thiol ene click hydrogels for therapeutic deliverykinampark.com/ddsref/files/kharkar 2016,...

Thiol−ene Click Hydrogels for Therapeutic DeliveryPrathamesh M. Kharkar,† Matthew S. Rehmann,‡ Kelsi M. Skeens,‡,§ Emanual Maverakis,⊥

and April M. Kloxin*,†,‡

†Department of Materials Science and Engineering, University of Delaware, 201 DuPont Hall, Newark, Delaware 19716, UnitedStates‡Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, Delaware 19716,United States⊥Department of Dermatology, School of Medicine, University of California, Davis, 3301 C Street, Suite 1400, Sacramento, California95816, United States

ABSTRACT: Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For use as a deliveryvehicle, hydrophilic precursors are usually laden with bioactive moieties and then directly injected to the site of interest for in situgel formation and controlled release dictated by precursor design. Hydrogels formed by thiol−ene click reactions are attractivefor local controlled release of therapeutics owing to their rapid reaction rate and efficiency under mild aqueous conditions,enabling in situ formation of gels with tunable properties often responsive to environmental cues. Herein, we will review the widerange of applications for thiol−ene hydrogels, from the prolonged release of anti-inflammatory drugs in the spine to the release ofprotein-based therapeutics in response to cell-secreted enzymes, with a focus on their clinical relevance. We will also provide abrief overview of thiol−ene click chemistry and discuss the available alkene chemistries pertinent to macromoleculefunctionalization and hydrogel formation. These chemistries include functional groups susceptible to Michael type reactionsrelevant for injection and radically mediated reactions for greater temporal control of formation at sites of interest using light.Additionally, mechanisms for the encapsulation and controlled release of therapeutic cargoes are reviewed, including (i) tuningthe mesh size of the hydrogel initially and temporally for cargo entrapment and release and (ii) covalent tethering of the cargowith degradable linkers or affinity binding sequences to mediate release. Finally, myriad thiol−ene hydrogels and their specificapplications also are discussed to give a sampling of the current and future utilization of this chemistry for delivery oftherapeutics, such as small molecule drugs, peptides, and biologics.

KEYWORDS: hydrogels, thiol−ene, Michael-type reactions, click chemistry, drug delivery, controlled release, biologics delivery

1. INTRODUCTION

Significant advancements have been made in the past decade todevelop new therapeutics with the potential to improve thetreatment of a variety of diseases, from small molecular weighthydrophilic and hydrophobic drugs to larger peptides andbiologics (e.g., therapeutic proteins and antibodies). Inparticular, biologics have been a major area of growth for thepharmaceutical industry with the worldwide sales of biologicsexceeding $92 billion in 2009.1 Despite this increasing demand,costs of drug development and production remain high.2,3

Delivery of therapeutics at a controlled rate to a targeted siteaffords opportunities to both improve treatment efficacy andreduce total treatment costs. However, successful developmentof drug carriers with appropriate therapeutic retention and

release characteristics for clinical use remains a challenge and anarea of active research. Tremendous progress has been made inthe design of novel drug carriers, including liposomes,4,5

nanoparticles,6−8 polymersomes,9−11 dendrimers,12,13 micro-particles,14,15 and hydrogels,16,17 with improved efficacy,prolonged drug action in vivo, reduced drug toxicity, anddecreased drug-associated costs. Among these drug carriers,hydrogels have emerged as promising delivery vehicles, especiallyfor biologics, because of their high cargo loading efficiency andtheir ability to retain cargo bioactivity.18

Received: October 4, 2015Accepted: January 10, 2016Published: January 11, 2016

Review

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Hydrogels, or hydrophilic polymer networks that imbibe andretain large amounts of water, have been fabricated for controlledrelease applications using a range of natural and syntheticpolymers as their base building blocks. Because of their inherentbiocompatibility and bioactivity, natural polymers, such ashyaluronic acid,19 chitosan,20 heparin,21 silk,22 and alginate,23

often provide synergistic interactions with cargo molecules andwith cells in vivo. For example, Elia and co-workers recentlydemonstrated the delivery of small molecular weight drugs(cortisone and hydrocortisone) using hyaluronic acid-silk basedhydrogels.24 On the other hand, synthetic biocompatiblepolymers, including poly(ethylene glycol) (PEG) and poly(vinylalcohol) (PVA), generally offer greater flexibility for chemicalmodification, improved tunability over mechanical properties,facile incorporation of degradable functional groups, and limitedbatch-to-batch variation.18 For example, Sinko and co-workersinvestigated the use of PEG hydrogels for ocular drug delivery toinjured rabbit corneas; sustained delivery of doxycycline over 7days using these hydrogels resulted in accelerated wound healingcompared to direct doses of doxycycline.25

Control over the formation of these hydrogels is essential fordelivery applications since the connectivity (e.g., cross-linkdensity) and mechanical properties of the network dictate masstransport and therapeutic release. For example, increasing thecross-link density decreases the distance between cross-links inthe hydrogel (i.e., mesh size), often resulting in a decreased initialburst of cargo.26,27 Yang and co-workers demonstrated meshsize-dependent release of the model protein bovine serumalbumin (BSA) from PEG-based hydrogels with mesh sizesranging from 4 to 14 nm.26 For hydrogel compositions with amesh size below 6 nm, only about ∼15% of encapsulated BSAwas released. In contrast, for the hydrogel with a mesh size ofapproximately 15 nm, ∼100% of encapsulated BSA was releasedwithin 2 h.Hydrogels have been formed by (i) physical cross-linking (i.e.,

noncovalent interactions), including ionic, electrostatic, orhydrophobic interactions between the polymeric macromers,or (ii) chemical cross-linking by reactive functional groups toform covalent linkages.28 Physically cross-linked hydrogels offeradvantages for injectable formulations, including dynamic cross-links for gel dissolution and therapeutic release, shear thinningfor injection, and in situ formation without initiators orcatalysts.29 However, covalently cross-linked hydrogels providebetter control over cross-link density and allow easierincorporation of labile functional groups for stimuli-responsivedegradability and release from the delivery vehicle.30 Amongcovalent cross-linking chemistries, click reactions, includingcopper-free azide−alkyne cycloadditions, Diels−Alder, thiol−ene, and oxime reactions, are attractive for therapeutic deliveryapplications due to their fast reaction kinetics under mildconditions, permitting rapid formation in situ in the presence ofcargo molecules and tissues.28,31,32 In particular, thiol−ene clickreactions have been broadly applied for the design of drugdelivery carriers.33−35

In this review, we highlight recent work utilizing thiol−eneclick reactions for controlled drug delivery applications. Drugincorporation and release mechanisms (section 2) and thiol−enereactions for hydrogel formation (section 3) are discussed alongwith recent examples of material chemistries used for therapeuticdelivery. We subsequently overview how thiol−ene hydrogelshave been used for delivery of small molecular weight drugs,therapeutic peptides, and proteins (section 4) and examinefuture directions in the field (section 5).

