Stimuli-Responsive DNA-Based Hydrogels: From Basic Principles to

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Stimuli-Responsive DNA-Based Hydrogels: From Basic Principles to Applications Published as part of the Accounts of Chemical Research special issue “Stimuli-Responsive Hydrogels”. Jason S. Kahn,# Yuwei Hu,# and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel CONSPECTUS: The base sequence of nucleic acids encodes structural and functional information into the DNA biopolymer. External stimuli such as metal ions, pH, light, or added nucleic acid fuel strands provide triggers to reversibly switch nucleic acid structures such as metal-ion-bridged duplexes, imotifs, triplex nucleic acids, G-quadruplexes, or programmed double-stranded hybrids of oligonucleotides (DNA). The signal-triggered oligonucleotide structures have been broadly applied to develop switchable DNA nanostructures and DNA machines, and these stimuli-responsive assemblies provide functional scaffolds for the rapidly developing area of DNA nanotechnology. Stimuli-responsive hydrogels undergoing signal-triggered hydrogel-to-solution transitions or signal-controlled stiffness changes attract substantial interest as functional matrices for controlled drug delivery, materials exhibiting switchable mechanical properties, acting as valves or actuators, and “smart” materials for sensing and information processing. The integration of stimuliresponsive oligonucleotides with hydrogel-forming polymers provides versatile means to exploit the functional information encoded in the nucleic acid sequences to yield stimuli-responsive hydrogels exhibiting switchable physical, structural, and chemical properties. Stimuli-responsive DNA-based nucleic acid structures are integrated in acrylamide polymer chains and reversible, switchable hydrogel-to-solution transitions of the systems are demonstrated by applying external triggers, such as metal ions, pH-responsive strands, G-quadruplex, and appropriate counter triggers that bridge and dissociate the polymer chains. By combining stimuli-responsive nucleic acid bridges with thermosensitive poly(N-isopropylacrylamide) (pNIPAM) chains, systems undergoing reversible solution ↔ hydrogel ↔ solid transitions are demonstrated. Specifically, by bridging acrylamide polymer chains by two nucleic acid functionalities, where one type of bridging unit provides a stimuli-responsive element and the second unit acts as internal “bridging memory”, shape-memory hydrogels undergoing reversible and switchable transitions between shaped hydrogels and shapeless quasi-liquid states are demonstrated. By using stimuli-responsive hydrogel cross-linking units that can assemble the bridging units by two different input signals, the orthogonally-triggered functions of the shapememory were shown. Furthermore, a versatile approach to assemble stimuli-responsive DNA-based acrylamide hydrogel films on surfaces is presented. The method involves the activation of the hybridization chain-reaction (HCR) by a surface-confined promoter strand, in the presence of acrylamide chains modified with two DNA hairpin structures and appropriate stimuli-responsive tethers. The resulting hydrogel-modified surfaces revealed switchable stiffness properties and signal-triggered catalytic functions. By applying the method to assemble the hydrogel microparticles, substrate-loaded, stimuli-responsive microcapsules are prepared. The signaltriggered DNA-based hydrogel microcapsules are applied as drug carriers for controlled release. The different potential applications and future perspectives of stimuli responsive hydrogels are discussed. Specifically, the use of these smart materials and assemblies as carriers for controlled drug release and as shape-memory matrices for information storage and inscription and the use of surface-confined stimuli-responsive hydrogels, exhibiting switchable stiffness properties, for catalysis and controlled growth of cells are discussed.

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INTRODUCTION Besides the key role of the DNA base sequence as a genetic information carrier and as the translational code for the synthesis of proteins, research efforts in the past two decades have highlighted the use of DNA as a functional material for numerous applications in chemistry, physics, computer science, and medicine. The base sequence in DNA encodes substantial structural and functional information into the biopolymers.1 In addition to the formation of Watson−Crick double-strand © 2017 American Chemical Society

helices, formed by complementary base pairings, various other DNA structures, such as the pH-driven formation of C−G·C+ or T−A·T triplexes, are known.2 Similarly, the pH stimulated assembly of cytosine-rich strands into i-motif configurations,3 the K+-stimulated assembly of G-rich strands into Gquadruplexes,4,5 and the cooperative stabilization of duplex Received: October 31, 2016 Published: March 1, 2017 680

