molecules
Review Thiol- and Disulfide-Based Stimulus-Responsive Soft Materials and Self-Assembling Systems
Danielle M. Beaupre 1 and Richard G. Weiss 1,2,*
1 Department of Chemistry, Georgetown University, Washington, DC 20057, USA; [email protected] 2 Institute for Soft Matter Synthesis and Metrology, Georgetown University, Washington, DC 20057, USA * Correspondence: [email protected]; Tel.: +1-202-687-6013
Abstract: Properties and applications of synthetic thiol- and disulfide-based materials, principally polymers, are reviewed. Emphasis is placed on soft and self-assembling materials in which inter- conversion of the thiol and disulfide groups initiates stimulus-responses and/or self-healing for biomedical and non-biomedical applications.
Keywords: stimulus-responsive materials; self-healing; redox-responsive; drug delivery; thiol- disulfide exchange; disulfide-disulfide metathesis
1. Introduction 1.1. Thiols and Disulfides in Materials Chemistry Thiols (RSH) and disulfides (RSSR) are components of many proteins, biopolymers, and biomolecules. In nature, thiols and disulfides contribute to key biological functions related to cell signaling, protein conformation and folding processes, redox homeostasis, Citation: Beaupre, D.M.; Weiss, R.G. and biopolymer secondary structure development. Thiol groups and/or disulfide linkages Thiol- and Disulfide-Based can be incorporated into polymers to produce stimulus-responsive and/or self-healing Stimulus-Responsive Soft Materials materials. They owe their stimulus-responsiveness to covalent disulfide bonds, which can and Self-Assembling Systems. be broken and re-formed under mild conditions. The extent of reactivity, and thus the Molecules 2021, 26, 3332. https:// stimulus-responsive behavior depends on the nature of the groups flanking the disulfide doi.org/10.3390/molecules26113332 bond, as well as interactions with stimuli such as light, heat, mechanical force, and changes in the pH or redox state [1]. The ease with which thiols can be converted to disulfides, and Academic Editor: Feihe Huang vice versa, renders thiol-containing polymers (thiomers) similarly dynamic. This review will focus on the properties and applications of synthetic thiomers and Received: 4 May 2021 disulfide-containing polymers, with an emphasis on soft and self-assembling materials. Accepted: 25 May 2021 Published: 1 June 2021 Common thiol-containing pendant groups, as well as the synthetic methodologies for their incorporation into polymers, have been discussed extensively in a 2019 review of
Publisher’s Note: MDPI stays neutral biomedically relevant thiomers, which includes a discourse on thiomer design strategies [2]. with regard to jurisdictional claims in A recent review by Bej and coworkers covered responsive aggregation of both small published maps and institutional affil- molecule and polymer amphiphiles containing disulfide bonds, with an emphasis on iations. biomedical applications [3]. Additionally, thiol and disulfide-based self-healing materials were reviewed by Yang [4], Wang [5], Utrera-Barrios [6], and their coworkers. Here, we discuss polymers in which the thiol and disulfide groups are important to their function. In addition to the types of systems previously reviewed, many examples of disulfide-based, self-healing polymers, many of which are elastomers, will be mentioned. However, to Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. the best of our knowledge, no review devoted specifically to self-healing disulfides has This article is an open access article been published. distributed under the terms and After a brief overview of reactions between (and among) thiols and disulfides, a conditions of the Creative Commons variety of thiol- and disulfide-based stimulus-responsive systems will be reviewed. The Attribution (CC BY) license (https:// examples will be classified by the structures of the materials and the stimuli employed. creativecommons.org/licenses/by/ The processes include thiol-disulfide and disulfide-disulfide exchange reactions, and redox 4.0/). reactions, as well as multiple responses by materials that contain additional functionalities
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whichas multiple trigger responses “cascade” by materials reactions that (such contai as decomposition).n additional functionalit The connectionsies which trigger between “cas- thecade” applications reactions (such and the as decomposition). structures of the The materials connections and the between nature the of theirapplications reactions and will the bestructures emphasized. of the materials and the nature of their reactions will be emphasized.
1.2.1.2. SomeSome ImportantImportant ReactionsReactionsof ofThiols Thiolsand and Disulfides Disulfides Thiol-disulfideThiol-disulfide exchange exchange reactions reactions can can occur occurbetween betweendeprotonated deprotonated thiols thiols(thiolates) (thiolates) andand disulfides. disulfides. Thiol-disulfide Thiol-disulfide exchange exchange proceeds proceeds via via substitution-type substitution-type pathways, pathways, with with the thiolatethe thiolate anion anion (RS− (RS) functioning-) functioning as the as nucleophilethe nucleophile (1 in (1 Figure in Figure1). In 1). addition, In addition, thiols thiols can becan oxidized be oxidized to disulfides to disulfides (2 through (2 through 10 in Figure10 in Figure1), and 1), disulfides and disulfides can be reducedcan be reduced to thiols. to Althoughthiols. Although the products the products of oxidation of oxidation or reduction or reduction reactions reactions are often are the often same the asthose sameof as thiol-disulfidethose of thiol-disulfide exchange exchange reactions, reactions, the pathways the pathways are mechanistically are mechanistically different (as different shown in(as Figureshown1). in There Figure are 1). well-documented There are well-documented multi-step examples multi-step of examples both one electronof both one (7 through electron 10(7 inthrough Figure1 10) and in Figure two electron 1) and (2 two through electron 6 in (2 Figure through1) oxidation 6 in Figure pathways 1) oxidation within pathways cells [ 7]. Thiylwithin radicals cells [7]. (RS Thiyl·) can radicals be generated (RS⋅) fromcan be thiols generated or disulfides from thiols using or a varietydisulfides of reagents using a va- or stimuli.riety of Thiylreagents radicals or stimuli. play a Thiyl crucial radicals role in play biological a crucial systems role in and biological are important systems reagents and are inimportant organic synthesis.reagents in Thiyl organic radicals synthesis. are intermediates Thiyl radicals in are disulfide-disulfide intermediates in disulfide-di- metathesis reactions,sulfide metathesis also known reactions, as disulfide-disulfide also known as disulfide-disulfide exchange reactions, exchange which reactions, proceed which via a radicalproceed pathway via a radical (11 in Figurepathway1). Because(11 in Figure disulfide 1). Because linkages disulfide are often thelinkages weakest are covalentoften the bondsweakest in ancovalent organic bonds molecule, in an theyorganic provide molecule, an avenue they provide to tune thean avenue properties to tune of a the material prop- undererties mildof a material reaction under or stimulus mild reaction conditions. or st Pleaseimulus note conditions. that the Please product note distributions that the prod- of bothuct distributions thiol-disulfide of andboth disulfide-disulfide thiol-disulfide and exchangedisulfide-disulfide depend upon exchange the ratios depend of reactants upon the andratios the of nature reactants of the and R groups,the nature which of the determine R groups, product which stabilities.determine product stabilities.
Figure 1. Reactions of thiols (RSH) and disulfides (RSSR): 1.1 SN2-mediated thiol-disulfide exchange; Figure 1. Reactions of thiols (RSH) and disulfides (RSSR): 1.1 SN2-mediated thiol-disulfide exchange; 1.2–1.6. Thiol oxidation with two-electron oxidants, where RSX is a sulfenyl halide, RSOH is a sul- 1.2–1.6. Thiol oxidation with two-electron oxidants, where RSX is a sulfenyl halide, RSOH is a sulfenic fenic acid, HOX is a halohydrin, and HOx is the reduced oxidizing agent; 1.7–1.10 Thiol oxidation acid, HOX is a halohydrin, and HOx is the reduced oxidizing agent; 1.7–1.10 Thiol oxidation with with one-electron oxidizing agents; 1.11 disulfide-disulfide metathesis. one-electron oxidizing agents; 1.11 disulfide-disulfide metathesis.
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1.3.1.3. Biological Biological Relavance Relavance of of Thiols Thiols and and Disulfides Disulfides SomeSome molecules molecules of of biological biological relevance relevance that that exploit exploit the the disulfide disulfide bond bond are are shown shown in in FigureFigure2. 2. For For example, example, processes processes involving involving cystine cystine (Cys (Cys2)2) and and cysteine cysteine (Cys) (Cys) interconver- interconver- sionssions have have structural structural and and functional functional consequences consequences on on the the macroscopic macroscopic and and microscopic microscopic scale.scale. On On a a macroscopic macroscopic level, level, disulfide disulfide bonds bonds in in Cys Cys2 stabilize2 stabilize the the secondary secondary structure structure of of keratin-basedkeratin-based materials, materials, such such as as hair, hair, wool, wool, and and nails. nails. The The cleavage cleavage of of disulfide disulfide bonds bonds in in keratinkeratin results results in in a a dramatic dramatic decrease decrease of of strength strength and and elasticity elasticity of of these these materials. materials. In In that that regard,regard, the the redox redox states states and and pH pH of of intracellular intracellular and and extracellular extracellular environments environments control control the the reversiblereversible oxidation oxidation of of Cys Cys to to Cys Cys2 in2 in polypeptides. polypeptides. As As such, such, disulfide disulfide bonds bonds are are keys keys to to thethe protein protein folding folding process, process, and and thus thus strongly strongly dictate dictate protein protein conformation. conformation.
FigureFigure 2. 2.Structures Structures of of some some biologically biologically important important thiols thiols and and disulfides. disulfides.
1.4.1.4. Thiols Thiols and and Disulfides Disulfides in in Materials Materials Science Science NatureNature has has shown shown thatthat thiol-disulfidethiol-disulfide exchangeexchange and redox reactions reactions can can be be used used to tointerconvert interconvert thiols thiols and and disulfides disulfides reversibly reversibly in response in response to changes to changes in pH in pHor redox or redox state. state.The dynamic The dynamic nature nature of the of disulfide the disulfide bond bondhas been has beenextended extended to materials to materials science science to pro- toduce produce a wide a wide range range of stimulus-responsive of stimulus-responsive materials. materials. The reviews The reviews of Seidi of Seidi[8] and [8 ]Zhang and Zhang[9] include [9] include recent recent examples examples of controlled of controlled drug drug release, release, with with an anemphasis emphasis on on stimulus-re- stimulus- responsivesponsive and and redox-triggered drug release.release. TheseThese reviewsreviews includeinclude several several examples examples of of thiol-thiol- and and disulfide-based disulfide-based materials. materials. Thiol-disulfideThiol-disulfide exchange exchange is is often often used used to to bind bind thiomers thiomers to to disulfide-containing disulfide-containing sub- sub- strates,strates, and and to to trigger trigger the the release release of of disulfide disulfide bound bound “cargo,” “cargo,” such such as as drugs drugs or or dyes, dyes, from from a polymer upon exposure to high concentrations of thiols and/or changes in pH. Thiol a polymer upon exposure to high concentrations of thiols and/or changes in pH. Thiol oxidation and disulfide reduction are commonly used to form and break covalent disulfide oxidation and disulfide reduction are commonly used to form and break covalent disul- crosslinks in polymeric networks, respectively. The scission and reformation of disulfide fide crosslinks in polymeric networks, respectively. The scission and reformation of disul- crosslinks in situ via reduction and oxidation, respectively, is often reported as a means for fide crosslinks in situ via reduction and oxidation, respectively, is often reported as a reversible sol-gel transitions. Commonly, dramatic rheological changes are observed over means for reversible sol-gel transitions. Commonly, dramatic rheological changes are ob- several reversible sol-gel redox cycles. Self-healing behavior has been widely reported for served over several reversible sol-gel redox cycles. Self-healing behavior has been widely a variety of disulfide-containing polymers, due to disulfide-disulfide shuffling reactions, reported for a variety of disulfide-containing polymers, due to disulfide-disulfide shuf- which occur after breakage. fling reactions, which occur after breakage. 1.5. Applications of Thiomers and Disulfide-Containing Polymers 1.5. Applications of Thiomers and Disulfide-Containing Polymers Drug delivery is among the most common biomedical applications of thiomers and disulfide-containingDrug delivery polymers. is among Thethe most prototypical common drug biomedical delivery systemapplications releases of athiomers conjugated and drugdisulfide-containing molecule after being polymers. exposed The to prototypic a cellularal trigger. drug Fordelivery example, system solid releases tumors a haveconju- highergated levelsdrug ofmolecule GSH and after lower being pH exposed levels than to a healthy cellular tissues. trigger. Chemotherapy For example, solid employing tumors ahave drug higher delivery levels system of GSH can and be tunedlower topH release levels thethan drug healthy when tissues. exposed Chemotherapy to the unique em- chemicalploying environmenta drug delivery of asystem cancer cell;can be more tune targetedd to release drug the delivery drug resultswhen exposed in fewer to side the effectsunique [3 ].chemical Figure3 environmentdemonstrates of the a widecancer variety cell; more of drug targeted delivery drug systems delivery that results rely on in disulfidefewer side bonds effects for controlled[3]. Figuredelivery 3 demonstrates of nucleic the acid wide therapeutics variety of[ 10drug]. delivery systems that rely on disulfide bonds for controlled delivery of nucleic acid therapeutics [10].
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FigureFigure 3. 3. TypesTypes of drug delivery delivery systems systems that that rely rely on on the the presence presence of disulfide of disulfide bonds bonds for the for tar- the geted delivery of nucleic acids. Reprinted with permission from [10]. Copyright (2021) American targeted delivery of nucleic acids. Reprinted with permission from [10]. Copyright (2021) American Chemical Society. Chemical Society.
TheThe reviews reviews of of Dutta Dutta [10 [10],], Wang Wang [11 [11],], and and Saitoa Saitoa [12 [12]] focus focus on on disulfide-based disulfide-based drug drug deliverydelivery systems. systems. Another Another notable notable application application is is in in the the field field of of biomedicine, biomedicine, which which merges merges biologicalbiological and and physiological physiological principles principles within within clinical clinical science. science. Biomedicine Biomedicine often dealsoften withdeals mucoadhesivewith mucoadhesive materials, materials, which which contain contain thiols and/orthiols and/or disulfides disulfides that penetrate that penetrate mucosal mu- membranescosal membranes by binding by binding with free with thiol free side thiol chains side fromchains Cys from residues Cys residues [2]. [2]. TheThe applications applications of thiomers of thiomers and disulfide-containingand disulfide-containing polymers polymers extend beyond extend biomedicine beyond bio-. Amedicine. wide range A wide of dynamic range of materials, dynamic such materials, as self-healing such as self-healing elastomers elastomers [4,13], shape [4,13], memory shape polymersmemory [ 14polymers] and stimulus-responsive [14] and stimulus-responsiv polymers [9e, 15polymers] have been [9,15] designed have been as coatings designed [16 ],as adhesivecoatings materials [16], adhesive [17], and materials sensors [17], [2,18 and]. Incorporation sensors [2,18]. of Incorporation thiols and/or disulfidesof thiols and/or into polymersdisulfides that into contain polymers other that stimulus-responsive contain other stimulus-responsive moieties is an emerging moieties technique is an emerging that has beentechnique used to that fabricate has been multi used stimulus-responsive to fabricate multi materials stimulus-responsive that may enhance materials specificity that formay bothenhance biomedical specificity [15] andfor both non-biomedical biomedical applications[15] and non-biomedical [6,14,18] For applications example, drug [6,14,18] delivery For systemsexample, incorporating drug delivery multi-stimulus-responsive systems incorporating multi-stimulus-responsive materials can increase the materials amount ofcan drugincrease reaching the amount the targeted of drug cells reaching [19,20]. the Some targeted multi stimulus-responsivecells [19,20]. Some multi materials stimulus-re- have beensponsive designed materials to release have a drugbeen onlydesigned after theto release application a drug of only two orafter more the stimuli application associated of two withor more the targetstimuli environment. associated with Other the target multi environment. stimulus-responsive Other mult drugi stimulus-responsive delivery systems incorporatedrug delivery functional systems groups incorporate designed functional to enhance groups targeting designed specific to enhance types of targeting cells. spe- cific types of cells. 2. Factors Influencing the Reactivity of Thiols and Disulfides 2.1.2. Factors InfluencingInfluencing the the Bond Reactivity Strengths of of Thiols Thiols andand Disulfides Disulfides 2.1.The Factors strengths Influencing of both the Bond the RS-H Strengths and of RS-SR Thiols bonds and Disulfides are highly dependent upon the natureThe of the strengths attached of R both groups. the RS-H Thus, and changing RS-SR the bonds R group are highly provides dependent a means upon to tune the the na- reactivityture of the and, attached therefore, R groups. the stimulus-responsiveness Thus, changing the R group of thiol- provides and disulfide-containing a means to tune the materials. Alkanethiols and thiophenols have homolytic S-H bond dissociation energies reactivity and, therefore, the stimulus-responsiveness of thiol- and disulfide-containing of ca. 87 and 79 kcal/mol, respectively [21]. Disulfides generally have lower homolytic materials. Alkanethiols and thiophenols have homolytic S-H bond dissociation energies S-S bond dissociation energies, ranging from 50-65 kcal/mol [21]. Aryl thiols, such as of ca. 87 and 79 kcal/mol, respectively [21]. Disulfides generally have lower homolytic S- thiophenol, generally have the lowest homolytic S-H bond dissociation energies, especially S bond dissociation energies, ranging from 50-65 kcal/mol [21]. Aryl thiols, such as thio- when an electron donating aryl substituent is present at the para position [22]. The relatively phenol, generally have the lowest homolytic S-H bond dissociation energies, especially
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when an electron donating aryl substituent is present at the para position [22]. The rela- tively low homolytic bond dissociation energies of thiols and disulfides facilitate im- portant radical reactions, and thiols are very popular initiators for free radical polymeri- zation reactions [22]. Molecules 2021, 26, 3332 5 of 44 2.2. Reactivity of Thiols and Disulfides in Thiol-Disulfide Exchange Reactions
low homolyticThiol bondpKa dissociation values span energies a large of thiols range, and disulfides from 3 facilitate to 11, important with values contingent upon the radicalnature reactions, of the and R thiolsgroup are attached very popular to initiators the sulfur for free atom radical [23]. polymerization Often, pKa values can be used to reactionspredict [22 ].whether a reaction will favor the reactants or products in a thiol-disulfide ex- 2.2.change Reactivity (1 of in Thiols Figure and Disulfides 1). The in Thiol-Disulfidethiolate anion Exchange RS Reactions- reacts about 1010 times faster than the corre- spondingThiol pKa valuesthiol spanin a a largethiol-disulfide range, from 3 to exchange 11, with values reaction. contingent However, upon the because thiol-disulfide nature of the R group attached to the sulfur atom [23]. Often, pKa values can be used to predictexchange whether is a reactionknown will to favor be the a reactantskinetically or products driven in a thiol-disulfideprocess, equilibrium exchange parameters are some- (1times in Figure not1). The good thiolate predictors anion RS − reacts [7]. about 1010 times faster than the corresponding thiol in a thiol-disulfide exchange reaction. However, because thiol-disulfide exchange is known toWhen be a kinetically the pKa driven of process, the thiol equilibrium is ~7, parameters the thiol-disulfide are sometimes not exchange good rate constant is at its predictorsmaximum [7]. value (Figure 4; [7]). The rate constant decreases as thiol pKa decreases below When the pKa of the thiol is ~7, the thiol-disulfide exchange rate constant is at its maximum7, or increases value (Figure above4;[ 7]). 7, The due rate constantto a decrease decreases in as thiolthiolate pKa decreases nucleophilicity below and in the fraction of 7,thiolate or increases in above solution. 7, due to In a decreaseaddition in thiolate to pKa nucleophilicity values, aand comparison in the fraction of of the redox potentials of the thiolatethiol inreactant solution. Inand addition product to pKa values,is generally a comparison a succe of thessful redox potentialsmethod of to the predict whether a reaction thiol reactant and product is generally a successful method to predict whether a reaction is favoredis favored [7,24]. [7,24].
FigureFigure 4. Calculated 4. Calculated apparent apparent rate constant rate for thiol-disulfideconstant for exchange thiol-di in asulfide solution exchange at pH 7 as a in a solution at pH 7 as a functionfunction of reactant of reactant thiol pKa thiol Reprinted pKa with Reprinted permission fromwith [7 ].perm Copyrightission Mary from Ann Liebert,[7]. Copyright Inc. Mary Ann Liebert, Inc. However, considering only the nature of the R groups of the thiol reactant and product tends toHowever, be a pragmatic considering (rather than critically only planned) the nature approach of when the the R reactants groups and/or of the thiol reactant and prod- products are polymers. The nature of monomers and co-monomers along the polymer backboneuct tends must to also be be considered.a pragmatic All else (rather being equal, than a thiomercritically comprised planned) of a flexible, approach when the reactants lowand/or glass transition products temperature are polymers. (Tg) polymer The backbone nature will of be monomers more reactive thanand one co-monomers along the pol- with a strong, rigid polymer backbone because the thiol and disulfide groups in the former areymer more likelybackbone to adopt must the required also transitionbe considered. state geometry All (Figure else 5being) when theyequal, are a thiomer comprised of a nearer.flexible, For similar low reasons,glass similartransition polymers temperature with greater degrees (Tg) ofpolymer thiolation arebackbone more will be more reactive reactivethan toone a disulfide with a than strong, those with rigid lower polymer degrees of backbone thiolation. because the thiol and disulfide groups in the former are more likely to adopt the required transition state geometry (Figure 5) when they are nearer. For similar reasons, similar polymers with greater degrees of thiolation are more reactive to a disulfide than those with lower degrees of thiolation.
