Stiffness‐Switchable, Biocatalytic pH‐Responsive DNA‐Functionalized Polyacrylamide Cryogels and their Mechanical Applications

The large‐pore interconnected channels in cryogels, allow convectional flow and rapid mass‐transport of solute constituents between the solution and cryogel polymer framework, as compared to slower, diffusionally‐controlled mass‐transport in small‐pore hydrogels. These features are applied to develop enzyme‐loaded polyacrylamide (pAAm) cryogels, and glucose oxidase (GOx)‐loaded pH‐responsive DNA‐based pAAm cryogels, revealing enhanced biocatalytic transformations, enhanced temporal stiffness changes, and mechanical bending functions, as compared to analog hydrogels. DNA‐based pAAm cryogel/hydrogel matrices, revealing pH‐switchable stiffness properties through reversible reconfiguration of DNA‐bridging units into i‐motif structures, are introduced. Enhanced switchable stiffness changes of DNA‐based pAAm cryogels, as compared to analog hydrogels, are demonstrated upon subjecting the cryogel/hydrogel matrices to auxiliary pH‐changes, or by integration of GOx into the frameworks, and driving pH‐changes through GOx‐catalyzed aerobic oxidation of glucose to gluconic acid. Enhanced stiffness changes of pAAm cryogels represent a major advance to control the mechanical properties of cryogels and are attributed to the convectionally‐controlled mass‐transport in the cryogel matrices. Moreover, bilayer constructs consisting of poly‐N‐isopropylacrylamide (pNIPAM) cryogels and pH‐responsive DNA‐based pAAm cryogel or hydrogel structures are constructed. Enhanced pH/glucose triggered mechanical bending rates of the pNIPAM cryogel/pAAm cryogel or pNIPAM cryogel/GOx‐loaded pAAm cryogel, as compared to analog pNIPAM cryogel/pAAm hydrogel frameworks are demonstrated.

[60] The cryogels are prepared at freezing temperatures by analog protocols to the synthesis of hydrogels.The cryo conditions lead, however, to crystalline solvent domains coated by concentrated crosslinked polymer matrices that lead, upon defrosting of the matrices to permanent structures consisting of interconnected solute macropores embedded in the crosslinked polymer framework.The interconnected macropores comprising the cryogels introduce important structural and functional features, as compared to analog hydrogels matrices.These are reflected by different mechanical strengths [61,62] and compressibilities [63] of the cryogels originating from the higher polymer content in the framework boundaries, and the solute squeezability, [64,65] of the interconnected macropores comprising the cryogels.In addition, the large interconnected solute macropores, allow the convectional transport of solute in the cryogels, [66][67][68][69][70] as compared to diffusionally-hindered transport in hydrogels, leading to enhanced mass-transport of the solute inside the cryogel and between the cryogel and its surrounding.These features of cryogels lead to enhanced physical and chemical responses of the cryogels as compared to hydrogels.Indeed, the superior physiochemical properties of cryogels were employed for diverse applications including separation [65,71,72] and bioseparation processes, [73][74][75] functional matrices for encapsulation of cells, [76][77][78] and cell culturing, [76,[79][80][81] therapeutics such as drug-delivery [82,83] or injectable cryogel microspheres for bone regeneration, [84] and to whole cell-based cancer vaccination. [85]Also, by entrapment of enzymes in cryogels, enhanced reactivity and stability of the biocatalysts were