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Figure 1. (A) Synthesis of a polyacrylamide/nucleic acid-tethered stimuli-responsive hydrogel undergoing cyclic hydrogel↔solution transitions in the presence of Ag+-ions and cysteamine as triggers. (B) Rheometry characterization of the Ag+-ions/cysteamine-triggered acrylamide hydrogels. (C) Synthesis of the catalytic switchable hemin/G-quadruplex HRP-mimicking DNAzyme hydrogel. (D) Cyclic ON/OFF activation of the catalytic functions of the hemin/G-quadruplex-functionalized hydrogel for the oxidation of ABTS2− by H2O2, upon (a) addition of K+-ions and (b) addition of crown-ether. (E) Photoinduced reversible hydrogel-to-solution transitions using photoisomerizable trans-azobenzene units as duplex-stabilizing components and cis-azobenzene units as duplex destabilizing constituents. (F) Volume changes of the photoresponsive DNA-based hydrogel upon controlled stiffening in the presence of (a) cis-azobenzene units or (b) trans-azobenzene units. Reprinted with permission from refs34 (copyright 2014 Royal Society of Chemistry), 35 (copyright 2013 American Chemical Society), and 38 (copyright 2012 American Chemical Society).

DNA by metal ion bridges, such as C−Ag+−C or T−Hg2+−T bridges,6,7 represent sequence-dependent structural motifs that dictate various spatial organizational motifs of DNA. These different structures can be dissociated by their respective counter signals.8 Beyond these structural motifs, there are further sequencedictated functions of DNA. These include sequence-specific recognition of low-molecular-weight substrates (aptamers),9,10 catalytic functions, for example, metal-ion sequence-dependent hydrolysis of ribonucleobase-containing substrates11,12 or the hemin/G-quadruplex horseradish peroxidase (HRP) mimicking DNAzyme,13 and the capability to conduct enzyme-catalyzed sequence-dictated reactions, for example, nicking enzymes or endonucleases.14 The sequence-dictated structural and functional properties of oligonucleotides have led to the implementation of this biopolymer as a functional material for the assembly of programmed DNA structures,15 DNA switches,16 and DNA machines.17,18 In addition, the unique sequence-controlled functions of DNA have been used to

develop DNA-based logic gates and computational circuits,19 novel catalytic structures,20 and stimuli-responsive nanoparticle assemblies.21 Different applications of the structural and functional properties of DNA and DNA-hybrid systems for sensing,22 drug delivery,23 and fabrication of nanoscale devices24 have been demonstrated. Hydrogels represent a broad class of materials consisting of highly cross-linked hydrophilic polymer networks that adsorb high quantities of water, leading to swelling of the hydrogel matrices. Numerous hydrogels, composed of natural or synthetic cross-linked matrices, are known. Stimuli-responsive hydrogels represent an important class within this field;25 different external triggers such as pH,26 light,27 temperature,28 redox reactions,29 or chemical triggers30 have been used to induce reversible hydrogel-to-solution or hydrogel-to-solid transitions. Different applications of stimuli-responsive hydrogels for sensing, separation of material, controlled release, and catalysis have been suggested. Advances in the synthesis and application of stimuli-responsive hydrogels were summarized in 681

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Figure 2. Synthesis of thermosensitive DNA-based stimuli-responsive pNIPAM hydrogels: (A) Using pH-responsive polymer chains. (B) Cyclic transition of the matrix across solution ↔ hydrogel ↔ solid states. (C) Images corresponding to the Ag+-ion/thermal transitions of the hydrogel using C−Ag+−C bridged chains. Reprinted with permission from ref 39. Copyright 2014 Wiley-VCH.

several recent review articles.31,32 The “tool-box” of triggers to reversibly switch the structure of DNA units and the unique recognition function of oligonucleotides suggest that crosslinking of polymer matrices by means of stimuli-responsive DNA units could provide a versatile means to design signaltriggered DNA-based hydrogels. This Account addresses recent advances from our laboratory toward the development of stimuli-responsive DNA hydrogels. Besides the use of nucleic acids as a versatile “toolbox” to assemble stimuli-responsive hydrogels as new functional materials, we address novel applications of these systems. Specifically, we introduce the development of shape-memory hydrogels and discuss a method to functionalize surfaces with thin layers of stimuli-responsive hydrogels.