Figure 5. The orientation of a thiol and disulfide during thiol-disulfide exchange. Sn is the nucleo- philic sulfur of the thiolate reactant; Sc and Slg are the central and leaving group sulfur atoms of the disulfide reactant Adapted with permission from [23]. Copyright (2013) Elsevier B.V.
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when an electron donating aryl substituent is present at the para position [22]. The rela- tively low homolytic bond dissociation energies of thiols and disulfides facilitate im- portant radical reactions, and thiols are very popular initiators for free radical polymeri- zation reactions [22].
2.2. Reactivity of Thiols and Disulfides in Thiol-Disulfide Exchange Reactions Thiol pKa values span a large range, from 3 to 11, with values contingent upon the nature of the R group attached to the sulfur atom [23]. Often, pKa values can be used to predict whether a reaction will favor the reactants or products in a thiol-disulfide ex- change (1 in Figure 1). The thiolate anion RS- reacts about 1010 times faster than the corre- sponding thiol in a thiol-disulfide exchange reaction. However, because thiol-disulfide exchange is known to be a kinetically driven process, equilibrium parameters are some- times not good predictors [7]. When the pKa of the thiol is ~7, the thiol-disulfide exchange rate constant is at its maximum value (Figure 4; [7]). The rate constant decreases as thiol pKa decreases below 7, or increases above 7, due to a decrease in thiolate nucleophilicity and in the fraction of thiolate in solution. In addition to pKa values, a comparison of the redox potentials of the thiol reactant and product is generally a successful method to predict whether a reaction is favored [7,24].
Figure 4. Calculated apparent rate constant for thiol-disulfide exchange in a solution at pH 7 as a function of reactant thiol pKa Reprinted with permission from [7]. Copyright Mary Ann Liebert, Inc.
However, considering only the nature of the R groups of the thiol reactant and prod- uct tends to be a pragmatic (rather than critically planned) approach when the reactants and/or products are polymers. The nature of monomers and co-monomers along the pol- ymer backbone must also be considered. All else being equal, a thiomer comprised of a flexible, low glass transition temperature (Tg) polymer backbone will be more reactive than one with a strong, rigid polymer backbone because the thiol and disulfide groups in Molecules 2021, 26, 3332the former are more likely to adopt the required transition state geometry (Figure 5) when 6 of 44 they are nearer. For similar reasons, similar polymers with greater degrees of thiolation are more reactive to a disulfide than those with lower degrees of thiolation.
Figure 5. The orientation of a thiol and disulfide during thiol-disulfide exchange. Sn is the nucleo- Figure 5. The orientation of a thiol and disulfide during thiol-disulfide exchange. Sn is the nucleophilic sulfur of the thiolate philic sulfur of the thiolate reactant; Sc and Slg are the central and leaving group sulfur atoms of the Moleculesreactant; 2021, S 26c ,and x FOR S PEERare theREVIEW central and leaving group sulfur atoms of the disulfide reactant Adapted with permission6 of 45 disulfidelg reactant Adapted with permission from [23]. Copyright (2013) Elsevier B.V. from [23]. Copyright (2013) Elsevier B.V.
Disulfide products are stabilized by electron donating groups, the presence of nega- Disulfide products are stabilized by electron donating groups, the presence of nega- tively charged groups, and hydrogen bonding in the overall structure [2]. The placement tively charged groups, and hydrogen bonding in the overall structure [2]. The placement and type of electrostatic charges on the backbone and the groups surrounding the thiol or and type of electrostatic charges on the backbone and the groups surrounding the thiol or disulfide influence the reactivity in the exchange reaction. Reactivity is inhibited if the thiol disulfide influence the reactivity in the exchange reaction. Reactivity is inhibited if the or disulfide moieties are flanked by groups with the same charge type. Similarly, if both thiol or disulfide moieties are flanked by groups with the same charge type. Similarly, if the thiol and the disulfide have the same charge, the rate of exchange will be impeded by both the thiol and the disulfide have the same charge, the rate of exchange will be impeded electrostatic repulsion (Figure6;[2]). by electrostatic repulsion (Figure 6; [2]).
Figure 6. Cartoon showing the influence of electrostatic effects of R groups flanking thiols on their relative rates of thiol- Figure 6. Cartoon showing the influence of electrostatic effects of R groups flanking thiols on their relative rates of disulfide exchange reactions. Adapted with permission from [2]. Copyright (2019) Elsevier B.V. thiol-disulfide exchange reactions. Adapted with permission from [2]. Copyright (2019) Elsevier B.V. Some thiol reactants can yield cyclic disulfides in thiol-disulfide exchange reactions. Some thiol reactants can yield cyclic disulfides in thiol-disulfide exchange reactions. Likewise, some disulfide reactants can yield thiols that tautomerize into forms such as Likewise, some disulfide reactants can yield thiols that tautomerize into forms such as thiones. In either case, the reverse reaction is substantially slowed and effectively blocked, thiones. In either case, the reverse reaction is substantially slowed and effectively blocked, resulting in a near quantitative thiol-disulfide exchange [23,24]. Thiols and disulfides, resulting in a near quantitative thiol-disulfide exchange [23,24]. Thiols and disulfides, such as dithiothreitol (DTT) and 4,4′dithiodipyridine, are therefore favored reactants for such as dithiothreitol (DTT) and 4,40dithiodipyridine, are therefore favored reactants for the reduction of disulfides and the oxidation of thiols, respectively [23]. Although these the reduction of disulfides and the oxidation of thiols, respectively [23]. Although these reagents participate mechanistically in thiol-disulfide exchange reactions, the products reagents participate mechanistically in thiol-disulfide exchange reactions, the products are are formed in nearly quantitative yields; thus, the terms “oxidation” and “reduction” are formed in nearly quantitative yields; thus, the terms “oxidation” and “reduction” are far far more common in the literature. Conversely, the term “thiol-disulfide exchange” is more common in the literature. Conversely, the term “thiol-disulfide exchange” is more more commonly used to describe reactions that are more dynamic and not quantitative. commonly used to describe reactions that are more dynamic and not quantitative.
2.3.2.3. FactorsFactors InfluencingInfluencing the Redox Chemistry of of Thiols Thiols and and Disulfides Disulfides 2.3.1.2.3.1. ReductionReduction ofof DisulfidesDisulfides with Thiols by by Exchange Exchange Reactions Reactions ReactionsReactions ofof disulfidesdisulfides with redox active active th thiols,iols, such such as as DTT, DTT, are are frequently frequently described described inin thethe literatureliterature as as being being reductions, reductions, but but mech mechanistically,anistically, they they are areoften often thiol-disulfide thiol-disulfide ex- exchangechange reactions reactions [7]. [ 7Generally,]. Generally, the term the term “reduc “reduction”tion” is applied is applied to thiol-disulfide to thiol-disulfide exchange ex- changereactions reactions that are that occur are in occur nearly in quantitative nearly quantitative yields. This yields. condition This condition can be achieved can be achievedby em- byploying employing excess excessthiol reagent thiol reagentand using and specific using thiols specific that thiols have higher that have reduction higher potentials reduction potentialsor that form or cyclic that form rings cyclic (such ringsas me (suchrcaptoethanol as mercaptoethanol (ME) or DTT; (ME) Figure or 2). DTT; Figure2). DTTDTT andand GSHGSH areare the most popular reagents reagents fo forr the the in in situ situ reduction reduction of of disulfide disulfide gels gels toto thiol thiol sols sols InIn softsoft matter,matter, andand manymany studies have have shown shown that that thiol thiol sols sols can can be be reversibly reversibly oxidizedoxidized toto disulfidedisulfide gels,gels, thenthen reduced with DTT or or GSH GSH to to regenerate regenerate the the thiol thiol sol. sol. This This processprocess cancan often be be repeated repeated several several times times with with little little loss of loss disulfide of disulfide gel strength gel strength in some in somecases cases[25,26]. [25 As,26 such,]. As such,it is ideal it is for ideal applications for applications related related to fast toand fast reversible and reversible in situ gela- in situ tion. However, in depth analysis of the gels and sols is not possible if the by-products are not removed between the gel-sol transformation steps.
2.3.2. Direct Reduction of Thiols with Non-Thiol Reducing Agents Facile, direct reduction of disulfides to thiols can be achieved with several non-thiol reducing agents, such as borohydrides, zinc and dilute acid, nascent hydrogen, and phos- phines [24,27]. The direct reduction of disulfides with non-thiol reductants does not re- quire the removal of disulfide by-products or residual thiol reductants in the reaction mix- ture. As indicated in the previous section, by-products that cannot be easily removed can
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gelation. However, in depth analysis of the gels and sols is not possible if the by-products are not removed between the gel-sol transformation steps.
2.3.2. Direct Reduction of Thiols with Non-Thiol Reducing Agents Facile, direct reduction of disulfides to thiols can be achieved with several non-thiol reducing agents, such as borohydrides, zinc and dilute acid, nascent hydrogen, and phos- phines [24,27]. The direct reduction of disulfides with non-thiol reductants does not require the removal of disulfide by-products or residual thiol reductants in the reaction mixture. As indicated in the previous section, by-products that cannot be easily removed can often complicate analysis. It is also possible to reduce one disulfide to one equivalent of the corresponding thiol and one equivalent of an S-substituted derivative using sulfite or cyanide. Although the atom efficiency of such reactions is low, they are used often for analytical purposes [24].
2.4. Oxidation of Thiols to Disulfides The oxidation of thiols can produce both symmetrical and unsymmetrical disulfides, the latter of which are sought for a variety of applications [28,29]. The formation of unsymmetrical disulfides has been extensively used to determine important members of dynamic combinatorial libraries comprised of thiol and disulfide mixtures. The knowledge gained from studying dynamic combinatorial libraries of thiols and disulfides can be used to promote specific reaction products, and interesting results have been reported for many unsymmetrical systems [1,30]. The groups attached to a thiol determine how easily it can be oxidized to a disulfide. The ease of oxidation increases from tertiary to primary R groups attached to sulfur, and aryl thiols are easier to oxidize than aliphatic ones. Aryl thiols can often be oxidized by molecular oxygen, even in air [28]. However, over-oxidation of thiols to sulfonic acids, sulfinc acids, etc. can occur with some oxidants. Iodine and disulfide-based reagents have been found less likely to over-oxidize thiols beyond the disulfide form. The oxida- tion of tertiary thiols generally requires very harsh conditions that are not tolerated by most functional groups. Conversely, the aerobic oxidation of aryl and primary thiols to the corresponding disulfides can often be achieved in high yields in the presence of a silica-supported iron, manganese, or cobalt salt catalysts [28]. Unlike air, diethyl azodicar- boxylate (DEAD) and diisopropyl azodicarboxylate (DIAD) can be used to selectively form unsymmetrical disulfides, RSSR’ from two thiols, RSH and R’SH [28,29]. There are many reports of materials based on aryl disulfides that exhibit exceptional self-healing abilities [4,13]. Overall, aryl and primary disulfides, as well as a wide vari- ety of non-aliphatic disulfides, are favored in self-healing applications because they are sufficiently reactive to undergo facile oxidation or thiol-disulfide exchange reactions. The mild conditions needed for aryl disulfide S-S bond scission generally tolerate most of the common polymer backbone functional groups. Thus, aryl disulfides are well suited for incorporation into multi stimulus-responsive materials, which often contain other func- tional groups. Many other materials incorporating aryl disulfides are more sensitive to applied stimuli than are aliphatic disulfides [13]. For that reason, employing aryl disulfides facilitates finding conditions appropriate for provoking material response. A potential problem of incorporating aryl disulfide groups into materials is the possibility of decom- position via hydrolysis at higher pH values [24]. As pointed out by Leichner [2], thiol- or disulfide-containing polymers behave optimally when two competing factors—strong re- sponses to applied stimuli in the target environment, and stability before entering the target environment—are balanced. If thiomers or disulfides are too reactive, they may prema- turely react with non-target stimuli, or decompose before reaching target sites in the body.
2.5. Radical Reactions and Radical Coupling of Thiols and Disulfides Disulfide-disulfide metathesis (11 in Figure1) is the primary reaction responsible for the self-healing processes of many disulfide-based polymers and elastomers [4]. Although Molecules 2021, 26, 3332 8 of 44
many such polymers have been reported to be self-healing, the mechanistic process for it is not well understood. Disulfide-disulfide metathesis is also an important side reaction in many disulfide- containing polymers [31]. When materials containing disulfide bonds are mechanically fractured, thiyl radicals are generated [4,32]. Self-healing of fractured disulfide-containing polymers requires thiyl radical chain ends to approach one another, after which radical recombination and self-healing become possible [4]. Self-healing is favored within flex- ible polymers with low Tg values, and recent research has considered various methods to incorporate disulfides into high Tg polymers to produce stronger materials that can adequately self-heal [13]. Disulfide-containing polymers that are slow to self-heal under ambient conditions can be exposed to ultraviolet (UV) light or heat to accelerate the rate and efficiency of self-healing.
3. Reaction Types and Applications Thiols and disulfides have been incorporated into a wide variety of polymer backbones to impart stimulus-responsiveness. In many cases, an applied stimulus prompts either a thiol-disulfide exchange reaction or a redox reaction. The review of Leichner contains examples of common polymer backbones and common thiol-containing pendant groups which can be appended to them to generate thiomers [2]. The wide array of polymer back- bones reported in the literature reflects why thiomers and disulfide-containing polymers are so ubiquitous in materials science: thiols and disulfides can be incorporated into many polymers with relative ease by a variety of techniques [1]. Additionally, the reactivity of the thiol and disulfide groups can be tailored easily by altering the connectivity (changing R groups). Thiols and disulfides are ubiquitous components of living systems, which have evolved sophisticated and specific reactivity pathways for in vivo stimulus-responsiveness. Thiomers and disulfide-containing polymers are ideal candidates for applications where biological stimuli are desired to trigger a response [2]. The following sections are divided to show key methods in which stimuli have been used to prompt thiol-disulfide exchange, disulfide-disulfide exchange, and/or redox reactions for specific applications.
3.1. Thiol-Disulfide Exchange-Based and Redox Reactions 3.1.1. Natural Keratin Protection and Regenerated Keratin Enhancement There is much interest in finding methods by which keratin waste products can be processed into high quality materials. Keratin is a ubiquitous, crosslinked biopolymer protein that forms a variety of materials, including animal horns, feathers, nails, and hair [33]. The animal butchery and textile industries produce large volumes of keratinous waste materials that usually are not repurposed due to a large decrease in the mechanical properties suffered during processing [34]. That is unfortunate because keratin is one of the strongest known natural materials. The currently employed conditions are harsh, in that they rupture disulfide bonds and destroy secondary structures [35,36]. A large part of the strength of keratin is a consequence of the large number of disulfide bonds between Cys groups of its amino acid residues [37]. In combination with strong hydrogen bonding, the disulfide bonds in keratin enable the formation of very strong and stable secondary structures, such as fibers. Mi et al. demonstrated that the secondary structures of waste keratinous materials can be restored after processing by crosslinking it with a dithiol, dithiothreitol (DTT), which functions as a “disulfide chain extender” [38]. In their work, the disulfide bonds of keratin were reduced, after which either a “disulfide capping agent” (Cys) or a “disulfide chain extender” (DTT) was added to regenerate disulfide bonds (Figure7). Both wet and dry thin films fabricated from the disulfide chain extended keratin exhibited superior mechanical properties, relative to the capped keratin films. MoleculesMolecules2021 2021, 26, 26, 3332, x FOR PEER REVIEW 9 of9 of45 44
FigureFigure 7. 7.Structures Structures of of disulfide disulfide extenders extenders (in(in red,red, DTT)DTT) and cappingcapping agents (in (in blue, blue, Cys) Cys) that that were were added added to to processed processed keratinkeratin to to restore restore disulfide disulfide bonds. bonds. Adapted Adapted withwith permissionpermission from [[38].38]. Copy Copyrightright (2020) (2020) American American Chemical Chemical Society. Society.
TheOther capping groups agents have adopted and disulfide different extending approa groupsches to wereinvestigate incorporated how to byprotect reacting kera- the freetin by thiol adding (Cys) a groupsthiomer of capping processed agent. keratin Thiol with groups individual are an ideal Cys target molecules site for or attachment DTT. Unlike theof groups capped that keratin, can protect the disulfide or restore extended hair, because keratin hair can contains form extended between networks 7–20% Cys because [37], onemost molecule of which of is DTT present can in react the withoxidized itself Cys and/or2 form the [39]. Cys Leichner groups inand keratin, coworkers forming [39] DTT-re- basedduced disulfide keratin from bridge human crosslinks hair, generating of variable a lengths. form with Mi etfree al. Cys hypothesize thiol groups that (keratin- the chain extended,SH). They crosslinkedfurther derivatized keratin networkkeratin-SH is dynamic,with oxidized and that2-mercaptonicotinic disulfide-disulfide acid metathesis (MNA), facilitatesgenerating crosslink a keratin-MNA reorganization derivative during (Figure stretching 8). (thus making the elasticity and tensile strength of the disulfide extended keratin film greater than the capped keratin film). The different bridge lengths are hypothesized to be the main reason for the dynamic nature of the disulfide extended network. Other groups have adopted different approaches to investigate how to protect keratin by adding a thiomer capping agent. Thiol groups are an ideal target site for attachment of groups that can protect or restore hair, because hair contains between 7–20% Cys [37], most of which is present in the oxidized Cys2 form [39]. Leichner and coworkers [39] reduced keratin from human hair, generating a form with free Cys thiol groups (keratin-SH). They further derivatized keratin-SH with oxidized 2-mercaptonicotinic acid (MNA), generating a keratin-MNA derivative (Figure8). Natural and bleached hair strands were soaked in an aqueous solution at pH 8 containing 1% of either keratin, keratin-SH, or keratin-MMA, to determine the binding ability of the keratins to hair. Keratin-MMA had the highest binding affinity to both natural and bleached hair fibers, relative to underivatized keratin and keratin-SH. The increase in the binding ability of keratin-MMA was especially pronounced for bleached hair strands, which were able to bind approximately three times more keratin-MMA than underivatized keratin, regardless of whether the strands were exposed to one or five treatments noted above. Fernandes [40], Roddick-Lanzilotta [41], and Barba [42] also investigated alternative
hair treatments designed to avoid directly breaking and then reforming disulfide bonds Figure 8. Reduction of keratin, to generate keratin-SH, and subsequent reaction with oxidized in keratin. MMA to generate keratin-MMA via a thiol-disulfide exchange reaction Reprinted with permission from [39]. Copyright (2019) Elsevier B. V.
Natural and bleached hair strands were soaked in an aqueous solution at pH 8 con- taining 1% of either keratin, keratin-SH, or keratin-MMA, to determine the binding ability of the keratins to hair. Keratin-MMA had the highest binding affinity to both natural and
Molecules 2021, 26, x FOR PEER REVIEW 9 of 45
Figure 7. Structures of disulfide extenders (in red, DTT) and capping agents (in blue, Cys) that were added to processed keratin to restore disulfide bonds. Adapted with permission from [38]. Copyright (2020) American Chemical Society.
Other groups have adopted different approaches to investigate how to protect kera- tin by adding a thiomer capping agent. Thiol groups are an ideal target site for attachment of groups that can protect or restore hair, because hair contains between 7–20% Cys [37], most of which is present in the oxidized Cys2 form [39]. Leichner and coworkers [39] re- Molecules 2021, 26, 3332 duced keratin from human hair, generating a form with free Cys thiol groups (keratin-10 of 44 SH). They further derivatized keratin-SH with oxidized 2-mercaptonicotinic acid (MNA), generating a keratin-MNA derivative (Figure 8).
Figure 8. Reduction of keratin, to generate keratin-SH, and subsequent reaction with oxidized Figure 8. Reduction of keratin, to generate keratin-SH, and subsequent reaction with oxidized MMA MMA to generate keratin-MMA via a thiol-disulfide exchange reaction Reprinted with permission to generate keratin-MMA via a thiol-disulfide exchange reaction Reprinted with permission from [39]. from [39]. Copyright (2019) Elsevier B. V. Copyright (2019) Elsevier B. V. Natural and bleached hair strands were soaked in an aqueous solution at pH 8 con- Fundamentally, both the protection and regeneration of keratinous materials relies taining 1% of either keratin, keratin-SH, or keratin-MMA, to determine the binding ability on an initial reduction followed by a binding step that establishes new disulfide bond crosslinksof the keratins between to hair. the CysKeratin-MMA groups of keratinhad the and highest thiol binding groups ofaffinity the added to both material. natural and
3.1.2. Mucoadhesion Thiomers and disulfide-containing polymers have garnered interest as potential mu- coadhesive drug delivery carriers. The use of thiomers and disulfide-containing polymers, also-called “S-protected” polymers, as mucoadhesives has been the subject of several re- views in the last decade [43–46], and drugs formulated with mucoadhesive thiomers have been successfully developed and marketed. In 2014, Lacrimera® eyedrops, formulated with thiolated chitosan, were introduced to European markets [47]. Mucosal membrane surfaces are coated with cysteine-rich mucin glycoproteins at a variety of key sites in the body, including the mouth and nose. Permeation enhancing materials can be added to in- crease drug permeation through membranes [48], and the size of the permeation enhancer determines whether it will be absorbed through the mucosa and taken into the bloodstream along with the drug—which is the case for small molecules—or whether it will permeate the mucosal layer and remain bound to it during, and after, drug absorption—which is the case with polymers. Macromolecular mucoadhesive materials are less invasive because they do not enter the blood stream. Mucoadhesion was achieved through a variety of non- covalent interactions, such as van der Waals forces, hydrogen bonding, and hydrophobic interactions [49]. Thiomers and disulfide-containing mucoadhesive polymers; however, covalently bind to mucosa through the formation of new disulfide bonds between the Cys and/or Cys2 of mucin and disulfides and/or thiols on the polymer. Thiol-disulfide exchange reactions between the polymer and mucosa prompt in situ gelation that binds the polymer to the membrane [50]. Like the systems described in Section 3.1.1, the key factor that allows thiomers to function in this application is binding of a thiomer to a protein-rich substrate via a disulfide bond formed in a thiol-disulfide exchange reaction (Figure9). The newly formed disulfide bonds can also participate in disulfide-disulfide metathesis reactions, affecting the quality of membrane adhesion through the formation of various inter- and intra-molecular disulfide crosslinks. Oxidation of thiols by GSH, which is present in mucosal membrane cells, is also important to the binding of thiomers to mucosa. The Molecules 2021, 26, 3332 11 of 44
Molecules 2021, 26, x FOR PEER REVIEW reactivity of thiomers and S-protected thiomers changes as a function of11 theof 45 penetration
depth into the mucosal membrane, as a response to decreasing pH [51].