demonstrated, [86][87][88] and the systems were applied as bioreactors for effective waste-water treatment. [89]In addition, stimuli-responsive cryogels were reported and their activation by light, [90] temperature, [91,92] ultrasound waves, [93] and pH [94] were reported and diverse fast-responsive matrices acting as actuators [95,96] and drug-release systems [97] were demonstrated.The integration of nucleic acids within cryogel networks is, however, less explored.While earlier studies evaluated the effects of interpenetrated nucleic acids into cryogels on the physical properties and stabilities of the matrices, [98] only recently, nucleic acids were integrated as functional constituents within polyacrylamide (pAAm) cryogel frameworks. [99]he superior mass-transport features of chemical agents between the solute channels and the polymeric backbone were utilized to demonstrate effective biocatalytic cascades activated between the enzyme (glucose oxidase), solubilized in the aqueous channels and a hemin/G-quadruplex DNAzyme localized in the polymer backbone. [100]Also, the squeezability and convectional mass-transport within a DNAzyme-modified thermoresponsive poly-N-isopropylacrylamide (pNIPAM) cryogel were applied for the effective uptake, cleavage, and release of the DNAzyme substrate, upon the solid-gel transitions of the cryogel framework. [100]Nonetheless, the design of stimuli-responsive, reconfigurable nucleic acid-functionalized cryogels, and their effect on the cryogel stiffness is, at present, unprecedented.Moreover, while previous studies introduced enzyme biocata-lysts into the cryogel solute phase, the incorporation of enzymes into DNA-based cryogel frameworks, and particularly, the coupling between the enzymes and stimuli-responsive nucleic acid constituents embedded in the polymer network is unknown.The coupling between biocatalysts and DNA contituents within the polymer backbone could provide an important path to activate and control the cryogel properties, functions, and possible applications.
Here we wish to report on the integration of multienzyme assemblies into cryogel frameworks and the advantages of the biocatalytic cascades in the cryogel matrices over analog hydrogels.Moreover, we address the preparation of enzyme (glucose oxidase, GOx)-loaded pH-responsive nucleic acid-functionalized pAAm cryogels (CryoPAAm-DNA-GOx) and discuss their temporal stiffness changes upon the acidification of the cryogel or upon the acidification of the CryoPAAm-DNA-GOx matrices by the biocatalyzed aerobic oxidation of glucose.The pH-stimulated stiffness changes of the cryogels are compared to analog GOx-loaded pHresponsive DNA-based hydrogel (HydroPAAm-DNA-GOx) matrices.We demonstrate that the structural features of the cryogels lead to substantially enhanced stiffness-changes, as compared to hydrogels, as a result of convection-stimulated mass-transport in the cryogel framework.In addition, we assemble bilayer constructs consisting of thermo-responsive pNIPAM cryogel (CryoPNIPAM), and DNA-based GOx-loaded pH-responsive pAAm cryogel (CryoPNIPAM/CryoPAAm-DNA-GOx), and CryoPNIPAM conjugated to GOx-loaded pH-responsive DNAbased pAAm hydrogel (CryoPNIPAM/HydroPAAm-DNA-GOx), and examine the rates of glucose-stimulated, pH-induced, mechanical bending of the bilayer constructs.We demonstrate that the enhanced stiffness changes of the cryogel layers lead to substantially enhanced bending rates of the CryoPNIPAM/CryoPAAm bilayer construct, as compared to the bending rates of the CryoPNIPAM/HydroPAAm bilayer constructs.