2. DRUG INCORPORATION AND RELEASEMECHANISMS

Strategies for incorporation of therapeutics into hydrogelsgenerally fall into three categories: (1) encapsulation, wheretherapeutics are entrapped within the cross-links of the polymernetwork (Figure 1A); (2) tethering, where drugs of interest arecovalently bound to the polymer network (Figure 1B); and (3)affinity binding, where hydrophobic, ionic, or peptideinteractions are utilized to retain therapeutics within the hydrogelnetwork (Figure 1C).36 To design effective drug deliveryvehicles, these strategies are combined with appropriate releasemechanisms to suit the application of interest; several examplesare shown in the right-hand column of Figure 1. The release oftherapeutics entrapped within or tethered to the matrix can becontrolled by (i) Fickian diffusion, (ii) degradation of tetheredlinkages in response to relevant biological stimuli, or (iii)combinations of both. With affinity binding, a ligand is added tothe hydrogel with affinity for therapeutic(s) of interest. In thisway, release is controlled by the reversible binding of the ligandto the therapeutic in combination with diffusion of the freespecies or matrix degradation.37 In this section, we will overviewtherapeutic delivery strategies specifically for thiol−ene hydrogelsystems with relevant examples.For the majority of applications, hydrogels are loaded with

therapeutics of interest by physical encapsulation (Figure 1A),mitigating the need to chemically modify the bioactive cargo,which can impact FDA approval.38 Loading strategies includemixing the therapeutic into the precursor solution prior toforming the hydrogel39 or diffusing therapeutics into thehydrogel after cross-linking.40 In addition, smaller therapeuticscan be encapsulated within nano- or microgels (i.e., hydrogelparticles on the size scale of nanometers or micrometers). Thesenano- or microgels are incorporated within hydrogels to restrictuncontrolled drug diffusion out of the bulk hydrogel immediatelyafter formation.41 Diffusion dictated release occurs when thenetwork mesh size is tuned to slightly larger than the diameter ofthe therapeutic of interest, benefiting patients by maintaining apotent therapeutic concentration in vivo over the course of hoursor days. Hydrogels with inherent or incorporated degradabilityhave been used for applications that require increased control ofrelease over time or at a specific location, generally exploitinglight-mediated, enzymatic, or hydrolytic degradation to releasethe drug of interest. Approaches for imparting degradability arediscussed further in section 3.4.42 In addition to degradation,stimuli-responsive hydrogels whose swelling changes in responseto a microenvironment stimulus have been used for thecontrolled release of entrapped therapeutics; increased swellingwithin the desired microenvironment leads to increased meshsize and consequently release. A recent review by He et al.highlights pH and temperature responsive drug delivery from avariety of materials, including stimuli-responsive thiol−enehydrogels.43

Often, covalent conjugation (Figure 1B) is required when thesize of the therapeutic is too small to allow entrapment within thehydrogel or when diffusion of the therapeutic from the hydrogelis too rapid for a desired application. The therapeutic typically ismodified for covalent attachment to the hydrogel and released bycleavage of the covalent linkage or by complete hydrogeldegradation. With linker cleavage (e.g., enzymatic cleavage of apeptide linker), tethered molecules are released without alteringthe greater network structure.44 When therapeutics areincorporated into the polymer backbone, rather than as a

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DOI: 10.1021/acsbiomaterials.5b00420ACS Biomater. Sci. Eng. 2016, 2, 165−179

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pendant group, the matrix itself is degraded to facilitate release.Van Hove et al. applied the latter approach for the release ofpeptide-based drugs that promote angiogenesis: the drugs were

flanked by enzymatically degradable peptides that cross-linkedthe hydrogel (IPES↓LRAG, which degrades in response tospecific matrix metalloproteinases [MMPs]), and the drug wasreleased upon MMP-mediated hydrogel degradation.45

The third major class of techniques for therapeuticincorporation within thiol−ene hydrogels is affinity binding(Figure 1C). Hydrophobic interactions, hydrogen bonding, andionic inclusion complexes all have been used to retaintherapeutics in hydrogel networks.46,47 Cleaving hydrophobicgroups from the network decreases the ability of a gel to retainhydrophobic drugs, and ionic networks have been used to releasetherapeutics in response to changes in pH. For example, Arslan etal. designed a PEG-based hydrogel in which cyclodextrin-containing cross-linkers form an inclusion complex with thehydrophobic drug puerarin, which is traditionally used fortreating glaucoma.48 The amount of puerarin loaded into eachhydrogel was maximized by decreasing the molecular weight ofPEG, which made the polymer and resulting hydrogel lesshydrophilic, and by increasing the concentration of cyclodextrin,which has a hydrophobic interior cavity that entraps and releaseshydrophobic drugs.

3. THIOL−ENE REACTIONS FOR HYDROGELFORMATION

Broadly, thiol−ene click chemistry refers to the reaction of thiol-containing compounds with alkenes, or “enes”, with close to onehundred percent conversion. Interest in thiol−ene chemistry forhydrogel design has arisen from several advantageous attributesof the chemistry for biological applications: (i) thiol−enereactions often proceed rapidly under mild conditions (e.g.,water and buffers) that are compatible with cells and otherbiological molecules; (ii) thiol−ene reactions have well-definedand well-characterized reaction mechanisms and products; and(iii) the process of introducing thiols and alkenes to macro-molecules for hydrogel synthesis is relatively simple compared tothe introduction of other functional groups (e.g., strainedcyclooctynes).49,50 In this section, we review some of the keyaspects of thiol−ene reactions for their use in designinghydrogels for therapeutic release: the thiol−ene reactionmechanisms, the introduction of thiol groups onto macro-molecules to generate macromers, and the characteristics ofalkenes that can be used for thiol−ene chemistry to formhydrogels. This section concludes with a brief discussion ofengineering thiol−ene hydrogels to control their degradationand release profiles.

3.1. Thiol−ene Reaction Mechanism. Reactions of thiolswith alkenes typically occur by two different mechanisms: thiol-Michael type reactions and radically mediated thiol−enereactions. Selection of the reactive functional groups andsolution conditions, as discussed in section 3.2, dictates thereaction mechanism and important characteristics relevant forconsideration when designing hydrogels for therapeutic delivery(e.g., reaction initiation, time, and byproducts). Although thereaction mechanisms are briefly discussed here, interestedreaders are referred to recent articles on thiol−ene chemistryfor a comprehensive review.33,34,49,51

The thiol-Michael type reaction typically proceeds at roomtemperature without interference from water protons, making itamenable to hydrogel synthesis, which usually takes place inaqueous solutions. Generally, in thiol-Michael type reactions, abase (B) abstracts a proton from a thiol, forming a thiolate anionthat acts as a nucleophile (Figure 2A). The thiolate anion attacksthe electrophilic ß-carbon of an alkene to form a carbon-centered

Figure 1. Therapeutic loading and release mechanisms from hydrogel-based delivery vehicles. Hydrogels, including those formed by thiol−eneclick chemistry, can be formed in vitro or in vivo for therapeutic deliveryapplications. Stoichiometric reactions between functional groups onmultiarm poly(ethylene glycol) (PEG) macromers functionalized withspecific alkenes or thiols, respectively, have been reported, producingthiol−ene hydrogels with nearly ideal network structure (shown here);these are of growing interest for delivery applications toward providing awell-defined and predictable mesh size. However, macromolecules ofvaried length and functionality, both of synthetic and natural origin anddecorated with various alkenes or thiols, broadly have been utilized forthe formation of thiol−ene hydrogels. Engineering of material structureand chemistry provide handles for controlling release profiles. (A) Forexample, therapeutics frequently have been encapsulated within thehydrogel network, where the cargo is entrapped if the average pore size(e.g., mesh size) of the hydrogel is smaller than the drug; degradation ofthe network, which increases mesh size, controls release. (B) Smallmolecule drugs or peptides, which can be difficult to entrap withinhydrophilic highly swollen hydrogels, have been tethered to the networkand released upon tether cleavage (or complete network degradation);here, cleavage by a cell-secreted enzyme is depicted. (C) Ligands for atherapeutic of interest also have been incorporated within hydrogels forcontrolling retention and release by affinity binding; reversible bindingof the ligand dictated by kon/koff determines the fraction of bound/freetherapeutic, where diffusion of the free species (or matrix degradation)controls release. These therapeutic loading and release mechanisms canbe used in different combinations than those depicted here and havebeen used in thiol−ene hydrogels.