Molecules 2021, 26, x FOR PEER REVIEW 11 of 45
FigureFigure 9. Cartoon 9. Cartoon showing showing key reactions key reactions and processes and proces occurringses occurring during mucoadhesion. during mucoadhesion. Reprinted withRe- permission from [2printed]. Copyright with (2019) permission Elsevier from B. V. [2]. Copyright (2019) Elsevier B. V.
Partenhauser andThiolation coworkers of polymer attached backbones thiol pendant derived groups from naturally to poly(dimethylsilox- occurring biopolymers, like ane)-co-(3-aminopropyl)methylsiloxanehyaluronic acid [52], chitosan by forming [53,54], alginateamide bonds [55], gellan between gum the [56, 57carboxylic], starch [58 ], glyco- acid group of a genthiol [59 “ligand”], cellulose precursors—either [60,61], arabinoxylan 3-mercaptopropionic [62], xanthan gum [63,64 ],acid and (MPA)cyclodextrins or [65–67] Cys—and the primaryhave been amine studied groups as mucoadhesive of the poly(siloxane) materials. The(Figure mucoadhesive 10; [72]). properties of thiolated syn- thetic polymers have been studied, also. Synthetic polymer backbones for this purpose include: poly(acrylicFigure 9. Cartoon acid) [showing68,69], poly(methacrylate) key reactions and [ 70proces], poly(acrylamide)ses occurring during [71], silicone mucoadhesion. oil [72,73 ],Re- poly(ethyleneprinted with permission glycol) (PEG) from [74 ,[2].75], Copyright poly(aspartamide) (2019) Elsevier [76], and B. poly(allylamine) V. [77]. Thiola- tion of both hydrophobic and hydrophilic polymers generally increases mucoadhesivity [2]. PartenhauserPartenhauser and and coworkers coworkers attached attached thiol pendant thiol pendant groups to groups poly(dimethylsiloxane)- to poly(dimethylsilox- ane)-co-(3-aminopropyl)methylsiloxaneco-(3-aminopropyl)methylsiloxane by forming by amideforming bonds amide between bonds the between carboxylic the acid carboxylic acidgroup group of a thiol of “ligand”a thiol precursors—either“ligand” precursors—either 3-mercaptopropionic 3-mercaptopropionic acid (MPA) or Cys—and acid (MPA) or Cys—andthe primary the amine primary groups amine of the groups poly(siloxane) of the (Figurepoly(siloxane) 10;[72]). (Figure 10; [72]).
Figure 10. Thiolated silicone oils bearing 3-mercaptopropionic acid (MPA) or cysteine (Cys) pen- dant groups synthesized by Partenhauser et al. [72].
Mucoadhesion between a silicone oil and excised porcine mucosa was measured for silicone oil-MMA and silicone oil-Cys, as well as a non-thiolated silicone oil control (Fig- ure 11). The maximum detachment force required to separate the mucosa from the silicone oil, as well as the total work of adhesion values demonstrate that the thiolated silicone oils bond more stronglyFigure to the 10.10.Thiolated mucosaThiolated silicone than silicone does oils oils bearing the bearing non-thiolated 3-mercaptopropionic 3-mercaptopr control.opionic acid (MPA) acid or(MPA) cysteine or (Cys)cysteine pendant (Cys) pen- dantgroups groups synthesized synthesized by Partenhauser by Partenhauser et al. [72]. et al. [72].
MucoadhesionMucoadhesion between between a siliconea silicone oil oil and and excised excised porcine porcine mucosa mucosa was was measured measured for siliconefor silicone oil-MMA oil-MMA and and silicone silicone oil-Cys, oil-Cys, as as well well as aa non-thiolatednon-thiolated silicone silicone oil oil control control (Fig- (ureFigure 11). 11 The). The maximum maximum detachment detachment force force required required to to separate separate the the mucosa mucosa from from the silicone silicone oil, as well as the total work of adhesion values demonstrate that the thiolated oil,silicone as well oils as bond the more total stronglywork of to adhesion the mucosa values than demonstrate does the non-thiolated that the thiolated control. silicone oils bond more strongly to the mucosa than does the non-thiolated control.
Figure 11. Maximum detachment force (MDF, gray bars) and total work of adhesion (TWA) were calculated as the area under the force versus displacement curves. Reprinted with permission [72]. Copyright (2015) Elsevier.
Figure 11. Maximum detachment force (MDF, gray bars) and total work of adhesion (TWA) were calculated as the area under the force versus displacement curves. Reprinted with permission [72]. Copyright (2015) Elsevier.
Molecules 2021, 26, x FOR PEER REVIEW 11 of 45
Figure 9. Cartoon showing key reactions and processes occurring during mucoadhesion. Re- printed with permission from [2]. Copyright (2019) Elsevier B. V.
Partenhauser and coworkers attached thiol pendant groups to poly(dimethylsilox- ane)-co-(3-aminopropyl)methylsiloxane by forming amide bonds between the carboxylic acid group of a thiol “ligand” precursors—either 3-mercaptopropionic acid (MPA) or Cys—and the primary amine groups of the poly(siloxane) (Figure 10; [72]).
Figure 10. Thiolated silicone oils bearing 3-mercaptopropionic acid (MPA) or cysteine (Cys) pen- dant groups synthesized by Partenhauser et al. [72].
Mucoadhesion between a silicone oil and excised porcine mucosa was measured for silicone oil-MMA and silicone oil-Cys, as well as a non-thiolated silicone oil control (Fig- ure 11). The maximum detachment force required to separate the mucosa from the silicone Molecules 2021, 26, 3332 oil, as well as the total work of adhesion values demonstrate that12 th ofe 44 thiolated silicone oils bond more strongly to the mucosa than does the non-thiolated control.
FigureFigure 11. 11.Maximum Maximum detachment detachment force (MDF, force gray (MDF, bars) and gray total workbars) of and adhesion total (TWA) work were of adhesion (TWA) were calculatedcalculated as the as areathe underarea theunder force the versus force displacement versus disp curves.lacement Reprinted curves. with permission Reprinted [72]. with permission [72]. Copyright (2015) Elsevier. Copyright (2015) Elsevier. The residence time of the silicone oils on porcine mucosa was measured after submerg- ing the samples in a phosphate buffer solution (pH 6.8). The non-thiolated silicone oil was completely washed away after about 2 h in the solution, whereas 40–60% of the thiolated silicone oil remained on the surface after 8 h for silicone oil-MPA and silicone oil-Cys.
3.1.3. Organic/Inorganic Hybrid Materials and Thiolated Organosilica Nanoparticles Thiol- and disulfide-containing organosilica materials have been used for other appli- cations, moreover mucoadhesion. For example, organosilica nanoparticles (ONPs) have been investigated as potential nanotherapeutics for anti-cancer drug delivery. ONP-based drug delivery systems tend to be physically stronger than other well-studied drug delivery system platforms based on liposomes or micelles [78]. The use of nanosized drug delivery systems for anticancer drug delivery offers advantages because cancer cells tend to accumu- late selectively nanosized materials and retain them to a greater extent than non-nanosized materials (possibly due to enhanced permeability and retention [79]). Additionally, the unique behavior of nanosized materials in magnetic fields, and in response to light, opens possibilities for manipulating nanomedicines in the body. Disulfide-containing materials are an attractive choice for improving controlled release of anticancer drugs in response to elevated levels of the reducing agent, glutathione (GSH; Figure2), in cancer cells. A drug delivery system should be sufficiently strong so that disulfide bonds linking the drug to the substrate do not break prematurely in cells that have normal cellular GSH levels. In an ideal system, disulfide bond scission will prompt anticancer drug release from the drug delivery system substrate only within the cancer cell. The concentration of GSH in cancer cells ranges between 2–10 mM, which is 100-1,000 times greater than in non-cancer cells [80]. Mekaru et al. used soft X-ray photoelectron spectroscopy (XPS) to study the biodegradability of two disulfide-containing ONPs formed from the hydrolysis of (3- mercaptopropyl)trimethoxysilane (MPMS) or (3-mercaptopropyl)methyldimethoxysilane (MPDMS) (Figure12;[81]). Molecules 2021, 26, x FOR PEER REVIEW 12 of 45
The residence time of the silicone oils on porcine mucosa was measured after submerg- ing the samples in a phosphate buffer solution (pH 6.8). The non-thiolated silicone oil was completely washed away after about 2 h in the solution, whereas 40–60% of the thiolated silicone oil remained on the surface after 8 h for silicone oil-MPA and silicone oil-Cys.
3.1.3. Organic/Inorganic Hybrid Materials and Thiolated Organosilica Nanoparticles Thiol- and disulfide-containing organosilica materials have been used for other ap- plications, moreover mucoadhesion. For example, organosilica nanoparticles (ONPs) have been investigated as potential nanotherapeutics for anti-cancer drug delivery. ONP- based drug delivery systems tend to be physically stronger than other well-studied drug delivery system platforms based on liposomes or micelles [78]. The use of nanosized drug delivery systems for anticancer drug delivery offers advantages because cancer cells tend to accumulate selectively nanosized materials and retain them to a greater extent than non-nanosized materials (possibly due to enhanced permeability and retention [79]). Ad- ditionally, the unique behavior of nanosized materials in magnetic fields, and in response to light, opens possibilities for manipulating nanomedicines in the body. Disulfide-con- taining materials are an attractive choice for improving controlled release of anticancer drugs in response to elevated levels of the reducing agent, glutathione (GSH; Figure 2), in cancer cells. A drug delivery system should be sufficiently strong so that disulfide bonds linking the drug to the substrate do not break prematurely in cells that have normal cel- lular GSH levels. In an ideal system, disulfide bond scission will prompt anticancer drug release from the drug delivery system substrate only within the cancer cell. The concen- tration of GSH in cancer cells ranges between 2–10 mM, which is 100-1,000 times greater than in non-cancer cells [80]. Mekaru et al. used soft X-ray photoelectron spectroscopy (XPS) to study the biodeg- radability of two disulfide-containing ONPs formed from the hydrolysis of (3-mercapto- Molecules 2021, 26, 3332 propyl)trimethoxysilane (MPMS) or (3-mercaptopropyl)methyldimethoxysilane13 of 44 (MPDMS) (Figure 12; [81]).
Figure 12. 12.Structures Structures of organosilica of organosilica nanoparticles nanoparticles (ONPs) derived from(ONPs) (3-mercaptopropyl)trimethoxysilane derived from (3-mercaptopropyl)tri- (MPMS)methoxysilane and (3-mercaptopropyl)methyldimethoxysilane (MPMS) and (3-mercaptopropyl)methyldimethoxysilane (MPDMS) precursors. Reprinted (MPDMS) with precursors. permissionReprinted from with [81 permission]. Copyright (2018)from American[81]. Copyright Chemical (2018) Society. American Chemical Society.
The sol-gel synthesis of MPMS ONPs via base hydrolysis resulted in ONPs with a greaterThe degree sol-gel of siloxane synthesis crosslinking of MPMS than theONPs MPDMS via ONPs,base hydrolysis because MPMS resulted can form in ONPs with a upgreater to three degree siloxane of bondssiloxane per monomer,crosslinking whereas than MPDMS the MPDMS can only ONPs, form one because siloxane MPMS can form bondup to per three monomer. siloxane Consequently, bonds per the MPMSmonomer, ONPs whereas were expected MPDMS to be more can extensively only form one siloxane crosslinked,bond per moremonomer. compact, Consequently, and more rigid than the MPDMS, MPMS but ONPs to have were fewer expected disulfide to be more crosslinks due to greater conformational restriction in the network. To demonstrate that the ONPs would decompose through disulfide bond scission, Meraku et al. incubated the ONPs in 10 mM GSH and monitored their degradation over 7 days using Raman spectroscopy and soft X-ray photoelectron spectroscopy (XPS), both of which are able to dif- ferentiate between disulfide and thiol bonds. Field-emission scanning electron microscopy (FE-SEM) images were obtained periodically and compared with the Raman and XPS data. The Raman data suggested that the proportion of disulfide bonds before degradation was much higher in the MPDMS ONPs, and much lower in the MPMS ONPs. The FE-SEM images of the MPDMS and MPMS ONPs after 7 days of GSH incubation showed extensive MPDMS degradation, but little to no MPMS degradation, suggesting that the cleavage of disulfide bonds by GSH is responsible for the observed decomposition. The XPS data indicated that oxidized GSH (GSSG) was present on the surface of the MPDMS ONP after 7 days incubation in GSH. which This observation provides indirect evidence for reduction of disulfide bonds in MPDMS by GSH. Doura and coworkers [82] synthesized three types of organosilica ONPs using the same thiolated MPMS and MPDMS precursors as Mekaru et al. [81]. A sol-gel NP synthesis was used to form MPDMS and MPMS ONPs, as well as an MPDMS-MPMS copolymer ONPs with varying ratios of MPDMS to MPMS. MPDMS-MPMS ONPs offer an approach to tune biodegradation, because the ratio of disulfide bonds to free thiols should be roughly proportional to the ratio of MPDMS to MPMS used to fabricate the ONPs. Raman spec- troscopy, thermal gravimetric analysis, and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine the ratio of MPMS to MPDMS and the pro- portion of thiol and disulfide groups in the ONPs. Rhodamine-b dye was loaded into the ONPs and fluorescence microscopy was used to track the GSH-triggered degradation of the nanoparticle suspensions over time. Transmission electron microscopy (TEM) and Molecules 2021, 26, 3332 14 of 44
SEM were used to track the degradation of the ONPs after incubation in a GSH solution for 2 to 7 days. Although only minor changes were observed in the shapes and sizes of the ONPs after this period, the SEM and TEM images showed a clearer trend after exposure to 40 mM GSH solution for 2 months: although MPMS ONPs were degraded to a negligible extent, the MPDMS ONPs were degraded completely. The extent of degradation of the MPMS-MPDMS copolymer ONPs was dependent upon the amount of MPDMS incorporated; more MPDMS resulted in more extensive degradation. It is unclear why the MPDMS ONPs in this study did not decompose after 7 days in 10 mM GSH, given that the MPDMS ONPs synthesized by Mekaru and coworkers did decompose extensively under similar conditions. Both Mekaru and Doura used a sol-gel methodology using base hydrolysis to synthesize their MPDMS-ONPs. Studies relating various aspects of ONP synthetic methodologies to the overall disulfide content and the degradation rate would be useful in the future.
3.1.4. Redox-Reversible Gelation In a technical sense, gelation through disulfide bond formation is responsible for the mucoadhesion of the materials described in Section 3.1.2. However, mucoadhesive systems are generally governed by thiol-disulfide exchange (1 in Figure1) and redox-based processes (2 through 10 in Figure1) that result in the formation of new disulfide bonds between the mucoadhesive polymer and the Cys-rich mucin glycoproteins (as in Figure9 ). Upon binding to a mucosal membrane, thiol- and disulfide-containing mucoadhesive materials undergo chemical reactions and gelation processes which are mechanistically different, and much more complicated than some of the redox-based examples that will be described below. Molecules 2021, 26, x FOR PEER REVIEW Reversible redox-triggered sol-to-gel transitions typically begin with the oxidation14 of of45 a thiomer in solution to form disulfide linkages that gel the solution in situ. The thiomer is reacting with itself (either intermolecularly, or intramolecularly), as opposed to a substrate, as in the mucoadhesion examples described in Section 3.1.2. Often, the gelled networks can networksbe reconverted can be to reconverted their sol phases to their by adding sol phases a reducing by adding agent a reducing to break the agent crosslinks. to break This the crosslinks.type of reaction This type can alsoof reaction be used can to formalso be more used complicated to form more networks complicated from networks poly-thiolated from poly-thiolatedmolecules, oligomers, molecules, and/or oligomers, polymers, and/or as shown polymers, in Table as shown1. in Table 1. Kamada and coworkerscoworkers usedused core-first core-first atom atom transfer transfer radical radical polymerization polymerization (ATRP) (ATRP) to tosynthesize synthesize star star polymers polymers whose whose arms arms contain contain disulfide disulfide crosslinks crosslinks (SS crosslinked(SS crosslinked star) star) [83]. [83].An initial An initial ATRP ATRP generated generated a star a polymer star polymer macroinitiator, macroinitiator, which which consisted consisted of a crosslinked of a cross- linkedpoly(ethylene poly(ethylene glycol diacrylate)glycol diacrylate) (polyEGDA) (polyEGDA) core surrounded core surrounded by linear by poly( linearn-butyl poly(n acry--bu- tyllate) acrylate) (polyBA) (polyBA) arms (polyEGDA-polyBA). arms (polyEGDA-polyBA). A second A chain-extensionsecond chain-extension ATRP was ATRP used was to usedincorporate to incorporate bis(2-methacryloyl-oxyethyl) bis(2-methacryloyl-oxyet disulfidehyl) disulfide (DSDMA) (DSDMA) on the ends on ofthe the ends star of arms, the starforming arms, the forming SS crosslinked the SS crosslinked star (Figure star 13). (Figure The disulfide 13). The bonds disulfide of DSDMA bonds wereof DSDMA scram- werebled uponscrambled their incorporationupon their incorporation into the SS crosslinked into the SS star crosslinked product. Resultsstar product. from dynamic Results frommechanical dynamic analysis mechanical (DMA) analysis indicate (DMA) that this indicate process that resulted this process in inter- resulted and intra-molecular in inter- and intra-moleculardisulfide crosslinks disulfide between crosslinks arms. between arms.
Figure 13.13. SyntheticSynthetic methodologymethodology to to incorporate incorporate DSDMA DSDMA on on polyEGDA-polyBA polyEGDA-polyBA stars stars and and their their redox redox behavior. behavior. ATRP ATRP was usedwas used to synthesize to synthesize polyEGDA-polyBA, polyEGDA-polyB generatingA, generating a core-crosslinked a core-crosslinked star polymer star polymer macroinitiator macroinitiator (synthesis (synthesis not shown). not Adaptedshown). Adapted with permission with permission from [83 ].from Copyright [83]. Co (2010)pyright American (2010) American Chemical Chemical Society. Society.
In the chain-extension ATRP step, the reaction solvent, dimethylformamide (DMF), was gelled as disulfide linkages formed. The redox responsiveness of the gels was studied in mixtures of tetrahydrofuran (THF) and chloroform (CHCl3). Addition of n-tributyl phosphine (n-Bu3P) cleaved the disulfide bonds to free thiols and converted the gel to a sol as the disulfide crosslinks were reduced at the peripheral ends of the star arms. Kamada et al. demonstrated the reversibility of this redox cycle by reconverting the sol to a gel upon the addition of an oxidizing agent (Table 1). Comparison of the relative sizes of the peaks associated with the S-S and C-SS bonds in the Raman spectra before and after each redox step, supported the mechanistic attribution of the sol-gel transitions to redox reactions. The temperature dependence of the storage (G’) and loss (G”) moduli for the different phases was assessed by DMA. The data suggest that the original and regen- erated SS crosslinked materials are structurally different. Both are different from the re- duced thiol form, which has a Tg close to that of polyBA. Yoon et al. studied the self-healing abilities of thin films fabricated from SS cross- linked polyEGDA-polyBA stars [[84]]. The mechanical properties and self-healing abilities were assessed for regenerated SS crosslinked films prepared by casting a dilute solution of reduced stars onto a silicon wafer. The kinetics of self-healing in the films was moni- tored after making cuts of various depths and widths using an AFM tip; the time-resolved tip-sample force vector was measured across the sample topology as the material healed at room temperature. Deep cuts that penetrated the film did not self-heal to the same ex- tent as shallower, non-penetrating cuts. The cuts were considered “healed” when the Young’s modulus of the cut region matched that of the virgin material. Figure 14 shows the proposed self-healing mechanism. Yoon et al. proposed that the self-healing limitations are due to the rigidity of the films: when cuts are deep and/or wide, there is insufficient flow around the fractured area to bridge the two sides of the cut. They also compared these results to the self-healing of polyEGDA-polyBA star polymers that were synthesized with a similar density of perma- nent crosslinks connecting the arms. The permanently crosslinked polyEGDA-polyBA
Molecules 2021, 26, 3332 15 of 44
In the chain-extension ATRP step, the reaction solvent, dimethylformamide (DMF), was gelled as disulfide linkages formed. The redox responsiveness of the gels was studied in mixtures of tetrahydrofuran (THF) and chloroform (CHCl3). Addition of n-tributyl phosphine (n-Bu3P) cleaved the disulfide bonds to free thiols and converted the gel to a sol as the disulfide crosslinks were reduced at the peripheral ends of the star arms. Kamada et al. demonstrated the reversibility of this redox cycle by reconverting the sol to a gel upon the addition of an oxidizing agent (Table1). Comparison of the relative sizes of the peaks associated with the S-S and C-SS bonds in the Raman spectra before and after each redox step, supported the mechanistic attribution of the sol-gel transitions to redox reactions. The temperature dependence of the storage (G’) and loss (G”) moduli for the different phases was assessed by DMA. The data suggest that the original and regenerated SS crosslinked materials are structurally different. Both are different from the reduced thiol form, which has a Tg close to that of polyBA. Yoon et al. studied the self-healing abilities of thin films fabricated from SS crosslinked polyEGDA-polyBA stars [84]. The mechanical properties and self-healing abilities were assessed for regenerated SS crosslinked films prepared by casting a dilute solution of reduced stars onto a silicon wafer. The kinetics of self-healing in the films was monitored after making cuts of various depths and widths using an AFM tip; the time-resolved tip-sample force vector was measured across the sample topology as the material healed at room temperature. Deep cuts that penetrated the film did not self-heal to the same extent as shallower, non-penetrating cuts. The cuts were considered “healed” when the Molecules 2021, 26, x FOR PEER REVIEW 16 of 45 Young’s modulus of the cut region matched that of the virgin material. Figure 14 shows the proposed self-healing mechanism.