polymer chains are modified with functional nucleic acids that, in the presence of appropriate triggers, cross-link the polymer chains, leading to the formation of stimuli-responsive hydrogels. Subjecting the hydrogel system to a counter trigger that separates the nucleic acid cross-linking units leads to the separation of the polymer chains and the formation of a polymer solution. Several examples of stimuli-responsive acrylamide−DNA hydrogels are displayed in Figure 1. In the first example, acrylamide chains were modified with acrydite nucleic acids (1), exhibiting self-complementarity and an internal C−C mismatch.34 While the number of selfcomplementary bases is insufficient to yield a stable crosslinked hydrogel structure, the addition of Ag+-ions cooperatively stabilizes the cross-linking units through the formation of C−Ag+−C bridges, Figure 1A. Formation of the hydrogel is evident in a high storage modulus, G′ ≈ 18 Pa, and a low loss modulus, G″ ≈ 2 Pa. Subjecting the hydrogel to cysteamine removed the Ag+-ions from the cross-linking units, resulting in the separation of the hydrogel into a polymer solution, a cycle that can be repeated through addition of the appropriate triggers, Figure 1B. The development of a G-quadruplex/18-crown-6-ether stimuli-responsive reversible acrylamide hydrogel system is presented in Figure 1C.35 Acrylamide copolymer chains, PA, composed of acrylamide chains functionalized with G-rich acrydite nucleic acid tethers (2) were used to assemble the switchable hydrogel matrix. In the presence of K+-ions, the modified polymer chains self-assemble into the K+-stabilized Gquadruplex-cross-linked hydrogel, with G′ ≈ 4 Pa and G″ ≈ 0 Pa. Treatment of the hydrogel with 18-crown-6-ether (CE) eliminated the K+-ions from the G-quadruplex units, leading to

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DISCUSSION Two general strategies are used to assemble stimuli-responsive DNA hydrogels undergoing reversible gel-to-solution transitions. By one strategy, all-DNA hydrogels are formed by the cross-linking of polydentate DNA units that can include within the cross-linking network stimuli-responsive components. Although some applications of all-DNA hydrogels have been reported, for example, the cross-linking of the hydrogel subunits by fluorescent nucleic acid stabilized Ag+-nanoclusters,33 the broad utility of stimuli-responsive hydrogels consisting of all DNA as a functional material is limited due to the cost of the nucleic acid constituents. A second approach to resolve the limitation associated with all-DNA stimuliresponsive nucleic acid-based hydrogels involves the tethering of functional nucleic acids to a polymer chain, for example, polyacrylamide, Figure 1. According to this approach, the 682

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reversible transitions between the solution and hydrogel states, Figure 2B. Heating the hydrogel matrix to 45 °C transforms the hydrogel into a shrunken solid, while cooling the solid to 25 °C leads to the recovery of the swollen hydrated hydrogel, Figure 2B. Similar transitions were demonstrated with pNIPAM copolymer chains functionalized with nucleic acids (6), being cross-linked by Ag+-ions, inducing formation of C−Ag+−C bridged duplexes between the tethers. The resulting hydrogel transformed to solid state upon heating to 45 °C and was dissociated to the solution state upon treatment with cysteamine, Figure 2C.39 Further applications of stimuli-responsive DNA-based hydrogels have included the use of functional hydrogels as sensing and controlled-release matrices. Representative examples of such systems include hydrogels generated by cross-linking of polymer chains with nucleic acid aptamer or metal ionsdependent DNAzyme/substrate complexes; these have been used for sensing proteins, for example, thrombin,30 or metal ions.40 The signal-triggered transition of hydrogels to a solution phase also provides a versatile means of release of macromolecules or molecular loads entrapped in the hydrogel. Cyclic dissolution of hydrogels by DNAzyme triggers were used to release enzyme loads and to activate enzyme cascades, Figure 3.40 Three enzymes, β-galactosidase (β-Gal), glucose oxidase