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anion intermediate, which subsequently abstracts a proton from aconjugate acid (BH+) to generate the thiol-Michael additionproduct, regenerating the original base. The reaction continues,usually rapidly, until one of the reactive functional groups isconsumed. Because of the mechanism, these reactions typicallyare free of byproducts and can occur with no or low amounts ofcatalyst, making thiol-Michael reactions attractive for injectablehydrogel applications.In radically mediated thiol−ene chemistry, the reaction also

proceeds under mild aqueous conditions but requires an initiatorto generate radicals that initiate the reaction between thiol and

select alkene functional groups (Figure 2B). Radical generationcan be achieved using a thermal, oxidation−reduction, orphotochemical process based on initiator selection.34 Afterinitiation, the generated radical attacks a thiol to form a thiylradical, which acts as an electrophile. Subsequently, the thiylradical attacks the CC double bond of the alkene, forming athiol−ene addition product and generating a new thiyl radical.The propagation process is highly efficient, resulting in a reactionof one thiol with one alkene. Unlike the traditional free radicalchain polymerization (e.g., acrylates or methacrylates), radicallymediated thiol−ene reactions are relatively oxygen-insensitive.33

Figure 2. Thiol−ene reaction mechanisms. Thiol−ene reactions take place by (A) Michael-type or (B) radically mediated mechanisms based on alkeneselection. Michael-type reactions occur upon mixing of the reactive macromers, where the rate of reaction can be increased by addition of a base catalystor increasing solution pH, making them well-suited for injectable hydrogel formation. Radically mediated thiol−ene reactions are initiated with theapplication of light and a photoinitiator, providing control over when and where hydrogels form.

Figure 3.Thiol−ene hydrogels. (A)Hydrogels are formed by thiol−ene reactions of alkene (“ene”) and thiol groups onmultifunctional macromers. (B)Thiol functional groups have been introduced on macromolecules to generate macromers by reacting (top to bottom) (i) a pendant amine with N-succinimidyl S-acetylthioacetate (SATA, aqueous condition in a nonamine containing buffer); (ii) Traut’s reagent (2-iminothiolane, aqueous bufferrange pH 7−10); (iii) carboxylic acid using carbodiimide chemistry; (iv) aldehyde and ketones with 2-acetamido-4-mercaptobutyric acid hydrazide(AMBH, under pH 7.4); or (v) by reducing disulfide linkages using dithiothreitol (DTT), dithioerythritol (DTE), or tris(2-carboxyethyl)phosphine(TCEP) under aqueous conditions. (C) Thiol-functionalized macromers have been reacted with various alkene-functionalized macromers to formhydrogels for therapeutic delivery applications, including (top to bottom) (i) vinyl sulfone (base catalyzed), (ii) maleimides (base catalyzed), (iii)norbornenes (radically mediated), (iv) acrylate groups (base catalyzed or radically mediated), and (v) allyl ethers (radically mediated).



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Photochemical processes are particularly attractive for design-ing thiol−ene hydrogels for biomedical applications as theyafford control of where, when, and how fast the polymerizationoccurs by controlling the application of light.52 Photoinitiatedthiol−ene reactions have been utilized to prepare various drugcarriers, including microgels and nanogels. Photoinitiators suchas Irgacure-2959,53 lithium acylphosphinate (LAP),54 and eosin-Y55 have been used extensively; initiator-free photopolymeriza-tion also has been reported but is less common.56 Thiol−enephoto-cross-linking reactions typically have been carried out inthe presence of biological cargoes (e.g., proteins, cells) with low,cytocompatible doses of light (e.g., ∼5 to 10 mW/cm2 of longwavelength UV light at 365 nm for ∼2−5 min). Visible-lightlight-mediated polymerizations of thiol−ene hydrogels also havebeen reported (e.g., 70 000 l× at 400−700 nm for ∼30 s).55 Fortranslation to the clinic, both the cytocompatibility and the depthof light penetration must be considered. While hydrogels may beformed in situ during surgical procedures without concern oflight penetration,57 formation of hydrogels by irradiationthrough tissues currently is limited to superficial depths by thescattering of light by tissues (e.g., penetration depths of∼0.5 mmat 365 to 500 nm through skin).58−60 Toward increasing therange of wavelengths available for photopolymerizations inwater, Grutzmacher and co-workers recently reported thesynthesis of highly efficient, water-soluble visible light photo-initiators using monoacylphosphineoxide and bisacylphosphine-oxide salts that are promising for biological applications based ontheir response to low doses of blue light (465 nm LED for <30min).61,62 In addition, cyclic benzylidene ketone-based initiatorsthat are water-soluble and responsive to two-photon near-infrared light (780 nm,∼60 to 400 mW, 100 fs pulse) may proveuseful for thiol−ene photopolymerizations in biomedicalapplications requiring increased light penetration in situ.63

3.2. Introduction of Thiol Functional Groups onMacromolecules. Thiol−ene hydrogels are prepared bycovalent cross-linking of hydrophilic polymers containingreactive thiols and alkenes; these reactive macromolecules alsoare referred to as macromolecular monomers or macromers(Figure 3A). Thiol functional groups (also known as sulfhydryls)are generally introduced on a macromer by reactions withfunctional groups commonly found on natural and syntheticpolymers, including amines, carboxylic acid groups, aldehydes,ketones, or reduced disulfides (Figure 3B). The reactivity of thethiol can vary depending on the functional group(s) proximate toit, which affects the pKa.

34 A brief overview of incorporation ofthiol groups on macromers is provided here; however,commonly used synthetic macromers, such as poly(ethyleneglycol) functionalized with thiols, are commercially available.For polymers and proteins containing primary amine groups,

thiols have been introduced by reacting primary amines using 2-iminothiolane (Traut’s reagent), N-succinimididyl S-acerylth-ioacetate, or succinimidyl acetyl-thiopropropionate.64 Forexample, Stevens and co-workers thiolated primary amines ona latent form of recombinant transforming growth factor (TGF-ß1) using 2-iminothiolane and subsequently reacted thethiolated growth factor with acrylate-functionalized PEG byradically mediated thiol−ene reaction (see 3.3.4 for mechanisticdetails).65 The resulting hydrogel contained the latent form ofTGF-ß1, resulting in increased metabolic activity and proteindeposition of encapsulated chondrocytes compared to a negativecontrol while maintaining the bioactivity and stability of thereleased TGF-ß1 over a 34 day time period.

Using carbodiimide chemistry, thiol groups have beenintroduced on a polymer or a protein by reaction of carboxylicacid groups with cystamine or similar reagents that contain aprimary amine and a thiol. For example, Revzin and co-workersincorporated thiol functional groups on heparin usingcarbodiimide chemistry to modify 40% of the available carboxylicacid groups.66 Heparin is a highly charged polysaccharide oftenused in hydrogels to mimic heparan sulfate that is found withinthe extracellular matrix and binds various proteins (e.g., growthfactors or cytokines), mediating their release.21 Here, hydrogelmicrospheres cross-linked with the thiolated-heparin wereformed by reaction of the thiol functional groups with acrylateend-functionalized PEG. The hydrogels containing heparinretained hepatocyte growth factor (HGF) over approximately5 days, whereas control hydrogels without heparin containedminimal levels of HGF. This example highlights that thiolationand subsequent incorporation of macromolecules such asheparin can be useful in designing thiol−ene hydrogels fortherapeutic delivery.In addition, aldehyde or ketone groups on macromolecules

have been modified using acetamido-4-mercaptobutyric acidhydrazide, a hydrazide derivative, to form reactive thiolfunctional groups.64 For macromolecules containing disulfidelinkages, disulfide groups can be reduced to introduce free thiolgroups for reaction with alkenes.64 For biomacromolecules,though, reduction of disulfides also may result in changes tonative structure and reduced bioactivity.Although thiol−ene polymerization conditions are typically

chosen to minimize side reactions, disulfide formation still canpresent a challenge in the consistent formation of thiol−enehydrogels: thiol-functionalized macromers can react with eachother to form disulfide linkages, making them inaccessible forsubsequent reaction with alkenes.67 Additionally, thiols onmacromers can react with various functional groups that arepresent on biologics (i.e., off-target reactions leading to oxidationof cysteine residues on proteins).68 Uncontrolled reactions cancontribute to loss of bioactivity and degradation of therapeuticpeptides and proteins and hence generally are undesirable. Evenso, this approach has been utilized to conjugate biologics topolymeric drug carriers.69,70