Figure 14. Proposed self-healing mechanism for SS crosslinked polyEGDA-polyBA stars. Re- Figure 14. Proposed self-healing mechanism for SS crosslinked polyEGDA-polyBA stars. Reprinted printed with permission from [84]. Copyright (2012) American Chemical Society. with permission from [84]. Copyright (2012) American Chemical Society.
YoonNaga et and al. proposed coworkers that studied the self-healing the redox-reversible limitations are duegelation to the of rigidity networks of the formed from films:the crosslinking when cuts are of deep four and/or different wide, multi-thio there is insufficientl-containing flow around monomers the fractured with areavarious degrees toof bridge branching: the two trimethylolpropane sides of the cut. They alsotris (3-mercaptopropionate) compared these results to the (TMMP), self-healing tris[(3-mercapto- of polyEGDA-polyBA star polymers that were synthesized with a similar density of permanent propionyloxy-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis (3-mercaptopropio- crosslinks connecting the arms. The permanently crosslinked polyEGDA-polyBA control wasnate) not PEMP, able to healand appreciably, dipentaerythritol indicating hexakis that the SS(DPMP) crosslinked (Figure films rely15; on[25]). both Oxidation flow of the andthiol-containing chemical reactions monomers between by the dimethyl bridged sides sulfoxide to self-heal. (DMSO), Kinetic which studies, was such also as the solvent these,(Table proposing 1), resulted molecular-level in gelation. mechanisms can help assess the suitability of self-healing materials for various applications. Molecular-level self-healing processes often determine the healing efficiency because, if effective, they prevent deformations which are the source of macroscopic fracture formation [85].
Figure 15. Redox behavior and synthesis of extended networks from multi-thiolated branched monomers—trimethylolpropane tris (3-mercaptopropionate) (TMMP), tris[(3-mercaptopropio- nyloxy-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis (3-mercaptopropionate) (PEMP), and dipentaerythritol hexakis (DPMP)--to generate disulfide linkages. Adapted with permission from [25]. Copyright (2017) Wiley Periodicals, Inc.
Compression tests revealed a correlation between the degree of monomer branching (either three, four, or six), monomer concentration, and gel strength. Increasing the
Molecules 2021, 26, 3332 16 of 44
Table 1. Redox-Reversibility in Disulfide-Crosslinked Polymers.
Polymer Backbone Thiol or Gelated Solvents Oxidizing Agent(s) Reducing Agent(s) Number of Reference Disulfide Ligand Reversible Cycles 1 Poly(acrylate)-based core-crosslinked star copolymer DMF, FeCl3 and O2 or DSDMA n-Bu3P One full cycle [83,84] (polyEGDA-polyBA) CHCl3,THF I2 and O2 (Figure 13) Branched trithiols: TMMP and TEMPIC Branched tetrathiol: N/A, disulfides formed DMSO or PEMP during oxidation of DMSO Albright-Goldman DTT One full cycle [25] Branched hexathiol: branched monomers oxidation 2 DPMP (Figure 15) Poly tris(2-carboxyethyl) (2(dimethylamino) Aqueous phosphate 1,2,3 triazole-based Heating in air phosphine Five cycles [86] ethyl methacrylate) buffer hydrochloride (Figure 17b) BDS-functionalized or PEG functionalized chitosan disulfide-conjugated to n/a, formed Water DTT One direction [87] (Figure 19) thiolated methyl red as disulfides dye or GSH Poly(styrene-co-4-vinylbenzene [EMI][TFSA] chloride) and PEG 1,2,3-triazole derivative ionic liquid Heated in air DTT Six redox cycles [26,88] triblock copolymer with DCM Poly(benzyl ether)- PEG copolymer (ScIP) Pyridine disulfide DMF n/a DTT Non-reversible [89] (Figure 23) 1 One cycle—disulfide was reduced to the thiol; one full cycle—disulfide was reduced to the thiol and back; non-reversible—the material could not be reconverted. 2 Albright-Goldman oxidation conducted in DMSO with acetic anhydride.
The research of Kamada and Yoon on thiol- and disulfide-containing polyEGDA- polyBA star polymers highlights the range of applications possible for redox-reversible thiol-disulfide-containing materials. In addition, the SS crosslinked thin films were stable and self-healing [84]. However, as noted, the addition of reducing agents—such as n- Bu3P—to the SS crosslinked organogels is effective in converting them to sols [83]. Thus, thin films swollen in organic solvents or organogels could potentially be removed from surfaces in this way. This methodology would have benefits for manipulating well-defined architectures, compact structures, and site-specific functional group placements in fields as diverse as drug delivery [90,91], coatings [92], and lithography [93]. Naga and coworkers studied the redox-reversible gelation of networks formed from the crosslinking of four different multi-thiol-containing monomers with vari- ous degrees of branching: trimethylolpropane tris (3-mercaptopropionate) (TMMP), tris[(3-mercaptopropionyloxy-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis (3-mercaptopropionate) PEMP, and dipentaerythritol hexakis (DPMP) (Figure15;[ 25]). Oxidation of the thiol-containing monomers by dimethyl sulfoxide (DMSO), which was also the solvent (Table1), resulted in gelation. Compression tests revealed a correlation between the degree of monomer branch- ing (either three, four, or six), monomer concentration, and gel strength. Increasing the monomer weight percentage during synthesis, and/or decreasing the degree of monomer branching resulted in stronger organogels. The gel networks could be reconverted to sols containing the free monomers via reduction with DTT. Heating the sols at 85 ◦C for 8 h was sufficient to oxidize the monomers and regenerate the gels. The TMMP, PEMP, and DPMP monomers were also copolymerized with a dithiol-containing derivative of tetra ethylene glycol (EGMP-4; Figure 16). Copolymerization of the monomers with a linear dithiol produced networks with mesh sizes of about 0.5 nm. Molecules 2021, 26, x FOR PEER REVIEW 16 of 45
Figure 14. Proposed self-healing mechanism for SS crosslinked polyEGDA-polyBA stars. Re- printed with permission from [84]. Copyright (2012) American Chemical Society.
Naga and coworkers studied the redox-reversible gelation of networks formed from the crosslinking of four different multi-thiol-containing monomers with various degrees of branching: trimethylolpropane tris (3-mercaptopropionate) (TMMP), tris[(3-mercapto- propionyloxy-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis (3-mercaptopropio- Molecules 2021, 26, 3332 nate) PEMP, and dipentaerythritol hexakis (DPMP) (Figure 15; [25]). Oxidation 17of of the 44 thiol-containing monomers by dimethyl sulfoxide (DMSO), which was also the solvent (Table 1), resulted in gelation.
Molecules 2021, 26, x FOR PEER REVIEW 17 of 45
monomer weight percentage during synthesis, and/or decreasing the degree of monomer FigurebranchingFigure 15.15. Redox Redoxresulted behavior behavior in stronger and and synthesis synthesisorganogels. of ofextended extendedThe gel networks networks networks from could from multi-thiolated multi-thiolatedbe reconverted branched branchedto sols monomers—trimethylolpropane tris (3-mercaptopropionate) (TMMP), tris[(3-mercaptopropio- monomers—trimethylolpropanecontaining the free monomers tris via (3-mercaptopropionate) reduction with DTT. (TMMP), Heating tris[(3-mercaptopropionyloxy- the sols at 85 °C for 8 h nyloxy-ethyl]-isocyanurate (TEMPIC), pentaerythritol tetrakis (3-mercaptopropionate) (PEMP), ethyl]-isocyanuratewas sufficient to oxidize (TEMPIC), the pentaerythritolmonomers and tetrakis regenerate (3-mercaptopropionate) the gels. The TMMP, (PEMP), PEMP, and and dipen- and dipentaerythritol hexakis (DPMP)--to generate disulfide linkages. Adapted with permission DPMP monomers were also copolymerized with a dithiol-containing derivative of tetra taerythritolfrom [25]. Copyright hexakis (DPMP)–to (2017) Wiley generate Periodicals, disulfide Inc. linkages. Adapted with permission from [25]. Copyrightethylene glycol (2017) Wiley(EGMP-4; Periodicals, Figure Inc. 16). Copolymerization of the monomers with a linear dithiol produced networks with mesh sizes of about 0.5 nm. Compression tests revealed a correlation between the degree of monomer branching (either three, four, or six), monomer concentration, and gel strength. Increasing the
FigureFigure 16. 16.Formation Formation of of crosslinked crosslinked copolymerscopolymers ofof PEMPPEMP and EGMP-4.EGMP-4. Adapted Adapted with with permission permission from from [25]. [25 ].Copyright Copyright (2017)(2017) Wiley Wiley Periodicals, Periodicals, Inc. Inc.
MocnyMocny and and coworkers coworkers examined examined redox-reversible redox-reversible disulfide disulfide crosslinking crosslinking of thiol-con- of thiol- containingtaining polymer polymer brushes brushes [86] [86 that] that are are thin thin films films formed formed by by end-grafting end-grafting linear linear polymer polymer chains.chains. Unlike Unlike the the sol-gel sol-gel examples examples above, above, po polymerlymer brushes brushes are aregraft grafteded to a tosubstrate, a substrate, so sothe the polymer polymer “gels” “gels” in this in this case case are areswollen swollen thin thin films films attached attached to a substrate, to a substrate, and the and re- the reductionduction of of the the disulfide disulfide crosslinks crosslinks merely merely changes changes the the brush brush density, density, instead instead of forming of forming a asol. sol. The The strength strength of the of thethin thin films films can be can correlated be correlated with brush with brushdensity, density, which can which be mod- can be modifiedified by incorporating by incorporating irreversible-covalent irreversible-covalent crosslinks. crosslinks. Mocny Mocny et al. incorporated et al. incorporated thiol thiolpendant pendant groups groups on poly(2-(dimethylamino)ethyl on poly(2-(dimethylamino)ethyl methacrylate) methacrylate) copolymer copolymer brushes brushesusing 1,2,3-triazole linkages in a copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) reac- tion (Figure 17a). The polymer brush density was reversibly changed through the oxida- tive crosslinking of the thiols (Figure 17b).
(a) (b) Figure 17. (a) Cartoon illustrating the reversible formation of disulfide crosslinks between polymer brush chains. (b) The composition of the polymer brushes studied by Mocny et al. Reprinted with permission from [86]. Copyright (2020) Amer- ican Chemical Society.
The swelling and dissipative properties of the polymer brushes were measured using ellipsometry and a quartz crystal microbalance, respectively, over five cycles of reduction and oxidation. Both techniques suggested that the crosslinking was reversible for two full
Molecules 2021, 26, x FOR PEER REVIEW 17 of 45
monomer weight percentage during synthesis, and/or decreasing the degree of monomer branching resulted in stronger organogels. The gel networks could be reconverted to sols containing the free monomers via reduction with DTT. Heating the sols at 85 °C for 8 h was sufficient to oxidize the monomers and regenerate the gels. The TMMP, PEMP, and DPMP monomers were also copolymerized with a dithiol-containing derivative of tetra ethylene glycol (EGMP-4; Figure 16). Copolymerization of the monomers with a linear dithiol produced networks with mesh sizes of about 0.5 nm.
Figure 16. Formation of crosslinked copolymers of PEMP and EGMP-4. Adapted with permission from [25]. Copyright (2017) Wiley Periodicals, Inc.
Mocny and coworkers examined redox-reversible disulfide crosslinking of thiol-con- taining polymer brushes [86] that are thin films formed by end-grafting linear polymer chains. Unlike the sol-gel examples above, polymer brushes are grafted to a substrate, so the polymer “gels” in this case are swollen thin films attached to a substrate, and the re-
Molecules 2021, 26, 3332 duction of the disulfide crosslinks merely changes the brush density, instead of forming18 of 44 a sol. The strength of the thin films can be correlated with brush density, which can be mod- ified by incorporating irreversible-covalent crosslinks. Mocny et al. incorporated thiol pendant groups on poly(2-(dimethylamino)ethyl methacrylate) copolymer brushes using using1,2,3-triazole 1,2,3-triazole linkages linkages in a copper(I)-catalyzed in a copper(I)-catalyzed azide azide−alkyne−alkyne cycloaddition cycloaddition (CuAAC) (CuAAC) reac- reactiontion (Figure (Figure 17a). 17 Thea). polymer The polymer brush brush density density was reversibly was reversibly changed changed through through the oxida- the oxidativetive crosslinking crosslinking of the of thiols the thiols (Figure (Figure 17b). 17b).
(a) (b)
FigureFigure 17.17. ((aa)) CartoonCartoon illustratingillustrating thethe reversiblereversible formationformation ofof disulfidedisulfide crosslinkscrosslinks betweenbetween polymerpolymer brushbrush chains.chains. ((bb)) TheThe composition of the polymer brushes studied by Mocny et al. Reprinted with permission from [86]. Copyright (2020) Amer- composition of the polymer brushes studied by Mocny et al. Reprinted with permission from [86]. Copyright (2020) ican Chemical Society. American Chemical Society.
The swelling and dissipative properties ofof the polymer brushes were measured usingusing ellipsometry and a quartz crystal microbalance,microbalance, respectively, overover fivefive cyclescycles ofof reductionreduction and oxidation. Both Both techniques techniques suggested suggested that that the the crosslinking crosslinking was was reversible reversible for fortwo two full full redox cycles. Thereafter, a decrease in the crosslinking density of the oxidized form was noted. It was attributed to the irreversible formation of a small amount of non- disulfide oxidation products. This was the first study to assess the reversibility of disulfide pendant crosslinks in polymer brush films, and it demonstrates that the polymer brush densities, and consequently the swelling and dissipative properties, can be controlled in a reversible manner. Further work will be required to assess what types of polymer backbones, thiol pendant groups, and redox reagents can be combined to provide systems that are redox-reversible over many cycles. The thiol groups on the brushes studied by Mocny and coworkers were easily crosslinked by heating (60 ◦C). However, crosslinking a more oxidation-resistant thiol might be a better option if repeated redox cycles are desired. Wei and coworkers also used a CuAAC reaction to tether a thiol-containing pen- dant group to an ABA block copolymer via a 1,2,3-triazole linkage [26,88]. The CuAAC “click” reaction is very popular for tethering thiol- or disulfide-containing groups to poly- mer backbones because it is efficient, functional group tolerant, and high yielding [94]. Wei and coworkers studied the redox-reversible gelation of an ionic liquid, 1-ethyl-3- methylimidazolium bis-(trifluoromethyl) sulfonyl amide ionic liquid ([EMI][TFSA]), by ABA block copolymers comprised of a thiol-containing, polystyrene A block that was insoluble in ionic liquids, and a PEG B block that was soluble in them (Table1). Ion gels using ionic liquids as the solvent have been proposed for use in a variety of applications, including the fabrication of electrochemical devices. These gels have been fabricated into flexible and transparent thin films with desirable properties for electronics applications because of their high ionic conductivity, low volatility, and low flammabil- ity [95–97]. After six gel redox cycles, the material exhibited less than 10% change in mechanical properties [26]. Sols of the ionic liquids were oxidatively crosslinked by heat- ing, and disulfide-disulfide metathesis was proposed to be crucial to the reshaping and restructuring of the gels after reduction and re-oxidation. The same group demonstrated by mass spectrometry of small molecule analogs that disulfide-scrambling probably occurred during oxidation [88]. They suspect that residual copper salts from the CuAAC click reaction were present and catalyzed disulfide-disulfide scrambling in the molecules during Molecules 2021, 26, x FOR PEER REVIEW 18 of 45
redox cycles. Thereafter, a decrease in the crosslinking density of the oxidized form was noted. It was attributed to the irreversible formation of a small amount of non-disulfide oxidation products. This was the first study to assess the reversibility of disulfide pendant crosslinks in polymer brush films, and it demonstrates that the polymer brush densities, and consequently the swelling and dissipative properties, can be controlled in a reversible manner. Further work will be required to assess what types of polymer backbones, thiol pendant groups, and redox reagents can be combined to provide systems that are redox- reversible over many cycles. The thiol groups on the brushes studied by Mocny and coworkers were easily crosslinked by heating (60 °C). However, crosslinking a more oxi- dation-resistant thiol might be a better option if repeated redox cycles are desired. Wei and coworkers also used a CuAAC reaction to tether a thiol-containing pendant group to an ABA block copolymer via a 1,2,3-triazole linkage [26,88]. The CuAAC “click” reaction is very popular for tethering thiol- or disulfide-containing groups to polymer backbones because it is efficient, functional group tolerant, and high yielding [94]. Wei and coworkers studied the redox-reversible gelation of an ionic liquid, 1-ethyl-3-me- thylimidazolium bis-(trifluoromethyl) sulfonyl amide ionic liquid ([EMI][TFSA]), by ABA block copolymers comprised of a thiol-containing, polystyrene A block that was insoluble in ionic liquids, and a PEG B block that was soluble in them (Table 1). Ion gels using ionic liquids as the solvent have been proposed for use in a variety of applications, including the fabrication of electrochemical devices. These gels have been fabricated into flexible and transparent thin films with desirable properties for electronics applications because of their high ionic conductivity, low volatility, and low flammability [95–97]. After six gel redox cycles, the material exhibited less than 10% change in mechan- ical properties [26]. Sols of the ionic liquids were oxidatively crosslinked by heating, and disulfide-disulfide metathesis was proposed to be crucial to the reshaping and restructur- ing of the gels after reduction and re-oxidation. The same group demonstrated by mass Molecules 2021, 26, 3332 spectrometry of small molecule analogs that disulfide-scrambling probably occurred19 ofdur- 44 ing oxidation [88]. They suspect that residual copper salts from the CuAAC click reaction were present and catalyzed disulfide-disulfide scrambling in the molecules during oxida- oxidation.tion. Control Control studies studies using using thethe small small molecule molecule analogs analogs indicated indicated no disulfide-disulfidedisulfide-disulfide metathesismetathesis inin thethe absenceabsence ofof thethe salts.salts.
3.1.5.3.1.5. Redox-TriggeredRedox-Triggered DrugDrug Release Release and and Disulfide-Diselenide Disulfide-Diselenide Chemistry Chemistry ZhangZhang andand coworkerscoworkers conductedconducted redoxredox reactionsreactions andand redoxredox reactionsreactions withwith triblocktriblock copolymerscopolymers comprisedcomprised ofof aa hydrophobichydrophobic poly(poly(εε-caprolactone)-caprolactone) (PCL) (PCL) “B” “B” blockblock flankedflanked by by twotwo hydrophilic hydrophilic PEG PEG “A” “A” blocks blocks [80 [80].]. A A disulfide disulfide or or diselenide diselenide linkage linkage present present at theat the center cen- ofter the of Bthe blocks B blocks produced produced copolymers copolymers of the of form the form AB-S-S-BA AB-S-S-BA or AB-Se-Se-BA, or AB-Se-Se-BA, respectively respec- (Figuretively (Figure 18). 18).
FigureFigure 18.18. StructuresStructures ofof disulfide-disulfide- andand diselenide-linkeddiselenide-linked PCLPCL andand PEGPEG triblocktriblock copolymerscopolymers synthe-syn- sizedthesized by Zhangby Zhang et al. et [ 80al.]. [80].