the separation of the hydrogel. The G-quadruplex units also act as structural scaffolds for the generation of the hemin/Gquadruplex HRP-mimicking DNAzyme.13 The DNAzyme catalyzes the H2O2-oxidation of ABTS2−, 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid), to the colored radical anion, ABTS•−, as well as the generation of chemiluminescence by the oxidation of luminol by H2O2.36 Indeed, the hemin/Gquadruplex hydrogel revealed stimuli-triggered HRP-mimicking DNAzyme functions. Figure 1D depicts the “ON”/”OFF” switchable oxidation of ABTS2− by H2O2 upon the formation of the hemin/K+-stabilized G-quadruplex cross-linked hydrogel (switch-ON) and upon its separation in the presence of 18crown-6-ether (switch-OFF).35 Besides the tailoring of reversible hydrogel ↔ solution systems, nonreversible hydrogels undergoing gel-to-solution transitions were also developed. For example, acrylamide hydrogel systems cross-linked by aptamer cross-linkers were separated by the formation of aptamer−ligand complexes.30 Similarly, acrylamide hydrogels cross-linked by metal-dependent DNAzyme/substrate bridges were prepared and separated into the solution state in the presence of the respective ions.37 Such systems demonstrate potential value as stimuli-triggered drug carriers and sensor systems. Similarly, light-responsive DNA-based hydrogels that use photoisomerizable azobenzene units were prepared, shown in Figure 1E.38 An acrylamide hydrogel of low stiffness was prepared by cross-linking the acrylamide polymer chains, PB and PC, with a low content of bis-acrylamide cross-linker. The polymer PB was modified with nucleic acid tethers functionalized with cis-azobenzene units (3) and the polymer chain PC included complementary tethers (4). It is well established that trans-azobenzene units intercalate into duplex nucleic acids, resulting in the stabilization of the hybrid structure, while cisazobenzene lacks affinity toward double-stranded nucleic acids. Accordingly, in the presence of the cis-azobenzene units, the duplex 3/4 was insufficiently stable to cross-link the acrylamide hydrogel. Photoisomerization of the cis-azobenze units to transazobenzene units (λ > 420 nm) resulted in the stabilization of a substantially stiffer duplex-bridged acrylamide hydrogel.38 By the reverse photoisomerization of the trans-azobenzene units to the cis-isomer state (λ = 365 nm), the duplex-bridging units separated to yield the less stiff hydrogel, and the system can again be cycled between higher stiffness (low volume) and lower stiffness (higher volume) configurations, Figure 1F. The ability to trigger stimuli-responsive nucleic acid-based hydrogels by two signals enables controlled reversible hydrogel↔solution transitions and stimuli-dictated volume transitions over multiple inputs and conditions. The poly(Nisopropylacrylamide), pNIPAM, is a thermosensitive polymer that undergoes reversible hydrogel↔solid transition at 32 °C. In contrast to covalently cross-linked pNIPAM hydrogels that undergo only reversible gel-to-solid transitions, DNA-functionalized pNIPAM allows reversible transitions across solution ↔ hydrogel ↔ solid states.39 This is exemplified in Figure 2A, with the use of NIPAM chains functionalized with an acrydite cytosine-rich tether (5). At pH = 7.5, the polymer exists in the solution state; acidification of the system to pH = 5.2 leads to the cross-linking of the pNIPAM chains by the configuration of the cytosine-rich tethers into i-motif units, resulting in the hydrogel state (G′ ≈ 220 Pa, G″ ≈ 0 Pa). The neutralization of the system leads to the separation of the cross-linking i-motif bridges and regenerates the solution state, and the cyclic treatment of the system at pH = 7.5 and pH = 5.2 shows

Figure 3. Activation of a three-enzyme catalytic cascade by the metalion dissociation of three different hydrogels: (A) Constituents of the metal-ion cross-linked hydrogels. (B) Three-enzyme cascade. (C) Time-dependent formation of ABTS•− by (a) the three-ion (Zn2+, Mg2+, Cu2+)-dependent DNAzymes or (b−d) in the presence of only two of the three ions. Reprinted with permission from ref 40. Copyright 2015 American Chemical Society.

(GOx), and HRP, were loaded in three different stimuliresponsive acrylamide DNA hydrogels cross-linked by three different metal ion-dependent DNAzyme/substrate bridges, Figure 3A. In a specific example, β-Gal was immobilized in a hydrogel stabilized by the Zn2+-dependent DNAzyme, GOx by the Mg2+-dependent DNAzyme, and HRP by the Cu2+dependent-DNAzyme. In the presence of lactose as substrate 683

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Accounts of Chemical Research and ABTS2−, and upon the addition of all three metal ions Zn2+, Mg2+, and Cu2+, the three enzyme cascade is activated, Figure 3B. The resulting colored ABTS•− provides the readout signal for the biocatalytic cascade, Figure 3C. Stimuli-responsive DNA-based hydrogels offer functional materials for tailoring shape-memory systems. Substantial research efforts have been directed toward the development of smart shape-memory materials. In these systems, the polymer constituents are first organized into a shape stabilized by auxiliary or internal triggers. Subjecting the shaped polymer to a counter-trigger yields a shapeless material configuration that stores the code (“memory”) to recover the original permanent shape in the presence of the original triggering signal. Different applications of shape-memory materials have been supported, including their use as sensors,41 drug-release matrices,42 actuators of microdevices,43 and functional materials for inscription and information storage.44 The diverse triggers to reversibly reconfigure nucleic acid structures provide a rich arsenal for designing shape-memory DNA-based hydrogel matrices as exemplified in Figure 4A, showing the synthesis of a pH-responsive shape-memory hydrogel.45 The acrylamide copolymer chains are functionalized with the cytosine-rich