3.3. Types of Alkenes Used for Thiol−ene HydrogelFormation. 3.3.1. Vinyl Sulfone. Vinyl sulfones are electronpoor alkenes that react with thiols in slightly alkaline conditions(e.g., pH ∼8) to give stable ß-thiosulfonyl linkages, known asthioether bonds (Figure 3C).64 Vinyl sulfone groups generallyare stable for extended periods of time under aqueous conditionsnear neutral pH. Vinyl sulfone groups have been introduced onmacromers by reaction with free amine and hydroxyl functionalgroups under basic pH.71,72 For example, in seminal work, Lutolfand Hubbell reported the reaction of hydroxyl end-function-alized PEG with divinyl sulfone in the presence of sodiumhydride to synthesize vinyl sulfone end-functionalized PEGs foruse in hydrogel formation.71

Hydrogels prepared using thiol-vinyl sulfone cross-linking areattractive for drug delivery applications and as tissue engineeringplatforms owing to their mild reaction conditions.72,73 Hubbelland co-workers have demonstrated applications of vinyl sulfone-based hydrogels for therapeutic delivery, including the delivery ofbone morphogenetic protein 2 (BMP-2) for bone regener-ation.74,75 Building upon this, Peng et al. recently reported thesynthesis of vinyl sulfone-functionalized dextran hydrogels fordelivery of BMP-2 in vivo.73 Vinyl sulfone propionic acid wasreacted with hydroxyl groups of dextran via esterification in a



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one-pot synthesis to yield a series of macromers with varyingdegrees of substitution (DS ≈ 2−12.5). In vivo studies wereperformed in mice where hydrogels loaded with recombinanthuman bone morphogenetic protein-2 (rhBMP-2) wereimplanted in muscle pouch. Hydrogels prepared from macro-mers with higher degrees of substitution (DS = 12.5) exhibitedslower degradation and higher ectopic bone formation (318 mg)compared to macromers with lower degrees of substitution (DS= 9, ectopic bone formation = 226 mg). This finding can becorrelated to slower degradation of these thiol−ene hydrogels,resulting in sustained release of rhBMP-2 for bone formation.3.3.2. Maleimides. Maleimides are electron poor alkenes and

readily react with thiols by Michael-type additions to formsuccinimide thioether linkages (Figure 3C). Maleimide groupsoften are incorporated onto macromolecules by carbodiimidechemistry (e.g., reaction of activated carboxylic acids with aminesto introduce maleimide groups). Thiol-maleimide reactions offera number of advantages: (1) at neutral pH, maleimides react withhigh selectivity for thiols; (2) thiol-maleimide reactions occurrapidly under physiological conditions; and (3) the thiol-maleimide linkage formed with aryl thiols can undergo retro-Michael reaction under reducing conditions for controlleddegradation and release applications.76,77 However, it isimportant to note that maleimide groups undergo ring hydrolysisunder aqueous conditions, yielding maleamic acid that is notreactive with thiols.64 Solution pH, temperature, neighboringfunctional groups, and hydroxyl ion concentration affect the rateof ring hydrolysis (k = 500−1600 M−1 s−1).78 Althoughmaleimide ring hydrolysis after formation of succinimidethioether linkages will not significantly change the propertiesof an existing hydrogel, ring hydrolysis in the precursor solutionbefore hydrogel preparation can significantly increase networkdefects; such defects typically increase mesh size and reducenetwork retention of loaded therapeutics, affecting releasecharacteristics.64 In addition, because unreacted small-moleculemaleimides can be cytotoxic, thorough purification of maleimide-functionalized macromers after synthesis is needed.79

Thiol-maleimide reactions have successfully been used todesign various cell-compatible hydrogels for tissue engineeringand drug delivery applications.80−83 For example, Fu and Kaoreported semi-interpenetrating thiol-maleimide hydrogelsformed in situ for controlled release.81 Thiol and maleimideend-functionalized PEGwere reacted in the presence of gelatin at37 °C to form a semi-interpenetrating network. A modelmacromolecule, fluorescein isothiocyanate-labeled dextran (Mw≈ 40 000 g/mol), was encapsulated in the network duringhydrogel formation by mixing with the precursor solutions. Inthe absence of gelatin, 80% of the dextran cargo was released after1 h, whereas the incorporation of gelatin slowed release of thecargo molecule to 25 h (100% release). The difference in therelease profiles was attributed to reduced mesh size as a result ofadditional physical entanglements caused by gelatin incorpo-ration.3.3.3. Norbornene.Norbornene is a strained cyclohexane ring

with a methylene bridge that reacts with thiols in the presence offree radicals in a 1:1 ratio, suggesting limited to no norbornenehomopolymerization under gelation conditions (Figure 3C).84

For hydrogel formation, the thiol-norbornene reaction has beencarried out in minutes using low, cytocompatible doses of longwavelength ultraviolet light with the photoinitiators Irgacure-2959 and LAP (10 mW/cm2 at 365 nm)84 or using visible lightwith the photoinitiator eosin-Y (70000 Lux at 400−700 nm).55,85Because norbornene groups are relatively stable, high con-

versions have been achieved when stoichiometric amounts ofreactive functional groups are used.86 The rate of reaction hasbeen controlled by varying the light intensity, wavelength, andconcentration of photoinitiator,53,84,87,88 as has been similarlydone for other radically mediated thiol−ene reactions notedbelow (section 3.3.4). For a comprehensive review of hydrogelsprepared using thiol-norbornene reactions, readers are referredto a recent review by Lin and co-workers.88 Broadly, the ability tocross-link hydrogels with light using radically mediated thiol−ene reactions (e.g., thiol-norbornene, thiol−acrylate) providescontrol over hydrogel formation at positions and times of interest(i.e., spatiotemporal control), which is attractive for theformation of hydrogels in complex geometries, including tissuedefects (e.g., critically sized bone defects), and for stabilizinghydrogel microparticles.89,90

3.3.4. Other Alkenes. Acryloyl groups (H2CCH−C(O)−) contain double bonds that are electron poor in nature andreact with thiols to form stable thioether linkages (Figure 3C) viaa step-growth mechanism under basic conditions or in thepresence of free radicals. However, in the presence of freeradicals, acryloyl groups also react with each other via free radicalchain polymerization resulting in an overall mixed modepolymerization: thiyl radicals react with acrylates (1:1) by stepgrowth polymerization, whereas acrylates react with each otherby a chain growth polymerization.91,92 These reactions of thiolswith acryloyl groups have been used to design drug deliverycarriers.93−95 Vermonden and co-workers reported the reactionof thiols andmethacrylates to form thermosensitive hydrogels forcontrolled delivery of the model therapeutic peptide bradykinin(BK).95 Briefly, a thermoresponsive macromer functionalizedwith acrylates (ABA-triblock polymer containing N-isopropyla-crylamide) was reacted with thiolated hyaluronic acid under basicconditions to form hydrogels with different polymer concen-trations and different mesh sizes. First order release kinetics wereobserved in vitro, indicating diffusion-mediated release of theencapsulated BK, and the rate of release was controlled by thenetwork mesh size. In addition to acryloyl groups, thiols can alsoreact with vinyl ethers, allyl ethers, and allyloxycarbonyls byradically mediated thiol−ene reactions with a range of reportedrates (Figure 3C); to date, such hydrogels primarily have beenused for tissue engineering applications.96−98

Unsaturated double bonds within maleate or fumarate readilyreact with thiol functional groups under basic pH by a Michael-type reaction; such reactions of thiols with maleates or fumaratesrecently have been utilized for drug delivery.99,100 For example,Shen and co-workers reported the use of thiol−ene hydrogels forthe delivery of camptothecin, a hydrophobic anticancer drug.99

Poly[oligo(ethylene glycol) mercaptosuccinate] was reactedwith poly[oligo(ethylene glycol) maleate] to form hydrogel films(thickness ∼10−35 nm). Camptothecin was conjugated to thepolymer backbone with hydrolytically cleavable ester linkages forcontrolled drug release over short periods of time (∼24 h).