TheThe amphiphilicamphiphilic triblocktriblock copolymerscopolymers formedformed micellesmicelles inin phosphate-bufferedphosphate-buffered salinesaline (PBS)(PBS) solutions, solutions, and and micelles micelles were were formed formed and and loaded loaded by by dialysis dialysis with with the the anticanceranticancer drug drug doxorubicindoxorubicin (DOX).(DOX). TheThe releaserelease ofof DOXDOX fromfrom thethe disulfidedisulfide andand diselenidediselenide micellesmicelles inin thethe bufferedbuffered solutionsolution waswas measuredmeasured withwith GSHGSH afterafter thethe reductivereductive cleavagecleavage ofof thethe disulfidedisulfide and diselenide bonds. The release of DOX in the buffered solution with 1% H2O2 was also measured; cancer cells are known to contain a 100-fold higher concentration of H2O2 than normal ones [98]. The non-drug loaded micelles were exposed to various concentrations
GSH and the micellar polydispersity index values were calculated from dynamic light scattering measurements. As the disulfide and diselenide bonds break in the hydrophobic core, the micelles dissociate or change size. Thus, the polydispersity increases can be correlated with the degree of S-S or Se-Se bond cleavage. When exposed to 1 mM GSH, the polydispersity of diselenide micelles changes more rapidly, and increases to a greater extent, when compared with the disulfide micelles. This result indicates that Se-Se bond scission in the diselenide micelles occurs more rapidly and to a greater extent than S-S bond scission in the disulfide micelles. Solutions of disulfide and diselenide micelles were incubated with GSH at concentrations ranging from 20 µM to 2 mM. The two micelles exhibited comparable minor changes in polydispersity after being incubated for 6 h in 20 µM GSH. Conversely, after 6 h of exposure to 1 or 2 mM GSH, the diselenide micelle polydispersity increased much more than that of the disulfide micelles. Fluorescence spectroscopy was used to quantify the DOX release from the micelles as a function of time in a simulated blood environment with and without 10 mM GSH or 1% H2O2. The results demonstrated that the diselenide-linked micelles were more responsive to both redox conditions, releasing DOX more rapidly, and to a greater ex- tent, than the disulfide-linked micelles. Disulfide micelles exposed to 1% H2O2 in PBS at 37 ◦C for 32 h released the same percentage of DOX (40%) as the control sample (in PBS at 37 ◦C). However, the diselenide micelles released about 65% of the loaded DOX under the same oxidizing conditions, which was slightly greater than the 60% lost by the control sample in PBS (that is, without H2O2). After 32 h of exposure to 10 mM GSH in PBS at 37 ◦C, the DOX-loaded diselenide micelles had released about 90% of the initial amount of DOX, whereas the disulfide micelles released about 70%. However, the dif- ference between the mass of DOX released from disulfide and diselenide micelles upon exposure to GSH or H2O2 is even greater than the difference based on the percentages because more DOX can be loaded into the diselenide micelles. The DOX drug loading Molecules 2021, 26, 3332 20 of 44
content was greater for the diselenide micelles, possibly due to coordination between Se and the quinonyl ring of DOX [80]. In this regard, diselenide-linked micelles may be more effective for triggered anti-cancer drug release for types of cancer associated with relatively lower GSH concentrations, such as Hela, B16F10, and 4T1 cells [98]. The con- centration gradient between these types of cancer cells and surrounding non-cancerous cells is lower, relative to many other cancer cell types, which commonly have GSH con- centrations closer to 10 mM [99]. The diselenide-linked micelles are more responsive to GSH concentration changes in the low mM range, which suggests that they may be more effective for anti-cancer drug delivery for types of cancers associated with GSH concentra- tions in the low mM range. Lang et al. [100] also found that DOX-loaded diselenide-linked poly(ester)-co-poly(urethane) micelles were more sensitive to reduction, and had better antitumor activity than the analogous disulfide-linked micelles. Although disulfides are well suited for use in the body, due to the ubiquity of thiol and disulfide groups in nature, diselenides can undergo some of the same reactions as disulfides, and studies comparing the two may reveal possible niche applications for diselenide chemistry. Self-healing dise- lenide systems have been reported as well [6]. Recently, Perera et al. demonstrated that norbornene-terminated poly(ethylene glycol) and poly(2-hydroxypropyl methacrylate-stat- mercaptoethyl acrylate) polymeric hydrogels could participate in seleno-sulfide exchange reactions with 5,50-diselenide-bis(2-aminobenzoic acid) upon irradiation with UV light, resulting in reversible hydrogel softening and stiffening [101]. Unlike the redox-reversible systems in Section 3.1.4, which can undergo reversible sol-to-gel transitions in response to a redox stimulus, the hydrogel system described by Perera and coworkers can alter its stiffness under irradiation via seleno-sulfide exchange. Several additional examples of disulfide-based drug delivery systems will be consid- ered in Sections 3.1.6 and 3.1.7. However, the handful of examples covered here is a minute subset of the literature since disulfide-linked drugs were first approved for human use in the early 2000s [12]. As shown in Figure3, there is a wide variety of disulfide-based drug delivery systems for nucleic acid-based drug delivery [10]. For more information, please consult reviews by Dutta, Saitoa, Quinn, and Wang [10–12]; as well as several reviews on redox-responsive [9,102–105] or multi stimulus-responsive [15] drug delivery systems, in addition to general reviews on them [8,79,106].
3.1.6. Loading Small Molecule Cargo into Networks Thiol-disulfide exchange (1 in Figure1) and redox reactions (2 through 10 in Figure1) can be used to generate mixed disulfides, R’SSR, where R’ is a polymer backbone and R is a small “cargo” molecule derived from a thiol-containing precursor. Thiol- and disulfide- containing polymers loaded with ‘cargo’ have been explored as drug delivery systems, sensors, and as analytical tools. The chemical reactions that govern a small molecule’s reversible conjugation to—and subsequent release from—an extended network are the same as those mentioned in preceding sections. In comparison to redox-reversible gel systems, cargo binding systems can bind cargo to a site in the network via a disulfide linkage. In the following examples, disulfide bonds are used to regulate the binding of small molecules to a network, as opposed to regulating the crosslinking between and within an extended network. Arslan and coworkers synthesized a disulfide-containing poly(ethylene glycol) (PEG) functionalized chitosan (Figure 19;[105]). Amine groups were reacted in an aza-Michael addition with benzothiazole disulfide acrylate (BDSA), in the presence of poly(ethylene glycol) diacrylate (PEGDA), to furnish benzothiazole disulfide (BDS)-containing pendant groups and form permanent crosslinks between the PEGDA and the chitosan chains. Most thiolated small molecules readily exchange with BDS groups. In the hydrogel states of the PEG functionalized chitosan, thiol-disulfide exchange reactions were conducted between the BDSA and either thiolated methyl red dye or GSH. The thiol-disulfide exchange reactions were used to incorporate the methyl red dye or GSH into the hydrogel network via disulfide bonds. MoleculesMolecules 20212021, 26,, 26x ,FOR 3332 PEER REVIEW 21 of 44 21 of 45
FigureFigure 19. 19.Conjugation Conjugation of BDSA of BDSA to PEG to functionalizedPEG functionalized chitosan viachitosan an aza-Michael via an aza-Michael addition. A addition. A hydrogelhydrogel was was formed formed from from the BDS-containing the BDS-containing PEG functionalized PEG functionalized chitosan, and chitosan, a thiol-disulfide and a thiol-disulfide exchangeexchange reaction reaction tethered tethered the thiolated the thiolated cargo molecules cargo molecules (red dot) to (red it. Adapted dot) to with it. Adapted permission with permission fromfrom [87 [87].]. Copyright Copyright (2020) (2020) Elsevier Elsevier B.V. B.V.
As will be discussed in Section 3.1.7, disulfide bonds can crosslink polymer networks and bindWierzba small cargoand moleculescoworkers to thedesigned networks. and The synthesized resulting materials a vitamin are capable B12 (B of12) derivative for simultaneouslydirect conjugation being to degraded small molecules and releasing or cargopolymer in response networks to a trigger,via a disulfide such as a linkage (Figure reducing20; [107]). agent. B12 has been touted as a potential transport agent for drug delivery and in vivo imagingThe cargo agents molecules because released of its from high networks affinity can toward also be used transport as sensors. protei For example,ns, and its propensity the amount of released cargo can be related to the amount of analyte that has reacted in an to be accumulated in dividing cells [108–111]. B12 has a unique dietary uptake pathway, exchange reaction. Although this subject is outside the scope of this review, it is sufficiently relatedand the to incorporation warrant some mention. of B12 groups Thus, Tomei into anda drug coworkers delivery recently system fabricated can improve filter the efficacy paper-basedof drug delivery electrochemical by expanding cells that the can metabolic detect as little pathways as 60 µM concentrationsto include B12 of uptake. GSH in blood.Although The strips other detect metabolically cysteamine (a thiol) stable as a productB12 derivatives of the thiol-disulfide have been exchange synthesized, they do reaction with GSH based on selective oxidation at a carbon black/Prussian blue electrode not always retain a high affinity for transport proteins. The domains of B12 circled in gray in the strips [18]. in Figure 20 have been explored as conjugation sites. Amide-based conjugates formed at Wierzba and coworkers designed and synthesized a vitamin B12 (B12) derivative forthe direct starred conjugation positions to smallstill demonstrate molecules or polymer a strong networks binding via affinity a disulfide for linkage trafficking proteins ([108].Figure Most20;[107 conjugates]). B12 has beenare formed touted as with a potential ester, amide, transport or agent carbamate for drug linkages delivery [107], which— andunlikein vivo disulfideimaging bonds—are agents because not of itscleavable high affinity in cells. toward transport proteins, and its propensity to be accumulated in dividing cells [108–111]. B12 has a unique dietary uptake The same authors also developed a B12 derivative based on disulfide conjugation at the pathway, and the incorporation of B12 groups into a drug delivery system can improve the 12 efficacy5′ position of drug of deliverythe ring. by A expanding pyridyl thedisulfide metabolic precursor pathways to(B include-5′-SSPy) B12 uptake. was found to be highly reactive toward a variety of thiols in thiol-disulfide exchange reactions (Figure 20). Like ben- zothiazole disulfides, pyridyl disulfides are highly reactive in thiol-disulfide exchange reac- tions. Thus, they are excellent precursors of unsymmetrical disulfides used in cargo tether- ing applications, including cyclic peptide conjugates [112]. The addition of glutathione (GSH) was shown to result in disulfide bond scission for the R groups examined. In theory, when both B12 and a drug are conjugated to a network via disulfide linkages, GSH-induced reduction can be used to trigger concomitant release of the drug and B12. The research of Wierzba et al. provides an alternative perspective for the release of cargo molecules. Research focusing on synthetic modifications of molecules prior to con- jugation would expand the range of cargo capable of being used in disulfide-based drug delivery systems. Despite the effects of enhanced permeability and retention, which are afforded to any nanosized anti-cancer drug delivery system, disulfide-based nano-drug delivery systems still suffer from inefficient delivery and cellular uptake [11]. However, the quality of drug delivery and release can be enhanced by the addition of various bio- logical targeting groups, such as folic acid, biotin, and galactose.
Molecules 2021, 26, 3332 22 of 44 Molecules 2021, 26, x FOR PEER REVIEW 22 of 45
Figure 20. Synthesis of the pyridyl disulfide derivative of B12 (B12-5′-SSPy). Thiols were reacted with B12-5′-SSPy to form a Figure 20. Synthesis of the pyridyl disulfide derivative of B (B -50-SSPy). Thiols were reacted with B -50-SSPy to form a variety of disulfide-linked conjugates, B12-SS-R. The structur12 e at12 bottom-left is a synthetic nucleic acid 12analog. Adapted varietywith permission of disulfide-linked from [107]. conjugates, CopyrightB12-SS-R. (2016) American The structure Chemical at Society. bottom-left is a synthetic nucleic acid analog. Adapted with permission from [107]. Copyright (2016) American Chemical Society. 3.1.7. Thiol-Disulfide Exchange and Redox Reactions That Initiate Degradation or Cas- cadeAlthough Reactions other metabolically stable B12 derivatives have been synthesized, they do not always retain a high affinity for transport proteins. The domains of B circled in gray Several applications have been proposed for thiomers or disulfide-containing12 poly- in Figure 20 have been explored as conjugation sites. Amide-based conjugates formed at mers that can be triggered to react in a cascade-like fashion, after an initial thiol-disulfide the starred positions still demonstrate a strong binding affinity for trafficking proteins [108]. exchange (1 in Figure 1) or redox reaction (2 through 10 in Figure 1) occurs. Examples Most conjugates are formed with ester, amide, or carbamate linkages [107], which—unlike include reactions that ultimately result in the formation of new bonds, or the destruction disulfide bonds—are not cleavable in cells. of bonds in self-immolative (i.e., degradation) processes. Disulfide- and thiol-containing The same authors also developed a B12 derivative based on disulfide conjugation at polymers0 offer mild, yet targeted opportunities to initiate and trigger0 the cascade. the 5 Self-immolativeposition of the polymers ring. A pyridylhave been disulfide proposed precursor for addressing (B12-5 several-SSPy) biomedical was found and to be highlyenvironmental reactive problems. toward a varietyThey include of thiols the in form thiol-disulfideulation or fabrication exchange of reactions biomedical (Figure ma- 20). Liketerials, benzothiazole drug delivery disulfides, systems, tissue pyridyl scaf disulfidesfoldings, biodegradable are highly reactive polymers, in thiol-disulfide soil treat- exchangements and reactions. microelectronics Thus, they [113–116]. are excellent Functional precursors groups of unsymmetricalcontaining cleavable disulfides bonds, used insuch cargo as esters, tethering amides, applications, polysulfides, including etc., have cyclic been peptide used for conjugates self-immolative [112]. polymer The addition for- of glutathionemulations [117]. (GSH) In was that shownregard, to the result inherent in disulfide biological bond redox scission responsiveness for the R groups of disulfide- examined. Incontaining theory, when polymeric both networks B12 and a makes drug arethem conjugated ideal for self-immolative to a networkvia polymers disulfide for linkages,envi- GSH-inducedronmental and reduction biomedical can degradation be used to applications. trigger concomitant release of the drug and B12. TheDisulfide-linked research of Wierzbanetworks etmay al. degrade provides by antwo alternative basic scenarios. perspective In the simpler for the case, release ofdisulfide cargo molecules. linkages functioning Research as focusing crosslink on or syntheticbranch sites, modifications can be cleaved of to molecules cause network prior to conjugationdecomposition would into expandconstituent the monomers, range of cargo oligomers, capable and/or of being sub-branches used in disulfide-based upon expo- drugsure to delivery a stimulus. systems. Such Despitea case was the described effects of by enhanced Naga and permeability coworkers [25] and (Section retention, 3.1.4). which areNone afforded of their to examples, any nanosized however, anti-cancer achieved drug redox-triggered delivery system, decomposition disulfide-based of linear nano-drug pol- deliveryymers into systems constituent still suffer monomers. from inefficient delivery and cellular uptake [11]. However, the qualityMore of drug mechanistically delivery and complex release degradation can be enhanced pathways by the for addition self-immolative of various polymers biological targetingoccur in the groups, second such scenario. as folic In acid, these biotin, systems, and disulfide galactose. bond scission leads to a cascade reaction that results in the subsequent cleavage of other, non-disulfide, linkages in the 3.1.7.polymer. Thiol-Disulfide Polymers linked Exchange to pendant and Redoxgroups Reactions via disulfide That bonds, Initiate as well Degradation as systems or with Cascadedisulfide Reactions linkages along the main polymer backbone, can exhibit true self-immolation. Generally,Several these applications systems have have well-defined been proposed architectures. for thiomers Commonly, or disulfide-containing the cleavage of poly-a mersdisulfide that canbond be results triggered in a tocascade react indecompos a cascade-likeition process fashion, that after affects an initial adjacent thiol-disulfide groups exchange (1 in Figure1) or redox reaction (2 through 10 in Figure1) occurs. Examples include reactions that ultimately result in the formation of new bonds, or the destruction
Molecules 2021, 26, 3332 23 of 44
of bonds in self-immolative (i.e., degradation) processes. Disulfide- and thiol-containing polymers offer mild, yet targeted opportunities to initiate and trigger the cascade. Self-immolative polymers have been proposed for addressing several biomedical and environmental problems. They include the formulation or fabrication of biomedical materi- als, drug delivery systems, tissue scaffoldings, biodegradable polymers, soil treatments and microelectronics [113–116]. Functional groups containing cleavable bonds, such as esters, amides, polysulfides, etc., have been used for self-immolative polymer formulations [117]. In that regard, the inherent biological redox responsiveness of disulfide-containing poly- meric networks makes them ideal for self-immolative polymers for environmental and biomedical degradation applications. Disulfide-linked networks may degrade by two basic scenarios. In the simpler case, disulfide linkages functioning as crosslink or branch sites, can be cleaved to cause network decomposition into constituent monomers, oligomers, and/or sub-branches upon exposure to a stimulus. Such a case was described by Naga and coworkers [25] (Section 3.1.4). None of their examples, however, achieved redox-triggered decomposition of linear polymers into constituent monomers. More mechanistically complex degradation pathways for self-immolative polymers occur in the second scenario. In these systems, disulfide bond scission leads to a cascade reaction that results in the subsequent cleavage of other, non-disulfide, linkages in the polymer. Polymers linked to pendant groups via disulfide bonds, as well as systems with disulfide linkages along the main polymer backbone, can exhibit true self-immolation. Generally, these systems have well-defined architectures. Commonly, the cleavage of a disulfide bond results in a cascade decomposition process that affects adjacent groups which have labile bonds, such as ester linkages. If disulfide bonds are used to crosslink a network and tether cargo to it, simultaneous drug delivery and decomposition (into monomers) can be achieved under appropriate conditions. The biodegradability, biocom- patibility, and environmental impact of the decomposition products are key factors to consider in this regard, as are the decomposition mechanisms which are consequential in in vivo applications.
Cascade Reactions Triggering Decomposition Bej and coworkers [118] used a polycondensation reaction between a hydroxyl- containing dithiol and pyridyl disulfide to create a poly(disulfide) with a free hydroxyl group on each monomer unit. Post-polymerization modification was used to incorpo- rate the anticancer drug camptothecin (CPT) via an ester linkage. Then, a thiol-disulfide exchange between thiol-terminated PEG oligomers and terminal pyridyl disulfides of the polydisulfide was used to generate an amphiphilic ABA triblock copolymer prodrug (polydisulfide-CPT). The polydisulfide-CPT contained two disulfide bonds per polydisul- fide monomer directly along the polymer backbone (Figure 21a). The polydisulfide-CPT formed polymersomes in aqueous solutions, and exposure to 10 mM GSH resulted in the re- lease of CPT and polymer backbone degradation. In the proposed mechanism (Figure 21b), initial GSH-triggered disulfide bond scission breaks the triblock copolymer into constituent PEG and polydisulfide blocks and generates a free thiol group in the PDS block. The free thiol then participates in an intrachain nucleophilic attack of the polydisulfide-CPT ester linkage, resulting in bond scission and CPT drug release. Comparative studies among the polydisulfide-CPT, free CPT, and an analogous CPT- free triblock copolymer were conducted to gauge the degree of cellular internalization, toxicity to HeLa cancer cells, and toxicity to non-cancerous cells. Based on the half-maximal inhibitory concentration (IC50) values (3.1 and 5.7 µg/mL, respectively), treatment of the cancer cells with polydisulfide-CPT was more effective than treatment with free CPT. Additionally, after 36 h exposure to 300 µg/mL (based on CPT content), polydisulfide-CPT killed <25% and free CPT killed > 60% of the non-cancer cells. Because the non-CPT containing polymer exhibited negligible toxicity toward both cancer and non-cancer cell lines, the undesired toxicity of polydisulfide-CPT in normal cells appears to be due to the Molecules 2021, 26, x FOR PEER REVIEW 23 of 45
which have labile bonds, such as ester linkages. If disulfide bonds are used to crosslink a network and tether cargo to it, simultaneous drug delivery and decomposition (into mon- omers) can be achieved under appropriate conditions. The biodegradability, biocompati- bility, and environmental impact of the decomposition products are key factors to con- sider in this regard, as are the decomposition mechanisms which are consequential in in vivo applications.
Cascade Reactions Triggering Decomposition Bej and coworkers [118] used a polycondensation reaction between a hydroxyl-con- taining dithiol and pyridyl disulfide to create a poly(disulfide) with a free hydroxyl group on each monomer unit. Post-polymerization modification was used to incorporate the an- ticancer drug camptothecin (CPT) via an ester linkage. Then, a thiol-disulfide exchange between thiol-terminated PEG oligomers and terminal pyridyl disulfides of the polydi- sulfide was used to generate an amphiphilic ABA triblock copolymer prodrug (polydisul- fide-CPT). The polydisulfide-CPT contained two disulfide bonds per polydisulfide mon- Molecules 2021, 26, 3332 omer directly along the polymer backbone (Figure 21a). The polydisulfide-CPT formed24 of 44 polymersomes in aqueous solutions, and exposure to 10 mM GSH resulted in the release of CPT and polymer backbone degradation. In the proposed mechanism (Figure 21b), in- itial GSH-triggered disulfide bond scission breaks the triblock copolymer into constituent presencePEG and ofpolydisulfide CPT, rather blocks than theand polymer generates backbone. a free thiol By group extension, in the PDS the greaterblock. The toxicity free of polydisulfide-CPTthiol then participates towards in an theintrachain cancer nucleophilic cells can be attack attributed of the to polydisulfide-CPT the in situ release ester of CPT, andlinkage, not the resulting polymer in bond backbone scission blocks and themselves.CPT drug release.