acrydite nucleic acid 10 and the strand 11 exhibiting selfcomplementarity. The amorphous polymer solution was subjected to acidic pH (pH = 5.0) in a triangle-shaped mold, resulting in a hydrogel that is cooperatively stabilized by duplexes 11/11 and by i-motif cross-linking bridges 10/10. The hydrogel extracted from the mold retained its triangular shape and possessed rheological parameters characteristic of a hydrogel (G′ ≈ 50 Pa, G′′ ≈ 2 Pa). Treatment of the triangle-shaped hydrogel at pH = 8.0 resulted in the dissociation of the i-motif bridging units. The residual duplex bridging units are insufficient to stabilize a hydrogel network of the polymer matrix, leading to the formation of a shapeless, quasi-liquid state (G′ ≈ G′′ ≈ 2 Pa). The duplex units associated with the quasi-liquid state provide, however, the entanglement of the polymer chains that acts as “memory” for dictating the regeneration of the hydrogel shape upon the pHstimulated formation of the cooperatively stabilized system of 11/11 hybridization and 10/10 i-motif. That is, the duplexentangled quasi-liquid preserves the cytosine-rich tether in appropriate spatial orientations that re-form, under appropriate pH conditions, the original i-motif bridges that restore the macroscopic shape of the hydrogel. Figure 4B depicts the cyclic and reversible pH-stimulated transitions of the hydrogel between the shaped hydrogel and the shapeless quasi-liquid states. This concept of stimuli-triggered shape-memory DNA-based hydrogels was implemented to develop different shape-memory hydrogels triggered by other stimuli such as pH-triggered C−G· C+ or T−A·T triplex bridges,46 K+-stabilized G-quadruplex/ Kryptofix[2.2.2],47 Ag+-stabilized C−Ag+−C-bridged/cysteamine,45,47 or fuel/antifuel strands.47 Figure 4C exemplifies the assembly of a K+-ion/crown-ether-triggered shape-memory hydrogel. The triangle-shaped hydrogel is stabilized by cooperative duplex cross-linking units 12/12 and K+-ionstabilized G-quadruplex 13/13. The cyclic transformation between shaped and shapeless states, Figure 4D, is induced by the separation of the G-quadruplex units with crown-ether, while the readdition of the K+-ions regenerates the Gquadruplex bridges to restore the shaped hydrogel. The concept of shape-memory DNA hydrogels was further extended to assemble hybrid shape-memory hydrogels that allow the dictated triggered, reversible transitions of target within hybrid hydrogel structures, across shaped hydrogels and shapeless quasi-liquid domains. This assembly concept is exemplified in Figure 5 with the assembly of the three-arm arrowhead structure.47 All three arrowheads include the self-complementary duplex DNA bridges 14/14 as hydrogel bridging units that act as permanent memories for any transition of hydrogel from a stiff hydrogel to a shapeless quasi-liquid state. Arms I and II consist of hydrogels cooperatively stabilized by the duplex cross-linkers 15/16 and 19/20, respectively. These duplexes are separated through the strand-displacement principle, by counterstrand 17 or 21, to yield the respective quasi-liquid domains of I or II. Similarly, the arrowhead III is co-stabilized by the cytosine-rich subunits that form a cross-linking i-motif bridges 23/23 at pH = 5.0. Figure 5B depicts the stepwise assembly of the three arrowhead structure in a mold, using edge-cross-linking of strands 24/25 as interdomain bridges. Subjecting the three arrowhead structure to the respective fuel/ antifuel strands as well as the appropriate pH cyclically transforms the arms between shaped and shapeless states, shown in Figure 5C. Recently, pH-stimulated dissociation/

Figure 4. (A) Synthesis of a pH-responsive shape-memory acrylamide−DNA hydrogel. (B) Cyclic pH-induced transition of the system across triangle-shaped hydrogel and quasi-liquid shapeless states. (C) Synthesis and shape-memory properties of an acrylamide− DNA hydrogel cooperatively stabilized by G-quadruplex and duplex bridging units. (D) Cyclic transitions of the system between the shaped triangle hydrogel and shapeless quasi-liquid state upon treatment with K+ and crown-ether, respectively. Reprinted with permission from refs 45 (copyright 2015 Wiley-VCH) and 47 (copyright 2015 American Chemical Society). 684