3.4. Administration of Thiol−ene Hydrogels in Vivo.Thiol−ene hydrogels can be administered in vivo by (i)intramuscular101 or subcutaneous injection102,103 of reactiveprecursor solutions that form a gel in situ, (ii) application ofprecursor solutions at desired sites during surgical proceduresfollowed by in situ gelation,57 or (iii) surgical implantation ofpreformed gels.104 For in situ formation, liquid precursorsolutions need to polymerize rapidly under physiologicallyrelevant conditions at the site of interest (e.g., < 1min): if the rateof reaction is too slow, precursor solutions containingtherapeutic drugs may diffuse into surrounding tissues, resulting



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in poor hydrogel formation and potential immunogenic responseto unreacted monomers.105 However, if the rate of reaction is toorapid, premature gel formation may occur within the syringe forthiol-Michael type reactions, resulting in an uneven hydrogeldistribution within the tissue and large network defects thatproduce undesirable release profiles. One approach to mitigatingthis issue is using a double-barrel syringe to control reactiveprecursor mixing and achieve rapid and consistent in situpolymerizations.102,103

3.5. Degradation of Thiol−ene Hydrogels Relevant forControlled Release of Therapeutics. Degradation of thiol−ene hydrogels is advantageous for therapeutic deliveryapplications, allowing tunable release of entrapped cargocontrolled based on the rate of network degradation. In addition,degradation of hydrogel-based drug carriers after the usefullifetime of the hydrogel in the body avoids the need for removalby surgery. Degradation has been achieved by incorporatingdegradable groups, such as water-degradable ester linkages,106,107

enzymatically degradable linkages (especially peptides),108 andphotodegradable linkages,109 into the polymer backbone, cross-links, or pendant groups of the hydrogel (Figure 1).28 Thenumber and type of degradable linkages and the localenvironment surrounding the degradable moiety alter the rateof degradation, providing handles for controlling the release rateof the therapeutic. For example, the rate of degradation via esterhydrolysis in thiol−acrylate hydrogels is influenced by thenumber of carbons between the sulfide and ester groups (e.g., k =0.08 days−1 for one carbon atom and 0.02 days−1 for two carbonatoms) and the pH of the surrounding buffer (e.g., k = 0.07days−1 at pH 7.4 and k = 0.28 days−1 at pH 8).110

Incorporation of enzymatically degradable peptide cross-linkers is a versatile way to control hydrogel degradation in astimuli-responsive manner. For example, Patterson and Hubbellevaluated the rate of cleavage of a variety of different matrixmetalloproteinase (MMP)-sensitive peptides (including VPMS↓MRGG from combinatorial screening, GPQG↓IAGQ fromcollagen, and faster-degrading variant GPQG↓IWGQ) andsubsequently studied the use of select cross-linkers forcontrolling the degradation of thiol-vinyl sulfone PEG-basedhydrogels in response to MMP-1 and MMP-2.44 The rate ofhydrogel degradation in response to one or both MMPs wasfound to be dependent on the sequence of the peptide cross-linker, where sequences were identified to achieve hydrogeldegradation times ranging from 1 to 10 days when incubatedwith MMP-1 and MMP-2. These peptide sequences also havebeen incorporated within hydrogels formed by a variety of thiol−ene reactions, including thiol-norbornene, -methacrylate, and-maleimide, where gel degradation times have been tuned fromdays to weeks based on sequence selection and networkconnectivity.80,84,111 Aryl-thiol-based succinimide thioether link-ages formed using thiol-maleimide chemistry can undergodegradation via retro-Michael type reactions in thiol-richreducing microenvironments, such as that provided byglutathione found in elevated levels within tumors.77,112 Thisapproach has been used to tune the delivery of the anticoagulantheparin over a range of time scales, from 3 days to 49 days,depending on the reactivity of the thiol and the strength of thereducing microenvironment.82 For a comprehensive review ofdegradable and stimuli-responsive hydrogels for biologicalapplications, readers are referred to a recent review by Kharkaret al.28

4. DELIVERY OF BIOACTIVE CARGO IN THIOL−ENEHYDROGELS

4.1. Small Molecular Drug and Therapeutic PeptideDelivery. Thiol−ene hydrogels have been developed to controlthe release of small molecular drugs by rational design andcontrol of hydrogel mesh size.108,113,114 However, controlleddelivery of small cargo molecules using thiol−ene hydrogels facestwo major challenges: maintaining precise control over the meshsize during equilibrium swelling and incorporating hydrophobicdrugs within highly hydrophilic hydrogels. In this section, we willhighlight recent advances inmaterial chemistry for the delivery ofsmall-molecular-weight drugs and therapeutic peptides usingthiol−ene hydrogels.Swelling of the hydrogels has been controlled by careful

selection of macromers. For example, the hydrophilicity of PEGis increased by increasing the number of repeating units ofethylene glycol (e.g., increasing chain length). However, tocontrol the swelling of hydrogels and thus control hydrogel meshsize, an optimum balance between hydrophilic and hydrophobiccontent is needed. By selecting low molecular weight macromerprecursors, hydrogel syneresis has been achieved, leading towater loss from the hydrogel after formation and a reduction inhydrogel mesh size. With this approach, Langer and co-workersreported the synthesis of PEG-based thiol−ene hydrogels thatcan undergo syneresis in physiological conditions, reducing gelvolume by∼40%. They utilized these materials for the controlledrelease of methylprednisolone sodium succinate (MPSS), aglucocorticoid prodrug with anti-inflammatory and immunosup-pressant properties.113 Hydrogels were prepared using acrylateend-functionalized PEG and a commercially available trifunc-tional thiol macromer (ethoxylated trimethylolpropane tri-3-mercaptopropionate). Incorporation of MPSS within thehydrogel suppressed the rate of hydrolysis of the ester linkagebetween methylprednisolone and succinate groups (k = 3.12 ±0.10 × 10−3 h−1 encapsulated as compared to k = 5.98 ± 0.50 ×10−3 h−1 in PBS), demonstrating how hydrogels can increasetherapeutic stability. Sustained release of MPSS was achievedover ∼20 days independent of the initial drug loadingconcentration, providing an opportunity to change the drugconcentration based on individual patients’ needs withoutchanging the drug carrier volume.Incorporation of small molecular hydrophobic drugs within

hydrogels presents additional challenges because of their limitedwater solubility. Introduction of hydrophobic units within thehydrogel network have been used to improve drug-loadingefficiency and influence the swelling behavior of the hydrogels.For example, Harth and co-workers reported polycarbonate-based thiol−ene hydrogels for sustained delivery of paclitaxel, achemotherapeutic drug used to treat ovarian and breastcancer.114 To incorporate the hydrophobic drug and optimizeits residence time in the hydrogel-based drug carrier,polycarbonate was incorporated within the hydrogel as anadditional hydrophobic backbone component. A three compo-nent system (polycarbonate, poly(ethylene oxide), and semi-branched polyglycidols) was used to optimize the balancebetween hydrophilicity and hydrophobicity, controlling hydrogelswelling behavior and drug release kinetics. Hydrogels thatundergo ester hydrolysis were formed using light-mediatedthiol−ene reactions by reacting allyl-functionalized polycarbon-ates with linear thiol-functionalized poly(ethylene oxide).Paclitaxel was encapsulated within the network during hydrogelformation with high loading efficiency (>98%), and release of