(a) (b) Figure 21. (a) GSH-induced cascade degradation of polydisulfide-CPT polymersomes. (b) Proposed mechanism for the degra- Figure 21. (a) GSH-induced cascade degradation of polydisulfide-CPT polymersomes. (b) Proposed mechanism for dation of polydisulfide-CPT by GSH. Adapted with permission from [118]. Copyright (2019) American Chemical Society. the degradation of polydisulfide-CPT by GSH. Adapted with permission from [118]. Copyright (2019) American Chemical Society. Comparative studies among the polydisulfide-CPT, free CPT, and an analogous CPT- free triblock copolymer were conducted to gauge the degree of cellular internalization, Yin and coworkers [19] designed and synthesized PEG and poly(methyl methacrylate) toxicity to HeLa cancer cells, and toxicity to non-cancerous cells. Based on the half-maxi- (PMA) diblock copolymers (BCPs) conjugated to CPT as dual-responsive micellar drug mal inhibitory concentration (IC50) values (3.1 and 5.7 μg/mL, respectively), treatment of delivery systems (GR-BCPs). The GR-BCPs were designed for the dual-responsive release the cancer cells with polydisulfide-CPT was more effective than treatment with free CPT. ofAdditionally, CPT in response after 36 to h elevated exposure concentrations to 300 μg/mL of(based reactive on CPT oxygen content), species polydisulfide- (ROS) and/or GSHCPT (i.e.,killed concentrations < 25% and free found CPT killed in cancer > 60% cells). of the The non-cancer CPT was cells. conjugated Because to the the non-CPT PMA block ofcontaining the BCPs polymer via a labile exhibited thioether negligible bond thattoxici wasty toward cleavable both in cancer the presence and non-cancer of excess cell ROS orlines, glutathione. the undesired The toxicity GR-BCPs of polydisulfide-CP incorporated disulfideT in normal linkages cells appears that were to be designed due to the to be cleavedpresence in of the CPT, presence rather of than high the concentrations polymer back ofbone. GSH, By as extension, well as thioketal the greater bonds toxicity that of were designedpolydisulfide-CPT to be cleaved towards in the the presence cancer cells of highcan be concentrations attributed to the of in ROS. situ Single-responsiverelease of CPT, BCPsand not were the also polymer prepared backbone with eitherblocks disulfidethemselves. bonds (G-BCPs) selective for elevated GSH concentrations or thioketal bonds (R-BCPs) for response to elevated ROS concentrations. Dual-responsive systems designed to respond to the presence of either GSH or ROS can enhance drug release because overexpression of ROS and GSH occur non-homogenously within cancer cells [119]. Although it is possible to design successful drug delivery systems for the precise targeting of specific cellular regions—such as mitochondria, which are known to overexpress ROSs—this approach requires the design and synthesis of extremely complex drug delivery systems [120]. The observation by Yin et al. that GR-BCP micelles can enter all parts of the cell suggests that CMT can be released in any area where either ROS or GSH is sufficiently overexpressed. All three of the BCPs shown in Figure 22 formed nano-sized micelles in PBS solution. The release of CPT from the three micelles was studied in vitro, using HeLa cancer cells, as well as in vivo, by treating mice with tumors with intravenous BCP solution injections. The dual-responsive GR-BCP exhibited the best performance in vivo and in vitro: the IC50 values were 6.3 µM for BR-BCP, 17.8 µM for G-BCP, and 28.9 µM for R-BCP. The increase in mouse tumor size was smallest in mice injected with GR-BCP. Molecules 2021, 26, x FOR PEER REVIEW 24 of 45
Yin and coworkers [19] designed and synthesized PEG and poly(methyl methacrylate) (PMA) diblock copolymers (BCPs) conjugated to CPT as dual-responsive micellar drug de- livery systems (GR-BCPs). The GR-BCPs were designed for the dual-responsive release of CPT in response to elevated concentrations of reactive oxygen species (ROS) and/or GSH (i.e., concentrations found in cancer cells). The CPT was conjugated to the PMA block of the BCPs via a labile thioether bond that was cleavable in the presence of excess ROS or gluta- thione. The GR-BCPs incorporated disulfide linkages that were designed to be cleaved in the presence of high concentrations of GSH, as well as thioketal bonds that were designed to be cleaved in the presence of high concentrations of ROS. Single-responsive BCPs were also prepared with either disulfide bonds (G-BCPs) selective for elevated GSH concentra- tions or thioketal bonds (R-BCPs) for response to elevated ROS concentrations. Dual-responsive systems designed to respond to the presence of either GSH or ROS can enhance drug release because overexpression of ROS and GSH occur non-homoge- nously within cancer cells [119]. Although it is possible to design successful drug delivery systems for the precise targeting of specific cellular regions—such as mitochondria, which are known to overexpress ROSs—this approach requires the design and synthesis of ex- tremely complex drug delivery systems [120]. The observation by Yin et al. that GR-BCP micelles can enter all parts of the cell suggests that CMT can be released in any area where either ROS or GSH is sufficiently overexpressed. All three of the BCPs shown in Figure 22 formed nano-sized micelles in PBS solution. The release of CPT from the three micelles was studied in vitro, using HeLa cancer cells, as well as in vivo, by treating mice with tumors with intravenous BCP solution injections. Molecules 2021, 26, 3332 The dual-responsive GR-BCP exhibited the best performance in vivo and in vitro: the IC2550 of 44 values were 6.3 μM for BR-BCP, 17.8 μM for G-BCP, and 28.9 μM for R-BCP. The increase in mouse tumor size was smallest in mice injected with GR-BCP.
FigureFigure 22. 22.(a) (a Self-assembly) Self-assembly of of GSH- GSH- and and ROH-dual-responsive ROH-dual-responsive BCPs (GR-BCPs) (GR-BCPs) comprised comprised of of a PEG a PEG block block and and CPT-PMA CPT-PMA conjugateconjugate block block (PGRCPT; (PGRCPT; top). top). Disassembly Disassembl ofy of GR-BCP GR-BCP with with subsequent subsequent CPT CPT drug drug release release in in mouse mouse tumor tumor tissue tissue (bottom). (bot- tom). (b) Reversible addition-fragmentation chain transfer (RAFT) copolymerization of PGRCPT and PEG blocks, and (b) Reversible addition-fragmentation chain transfer (RAFT) copolymerization of PGRCPT and PEG blocks, and structures structures of R groups for dual-responsive BCPs (GR-BCPs), and single-responsive BCPs that respond to GSH (G-BCP) or of R groups for dual-responsive BCPs (GR-BCPs), and single-responsive BCPs that respond to GSH (G-BCP) or ROS (R-BCP). (c) GR-BCP structure before and after subsequent disassembly and CPT release for GSH- and RSH-triggered pathways. Reprinted with permission from [19]. Copyright (2020) American Chemical Society.
Although drug release in response to stimuli associated with intracellular redox chemicals has been widely studied, other stimuli can function as secondary triggers in multi stimulus-responsive drug delivery systems. In fact, any other differences between the microenvironments of cancerous and non-cancerous cells (e.g., pH, concentrations of specific enzymes, and oxygen levels (due to hypoxia) [121]), can, in principle, be exploited for selective drug release. Xiao and coworkers [89] placed pyridyl disulfide-containing pendant groups on poly(benzyl ether) (PBE) backbones to form side chain-immolative polymers (ScIPs; Figure 23) which can be degraded upon exposure to reducing agents, such as DTT. These pendant groups were then reacted in a thiol-disulfide exchange reaction with either a mono- mercapto- terminated PEG (HS-PEG) or a bis-mercapto-terminated PEG (HS-PEG-HS), to produce a PEG-grafted ScIP (ScIP-g-PEG) or an extended network crosslinked by PEG groups via disulfide bonds (Figure 24). The self-immolative behavior was studied in the solution, organogel, and solid states by gel permeation chromatography, 1H NMR, electrospray ionization-mass spectrometry, and molecular modeling. The data indicated that exposure of the ScIPs (in the solid and solution states) to DTT and 1,8-diazobicyclo [5.4.0]undec-7-ene (DBU) resulted in unidirectional self-immolation, triggered by DTT-induced cleavage of the pyridyl disulfide bonds (Figure 25). Molecules 2021, 26, x FOR PEER REVIEW 25 of 45
ROS (R-BCP). (c) GR-BCP structure before and after subsequent disassembly and CPT release for GSH- and RSH-triggered pathways. Reprinted with permission from [19]. Copyright (2020) American Chemical Society.
Although drug release in response to stimuli associated with intracellular redox chemicals has been widely studied, other stimuli can function as secondary triggers in multi stimulus-responsive drug delivery systems. In fact, any other differences between the microenvironments of cancerous and non-cancerous cells (e.g., pH, concentrations of specific enzymes, and oxygen levels (due to hypoxia) [121]), can, in principle, be exploited for selective drug release. Xiao and coworkers [89] placed pyridyl disulfide-containing pendant groups on poly(benzyl ether) (PBE) backbones to form side chain-immolative polymers (ScIPs; Fig- ure 23) which can be degraded upon exposure to reducing agents, such as DTT. These pendant groups were then reacted in a thiol-disulfide exchange reaction with either a Molecules 2021, 26, 3332 mono-mercapto- terminated PEG (HS-PEG) or a bis-mercapto-terminated PEG26 (HS-PEG- of 44 HS), to produce a PEG-grafted ScIP (ScIP-g-PEG) or an extended network crosslinked by PEG groups via disulfide bonds (Figure 24).
FigureFigure 23. 23.Synthesis Synthesis of PBE-basedof PBE-based ScIPs ScIPs bearing bearing pyridyl pyridyl disulfide disulfide pendant pendant groups, groups, and their and reaction their reac- withtion mono-or with mono-or bis-mercapto-terminated bis-mercapto-terminate PEG tod formPEG PEG-graftedto form PEG-grafted ScIPs (ScIP- ScIPsg-PEG) (ScIP- org organogel-PEG) or or- Molecules 2021, 26, x FOR PEER REVIEW 26 of 45 networksganogel withnetworks PEG-disulfide with PEG-disulfide crosslinks. crosslinks. Adapted with Adapted permission with permission from [89]. from Copyright [89]. Copyright (2019) American(2019) American Chemical Chemical Society. Society.
The self-immolative behavior was studied in the solution, organogel, and solid states by gel permeation chromatography, 1H NMR, electrospray ionization-mass spectrometry, and molecular modeling. The data indicated that exposure of the ScIPs (in the solid and solution states) to DTT and 1,8-diazobicyclo [5.4.0]undec-7-ene (DBU) resulted in unidi- rectional self-immolation, triggered by DTT-induced cleavage of the pyridyl disulfide bonds (Figure 25).
FigureFigure 24. 24.PBE-based PBE-based ScIP ScIP structure, structure, and and reactions reactions with with mono- mono- and and bis-mercapto-terminated bis-mercapto-terminated PEG. PEG. TheThe graftgraft polymer polymer (ScIP-g-PEG), (ScIP-g-PEG), organogel, organogel, and and ScIP ScIP can can all all be be degraded degraded by DTT.by DTT. Reprinted Reprinted with with permissionpermission from from [89 [89].]. Copyright Copyright (2019) (2019) American American Chemical Chemical Society. Society.
Self-immolation in this system is proposed to begin with DTT-triggered pyridyl di- sulfide bond rupture, which generates a sulfhydryl anion capable of backbiting the car- bonate group and generating a phenolate ion. Phenolate ions are known to trigger self- immolation of PBEs when initiated from an end group. Since the PBE-based ScIPs decom- pose from their pendant groups, full decomposition via depolymerization occurs unidi- rectionally along one side of the ScIP, producing monomers. The other side of the ScIP remains intact, as an oligomer. Because the pyridyl disulfide pendants on the intact oligo- mer continue to react with DTT over time, so the ScIP can be depolymerized almost com- pletely into constituent monomers or small oligomers.
Figure 25. Proposed mechanism for the unidirectional self-immolative depolymerization of the ScIP after exposure to DTT and DBU. Data from electrospray ionization-mass spectrometry were able to identify the two molecules on the far right as the predominant products Reprinted with permission from [89]. Copyright (2019) American Chemical Society.
Li et al. [122] synthesized disulfide-linked hyperbranched polystyrene (PS) networks (HB-(S-S-PS)) with well-defined architectures. “Seesaw”-like macroinitiators were pre- pared wherein dual azide-terminated PS pendant groups branched from a middle region that contained a disulfide linkage and a terminal alkyne (≡−S-S-(PS-N3)2) (Figure 26). Hy- perbranched networks were formed by reacting the PS-chains in a CuAAC click reaction (Figure 26). The objective was to synthesize a well-defined hyperbranched polymer with disulfide linkages as a “model”. The disulfide-linked hyperbranched polymers reported in previous literature lacked the well-defined architectures that are better for biomedical applications [123–127]
Molecules 2021, 26, x FOR PEER REVIEW 26 of 45
Figure 24. PBE-based ScIP structure, and reactions with mono- and bis-mercapto-terminated PEG. The graft polymer (ScIP-g-PEG), organogel, and ScIP can all be degraded by DTT. Reprinted with permission from [89]. Copyright (2019) American Chemical Society.
Self-immolation in this system is proposed to begin with DTT-triggered pyridyl di- sulfide bond rupture, which generates a sulfhydryl anion capable of backbiting the car- bonate group and generating a phenolate ion. Phenolate ions are known to trigger self- immolation of PBEs when initiated from an end group. Since the PBE-based ScIPs decom- pose from their pendant groups, full decomposition via depolymerization occurs unidi- rectionally along one side of the ScIP, producing monomers. The other side of the ScIP Molecules 2021, 26, 3332 remains intact, as an oligomer. Because the pyridyl disulfide pendants on the intact oligo-27 of 44 mer continue to react with DTT over time, so the ScIP can be depolymerized almost com- pletely into constituent monomers or small oligomers.
FigureFigure 25. 25.Proposed Proposed mechanism mechanism forfor thethe unidirectional self-immolative depolymerization depolymerization of of the the ScIP ScIP after after exposure exposure to toDTT DTT andand DBU. DBU. Data Data from from electrospray electrospray ionization-mass ionization-mass spectrometry spectrometry were were able able to to identify identify the the two twomolecules molecules onon thethe farfar rightright as as the predominant products Reprinted with permission from [89]. Copyright (2019) American Chemical Society. the predominant products Reprinted with permission from [89]. Copyright (2019) American Chemical Society.
Self-immolationLi et al. [122] synthesized in this systemdisulfide-linked is proposed hyperbranched to begin with polystyrene DTT-triggered (PS) networks pyridyl disulfide(HB-(S-S-PS)) bond with rupture, well-defined which generatesarchitecture as. sulfhydryl “Seesaw”-like anion macroinitiators capable of backbiting were pre- the carbonatepared wherein group dual and azide-terminated generating a phenolate PS pendant ion. groups Phenolate branched ions from are knowna middle to region trigger self-immolationthat contained a disul of PBEsfide whenlinkage initiated and a terminal from an alkyne end group. (≡−S-S-(PS-N Since the3)2) (Figure PBE-based 26). Hy- ScIPs decomposeperbranched from networks their pendantwere formed groups, by reacti full decompositionng the PS-chains via in depolymerizationa CuAAC click reaction occurs unidirectionally(Figure 26). The objective along one was side to ofsynthesize the ScIP, a producing well-defined monomers. hyperbranched The other polymer side with of the ScIPdisulfide remains linkages intact, as asa “model”. an oligomer. The Becausedisulfide-linked the pyridyl hyperbranched disulfide pendants polymers on reported the intact oligomerin previous continue literature to reactlacked with the DTTwell-defined over time, architectures so the ScIP that can are be depolymerizedbetter for biomedical almost completelyapplications into [123–127] constituent monomers or small oligomers. Li et al. [122] synthesized disulfide-linked hyperbranched polystyrene (PS) networks (HB-(S-S-PS)) with well-defined architectures. “Seesaw”-like macroinitiators were pre- pared wherein dual azide-terminated PS pendant groups branched from a middle region that contained a disulfide linkage and a terminal alkyne (≡−S-S-(PS-N3)2) (Figure 26). Hyperbranched networks were formed by reacting the PS-chains in a CuAAC click reaction (Figure 26). The objective was to synthesize a well-defined hyperbranched polymer with disulfide linkages as a “model”. The disulfide-linked hyperbranched polymers reported Molecules 2021, 26, x FOR PEER REVIEW 27 of 45 in previous literature lacked the well-defined architectures that are better for biomedical applications [123–127]
Figure 26. Structure and reaction of the seesaw-like macroinitiator (≡−S-S-(PS-N3)2) used to form Figure 26. Structure and reaction of the seesaw-like macroinitiator (≡−S-S-(PS-N3)2) used to form HB- (S-S-PS),HB-(S-S-PS), and by-productsand by-products of the of DTT-induced the DTT-induced degradation degradation of HB-(S-S-PS). of HB-(S-S-PS). Adapted Adapted with permission with per- mission from [122]. Copyright (2014) American Chemical Society. from [122]. Copyright (2014) American Chemical Society. The seesaw macroinitiator was designed so that a CuAAC could be used to form the network, ensuring high specificity during branch formation. The DTT-induced cleavage of the disulfide bonds was monitored by DLS. Data from kinetic studies conducted in DMF solutions suggest that the degradation of HB-(S-S-PS) by DTT proceeds by both faster (in peripheral regions) and slower (in interior regions) reaction pathways. The inte- rior disulfides react more slowly due to greater steric hinderance, restricted chain mobil- ity, and lowered reagent accessibility. Both reactions exhibited first-order dependence of on DTT concentration [128–130], but both rate constants are several orders of magnitude lower than those reported for the reactions of DTT with small molecule thiols [131]. Zhang and coworkers [132] took a similar approach when synthesizing amphiphilic PEG- and palmitate-grafted linear poly(urethane)s (PUs), with disulfide linkages along the main chain (Figure 27). The amphiphilic polymers formed micelles in solution that were degradable upon being reduced.
Figure 27. PEG-grafted PU amphiphilic polymers made by Zhang et al. [132]. The main chains are cleavable upon reduction.
Molecules 2021, 26, x FOR PEER REVIEW 27 of 45
Molecules 2021, 26, 3332 Figure 26. Structure and reaction of the seesaw-like macroinitiator (≡−S-S-(PS-N3)2) used to form28 of 44 HB-(S-S-PS), and by-products of the DTT-induced degradation of HB-(S-S-PS). Adapted with per- mission from [122]. Copyright (2014) American Chemical Society.
TheThe seesaw seesaw macroinitiator macroinitiator was designed so that a CuAAC could be used to form the network,network, ensuring ensuring high high specificity specificity during branch formation. The DTT-induced cleavage ofof the the disulfide disulfide bonds was monitored by DLS. Data from kinetic studies conducted in DMFDMF solutions suggestsuggest that that the the degradation degradation of HB-(S-S-PS)of HB-(S-S-PS) by DTTby DTT proceeds proceeds by both by fasterboth faster(in peripheral (in peripheral regions) regions) and slowerand slower (in interior (in interior regions) regions) reaction reaction pathways. pathways. The The interior inte- riordisulfides disulfides react react more more slowly slowly due todue greater to grea stericter steric hinderance, hinderance, restricted restricted chain mobility,chain mobil- and ity,lowered and lowered reagent reagent accessibility. accessibility. Both reactions Both re exhibitedactions exhibited first-order first-order dependence dependence of on DTT of onconcentration DTT concentration [128–130 [128–130],], but both but rate both constants rate constants are several are several orders orders of magnitude of magnitude lower lowerthan those than reportedthose reported for the for reactions the reactions of DTT of with DTT small with moleculesmall molecule thiols [thiols131]. [131]. ZhangZhang and and coworkers coworkers [132] [132] took took a similar approach when synthesizing amphiphilic PEG-PEG- and palmitate-graftedpalmitate-grafted linearlinear poly(urethane)s poly(urethane)s (PUs), (PUs), with with disulfide disulfide linkages linkages along along the themain main chain chain (Figure (Figure 27). 27). The The amphiphilic amphiphilic polymers polymers formed formed micelles micelles in solution in solution that werethat weredegradable degradable upon upon being being reduced. reduced.
Molecules 2021, 26, x FOR PEER REVIEW 28 of 45 Figure 27. 27. PEG-graftedPEG-grafted PU PU amphiphilic amphiphilic polymers polymers made made by by Zhang Zhang et etal al.. [132]. [132 The]. The main main chains chains are are cleavable cleavable upon upon reduction. reduction.
Cascade Reactions Resulting in Polymerization or Material Rearrangements Cascade Reactions Resulting in Polymerization or Material Rearrangements Zhang and Waymouth [31] synthesized ABA triblock copolymers comprised of PEG and polycarbonateZhang and Waymouth (PC), the [31] latter synthesized of which ABA was triblock appended copolymers with 1,2-dithiolane comprised of pendant PEG groups.and polycarbonate Two different (PC), 1,2-dithiolane the latter of containingwhich was molecules,appended TMCDTwith 1,2-dithiolane and TMCLA, pendant derived groups. Two different 1,2-dithiolane containing molecules, TMCDT and TMCLA, derived from methyl asparagusic acid and lipoic acid, respectively, were synthesized (Figure 28). from methyl asparagusic acid and lipoic acid, respectively, were synthesized (Figure 28). Triblock copolymers incorporating either TMCDT, TMCLA, or both were synthesized by Triblock copolymers incorporating either TMCDT, TMCLA, or both were synthesized by the ring opening polymerization (ROP) of the cyclic carbonate groups. the ring opening polymerization (ROP) of the cyclic carbonate groups.
FigureFigure 28. 28.Synthesis Synthesis of TMCDT TMCDT and and TMCLA, TMCLA, and and the ring the ringopening opening polyme polymerizationrization to formto PEG-PC form PEG-PCABA triblock ABA copol- triblock ymers with 1,2-dithiolane pendant groups. The block copolymer product on the right is for the ring opening polymeriza- copolymers with 1,2-dithiolane pendant groups. The block copolymer product on the right is for the ring opening tion of TMCDT with PEG. Reprinted with permission from [31]. Copyright (2017) American Chemical Society. polymerization of TMCDT with PEG. Reprinted with permission from [31]. Copyright (2017) American Chemical Society. In aqueous solution, both 1,2-ditholane-functionalized block copolymers (DBCPs) In aqueous solution, both 1,2-ditholane-functionalized block copolymers (DBCPs) formed micelles above their critical micelle concentrations, and further aggregated at formedhigher concentrations. micelles above Eventually, their critical they micelle formed concentrations, physical gels at and ~10–15 further wt% aggregatedconcentra- at highertions. This concentrations. type of concentration Eventually, dependence they formed is well-known physical gels for at ABA ~10–15 triblock wt% concentrations.copolymers with hydrophobic end blocks (Figure 29a) [133–137].