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Figure 5. (A,B) Schematic assembly of a three arrowhead shaped hybrid hydrogel structure that includes three triggerable domains undergoing reversible shape ↔ quasi-liquid transitions. (C) Domain-dictated shape ↔ quasi-liquid transitions in the presence of the appropriate trigger(s). Reprinted with permission from ref 47. Copyright 2015 American Chemical Society.

association of C−G·C+ and T−A·T triplex structures was applied to control a two-arrowhead hybrid system.48 The concept of DNA-based shape-memory hydrogel was further developed by designing shape-memory hydrogels that are triggered by two orthogonal triggers,49 Figure 6. In these systems, the shaped hydrogel is cross-linked by two cooperative motifs. One cross-linking element includes a self-complementary bridge, and the second cross-linking unit is separated and restored by two different orthogonal triggers. Figure 6A depicts a shape-memory hydrogel triggered by pH and Ag+ ions as orthogonal triggers. The polymer chain PD is functionalized with the self-complementary strands, 26, and with the cytosinerich tethers, 27. The triangle-shaped hydrogel is generated in a mold, at pH = 5.0, using the self-complementary duplexes 26/ 26 and the i-motif bridges 27/27 as cooperative functionalities stabilizing the shaped hydrogel matrix (Young’s modulus ≈ 1700 Pa). Neutralization of the system at pH = 7.4 dissociated the i-motif bridges, resulting in the shapeless quasi-liquid state (Young’s modulus ≈ 380 Pa). The duplex bridges present in the quasi-liquid state provide the permanent memory to reshape the triangle upon subjecting the system to pH = 5.0 which restores the i-motif bridge. The free C-rich tethers in the shapeless quasi-liquid phase allow, however, the cross-linking of the tether in the presence of Ag+-ions to form C−Ag+−C quadruplex bridges. Treatment of the resulting hydrogel with cysteamine, acting as a chelating ligand for the Ag+ ions, regenerates the shapeless quasi-liquid state that includes the duplex 26/26 as internal memory. The shapeless system regenerates the triangle shape in the presence of Ag+ or H+, pH = 5.0, as triggers.49 By the cyclic application of pH or Ag+/ cysteamine as orthogonal triggers, the hydrogel is reversibly transformed between shaped hydrogel and shapeless, quasiliquid states, Figure 6B. A related system used Pb2+ or Sr2+ ions as orthogonal triggers for the construction of a shape-memory hydrogel based on duplexes (permanent memory) and Gquadruplexes as cooperative cross-linking units that stabilize the shaped hydrogel structure, Figure 6C,D. The deposition of hydrogels onto surfaces, particularly stimuli-responsive hydrogels, is interesting for developing

hydrogel devices. A versatile method to assemble thin stimuliresponsive hydrogels on surfaces has used the hybridization chain reaction (HCR) process.50 This reaction involves the use of a promoter nucleic acid-initiated cross-opening of two hairpins HA and HB, where the promoter opens HA to yield a single-strand toehold for the opening of HB, and, in turn, the HB-opened product includes a toehold domain to open hairpin HA. The cross-opening of the hairpin structure has been used to cross-link nucleic acid-functionalized acrylamide chains to yield stimuli-responsive hydrogel films on surfaces, Figure 7A. Two acrylamide copolymer chains PF and PG were prepared, where PD was modified with hairpin HA, 30, and G-rich tether, 33, and polymer PE was functionalized with hairpin HB, 31, that is associated with the tether 32 (for controlling directionality) and with the G-rich tether, 33. The nucleic acid, 34, associated with the surface (e.g., Au electrode, glass) acts as the promoter for localizing the HCR process on the surface. The promoterinduced opening of hairpin HA associated with PF yield a toehold strand that hybridizes with HB associated with PG, and the opened hairpin HB includes a toehold strand that hybridizes with HA link to PD. Thus, the promoter strand initiates the cross-opening of the HA and HB and the assembly of a crosslinked hydrogel consisting of the duplex bridged polymer chains PF and PG. As the bridged hydrogel includes free G-rich tethers, these strands assemble, in the presence of K+ ions, into the G-quadruplex that cooperatively stabilized the hydrogel yielding a stiffer hydrogel. Treatment of the film with 18-crown6-ether removed the K+-ions from the matrix, resulting in a hydrogel film of decreased stiffness. Microindentation experiments followed the cyclic transitions of the hydrogel film between stiff and less-stiff states, Figure 7B (Young’s modulus 1770 Pa vs 460 Pa, respectively).50 Hemin binding to Gquadruplex units yielded electrocatalytic units for the reduction of H2O2, and thus by the assembly of hemin/G-quadruplexfunctionalized hydrogel thin films on an electrode surface and the subsequent crown-ether-stimulated separation of the hemin/G-quadruplexes, the cyclic ON/OFF electrocatalyzed reduction of H2O2 was demonstrated, Figure 7C. 685