Biocatalytic Transformations in Cryogel Matrices Versus Hydrogel Matrices
To examine the effect of the cryogels versus hydrogels structures on biocatalyzed transformations of entrapped enzymes in the responsive matrices, we synthesized pAAm cryo/hydro gels, revealing comparable biocatalyst loadings of one enzyme, glucose oxidase (GOx), two enzymes, GOx and horseradish peroxidase (HRP), and three enzymes GOx, HRP, and -galactosidase (-gal).The loading of the gels with the enzymes corresponded to 77.3%/76.8% in the GOx-loaded (cryogel/hydrogel), to 66.5%/52% in the GOx/HRP (cryogel/hydrogel), and to 78.8%/82.5% in the GOx/HRP/-Gal (cryogel/hydrogel) (Figures S1.1 and S1.2, Supporting Information).The biocatalytic transformations driven by the different biocatalyst-loaded gel matrices were followed in the presence of glucose as substrates (for the GOx and the GOx/HRP-loaded matrices) or lactose, in the GOx/HRP/-Gal assembly, and the biocatalytic transformations were probed by following the colored 2,2-azino-bis(3ethylbenzthiazoline)−6-sulfonic acid radical anion (ABTS •− ) (For the enhanced GOx-catalyzed aerobic oxydation of glucose in the cryogel, as compared to the GOx-loaded hydrogel, in the presence of different concentrations of glucose, see Figure S3.1 (Supporting Information) and accompanying discussion).Figure 1B, Panel II, shows the rate of the biocatalytic cascade driven by the GOx/HRP-loaded cryogel, curve (a), and the control GOx/HRP-loaded hydrogel, curve (b).In these systems, the GOxcatalyzed aerobic oxidation of glucose yields gluconic acid and H 2 O 2 , and the resulting H 2 O 2 is used to oxidize 2,2-azino-bis(3ethylbenzthiazoline)−6-sulfonic acid anion (ABTS 2− ), acting as the substrate in the bulk solution, to produce ABTS •− .The rate of ABTS •− formation by the GOx/HRP cascade in the cryogel is 2.5 fold higher as compared to biocatalytic cascades in the hydrogel.Figure 1B, Panel III, depicts the rates of the three-enzyme biocatalytic cascade driven by the -Gal/GOx/HRP-loaded cryogel, curve (a), and the hydrogel, curve (b).In these systems, lactose acts as the substrate solubilized in the bulk solution.The -gal catalyzed hydrolysis of lactose yields glucose and galactose, and the resulting glucose is aerobically oxidized to yield gluconic acid and H 2 O 2 , and the latter H 2 O 2 product acts as the substrate of HRP, oxidizing ABTS 2− to ABTS •− .The rate of the three-enzyme biocatalytic cascade in the cryogel is 2.7 fold higher as compared to the hydrogel matrix.The enhanced biocatalytic processes in the cryogel material are attributed to the structural features of the cryogel matrix.The large pores in the cryogel and the formation of interconnected channels enable the convectional flow of the solution across the channels, leading to effective substrate transfer into the crosslinked polymer matrices in which the biocatalysts are concentrated in confined reaction media, resulting in efficient biocatalysis and cascaded biocatalysis.In contrast, the small-pore structure of the crosslinked hydrogel distributes the biocatalysts between the small-sized pores boundaries, leading to substantially lower reaction rates.(For further SEM images demonstrating the different structural features of the cryogel/hydrogel matrices, see Figure S2, Supporting Information and accompanying discussion).

pH-Stimulated Dynamic Switchable Stiffness Changes in pH-Responsive DNA-based Cryogels (Cryo-PAAm-DNA) Versus Hydrogels (Hydro-PAAm-DNA)
The substantially enhanced mass-transport features demonstrated by the cryogel versus hydrogel matrices, were, then, applied to examine the dynamic stiffness properties of pHresponsive DNA-based pAAm cryo/hydro gels frameworks.Toward this goal, bulk cryo/hydro gels frameworks consisting of pAAm, cooperatively crosslinked by bis-acrylamide (bis-AAm) and nucleic acid duplexes (1)/(2), composed of the cytosinerich pH-responsive acrydite unit (1), an its complementary strand (2) contituent, were prepared (the gels were synthesized by ammonium persulfate (APS)/N,N,N′,N′-tetramethylethane-1, 2-diamine (TEMED)-induced radical polymerization