(a)
(b)
Molecules 2021, 26, x FOR PEER REVIEW 28 of 45
Cascade Reactions Resulting in Polymerization or Material Rearrangements Zhang and Waymouth [31] synthesized ABA triblock copolymers comprised of PEG and polycarbonate (PC), the latter of which was appended with 1,2-dithiolane pendant groups. Two different 1,2-dithiolane containing molecules, TMCDT and TMCLA, derived from methyl asparagusic acid and lipoic acid, respectively, were synthesized (Figure 28). Triblock copolymers incorporating either TMCDT, TMCLA, or both were synthesized by the ring opening polymerization (ROP) of the cyclic carbonate groups.
Figure 28. Synthesis of TMCDT and TMCLA, and the ring opening polymerization to form PEG-PC ABA triblock copol- ymers with 1,2-dithiolane pendant groups. The block copolymer product on the right is for the ring opening polymeriza- tion of TMCDT with PEG. Reprinted with permission from [31]. Copyright (2017) American Chemical Society.
Molecules 2021, 26, 3332 In aqueous solution, both 1,2-ditholane-functionalized block copolymers (DBCPs)29 of 44 formed micelles above their critical micelle concentrations, and further aggregated at higher concentrations. Eventually, they formed physical gels at ~10–15 wt% concentra- tions. ThisThis type type of of concentration concentration dependence is is well-known well-known for for ABA ABA triblock triblock copolymers copolymers with with hydrophobichydrophobic end end blocks blocks (Figure (Figure 2929a)a) [[133–137].133–137].
(a)
(b)
Figure 29. (a) ABA triblock copolymers, like DBCP, with hydrophobic end blocks and their self- assembly into flower micelles above their critical micelle concentrations and gelation at still higher concentrations. (b) Formation of chemically crosslinked gels from DBCPs after addition of dithiol 3,6-dioxa-1,8-octadecanethiol. Adapted with permission from [31]. Copyright (2017) American Chemical Society.
To crosslink chemically the DBCP micelles and form a hydrogel, a dithiol, 3,6-dioxa- 1,8-octadecanethiol (ODT), was added to solutions of the DBCP at concentrations above the critical micelle concentration. Zhang and Waymouth found that hydrogelation occurred when even a monothiol, 2-mercaptoethanol (ME), was added. Furthermore, the observation that DBCP hydrogels formed with ODT were physically and rheologically similar to those formed with ME indicates that gelation was a result of a thiol-initiated ring opening polymerization (Figures 29b and 30a). Molecules 2021, 26, x FOR PEER REVIEW 29 of 45
Figure 29. (a) ABA triblock copolymers, like DBCP, with hydrophobic end blocks and their self- assembly into flower micelles above their critical micelle concentrations and gelation at still higher concentrations. (b) Formation of chemically crosslinked gels from DBCPs after addition of dithiol 3,6-dioxa-1,8-octadecanethiol. Adapted with permission from [31]. Copyright (2017) American Chemical Society.
To crosslink chemically the DBCP micelles and form a hydrogel, a dithiol, 3,6-dioxa- 1,8-octadecanethiol (ODT), was added to solutions of the DBCP at concentrations above the critical micelle concentration. Zhang and Waymouth found that hydrogelation oc- curred when even a monothiol, 2-mercaptoethanol (ME), was added. Furthermore, the observation that DBCP hydrogels formed with ODT were physically and rheologically similar to those formed with ME indicates that gelation was a result of a thiol-initiated ring opening polymerization (Figures 29b and 30a). Unlike the physically crosslinked gels, the chemically crosslinked TMCDT- and TMCLA-based DBCP hydrogels were rheologically dissimilar. The chemically crosslinked TMCDT-based DBCP hydrogels were structurally dynamic, flowed under applied stress, and exhibited self-healing properties while the TMCLA-based hydrogels were rigid, not moldable, not deformable, and not self-healing. Gels from the mixed TMCDT-TMCLA DBCPs exhibited intermediate rheological properties: higher TMCLA contents led to more brittle and more rigid gels. Gelation was reversible and tunable based on temperature, polymer concentration, and pH. Thermodynamic and kinetic parameters associated with the ring opening polymeri- zation of TMCLA and TMCDT were determined and used to construct the energy profile in Figure 30b. It suggests that the ring opening polymerization of TMCDT is less favored thermodynamically than that of TMCLA, but it proceeds with a lower energy barrier. The authors proposed that TMCLA hydrogels are more dynamic than the rigid TMCDT gels because TMCLA can crosslink (via ring opening polymerization) and un-crosslink (via depolymerization) much more rapidly than TMCDT. Molecules 2021, 26, 3332 Studies in non-aqueous solutions demonstrated the role of self-aggregation to the30 ofring 44 opening polymerization-induced crosslinking. Thus, the DBCPs were unable to crosslink in acetone; an initial self-assembly step is required to crosslink the DBCP micelle cores.
(a)
(b)
Figure 30. (a) Ring opening polymerization of 1,2-ditholane pendant group in the hydrophobic micelle cores upon exposure to a small molecule thiol. (b) Ring opening polymerization energy profiles (relative) for TMCLA (green) and TMCDT (blue) monomers. Reprinted with permission from [31]. Copyright (2014) American Chemical Society.
Unlike the physically crosslinked gels, the chemically crosslinked TMCDT- and TMCLA-based DBCP hydrogels were rheologically dissimilar. The chemically crosslinked TMCDT-based DBCP hydrogels were structurally dynamic, flowed under applied stress, and exhibited self-healing properties while the TMCLA-based hydrogels were rigid, not moldable, not deformable, and not self-healing. Gels from the mixed TMCDT-TMCLA DBCPs exhibited intermediate rheological properties: higher TMCLA contents led to more brittle and more rigid gels. Gelation was reversible and tunable based on temperature, polymer concentration, and pH. Thermodynamic and kinetic parameters associated with the ring opening polymer- ization of TMCLA and TMCDT were determined and used to construct the energy profile in Figure 30b. It suggests that the ring opening polymerization of TMCDT is less favored thermodynamically than that of TMCLA, but it proceeds with a lower energy barrier. The authors proposed that TMCLA hydrogels are more dynamic than the rigid TMCDT gels because TMCLA can crosslink (via ring opening polymerization) and un-crosslink (via depolymerization) much more rapidly than TMCDT. Studies in non-aqueous solutions demonstrated the role of self-aggregation to the ring opening polymerization-induced crosslinking. Thus, the DBCPs were unable to crosslink in acetone; an initial self-assembly step is required to crosslink the DBCP micelle cores. Komaromy et al. [138] investigated the role of self-assembly on the distribution of oxidation products formed by small, amphiphilic dithiol building blocks. They showed that controlling the self-assembly pathway can control covalent disulfide bond formation. From analyses by ultra high-performance chromatography-mass spectrometry (UPHC-MS), it was possible to detect the influence of several experimental variables (including stirring) on the ring sizes which ranged from trimers to 51-mers (Figure 31). Molecules 2021, 26, x FOR PEER REVIEW 30 of 45
Figure 30. (a) Ring opening polymerization of 1,2-ditholane pendant group in the hydrophobic micelle cores upon exposure to a small molecule thiol. (b) Ring opening polymerization energy profiles (relative) for TMCLA (green) and TMCDT (blue) monomers. Reprinted with permission from [31]. Copyright (2014) American Chemical Society.
Komaromy et al. [138] investigated the role of self-assembly on the distribution of oxidation products formed by small, amphiphilic dithiol building blocks. They showed that controlling the self-assembly pathway can control covalent disulfide bond formation. Molecules 2021, 26, 3332 From analyses by ultra high-performance chromatography-mass spectrometry (UPHC-31 of 44 MS), it was possible to detect the influence of several experimental variables (including stirring) on the ring sizes which ranged from trimers to 51-mers (Figure 31).
FigureFigure 31.31. Oxidation and and subsequent subsequent self-assembly self-assembly of small of small dithiol dithiol amphiphiles amphiphiles with and with without and without stirring. Reprinted with permission from [138]. Copyright (2017) American Chemical Society. stirring. Reprinted with permission from [138]. Copyright (2017) American Chemical Society.
TheThe role of the the self-assembly self-assembly process process on onoxidation oxidation product product specificity specificity was examined was examined using stirred and un-stirred reaction conditions with different co-solvents. Some condi- using stirred and un-stirred reaction conditions with different co-solvents. Some conditions tions generated one ring type with high specificity, whereas others generated a diverse generated one ring type with high specificity, whereas others generated a diverse array of array of different ring sizes. For example, after stirring for 7 days, the mass spectrum of differentthe reaction ring mixture sizes.For in a example, 9:1 aqueous after borate stirring buffer:DMF for 7 days, solution the mass showed spectrum thiol oxidation of the reaction mixtureproducts in that a 9:1 included aqueous a trimer, borate a buffer:DMF tetramer, and solution a distribution showed of thiol large oxidation macrocyclic products rings that included(LMCs; Figure a trimer, 32, atop). tetramer, When andunstirred, a distribution the reaction of large mixture macrocyclic produced rings hexamers (LMCs; exclu-Figure 32, top).sively When (Figure unstirred, 32, bottom)! the These reaction results mixture demonstrate produced that hexamersself-assembly exclusively can dictate (Figure the 32, bottom)!type and Thesenature resultsof disulfides demonstrate formed during that self-assembly the oxidation of can multi-thiolated dictate the type amphiphiles and nature of disulfidesin solution. formed The highly during specific the oxidationproduction of of multi-thiolated the hexamers in amphiphilesthe stirred solution in solution. is an The highlyexample specific of exclusive production and autocatalytic of the hexamers form ination the of stirred one dynamic solution combinatorial is an example library of exclusive andmember. autocatalytic Additionally, formation because of any one examples dynamic of combinatorial disulfide-containing library micellar member. drug Additionally, deliv- becauseery systems any have examples been reported, of disulfide-containing the role of self-assembly micellar on drug their delivery reactivity systems may be have im- been portant when considering long-term storage and drug delivery. Molecules 2021, 26, x FOR PEER REVIEWreported, the role of self-assembly on their reactivity may be important when31 considering of 45 long-term storage and drug delivery.
FigureFigure 32.32. Mass spectra spectra of of dithiol dithiol amphiphiles amphiphiles from from unstirred unstirred (top) (andtop )stirred and stirred (bottom) (bottom solutions) solutions after exposure to an oxidant for one week. Reprinted with permission from [138]. Copyright (2017) after exposure to an oxidant for one week. Reprinted with permission from [138]. Copyright (2017) American Chemical Society. American Chemical Society. 3.2. Disulfide-Disulfide Metathesis-Based Systems 3.2.1. Introduction to Self-Healing Materials and Current Challenges in the Field Many examples of disulfide-based self-healing polymeric systems, including gels and elastomers, have been reported [4–6]. Here, we review briefly research that is histor- ically important and is currently being conducted to address the biggest challenges facing the development of new disulfide-based self-healing polymers. Self-healing polymers were reported first in the 1970s [139–141]. In the early exam- ples, the self-healing was driven by chain interdiffusion, which occurs at temperatures above the Tg [6,142]. A recent review by Utrera-Barrios et al. [6] identified four generations of self-healing polymers, beginning with the development of extrinsic self-healing mate- rials in the early 2000s. ‘Extrinsic’ self-healing materials include encapsulated healing re- agents that are released upon fracture to heal the material. Additionally developed in the early 2000s, second generation ‘intrinsic’ self-healing materials do not require additional reagents, although external stimuli are sometimes added to speed self-healing. Some intrinsically self-healing systems incorporate dynamic covalent bonds, such as disulfide, diselenide, ditelluride, alkoxyamine, oxime-carbamate, urea, imine, and boron- based bonds [5,6]. Intrinsic self-healing prompted by reversible reactions, such as transester- ification or Diels-Alder reactions, has also been explored. Intrinsic self-healing can also be achieved by strong non-covalent interactions, including dipole-dipole, hydrogen bonding, host-guest, metal-ligand, ionic interactions, and even shape memory responsiveness. It is challenging to find intrinsically self-healable materials with high healing efficien- cies and high tensile strengths. Intrinsic self-healing requires that fractured surfaces be bridged before healing. However, high tensile strength materials, often associated with higher Tg values, do not have adequate flow for this key step to occur. If the ambient tem- perature is below the Tg, chain-end mobility will be too restricted for interchain diffusion and/or chain end recombination, and the efficiency of self-healing will be low [143]. Con- sequently, most examples of intrinsically self-healing materials are elastomers. The most recent 4th generation of self-healing materials is based on the combination of two or more intrinsically self-healing motifs (the 3rd generation is not pertinent to this review). This approach circumvents problems associated with balancing healing ability and strength [6]. Self-healing materials combining dual-non-covalent, dual-covalent, and a combina- tion of non-covalent and covalent healing motifs have been reported.
Molecules 2021, 26, 3332 32 of 44
3.2. Disulfide-Disulfide Metathesis-Based Systems 3.2.1. Introduction to Self-Healing Materials and Current Challenges in the Field Many examples of disulfide-based self-healing polymeric systems, including gels and elastomers, have been reported [4–6]. Here, we review briefly research that is historically important and is currently being conducted to address the biggest challenges facing the development of new disulfide-based self-healing polymers. Self-healing polymers were reported first in the 1970s [139–141]. In the early examples, the self-healing was driven by chain interdiffusion, which occurs at temperatures above the Tg [6,142]. A recent review by Utrera-Barrios et al. [6] identified four generations of self-healing polymers, beginning with the development of extrinsic self-healing materials in the early 2000s. ‘Extrinsic’ self-healing materials include encapsulated healing reagents that are released upon fracture to heal the material. Additionally developed in the early 2000s, second generation ‘intrinsic’ self-healing materials do not require additional reagents, although external stimuli are sometimes added to speed self-healing. Some intrinsically self-healing systems incorporate dynamic covalent bonds, such as disulfide, diselenide, ditelluride, alkoxyamine, oxime-carbamate, urea, imine, and boron- based bonds [5,6]. Intrinsic self-healing prompted by reversible reactions, such as transester- ification or Diels-Alder reactions, has also been explored. Intrinsic self-healing can also be achieved by strong non-covalent interactions, including dipole-dipole, hydrogen bonding, host-guest, metal-ligand, ionic interactions, and even shape memory responsiveness. It is challenging to find intrinsically self-healable materials with high healing efficien- cies and high tensile strengths. Intrinsic self-healing requires that fractured surfaces be bridged before healing. However, high tensile strength materials, often associated with higher Tg values, do not have adequate flow for this key step to occur. If the ambient tem- perature is below the Tg, chain-end mobility will be too restricted for interchain diffusion and/or chain end recombination, and the efficiency of self-healing will be low [143]. Con- sequently, most examples of intrinsically self-healing materials are elastomers. The most recent 4th generation of self-healing materials is based on the combination of two or more intrinsically self-healing motifs (the 3rd generation is not pertinent to this review). This approach circumvents problems associated with balancing healing ability and strength [6]. Self-healing materials combining dual-non-covalent, dual-covalent, and a combination of non-covalent and covalent healing motifs have been reported. Dual-responsive, fourth generation self-healing materials have been made by pairing disulfides with shape memory materials [14,32,144–147], as well as iminie [17,148,149] hydrogen [143,150–158], and metal-ligand bonds [159]. Self-healing in the presence of a stimulus, such as heat or UV radiation, is commonly reported but a greater emphasis has been placed on developing systems that can heal in ambient conditions [13]. If the Tg of the material is above room temperature, an added stimulus will be necessary for self-healing. This is part of the trade-off that is often required if strong materials are desired. Since disulfide-disulfide metathesis generally drives material repair, aryl disulfides, which are more reactive in metathesis reactions, have been popular in self-healing material design.
3.2.2. Quantification of Self-Healing Behavior Healing time and healing efficiency are commonly monitored via optical techniques. The size of a scratch or crack can be monitored over time, and the ratio of the initial crack to the crack width at a time can be used to calculate the self-healing efficiency. Ultimate tensile strength (UTS) can be measured before and after self-healing to quantify self-healing efficiency using the method of Wool and O’Connor, which is expressed in equation (1), where x is a tensile property such as strength, elongation at break, or robustness [160]. x self − healing efficency = healed × 100% (1) xvirgin Comparing the self-healing efficiencies found from different studies, or finding using different methods in one study, can be problematic because many variables can influence Molecules 2021, 26, 3332 33 of 44
the result. For example, the size of the initial cut and positioning of the cut pieces can influence self-healing time and efficiency measurements. Additionally, for tensile studies, changes in the thin film thickness and stretching rate can result in different tensile property values. Some studies also report the stress at which the material breaks when stretched. The UTS of a material is always less than or equal to the yield strength, which is the stress above which permanent deformation occurs. When considering the self-healing data in Table2, these factors should be kept in mind.
3.2.3. Disulfide-Based Self-Healing The tensile strengths and strains at the break points of the pristine materials and pa- rameters related to self-healing are included in Table2. Those data demonstrate the variety of different disulfides that have been incorporated into self-healing polymer networks. They include aryl [13,14,17] and aliphatic [32,84,143,161] disulfides. More exotic disulfides, such as bis(2,2,6,6-tetramethylpiperidin-1-yl)disulfides (BiTEMPS) [162,163], and thiuram disulfide [164] have been incorporated into polymeric networks as well. Lv and coworkers [17] prepared dual-self-healing polydimethylsiloxane (PDMS) elas- Molecules 2021, 26, x FOR PEER REVIEW 33 of 45 tomers containing two types of dynamic covalent bonds, aromatic disulfide and imine linkages (Figure 33).
FigureFigure 33. 33.Dual Dual imine- imine- and and disulfide-functionalized disulfide-functionalized PDMS PDMS elastomers elastomers synthesized synthesized from from aminopropyl ami- poly(dimethylnopropyl poly(dimethyl siloxane), 1,3,5-triformyl siloxane), 1,3,5-triformyl benzene (TFB), benzene and either (TFB), 4-aminophenyl and either 4-aminophenyl disulfide (APDS) disul- or 4,4fide0-aminophenyl (APDS) or 4,4 methane′-aminophenyl (DADPM). methane Reaction (DADPM). with APDS Reaction resulted with in aAPDS dual-functionalized resulted in a dual-func- network, tionalized network, whereas reaction with DADPM resulted in a mono-functionalized imine net- whereas reaction with DADPM resulted in a mono-functionalized imine network. Reprinted with work. Reprinted with permission from [17]. Copyright (2017) Wiley Periodicals, Inc. permission from [17]. Copyright (2017) Wiley Periodicals, Inc.
ImineImine bondsbonds werewere incorporatedincorporated intointo thethe PDMSPDMS networknetwork asas temporarytemporary crosslinks,crosslinks, whereaswhereas disulfidedisulfide bondsbonds werewere incorporatedincorporated asas “sacrificial“sacrificial bonds”bonds” thatthat couldcould breakbreak andand reorganizereorganize in in response response to to mechanical mechanical stress. stress. The The elastomers elastomers were were reprocessable reprocessable and and could could self-healself-heal completely completely within within 4 h.4 h. Tensile Tensile testing testing for for the dual-functionalizedthe dual-functionalized PDMS PDMS elastomers elasto- showedmers showed strain strain at break at break values values of up of to up 2000% to 2000% if stretched if stretched slowly. slowly. The The strain strain at break at break of theof the dual-functionalized dual-functionalized PDMS PDMS was was higher higher than than a controla control PDMS PDMS containing containing only only imine imine groups.groups. The The disulfide disulfide bonds bonds appear appear to to function function as as sacrificial sacrificia bondsl bonds during during stretching stretching and and be responsiblebe responsible for thefor highthe high elasticity. elasticity. Cyclic Cyclic tensile tensile tests ontests the on dual-functionalized the dual-functionalized elastomers elas- revealedtomers revealed a pronounced a pronounced hysteresis hysteresis between subsequentbetween subsequent stress loading stress and loading unloading and unload- cycles. However,ing cycles. when However, the material when the was material left undisturbed was left undisturbed for 2 days for at 2 room days temperature,at room tempera- the initialture, the stress initial loading/unloading stress loading/unloading curve was curve reestablished. was reestablished. The self-healing ability of dual-functionalized PDMS was better than that of imine single-functionalized PDMS: 100% self-healing was attained in 4h for the dual-function- alized PDMS and 55% in 24h for the imine single-functionalized material. The dual-func- tionalized PDMS was reprocessed using cold-compression modeling, and the decrease in tensile strength was negligible after three repeat reprocessing steps. The dual-functional- ized PDMS was degraded due to imine bond scission when the pH was lowered, or when an aldehyde or alkoxyamine was added. Although not examined, the effects of disulfide bond cleavage reagents would be interesting to examine, because this system is probably sensitive to stimulus-induced degradation and self-healing. Jian and coworkers [143] examined dual-functionalized polyurethane (PU) that con- tained disulfide bonds with a strong hydrogen bonding motif for advanced self-healing applications (Figure 34a). Although the self-healing associated with the disulfides oc- curred over longer timescales than that associated with hydrogen bonding, it was key to full self-healing, as shown by comparison of the tensile curves of dual-functionalized and hydrogen bond mono-functionalized PUs (Figure 34b). However, hydrogen bonding in- teractions accounted for almost 50% of the early self-healing.