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Figure 6. Cyclic and reversible switching between shaped hydrogels and quasi-liquid states using the two orthogonal triggers that activate the shapememory hydrogels. (A) Reversible switching for the cytosine rich shape-memory hydrogel using pH and Ag+/cysteamine as triggers. (B) Images corresponding to shaped and shapeless states of the hydrogel subjected to the two orthogonal triggers. (C) Reversible switching of the G-rich shapememory hydrogel using Pb2+/DOTA and Sr2+/Kryptofix[2.2.2] as triggers. (D) Images corresponding to shaped and shapeless states of the hydrogel subjected to the two orthogonal triggers. Reprinted with permission from ref 49. Copyright 2016 Wiley-VCH.

The successful coating of surfaces with stimuli-responsive hydrogel matrices enables the development of a versatile method to prepare stimuli-responsive hydrogel microcapsules for the triggered release of drugs.51 The deposition of micrometer thick stimuli-responsive acrylamide−DNA hydrogel films has been used to synthesize substrate-loaded stimuliresponsive hydrogel microcapsules. This is exemplified in Figure 8A with the preparation of pH-responsive hydrogel microcapsules. Substrate-loaded CaCO3 microparticles were coated with a poly(allylamine hydrochloride) and poly(acrylic acid) layers. The single-stranded amino-modified nucleic acid

35 was covalently linked to the base polymer layer, and this acted as a promoter strand to initiate the HCR. In the presence of the polyacrylamide chain PH and PI modified with the hairpins HC and HD and the complementary nucleic acids 36 and 37, respectively, the HCR process induced by 35 resulted in the cross-opening of hairpins HC and HD leading to the cross-linking of the polymer chains and the formation of a hydrogel film on the CaCO3 core-template particles. The hydrogel coating is stabilized by cooperative duplex bridges formed upon the cross-opening of the hairpins HC/HD and by the complementary duplexes 36/37. The strand 36 was 686

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Figure 7. (A) Assembly of a G-quadruplex stimuli-responsive thin film hydrogel on Au-coated surfaces using the HCR. (B) Switchable control of the stiffness of the hydrogel film by cross-linking of the hydrogel with K+-stabilized G-quadruplexes and their dissociation in the presence of crown-ether. (C) Stimuli-triggered ON−OFF electrocatalyzed reduction of H2O2 by the hemin/G-quadruplex. Cyclic voltammograms (right) corresponding to (I) the background response in the absence of H2O2, (II) the electrocatalyzed reduction of H2O2, (III) the voltammetric response of the crownether-treated hydrogel in the presence of hemin and H2O2, where the DNAzyme is dissociated (switch-OFF), and (IV) the recovered electrocatalytic functions of the hemin/G-quadruplex-modified hydrogel upon readdition of K+-ions (switch-ON). Reprinted with permission from ref 50. Copyright 2015 American Chemical Society.

Figure 8. (A) Synthesis of substrate-loaded pH-responsive acrylamide−DNA hydrogel microcapsules. (B) Schematic pH-switchable release of a load by the pH-induced enhancement of the fluidity of the microcapsule shells via the reconfiguration of one of the hydrogel cross-linking elements into imotif units. (C) Time-dependent release of doxorubicin-modified dextran from the pH-responsive microgel microcapsules at (a) pH = 7.2 and (b) pH = 5.0 and the effect of different pH values on the release of the fluorescent load. Reprinted with permission from ref 51. Copyright 2017 Royal Society of Chemistry.

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bridges provide means to develop self-healing hydrogels. The possibility to combine two different hydrogels by self-healing principles, as well as the ability to control the stiffness of the hydrogels by external triggers, provide means to dictate switchable stress properties within composite hydrogels, thereby allowing the development of materials exhibiting reversible, stimuli-responsive, mechanical properties, such as, bending. In addition, the miniaturization of stimuli-responsive hydrogels into microcapsule structures introduces interesting fundamental topics. For example, probing the stiffness of single stimuli-responsive microcapsules using force measurements, imaging the interactions between modified microcapsules and cells, and the development of additional stimuli to control the permeability of microcapsule shells represent future challenges. Lastly, the broad application of stimuli-responsive hydrogel microcapsules for controlled release (e.g., drugs, fragrances, food additives) is an important path to follow.