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∼7% to ∼30% of the drug was observed over 7 days dependingon initial hydrogel swelling.As noted in section 2, control over release kinetics also can be

achieved by covalently conjugating the drug molecule to thehydrogel backbone. The rate of release of cargo molecules insuch cases depends on the rate of degradation of the covalentlinkage between the drug and the network. Covalently linkeddrug molecules can have preprogrammed drug release kinetics(e.g., different numbers of hydrolytically degradable esterlinkages with known rates of hydrolysis)115,116 or can cleave inresponse to the local microenvironment; both approaches havebeen used in drug delivery systems. For example, Anseth and co-workers reported the use of thiol−ene click hydrogels for cell-mediated delivery of dexamethasone (Dex), an anti-inflamma-tory and immunosuppressant steroid drug (Figure 4A).108 Dexwas covalently linked to a peptide cross-linker (KCGPQG↓

IAGQCK), which was susceptible to enzymatic cleavage byMMPs secreted by cells within tissues. The hydrogels wereformed by reacting norbornene end-functionalized PEG with thethiol groups of MMP-degradable peptide cross-linkers, and therelease of Dex was controlled by cleavage of tethers. Since Dex isknown to promote osteogenic differentiation of humanmesenchymal stem cells (hMSCs), released Dex bioactivitywas monitored by measuring hMSC alkaline phosphatase (ALP)activity, a marker of early osteogenic differentiation.117 hMSCsencapsulated within hydrogels containing Dex-conjugatedMMP-degradable peptide showed a significant increase in ALPactivity compared to hydrogels containing Dex conjugated to anondegradable linker, demonstrating both the release andbioactivity of Dex. Although such an approach can be translatedto deliver other small molecules using thiol−ene hydrogels, thebiological activity of drugs or released drug fragments may

Figure 4. Controlled release of small molecular weight drugs and therapeutic peptides. (A) Dexamethasone (Dex) was covalently conjugated to thebackbone of the hydrogel network using a thiol-functionalized MMP-sensitive peptide linker (KCGPQG↓IAGQCK). In the presence of cell-secretedMMP, the dexamethasone fragment (closed circle) was released and bioactive as demonstrated by enhanced alkaline phosphate (ALP) activity ofencapsulated stem cells compared to the negative control (open circle, peptide sequence with substitution ofD-isoleucine [QG(d-)I], making the linkerMMP-insensitive and nondegradable). Adapted with permission from ref 108. Copyright 2012 Elsevier. (B) Furan-functionalized peptide (integrin-binding RGDS) was incorporated in thiol-maleimide hydrogels by a Diels−Alder reaction with excess maleimides. The release of peptide was controlledby a retro Diels−Alder reaction, which increases in rate with elevated temperature as demonstrated by the increasing fraction of peptide released at 37,60, and 80 °C. Adapted with permission from ref 123. Copyright 2013 American Chemical Society. (C) Toward promoting angiogenesis, a vascularendothelial growth factor mimetic peptide (Qk) was tethered to a nondegradable thiol−ene hydrogel using an MMP-cleavage linker, and temporalcontrol of Qk release was achieved in vitro and in vivo by changing the MMP2-susceptible peptide tether (FL = Qk-PES↓LRAG-C-G, SL = Qk-VPLS↓LYSG-C-G). * p < 0.05; & p < 0.01; and # p < 0.0001 compared to buffer alone for each respective time point. Adapted with permission from ref 124.Copyright 2015 Wiley.



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change substantially after conjugation and release from thenetwork.118 For each drug of interest, tests must be run to ensurethe conjugated and released drug retains its desired biologicalactivity.In the past decade, with significant advances in solid-phase

synthesis and recombinant DNA technology, FDA-approved,peptide-based drugs have gained substantial importance for thetreatment of human diseases.119,120 For example, peptideformulations such as cilengitide, taltirelin hydrate, and ziconotideacetate are in clinical trials for treating diseases associated withthe central nervous system (i.e., spinocerebellar degeneration,ataxia, and severe chronic pain).121 Thiol−ene hydrogels havebeen used to deliver therapeutic peptides because of their abilityto maintain the bioactivity of cargo peptides and the ease ofpeptide encapsulation by facile cross-linking chemistries.122,123

Recently, Wang et al. demonstrated the release of a fluorescentlylabeled library of model peptide drugs using thiol−enehydrogels.122 Hydrogels were formed using a combination of athiol-containing protein (ubiquitin-like domain tetramer ofSATB1 with cysteine residues) and PEG-maleimide via aMichael-type cross-linking reaction. Controlled release ofmodel peptide drugs (∼50−80% release) was achieved withinapproximately 24 h, depending on the protein-peptide affinityinteractions (i.e., a higher binding affinity peptide released moreslowly). In another example, Koehler et al. reported the release ofa covalently linked peptide (integrin-binding RGDS) byreversible Diels−Alder reactions (Figure 4B).123 PEG-basedhydrogels were formed using thiol-maleimide chemisty, andtemperature sensitive Diels−Alder linkages were used to

conjugate the bioactive peptide functionalized with furan groupsby reaction with excess maleimide groups. The release rate ofpeptide was tuned by the incorporation of different numbers ofmaleimide tethering sites (varied by incorporation of themonofunctional thiol) in the hydrogels: ∼ 45−60% of peptidewas released by 35 h depending on the number of maleimidetethering sites, and the percent release could be further variedwith changes in local temperature (i.e.,∼40% cargo released at 37°C and ∼70% cargo released at 60 °C). In another example,Benoit and co-workers reported thiol−ene hydrogels to controlrelease of Qk, a proangiogenic peptide mimic of vascularendothelial growth factor using tissue-specific enzymaticactivity.124 Qk was tethered to the hydrogel using enzymaticallycleavable linkers, and variation in kcat/KM provided temporalcontrol over release kinetics (i.e., ∼70% release was achievedusing the sequence [Qk-PES↓LRAG-C-G], whereas only ∼15%release was achieved using the sequence [Qk-VPLS↓LYSG-C-G], Figure 4C). These examples, along with those mentioned insections 2 and 3, highlight approaches and methods for thedesign of thiol−ene hydrogels to deliver peptide-basedtherapeutics.