of acrylamide, bis-acrylamide, Cytosine-rich (1) acrydite, and complementary (2) acrydite at molar ratios of 156:3.125:1:1 at −18 °C (cryogel), and 4 °C (hydrogel) conditions).Under these conditions, and at neutral pH = 7.4, a pH-responsive (1)/(2) nucleic acids and bis-AAm crosslinked cryo/hydro gels are formed. [48]he loading of the nucleic acids integrated in the gel matrices correspond to 0.229 μmoles/129 mg of Cryo-PAAm-DNA and 0.227 μmoles/117 mg of Hydro-PAAm-DNA, which translates to ≈95.7% yield for the cryogel, and ≈94.6% yield for the hydrogel, of incorporated nucleic acids (1)/(2) into the corresponding frameworks.(For experimental details see Supporting Information, 4.2) At pH = 5.5 the cytosine-rich strand ( 1) is reconfigured into an i-motif structure, leading to the separation of the duplex units.Thus, by the cyclic treatment of the cryo/hydro gels at pH = 7.4 and pH = 5.5, the matrices are anticipated to be cycled between high and low-stiffness values.Moreover, the bisacrylamide and (1)/(2) duplexes cooperatively crosslinked pAAm cryo/hydro gels were loaded with GOx.In the presence of glucose, the aerobic oxidation of glucose, leads to the formation of gluconic acid and H 2 O 2 , resulting in acidification of the gel matrices by the biocatalytic process, rather than by auxiliary pHchanges.Thus, acidification of the gels through the biocatalytic, GOx-catalyzed aerobic oxidation of glucose is anticipated to affect the stiffness of the matrices.Accordingly, the dynamic stiffness changes of the cryo/hydro gel matrices upon subjecting the gels to auxiliary pH changes or upon treatment of the respective GOxloaded matrices with glucose were probed by rheometry.Figure 2A depicts schematically the pH-stimulated reconfiguration of the cooperatively crosslinked bis-acrylamide and (1)/(2) duplexes pAAm cryo/hydro gels and the resulting stiffness changes of the matrices.Nevertheless, the structural differences between the cryogel and hydrogel, and the mass-transport differences of the solute into the polymer frameworks, suggest that the dynamics of pH-stimulated uncaging of the duplex bridges (1)/(2) might be different in the cryo/hydro gels, and thus, the dynamics of stiffness changes of the matrices might be different.Figure 2B As stated, the large pore interconnected channeled structure of cryogels enables convectionally-controlled mass-transport within enzyme-loaded cryogel matrices, revealing enhanced biocatalytic transformations, as compared to analog biocatalytic processes in hydrogel matrices.The GOx-catalyzed aerobic oxidation of glucose yields gluconic acid and H 2 O 2 .Thus, integration of GOx into the pH-responsive nucleic acid-functionalized pAAm cryogel, cooperatively crosslinked by bis-AAm and (1)/(2) duplex nucleic acid, is anticipated to yield a functional cryogel in which the convectional mass-transport of glucose leads to the efficient biocatalytic aerobic oxidation of glucose to gluconic acid, resulting in the acidification of the cryogel matrix and the effective control over the stiffness of the cryogel framework.This is exemplified in Figure 3A, with the assembly of a GOx-loaded pHresponsive DNA-based pAAm cryogel (CryoPAAm-DNA-GOx) or hydrogel (HydroPAAm-DNA-GOx), cooperatively crosslinked by permanent bis-AAm bridges, and the pH-responsive (1)/(2) duplexes.At pH = 7.4, a stiff cryogel or hydrogel is formed (G' = 120 Pa, and G' = 105 Pa, respectively).Subjecting the gels to glucose results in the aerobic oxidation of glucose and the formation of gluconic acid and H 2 O 2 .Acidification of the gel matrices, results in the separation of the duplex (1)/(2), through the reconfiguration of the strand (1) into an i-motif structure, revealing lower-stiffness.Treatment of the bulk, lower-stiffness gels with a buffer solution at pH = 7.4 separates the i-motif units and restores the cooperatively crosslinked bis-acrylamide and (1)/(2)-bridges higher-stiffness gels.By the cyclic treatment of the gels with glucose/pH = 7.4 -solutions, the reversible switching of the gel matrices is expected.Figure 3B   switchable G'-values of the cryogel, curve (a), and hydrogel, curve (b), treated with glucose 20 mm/pH = 7.4 cycles for time-intervals of 180 min.The biocatalyzed, glucose-stimulated, temporal stiffness changes of the pH-responsive gels are controlled by the concentrations of glucose.Figure 3C, Panel I, depicts the temporal G'-changes of CryoPAAm-DNA-GOx, upon subjecting the cryogel to variable concentrations of glucose.As the concentrations of glucose increase, the temporal G'-values changes, reflecting the stiffness of the cryogel, are higher, consistent with the enhanced pH changes and formation of the enhanced content of imotif constituents.The parallel pH changes of the HydroPAAm-DNA-GOx, in the presence of variable concentrations of glucose, are displayed in Figure 3C, Panel II.Very slow temporal G'-values changes are observed for the hydrogels.That is, the temporal stiffness changes of the CryoPAAm-DNA-GOx matrices are substantially enhanced, as compared to the analog hydrogel matrices.(In all experiments, 23.9 μm, and 24.4 μm of GOx are integrated in the respective cryo/hydro gels, see Figure S4, Supporting Information).The enhanced biocatalyzed-driven stiffness changes of the cryogels, as compared to the hydrogel systems, are attributed to the superior convectionally-dictated masstransport-controlled delivery of the glucose substrate into the cryogel framework, allowing faster and efficient GOx-mediated oxidation of glucose, and faster stiffness changes.(For SEM and confocal microscopy images of large-pore interconnected channels of CryoPAAm-DNA-GOx versus small-pore structure of HydroPAAm-DNA-GOx, see Figure S5, Supporting Information).

Dynamic Bending of Bilayers Composites, Composed of pNIPAM Cryogel Layer/pH-Responsive DNA-based Cryogel Layer (CryoPNIPAM/CryoPAAm-DNA) Versus pNIPAM Cryogel Layer/pH-Responsive DNA-based Hydrogel Layer (CryoPNIPAM/HydroPAAm-DNA)
The improved pH-controlled stiffness changes demonstrated by DNA-based cryogels as compared to hydrogels, stimulated either by auxiliary pH changes or by glucose concentrationsguided pH changes induced by the GOx aerobic catalyzed oxidation of glucose, were then applied to fabricate gel-based devices undergoing macroscopic dynamic mechanical bending.Toward this goal, we assembled bilayer pH-responsive gel devices, as schematically presented in Figure 4A.In the first step, a thermoresponsive poly-N-isopropylacrylamide cryogel (CryoPNI-PAM) sublayer crosslinked by bis-acrylamide was polymerized in a rod-shape mold under cryo-polymerization.In the second step, a pH-responsive layer, consisting of GOx-loaded, DNAfunctionalized pAAm, cooperatively crosslinked by bis-AAm and (1)/(2) nucleic acid bridges, was polymerized onto the CryoP-NIPAM to yield a pAAm cryogel (CryoPNIPAM/CryoPAAm-DNA-GOx) (under cryo conditions −18 °C), or a hydrogel (CryoPNIPAM/HydroPAAm-DNA-GOx) (4 °C).Furthermore, the CryoPNIPAM layer was loaded with the Brilliant blue G dye to identify the resulting bilayer construct.The bilayer construct was extruded from the mold in the form of a stable rod-shape structure (for microscopic characterization of the bilayer constructs, vide infra.Note that in all systems, one layer was always composed of CryoPNIPAM, and the second layer was either in the form of a pH-responsive cryogel (CryoPNIPAM/CryoPAAm) or hydrogel (CryoPNIPAM/HydroPAAm), that allowed the pHstimulated control over the stiffness either by auxiliary pH changes or via the biocatalytic GOx-catalyzed aerobic oxidation of glucose, in the presence of variable concentrations of glucose.Moreover, the thermoresponsive CryoPNIPAM layer was selected as sublayer to induce rapid temperature-controlled stiffness changes as a result of thermal gel-solid phase-transitions, aimed to amplify slower pH-stimulated stiffness changes occurring in the adjacent DNA-based cryogel (or hydrogel), thus allowing to probe the dynamic, temporal, bending of the bilayer device.That is, the bending of the bilayer devices is guided by temperature-stimulated phase-transitions of the CryoPNIPAM layer and by pH/glucose-mediated stiffness changes of the pHresponsive cryo/hydro PAAm-DNA layer.