designed to include cytosine tethers that under acidic conditions (pH = 5.0) undergo reconfiguration into the energetically stabilized i-motifs, Figure 8B. The separation of the duplex units 36/37 decreased the stiffness of the hydrogel shell, resulting in the enhanced permeability of the microcapsule shells and the release of the encapsulated substrate. Figure 8C depicts the time-dependent release of the chemotherapeutic drug doxorubicin encapsulated in the microcapsules, upon their treatment at pH = 5.0. Indeed, preliminary experiments demonstrated that the acidic conditions present in cancer cells stimulated the release of doxorubicin in MD-MBA231 breast cancer cells, and the selective cytotoxicity of the drug toward these cells was observed. Furthermore, we note that the stimuli-responsive, DNAcross-linked, acrylamide microcapsules reveal several distinct properties: (i) The shells of the microcapsules retain their reservoir boundaries and undergo, in the presence of appropriate stimuli, reversible transitions between stiff and quasi-liquid states. (ii) Low molecular weight or macromolecular loads (e.g., enzymes) do not diffuse across the stiff shells to the bulk solution, yet freely permeate through the quasi-liquid shells. Thus, the stimuli-responsive DNA-based acrylamide hydrogel microcapsules complement functional microcarriers such as polymersomes (e.g., block copolymer liposome-like carriers) or microgels. Nonetheless, in contrast to these systems, that allow free in/out permeation of low molecular weight substrates and prohibited diffusion of macromolecular loads, the stimuli-responsive DNA-based microcapsules reveal switchable ON/OFF permeation across the microscapsule shells. These unique functions of the DNAbased hydrogel microcapsules are anticipated to reveal enhanced control over the release of the loads by allowing the release of molecular and macromolecular loads and by dictating dose-controlled release of the loads. These features represent important advantages of the DNA-based microgels for future medical applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +972-2-6585272. Fax: +972-2-6527715. ORCID

Itamar Willner: 0000-0001-9710-9077 Author Contributions #

J.S.K. and Y.H. contributed equally.

Notes

The authors declare no competing financial interest. Biographies Jason S. Kahn completed his Ph.D. studies at Cornell University in 2014. He is currently a postdoctoral fellow at The Hebrew University of Jerusalem. His research interests include nanoscale materials and organic−inorganic materials for medical and energy applications.

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Yuwei Hu obtained his Ph.D. jointly from Jilin University and Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, in 2013 and is a postdoctoral research fellow at The Hebrew University of Jerusalem. His research interests include DNA nanotechnology and stimuli-responsive DNA hydrogels.

CONCLUSIONS AND PERSPECTIVES The rich “tool-box” of switchable nucleic acid structures provides versatile means to construct stimuli-responsive hydrogels undergoing reversible hydrogel↔solution phase transitions to assemble shape-memory hydrogels and to design hydrogels of controllable stiffness properties. Major advances were demonstrated by the integration of the stimuli-responsive hydrogel with surfaces to yield thin films of signal-triggered hydrogels and microscale structures such as microscapsules. Different applications of stimuli-responsive DNA-based hydrogels are envisaged, including their application as functional matrices for controlled drug delivery, sensing, information storage, and inscription. Despite progress in developing DNA-based stimuli-responsive hydrogels, important and challenging scientific topics and possible applications of this class of materials can be identified. For example, the design of additional stimuli such as light or redox triggers for controlling the hydrogel properties could lead to new electrocatalytic or photocatalytic materials. Also, the integration of nanomaterials, such as metal or semiconductor nanoparticles or nanorods, or carbon materials (carbon nanotubes/graphene) with stimuli-responsive hydrogel could yield hybrid matrices of controllable stiffness properties, such as, interfaces for the dictacted growth of cells. Furthermore, the intrinsic properties of oligonucleotides to form duplexes or triplexes by base pair complementarity, metal-ion, or pH-driven

Itamar Willner completed his Ph.D. studies at The Hebrew University of Jerusalem in 1978 and, after postdoctoral research at U.C. Berkeley, USA, he joined The Hebrew University of Jerusalem and acts currently as a Professor of Chemistry. His research interests include bioelectronics, nanobiotechnology, nanomaterials, electrochemistry, and photochemistry. He is the recipient of the Israel Prize in Chemistry (2002), the Rothschild Prize (2008), and the EMET Prize (2008).

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ACKNOWLEDGMENTS Our research on stimuli-responsive hydrogels is supported by the Israel Science Foundation. REFERENCES

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