4.2. Therapeutic Protein Delivery. Protein therapeuticsare in development to treat cancer, autoimmune diseases, proteindeficiencies, and infectious diseases.125 The complexity ofproteins allows them to complete tasks that small moleculescannot easily achieve, such as catalyzing an enzymatic reaction orinhibiting a biological process in a specific manner.126 Forexample, the antibody rituximab binds specifically to CD20, acell-surface glycoprotein on B-cells, and has been approved to

Figure 5. Controlled release of therapeutic proteins. (A) Proteins of various sizes were released from PEG-based hydrolytically degradable thiol−vinylsulfone hydrogels. Lysozyme, the smallest protein, was released in less than 24 h, whereas Ig, the largest protein, was released upon complete geldegradation. Adapted with permission from ref 39. Copyright 2011Wiley. (B) Growth factor VEGF was loaded into microspheres containing a peptide-based ligand derived from VEGF receptor 2 (VBP). VEGF was bound and released from the microspheres containing the VEGF receptor mimic,whereas microspheres containing a scrambled inert sequence exhibited a low level of release corresponding with random diffusion of VEGF into and outof the microspheres. Adapted with permission from ref 133. Copyright 2012 Elsevier. (C) Lysozyme was exposed to free radicals in conditionsmimicking that of photoinitiated thiol−ene hydrogel polymerization. Higher radical concentrations would result from higher photoinitiatorconcentrations (10 mM), as compared to lower photoinitiator concentrations (0.1 mM), and from increased exposure time to light (plotted on x-axis).Increasing radical exposure decreased the bioactivity of lysozyme, indicating the importance of minimizing protein exposure to free radicals during gelformation. Bioactivity was increased upon introduction of reactive thiol and norbornene functional groups, supporting the protective effect of thiol−enechemistry during hydrogel formation relative to acrylate homopolymerization (data not shown). Adapted with permission from ref 135. Copyight 2012American Chemical Society. (D) FITC-labeled ovalbumin was released from thiol-functionalized ethoxylated polyol ester/PEG-diacrylate basedhydrogels of different compositions. By changing the ratio of the macromers within the hydrogel, the rate of release and the overall release profiles weretuned over a wide range of time scales. Adapted with permission from ref 42. Copyright 2015 Wiley.



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treat non-Hodgkin’s lymphoma.127 However, their complexityleads to additional challenges, as protein structure must bemaintained and proteolytic degradation must be avoided untilthe protein achieves the desired therapeutic effect either locallyor systemically.126 Controlled release of therapeutic proteinsfrom hydrogels offers the potential to maintain potentconcentrations over extended periods of time and to limitpremature degradation before the therapeutic reaches its desiredtarget.128

As with small molecule drugs, the mesh size of hydrogel-baseddelivery vehicles is an important consideration; however, themesh sizes of typical synthetic hydrogels and the size oftherapeutic proteins are often on the same scale (approximately1−10 nm). As a result, the protein often is encapsulated withinthe network and released by diffusion. If the protein size issmaller than the mesh size of the hydrogel, the protein can diffuseout of the hydrogel, usually at a slower rate than protein diffusionthrough water (and slower than uninhibited diffusion throughoutthe body). If the protein size is larger than the mesh size of thehydrogel, the protein is trapped until released by hydrogeldegradation or a stimulus-triggered change in mesh size. Bothapproaches have proven useful depending on the desired timescale for therapeutic release as noted in examples below anddiscussed more broadly within a relevant review.129 In manycases, model proteins of different sizes, such as lysozyme (14kDa) or BSA (66 kDa), are used. Although these are useful andinexpensive models for protein release experiments, it isimportant to note that the bioactivity of therapeutic proteinsmight be compromised during loading or release from hydrogeldelivery vehicles,130 and the compatibility of the hydrogel with aspecific therapeutic protein of interest needs to be verified foreach application.PEG-based thiol−ene hydrogels are some of the most

common types of hydrogels used in controlled release oftherapeutic proteins. For example, Buwalda et al. cross-linked 8-arm PEG-poly(L-lactide)-acrylate block copolymers with multi-functional PEG-thiols by Michael addition for encapsulation andrelease of the model proteins lysozyme and albumin.131 Proteindiffusion out of the hydrogel was slowed relative to diffusion inwater and took place on time scales of days to weeks,demonstrating controlled release. Zustiak and Leach used anentirely PEG-based network formed by thiol-vinyl sulfonechemistry with hydrolytically degradable ester bonds forcontrolled protein release.39 Lysozyme encapsulated in thehydrogel diffused out within 18 h, but the largest proteinencapsulated, immunoglobulin, remained in the gel untilcomplete hydrogel degradation at 1 week (Figure 5A). In bothof these cases, the diffusion rate was tuned by changing thenumber of ester bonds present in the network, affecting thedegradation rate of the hydrogel and protein release.Although Michael-type reactions have enabled successful

encapsulation and release of model proteins, gel formation inthese systems begins upon mixing of all components. In contrast,photopolymerization allows for spatial and temporal control ofpolymerization, enabling the use of photolithography andmicromolding for the formation of macroscale hydrogelgeometries and microscale particles.132 Impellitteri et al. used aphotopolymerized PEG-based thiol-norbornene reaction toencapsulate vascular endothelial growth factor (VEGF) inhydrogel microspheres containing a peptide mimic of theVEGF receptor (RTELNVGIDFNWEYP), serving as an affinitybinding sequence that mediated VEGF release.133,134 Themicrospheres were formed in a water-in-water emulsion: one

phase contained 4-arm PEG-norbornene, PEG-dithiol, and 0.5%w/w of the photoinitiator Irgacure 2959; the other phasecontained 40 kDa dextran. The PEG macromers were cross-linked into stable microspheres by irradiation (low dose of UVlight, 4 mW/cm2 for 8 min). Released VEGF was shown to bebioactive by its enhancement of human umbilical veinendothelial cell proliferation in vitro. (Figure 5B).McCall and Anseth compared the efficacy of photoinitiated

thiol-norbornene or acrylate only reactions for the encapsulationof a model protein lysozyme within PEG-based hydrogels.135 Atequal functional group concentrations (40 mM acrylate or 40mM norbornene with 40 mM thiol) and the same photoinitiatorconcentration (1 mM LAP, 10 mW/cm2 at 365 nm for <5 min),the rapid thiol-norbornene system maintained a higher level oflysozyme activity after encapsulation and release than the slower(oxygen-inhibited) acrylate-based chain-growth system. Thisresult was attributed to the protective effect of thiol−enechemistry during hydrogel formation, which may limit the overallprotein exposure to damaging free radicals in the gel-formingsolution compared to acrylate homopolymerization (Figure 5C).Hydrogel degradation rates can be engineered to respond to

microenvironment conditions (e.g., enzymes, reducing con-ditions, pH), allowing triggered release of a protein therapeutic,as discussed in section 3.5. For example, PEG-based hydrogelsformed by thiol-norbornene photopolymerization with cysteine-functionalized, enzyme-sensitive peptide cross-links weredeveloped and used for protein release by Anseth and co-workers.136 The cross-links contained a sequence (CGAAPV↓RGGGGC) that degrades in the presence of human neutrophilelastase (HNE), an enzyme upregulated at the site ofinflammatory disease and injury.137 The model protein BSAwas released from these hydrogels upon the application of humanneutrophil elastase.136 Notably, no protein release was observedin the absence of the enzyme, demonstrating that the proteinrelease was controlled by the degradation of the hydrogel in thepresence of HNE.In an alternative approach to localized protein release, Kharkar

et al. formed PEG-based hydrogels sensitive to glutathione,which is elevated in tumors, using thiol-maleimide chemis-try.83,109 Hydrogels encapsulating BSA were formed with PEG-maleimide and PEG functionalized with different thiols thatinfluenced hydrogel degradability. Approximately 40% of BSAwas released from all compositions as a result of initial swelling;however, when the thiol-containing macromer contained anelectron-withdrawing aryl group (PEG-4-mercaptophenylaceticacid), an additional 50% of the BSA was released in the presenceof 10 mM glutathione over the course of 7 days. Negligibleadditional release was observed over 7 days for the compositionsthat were glutathione-insensitive, collectively demonstratingpromise for tailoring protein release within tumor microenviron-ments.Although PEG-based materials are often commercially

available and a number of them are FDA-approved,138 there isa limit to the tunability one can achieve simply by varying the endgroups and functionality of PEG-based monomers. Langer andco-workers synthesized a library of thiol-functionalized ethoxy-lated polyol esters and reacted them with PEG-diacrylate to forma library of hydrogels with highly tailorable rates of degradation.42