Molecules 2021, 26, 3332 34 of 44
The self-healing ability of dual-functionalized PDMS was better than that of imine single- functionalized PDMS: 100% self-healing was attained in 4h for the dual-functionalized PDMS and 55% in 24h for the imine single-functionalized material. The dual-functionalized PDMS was reprocessed using cold-compression modeling, and the decrease in tensile strength was negligible after three repeat reprocessing steps. The dual-functionalized PDMS was degraded due to imine bond scission when the pH was lowered, or when an aldehyde or alkoxyamine was added. Although not examined, the effects of disulfide bond cleavage reagents would be interesting to examine, because this system is probably sensitive to stimulus-induced degradation and self-healing. Jian and coworkers [143] examined dual-functionalized polyurethane (PU) that con- tained disulfide bonds with a strong hydrogen bonding motif for advanced self-healing applications (Figure 34a). Although the self-healing associated with the disulfides occurred over longer timescales than that associated with hydrogen bonding, it was key to full self- Molecules 2021, 26, x FOR PEER REVIEWhealing, as shown by comparison of the tensile curves of dual-functionalized and hydrogen37 of 45 bond mono-functionalized PUs (Figure 34b). However, hydrogen bonding interactions accounted for almost 50% of the early self-healing.
(a) (b)
Figure 34. ((aa)) Structure Structure of of dual-functionalized dual-functionalized polyurethanes polyurethanes incorp incorporatingorating poly(tetramethylene poly(tetramethylene ether) ether) glycol glycol (PTMG). (PTMG). (b) (Tensileb) Tensile stressstress versus versus strain strain curves curves befo beforere and and after after self-healing self-healing for for dual-function dual-functionalizedalized PU PU and and a a control control material with only hydrogen bonding Adapted with permission from [143]. Copyright (2017) Wiley Periodicals, Inc. only hydrogen bonding Adapted with permission from [143]. Copyright (2017) Wiley Periodicals, Inc. The results of the groups of Lv [17] and Jian [143] suggest that disulfide bonds are more effective as self-healers than imine or hydrogen bonds. However, disulfide-based self-healing appears to occur over a longer time scale. Pairing disulfide bonds with the faster self-healing functionalities may improve overall self-healing and material longevity by lowering the risk of permanent deformation when rapid stress is applied. Kim and co-workers [13] examined how structure-property relationships influenced self-healing in thermoplastic polyurethanes (TPUs) containing aryl disulfide groups with variable molecular packing and rigidities. Thus, ‘hard’ para-substituted diaryl disulfide prepolymers were copolymerized with ‘soft’ poly(tetramethylene ether) glycol segments (PTMG) (Figure 35). Comparison of the results from the resultant copolymers suggests that the packing ability of the hard segment, which is determined by the R groups flanking the aryl disulfide, strongly influences self-healing. TPUs formed with an alicyclic isopho- rone diisocyanate-based hard segment (IP-SS) self-healed most efficiently (Table 2). The tensile strength of IP-SS was 6.8 MPa, which is nearly one order of magnitude higher than that reported for crosslinked PUs (0.8 MPa) [150]. This is one of the highest strengths re- ported among materials that can self-heal at room temperature. Kim and coworkers propose that the combination of the rigid diaryl disulfide and the alicyclic flanking groups provides sufficient chain mobility for efficient healing with- out weakening the material to an appreciable extent. By comparison, TPUs formed with symmetric alicyclic 4,4′-methylenebis(phenyl isocyanate) (M-SS) and linear aliphatic hex- amethylene diisocyanate hard segments (H-SS), exhibited little self-healing ability. The higher tensile strengths of M-SS and H-SS (Table 2) than that of IP-SS suggests that the rigidity of M-SS and the high packing ability of H-SS hinder self-healing. Although both M-SS and H-SS exhibited no self-healing in scratch tests, some recovery of tensile proper- ties was observed (especially for H-SS, see values in Table 2) when the materials were cut and subjected to pressure.
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Table 2. Self-Healing Polymers Incorporating Disulfide Linkages. Molecules 2021, 26, x FOR PEER REVIEW 34 of 45 TableTable 2. Self-Healing2. Self-HealingTable Polymers 2. PolymersSelf-Healing Incorporating Incorporating Polymers Disulfide IncorporatingDisulfide Linkages. Linkages. Disulfide Linkages. Secondary Self- External Self-Healing Time to Tensile Strength 3 3 Polymer Structure Secondary Self- 1Time to 2 3 Strain3 at Break 3Reference TableTable 2. 2. Self-Healing Self-HealingHealingSecondary PolymersSecondary Polymers Interaction Self- Incorporating Incorporating Self-External External Disulfide DisulfideSelf-Healing Linkages. Linkages.Self-Healing Self-Heal TensileTime to Strength Tensile(MPa) Strength Tensile3 Strength PolymerPolymerPolymer Structure Structure Structure ExternalStimuli StimuliEfficencySelf-Healing Efficency 1 Time to Self-HealStrain at2 Break Strain Reference at Break 3 StrainReference at Break 3 Reference HealingHealing Interaction Interaction Stimuli Efficency 1 Self-Heal1 2 (MPa)2 (MPa) SecondarySecondaryHealing Self- Self- Interaction ExternalExternal StimuliSelf-HealingSelf-Healing Efficency TimeTime to to TensileTensileSelf-Heal Strength Strength 3 3 (MPa) PolymerPolymer Structure Structure StrainStrain at at Break Break 3 3 ReferenceReference Table 2. Self-HealingHealingHealing Polymers Interaction Interaction Incorporating StimuliStimuli Disulfide Efficency Linkages.Efficency 1 1 Self-Heal Self-Heal 2 2 (MPa)(MPa) Secondary Self- External Self-Healing Time to Tensile Strength 3 Polymer Structure Strain at Break 3 Reference Healing Interaction Stimuli Efficency 1 Self-Heal 2 (MPa) none none 100% 4 [84] none none 100% VariableVariable 4 n/a n/a n/a n/a [84] nonenone nonenone 100% 100% Variable 4 Variablen/a4 n/an/a n/a[84] [84] nonenone nonenone 100%100% VariableVariable 4 4 n/an/a n/an/a [84][84]
none none 100% Variable 4 n/a n/a [84] polyEGDA-polyBApolyEGDA-polyBA star polymerspolymers
polyEGDA-polyBApolyEGDA-polyBApolyEGDA-polyBApolyEGDA-polyBA star star polymersstar star polymers polymers polyEGDA-polyBA star polymers 5 imineimine bonding bonding nonenone 5 95% 95% 4h 4h 0.14 0.14 2200% 2200% [16] [16] imineimineimine bonding bonding bonding nonenone 5 5 none 595%95% 4h4h 95% 0.140.14 4 h2200%2200% [16][16]0.14 2200% [16] imine bondingimine bonding none 5 95%none 5 4h 95% 0.14 4h 2200% 0.14 [16] 2200% [16] random PDMS-based co-polymer
randomrandomrandomrandom PDMS-based PDMS-basedPDMS-based PDMS-based co-polymer co-polymer co-polymerco-polymer random PDMS-based co-polymer H-bonding 60 °C 100% 6h 5.01 n/a [143] random PDMS-based co-polymer ◦ = PTMG H-bondingH-bondingH-bonding 6060 °C °C 60 °C60 C 100%100% 100% 6h6h 100% 6h 5.015.01 5.016 h n/an/a n/a[143][143]5.01 [143] n/a [143] H-bonding 60 °C 100% 6h 5.01 n/a [143]
= =PTMG= PTMG PTMG = PTMG H-bonding 60 °C 100% 6h 5.01 n/a [143] = PTMG none 25 °C 77–100% 2h 6.8 920% [12] ◦ nonenonenone nonenone 25 °C 25 25 °C °C 2577–100% °C25 77–100%C 77–100% 77–100% 2h 2h2h 77–100% 6.8 2h 6.86.8 920% 6.8 2 h920%920%[12] 920%[12][12]6.8 [12] 920% [12] IP-SS: co-polymerized with PTMG IP-SS:IP-SS:IP-SS: co-polymerized co-polymerizedco-polymerized co-polymerized withwith with PTMG withPTMG PTMG PTMG IP-SS: co-polymerized with PTMG none 25 °C 77–100% 2h 6.8 920% [12]
none 25 °C 70–89%◦ 2h 4.5 490% [12] IP-SS: co-polymerized with PTMG nonenone nonenone 25 °C 2525 °C °C70–89% 70–89% 70–89%25 2h C 2h2h 4.5 4.5 4.5 70–89% 490% 490% 490%[12] [12][12] 2 h 4.5 490% [12] 4,4’-methylenebis(phenyl4,4’-methylenebis(phenyl4,4’-methylenebis(phenyl isocyanate) isocyanate) isocyanate) based copolymerbased based copolymer copolymer of of of none 25 °C 70–89% 2h 4.5 490% [12] 4,4’-methylenebis(phenyl4,4’-methylenebis(phenylPTMG (HM-SS) isocyanate) isocyanate) based based copolymer copolymer of of PTMG (HM-SS) 4,4’-methylenebis(phenylPTMGPTMGPTMG isocyanate) (HM-SS) (HM-SS) (HM-SS) based copolymer of
none 25 °C ◦ 70–89% 2h 4.5 490% [12] PTMG (HM-SS) none none25 °C 0–4% 25 2hC 30.4 0–4% 940% [12] 2 h 30.4 940% [12] nonenone 252525 °C °C °C 0–4%0–4%0–4% 2h2h 2h 30.4 30.4 30.4 940% 940% 940% [12][12] [12] 4,4’-methylenebis(phenyl isocyanate) based copolymer of M-SS: co-polymerizedM-SS: co-polymerized with PTMGwith PTMG Molecules 2021, 26, x FOR PEERM-SS:M-SS: REVIEW co-polymerized with with PTMG PTMG none 25 °C 0–4% 2h 30.4 940% [12] 35 of 45 M-SS:M-SS: co-polymerized co-polymerizedPTMG (HM-SS) with with PTMG PTMG none none25 °C 4–30% 25 2h◦C 9.5 4–30% 750% [12] 2 h 9.5 750% [12] M-SS: co-polymerized with PTMG none 25 °C 4–30% 2h 9.5 750% [12] nonenone none25 25 °C °C 254–30% 4–30%°C 0–4%2h2h 9.52h 9.5 750% 750%30.4 [12][12] 940% [12] H-SS: co-polymerizedH-SS: co-polymerized with PTMG with PTMG
H-SS:H-SS: co-polymerized co-polymerized with with with PTMG PTMG PTMG none 25 °C 4–30% 2h 9.5 750% [12] M-SS: co-polymerized with PTMG none none 100% 7 3–30 min. n/a n/a [161] H-SS: co-polymerized with PTMG none none 100% 7 3–30 min. n/a n/a [161] none 25 °C 4–30% 2h 9.5 750% [12] co-polymerizedco-polymerized with with 2-ethylhexyl methacrylate methacrylate via disulfide via linkages disulfideH-SS: linkages co-polymerized with PTMG
100 °C shape memory 74–91% 6 10 min. 22.9–31.9 6 850–1160% 6 [13] (microwave)
co-polymerized with PTMG
180 °C 20% 3 h shape memory 0.015–0.12% 350% [32] RT, UV light 24% 3 h
disulfide-liquid crystal elastomer
table-top none 87–97% 1 min. 4.2 200% [163] lamp
thiuram disulfide crosslinked polytetra(ethylene glycol)
none 100 °C 86–93% 24 h 0.14 440% [162]
PU incorporating BiTEMPS
120 °C, 70 none 85–92% 24 h 7.6 280 [164] kPa
poly(hexyl methacrylate) incorporating BiTEMPS
Molecules 2021, 26, x FOR PEER REVIEW 35 of 45
Molecules 2021, 26, 3332 36 of 44 MoleculesMolecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 3535 ofof 4545 Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 35 of 45
Table 2. Cont. 7 none none 100% 3–30 min. n/a n/a [161] 77 3 co-polymerized with 2-ethylhexyl methacrylate via Secondarynonenone Self-nonenone 100%100% 77 3–303–30 min.min. n/an/a 1 n/a n/a [161] [161] 2 Tensile Strength 3 Polymer Structure none none 100%External Stimuli 3–303–30Self-Healing min.min. n/an/a Efficency n/a n/a Time toSelf-Heal[161] [161] Strain at Break Reference Healing Interaction (MPa) co-polymerizedco-polymerizedco-polymerizeddisulfide with withwith 2-ethylhexyl 2-ethylhexyl2-ethylhexyl linkages methacrylate methacrylatemethacrylate via viavia
disulfidedisulfide linkages linkages
100100◦ C°C shape memory 100100 °C°C 6 6 10 min. 6 6 6 6 [13] shapeshape memorymemoryshape memory 100 °C (microwave)74–91%74–91% 66 74–91%1010 min.min. 74–91% 22.9–31.922.9–31.910 min. 66 850–1160%850–1160%22.9–31.9 66 [13][13]22.9–31.9 850–1160% 850–1160%[13] shapeshape memorymemory (microwave) 74–91% 66 10 min. 22.9–31.9 66 850–1160% 66 [13][13] (microwave)(microwave)(microwave)(microwave)
co-polymerizedco-polymerized withwith PTMGPTMG co-polymerizedco-polymerizedco-polymerizedco-polymerized with withwith with PTMGPTMG PTMGPTMG
180180 °C°C 20%20% 33 hh 180 °C ◦20% 3 h shapeshape memorymemory 180 20%C 3 h 20% 0.0150.015––0.12%0.12% 350%3350% h [32][32] shapeshapeshape memorymemory memory RT,RT, UV UV lightlight 24% 3 h 0.015–0.12% 350% [32]0.015–0.12% 350% [32] RT, UV light RT,180 UV24% light24%°C 320%3 h h 24% 3 h 3 h shape memory 0.015–0.12% 350% [32] RT, UV light 24% 3 h
disulfide-liquid crystal elastomer disulfide-liquiddisulfide-liquiddisulfide-liquid crystal crystal crystal elastomer elastomer elastomer
disulfide-liquid crystal elastomer table-toptable-top nonenone table-toptable-top 87–97%87–97% 11 min.min. 4.24.2 200%200% [163][163] nonenone lamp table-top 87–97% lamp 1 min. 87–97%4.2 1200% min. [163][163] 4.2 200% [163] lamplamplamp
thiuramthiuram disulfidedisulfide crosslinkedcrosslinked polytetra(ethylenepolytetra(ethylene glycol)glycol) table-top thiuramthiuramthiuram disulfidedisulfide disulfide crosslinkedcrosslinked crosslinked polytetra(ethylenepolytetra(ethylene polytetra(ethylene glycol)glycol) glycol) none 87–97% 1 min. 4.2 200% [163] lamp
thiuram disulfide crosslinked polytetra(ethylene glycol) none 100 °C 86–93%◦ 24 h 0.14 440% [162] nonenonenone 100100 °C °C 10086–93%86–93%C 2424 h h 86–93% 0.140.14 24440%440% h [162][162][162] 0.14 440% [162]
PU incorporating BiTEMPS PUPU incorporating incorporating BiTEMPS BiTEMPS none 100 °C 86–93% 24 h 0.14 440% [162]
120120 °C,°C, 7070 ◦ nonenonenone 120 °C, 70 120 C,85–92%85–92% 70 kPa 2424 hh 85–92% 7.6 7.6 24 280 280 h [164] [164] 7.6 280 [164] none kPa 85–92% 2424 hh 7.6 7.6 280 280 [164] [164] PU incorporating BiTEMPS kPakPa
poly(hexylpoly(hexyl methacrylate) incorporating incorporating BiTEMPS BiTEMPS poly(hexylpoly(hexyl methacrylate) methacrylate) incorporating incorporating BiTEMPS BiTEMPS 1 Self-healing efficencies are reported as ranges in some cases because different self-healing method. 2 For healing at room temperature (RT), if more than one temperature tested. 3 For.pristine materials. 4 5 120 °C, 70 6 Self-healing time varried based upon the width and depth of a cut. Fornone cutting and reprocessing studies,85–92% pressure was 1024 MPa; h atmospheric 7.6 pressure for scratch 280 tests. Depended [164] on disulfide content. 7 Healing based on the change in the cut width. kPa
poly(hexyl methacrylate) incorporating BiTEMPS
Molecules 2021, 26, 3332 37 of 44
The results of the groups of Lv [17] and Jian [143] suggest that disulfide bonds are more effective as self-healers than imine or hydrogen bonds. However, disulfide-based self-healing appears to occur over a longer time scale. Pairing disulfide bonds with the faster self-healing functionalities may improve overall self-healing and material longevity by lowering the risk of permanent deformation when rapid stress is applied. Kim and co-workers [13] examined how structure-property relationships influenced self-healing in thermoplastic polyurethanes (TPUs) containing aryl disulfide groups with variable molecular packing and rigidities. Thus, ‘hard’ para-substituted diaryl disulfide prepolymers were copolymerized with ‘soft’ poly(tetramethylene ether) glycol segments (PTMG) (Figure 35). Comparison of the results from the resultant copolymers suggests that the packing ability of the hard segment, which is determined by the R groups flanking the aryl disulfide, strongly influences self-healing. TPUs formed with an alicyclic isophorone diisocyanate-based hard segment (IP-SS) self-healed most efficiently (Table2). The tensile strength of IP-SS was 6.8 MPa, which is nearly one order of magnitude higher than that Molecules 2021, 26, x FOR PEER REVIEWreported for crosslinked PUs (0.8 MPa) [150]. This is one of the highest strengths38 reported of 45 among materials that can self-heal at room temperature.
FigureFigure 35. 35.Synthesis Synthesis ofof diphenyldiphenyl disulfidedisulfide “hard segment” prepolymers prepolymers and and subsequent subsequent copolymerization copolymerization with with PTMG. PTMG. AdaptedAdapted with with permission permission fromfrom [[13].13]. CopyrightCopyright (2017) Wiley Periodicals, Inc. Inc.
KimAn Ashby and coworkers plot of self-healing propose time that at the room combination temperature of versus the rigid toughness diaryl disulfide (Figure 36) and thehighlights alicyclic the flanking superior groups performance provides of sufficientIP-SS compared chain mobilityto other intrinsically for efficient self-healing healing with- outmaterials. weakening As shown the material in Table to 2, an self-healing appreciable materials extent. with By comparison, comparable TPUsor higher formed tensile with symmetricstrengths have alicyclic been 4,4reported,0-methylenebis(phenyl but they require heat, isocyanate) pressure, (M-SS) and/or and light linear for comparably aliphatic hex- amethyleneefficient self-healing diisocyanate to occur hard [14,143,163]. segments (H-SS), exhibited little self-healing ability. The higher tensile strengths of M-SS and H-SS (Table2) than that of IP-SS suggests that the rigidity of M-SS and the high packing ability of H-SS hinder self-healing. Although both M-SS and H-SS exhibited no self-healing in scratch tests, some recovery of tensile properties was observed (especially for H-SS, see values in Table2) when the materials were cut and subjected to pressure.
Figure 36. Ashby plot of self-healing time at room temperature versus toughness. Adapted with permission from [13]. Copyright (2017) Wiley Periodicals, Inc.
Molecules 2021, 26, x FOR PEER REVIEW 38 of 45
Molecules 2021, 26, 3332 38 of 44 Figure 35. Synthesis of diphenyl disulfide “hard segment” prepolymers and subsequent copolymerization with PTMG. Adapted with permission from [13]. Copyright (2017) Wiley Periodicals, Inc. An Ashby plot of self-healing time at room temperature versus toughness (Figure 36) An Ashby plot of self-healing time at room temperature versus toughness (Figure 36) highlights the superior performance of IP-SS compared to other intrinsically self-healing highlights the superior performance of IP-SS compared to other intrinsically self-healing materials. As shown in Table2, self-healing materials with comparable or higher tensile materials. As shown in Table 2, self-healing materials with comparable or higher tensile strengths have been reported, but they require heat, pressure, and/or light for comparably strengths have been reported, but they require heat, pressure, and/or light for comparably efficient self-healing to occur [14,143,163]. efficient self-healing to occur [14,143,163].
FigureFigure 36.36. AshbyAshby plot of self-healing time time at at room room temperature temperature versus versus toughness. toughness. Adapted Adapted with with permissionpermission fromfrom [ 13[13].]. CopyrightCopyright (2017)(2017) Wiley Wiley Periodicals, Periodicals, Inc. Inc.
4. Conclusions Thiomers and disulfide-containing polymers have been studied for a wide variety of applications, due to their stimulus-responsive reactions. Many applications for these
materials have been proposed and realized. Since disulfide-based drug delivery systems were first approved for human use nearly 20 years ago, research on them and an expansion of their forms has led to a wide range of sustainable materials that exhibit self-healing and controllable biodegradation. Future research in the field of thiomer and disulfide- containing polymers as in drug delivery agents and self-healing materials appears to be ongoing at an ever-increasing pace. We expect that future research will explore the potential of multi stimulus-responsive materials that include other functionalities moreover disulfides and thiols.
Author Contributions: Conceptualization has been done by and methodologies were developed by both authors, as were writing/editing. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by the U. S. National Science Foundation through Grant CHE-1502856. Acknowledgments: We thank Jake Bimrose for technical assistance in some aspects of the preparation of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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