaterials and creating complex 3D microenvironments for tissue engineering. Gels 2018, 4, x FOR PEER REVIEW 16 of 22

Figure 19. TheThe non-radical non-radical nucleophilic nucleophilic addition addition reaction reaction between between multi-armed multi-armed PEG-thiol PEG-thiol and PEG- and PEG-alkynealkyne produced produced vinylene vinylene sulfide-linked sulfide-linked hydrogel hydrogel for encapsulation for encapsulation of MCF-7 of breast MCF-7 cancer breast cells. cancer An cells.extracellular An extracellular matrix mimicking matrix mimicking peptide CGRGDS peptide CGRGDS is incorporated is incorporated to the gel to through the gel through the same the type same of typeaddition of addition between between the thiol the group thiol groupin the cysteine in the cysteine and PEG-alkyne and PEG-alkyne during during the network the network formation. formation. The Thehydrogel hydrogel is degradable is degradable through through hydrolysis hydrolysis of ofthe the esters esters in in PEG-alkyne PEG-alkyne to to allow allow cell cell growth and formation ofof cellcell clusters.clusters. Images reproduced withwith permissionpermission fromfrom [[107].107]. CopyrightCopyright 20182018 Elsevier.Elsevier.

5. Summary and Perspectives Covalently crosslinked crosslinked hydrogels hydrogels have have structure structure stability stability and and biomechanical biomechanical properties properties that that are aretunable tunable in ain large a large range. range. Chemoselective Chemoselective crosslinking crosslinking strategies strategies are are necessary necessary for developing developing injectable,injectable, in situ-formed hydrogels in contact with the human body and encapsulated therapeutics. therapeutics. Although not completely bio-orthogonal, the thiol group is one ofof thethe mostmost utilizedutilized functionalitiesfunctionalities for selective crosslinking of polymers, producing hydrogels through a diversity diversity of of chemical chemical reactions, reactions, as discussed in this review. review. Kinetic control is necessary to limit the unwanted cross-reaction of polymers with a a system system of of study study and and further further improve improve the the chemoselectivity chemoselectivity and and compatibility compatibility of the of thethiol-mediated thiol-mediated hydrogel hydrogel crosslinking crosslinking in situ. in It situ. should It shouldbe noted be that, noted when that, thiol-containing when thiol-containing polymers polymersare present are at present high concentrations, at high concentrations, disulfide disulfide formation formation among thiols among is thiols unavoidable is unavoidable and always and alwaysresults in results additional in additional crosslinking crosslinking during and during after and the afterinitial the formation initial formation of hydrogel of hydrogelby a different by a differentreaction [35,118],reaction [ which35,118 ], may which make may it make difficult it difficult to precisely to precisely control control the crosslinking the crosslinking degree degree of gel of gelnetworks. networks. So far, studies of hydrogel hydrogel material material have have been been focused focused on on bulk bulk characteristics characteristics such such as asthe the rate rate of ofgelation, gelation, and and the the mechanical mechanical and/or and/or biological biological properties, properties, but but the the fundamental fundamental understanding understanding of ofpolymer polymer crosslinking crosslinking at the at molecular the molecular level level has not has received not received adequate adequate attention. attention. For instance, For instance, during duringhydrogel hydrogel network network formation, formation, the functionalities the functionalities of the of the polymer polymer backbone backbone that that are are not not directly involved in crosslinking crosslinking reactions reactions can gather gather and and compose compose a a local local environment, environment, influencing influencing the the continuation of crosslinking by H-bonding and acid–base catalysis (not only limitedlimited toto thiol-basedthiol-based reactions). While While the the PEG backbone may may be considered to be “inert” or “nondisturbing”“nondisturbing” to many crosslinking reactions, other types of polymers need to be examined depending on the specificspecific crosslinking reaction used used [119]. [119]. Investigation Investigation of of the the molecular molecular interaction interaction within within the the 3D 3D network network is ischallenging challenging and and requires requires the the use use of of advanced advanced instrumentation instrumentation and computer-aided molecular molecular simulation. The The knowledge knowledge from from future future studies studies in inthis this area area will will provide provide guidance guidance for the for design the design and andselection selection of polymers of polymers and and chemoselective chemoselective strategies strategies in in applications applications of of chemically crosslinked crosslinked hydrogels in biomedicalbiomedical researchresearch andand clinicalclinical practice.practice.

Funding: This research received no external funding.

Acknowledgments: The author acknowledges the Northeastern Illinois University Faculty Research Stipend Program for the support in completing this manuscript.

Conflicts of Interest: The author declares no conflict of interest.

Gels 2018, 4, 72 17 of 22

Funding: This research received no external funding. Acknowledgments: The author acknowledges the Northeastern Illinois University Faculty Research Stipend Program for the support in completing this manuscript. Conflicts of Interest: The author declares no conflict of interest.

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