By changing the thiol composition of the hydrogel from 100%thioglycolate-functionalized ethoxylated polyol ester to 100%thiolactate-functionalized ethoxylated polyol ester, the time forcomplete hydrogel degradation was varied from∼12 days to∼38days. The release of many model macromolecules (3, 10, 20, and



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40 kDa FITC-dextrans; FITC-labeled ovalbumin; and AlexaFluor 647 IgG) was controlled by changing the chemical identityof the polyol esters and by using multiple polyol esters within asingle hydrogel. For example, FITC-labeled ovalbumin wasreleased from the hydrogel over the course of ∼4 days to ∼25days, depending on which of the six thiol-containing polyol esterswas used to form the hydrogel (Figure 5D). In a follow-up work,trehalose, a disaccharide known for its protein-stabilizingproperties, was incorporated within these hydrogels.139

Trehalose diacrylate was mixed with PEG-diacrylate at variousratios and reacted with thiol-functionalized polyol esters for atotal of 25 wt % polymer in the final hydrogel. When 100% of theacrylate groups came from trehalose (i.e., 0% from PEG-diacrylate), nearly 100% of active horseradish peroxidaseencapsulated in the gel was recovered within ∼4 days. Incontrast, when 6.25% of the acrylate groups came from trehalose(i.e., 93.75% from PEG-diacrylate), less than 3% of thehorseradish peroxidase was recovered in active form within∼12 days. Covalent incorporation of trehalose also maintainedthe activity of two other model proteins, glucose oxidase and α-chymotrypsin. Although the mechanism of trehalose-mediatedprotein stabilization is not fully understood, trehalose isproduced by many plants and animals in response to osmotic,high temperature, and other stresses, and it is thought to stabilizeproteins by making protein unfolding more thermodynamicallyunfavorable.38 This concept may motivate additional approachesfor stabilization of proteins in hydrogels.Natural polymers and their derivatives also have been used to

make hydrogels for protein release. For instance, Peng et al.reacted dextran-maleimide with thiol-containing azobenzene toform a photoresponsive dextran hydrogel.140 The cis/transisomerization of azobenzene was used to release greenfluorescent protein in a light-responsive manner (100 W at 365nm). Beyond model proteins, hyaluronic acid−based hydrogelshave been used to deliver two growth factors, stromal cell-derivedfactor-1 (SDF-1) and BMP-2, to promote hMSC infiltration anddifferentiation for bone regeneration in an animal model.141

These hydrogels were formed in situ by a Michael-type reactionbetween hyaluronic acid-maleimide, a dithiol MMP-degradablepeptide (GCRDVPMS↓MRGGDRCG), and a thiol-function-alized RGD peptide (GCGYGRGDSPG). Limited release ofthese growth factors occurred by diffusion, but in the presence ofthe enzyme collagenase, the gel cross-links were degraded andgrowth factors were released from the hydrogel. Completedegradation was observed within 9 days with 1 U mL−1

collagenase and within 4 days with 2 U ml−2 collagenase.Toward modulating myofibroblast activities and tissue regener-ation after myocardial infarction, Burdick and co-workers alsocreated injectable, MMP-degradable, hyaluronic acid−basedhydrogels for delivery of a recombinant tissue inhibitor ofMMPs (rTIMP-3).142 The hydrogel was formed by conjugating athiol- and hydrazide-functionalized MMP-degradable peptide(GGRMSMPV) to maleimide-functionalized hyaluronic acid.The hydrazide-functionalized MMP-degradable hyaluronic acidmacromer was then reacted with aldehyde-functionalizedhyaluronic acid to form a hydrogel in situ upon injection.Additionally, aldehyde-functionalized dextran sulfate was in-corporated into the hydrogel to sequester encapsulated rTIMP-3through electrostatic interactions. The hydrogel/TIMP-3construct was delivered to a region of MMP overexpression ina porcine myocardial infarction model, resulting in significantlyreduced MMP activity and adverse left ventricular remodeling.The study demonstrated the ability to locally release an MMP-

inhibitor as needed in response to MMP-overexpressingpathologies.

4.3. Cell Delivery. The delivery of mammalian cells to thesite of injury or diseased tissues is an emerging therapeuticapproach, where the cell is able to exert positive effects on itssurroundings by a variety of mechanisms including thedeposition of ECM or the secretion of effector molecules (e.g.,cytokines, chemokines) that can influence surrounding cellbehavior.143 In this regard, hydrogels have been used to (i)improve the viability or decrease the immunogenicity oftransplanted cells, (ii) provide mechanical integrity to theconstruct of transplanted cells, and (iii) control the transport ofcells and biomolecules. In these applications, the material canprevent the uncontrolled release of cells throughout the body,limit contact between the transplanted cells and the immunesystem, and support (or prevent) the diffusion of nutrients andwaste to and from the transplanted cells.144 In particular,hydrogels formed by thiol−ene chemistry are advantageous forcell delivery because of their often cytocompatible nature; theirability to form in neutral pH, aqueous, oxygen-rich solutions;their often rapid reaction kinetics; and reaction specificity andhigh yield.145 Additional information on cell delivery fromhydrogels, including those formed by thiol−ene reactions, can befound in depth in several recent reviews.146−148

5. CONCLUSIONS AND FUTURE PERSPECTIVES

Significant progress has been made in the synthesis andcharacterization of thiol−ene hydrogels for the controlleddelivery of therapeutics. Many of the systems overviewed herehave shown promise with model cargo molecules, such as BSA,IgG, and lysozyme, and these studies provide well-definedformulations that now can be adapted for the delivery of specifictherapeutics in vivo. However, several challenges need to beaddressed toward broad clinical translation of thiol−enehydrogels. The ability to retain the bioactivity of cargo moleculesafter exposure to the hydrogel environment, whether duringformation or degradation, is one key area that further needs to bestudied and addressed. For example, incorporation of naturalpolymers, such as silk, trehalose, or heparin, can be used toinfluence the stability of bioactive cargoes within otherwisesynthetic hydrogels to provide protection during the encapsu-lation process.149 Maintaining precise control over cargo releaseis another persistent challenge that needs to be further explored.The ability to control release of cargo from thiol−ene hydrogelspostadministration for patient-specific treatment regimes (e.g.,triggered changes in material properties and release with light orenzymes) will be beneficial for efficient disease management.Furthermore, the light-directed control afforded by radical-mediated thiol−ene reactions may provide opportunities tocreate drug-releasing constructs of arbitrary shapes and sizes withrapidly evolving three-dimensional printing systems and otherphotolithographic processes, such as those recently demon-strated by DeSimone and co-workers.150 Taken together, thiol−ene hydrogels are promising therapeutic delivery vehiclesbecause of the range of facile, often biocompatible, chemistriesavailable for their formation and modification providingtunability over material mechanical properties and cargo releaseprofiles.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected]. Tel: +1 302-831-3009



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mailto:[email protected]


Present Address§K.M.S. is currently at Frontier Scientific Services, Inc. 601Interchange Blvd Newark, DE 19711

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors gratefully acknowledge support from the NationalInstitutes of Health (NIH) Chemistry-Biology Interface programat the University of Delaware (NIH T32GM008550), theDelaware COBRE program with a grant from the NationalInstitute of General Medical Sciences (NIGMS P20GM104316)from the NIH, the Burroughs Wellcome Fund, a grant from theCalifornia Institute for Regenerative Medicine (RN3-06460),and the University of Delaware Research Foundation for relatedwork in their laboratories. The authors thank Prof. ChristopherKloxin for helpful discussions on thiol−ene chemistry.

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