The fast thermally-induced bending processes of the CryoPNI-PAM layer proceed within two minutes, and after washing of the bent bi-layer construct at pH 7.4, small further structural changes are observed.This thermally-induced curvature is reversible, and upon cooling from 35 to 25 °C the linear rod-shape bi-layer structure is recovered (time scale of 15 min), Figure 4B.The rapid thermally-induced curved bi-layer structure, at pH = 5.5 undergoes, however, a continuous further temporal bending for 13 minutes to yield a highly compact coiled structure, Figure 4C, Panel I (that is not observed at pH = 7.4 on this time-scale!).Enhanced bending is observed at pH = 5.5, Figure 4C The bending processes are enhanced as the concentrations of glucose increase.Figure 4D, Panel II, quantifies the temporal curvature degrees, using Equation 1, where Y corresponds to the distance separating the ends of the curved structures, and X corresponds to the maximum height of the curved structure. [101,102]Evidently, the acidification of the pH-responsive pAAm cryogel layer through the aerobic oxidation of glucose stimulated the temporal bending of the device by the time-dependent decrease in the stiffness of the pAAm layer.As the concentrations of glucose increase this stiffness changes and accompanying temporal bending curvatures are enhanced.
In the next step, the pH-stimulated bending of the CryoP-NIPAM/CryoPAAm bilayer systems was compared to the Cry-oPNIPAM/HydroPAAm bilayer assemblies, using auxiliary pH changes or GOx/glucose biocatalyzed pH changes, Figure 5.The bilayer CryoPNIPAM/HydroPAAm-DNA assembled in the mold in a linear shaped configuration, underwent spontaneous slight bending into a bent configuration opposite to the bending direction upon acidification of the hydrogel layer.This spontaneous equilibrated structure is a result of stiffness differences dictated by the crosslinking and swelling degrees of the two layers, resulting in a slightly, stiffer pAAm layer as compared to the pNIPAM layer in the, as prepared, bilayer system.Heating the bilayer system increases the stiffness of the pNIPAM layer, resulting in the rapid stretching of the bent structure into a linear configuration, and cooling the system at 25 °C, recovers the bent bilayer configuration, Figure 5A, Panel I.By subjecting the system to cyclic temperature cycles between 35 °C (ii), and 25 °C (i), Figure 5A, Panel II, the bilayer device is cycled between the bent and linear structure.Figure 5B, Panel I, depicts the temporal structural changes of the CryoPNIPAM/HydroPAAm-DNA bilayer system, subjected at 35 °C to pH = 5.5 (time-interval of 15 min).The initially bent bilayer structure undergoes rapid transition into the linear structure (within two minutes) and then, a very slow bending into an opposite curvature structure within a time-interval of 15 minutes proceeds.This bending is attributed to slow pHstimulated stiffness changes in the pAAm hydrogel originating from the separation of the i-motif units within the pAAm matrix.(No bending of the linear configuration is observed at pH = 7.4 within this time scale).It should be noted, however, that significant bending of the CryoPNIPAM/HydroPAAm-DNA bilayer is observed on a time scale of 90 min, cf. Figure S7 (Supporting Information).Figure 5B, Panel II depicts the temporal bending of the CryoPNIPAM/CryoPAAm-DNA bilayer, curve (a) versus the bending of the CryoPNI-PAM/HydroPAAm bilayer, curve (b), subjected to pH = 5.5.Evidently, the pH-stimulated temporal bending curvatures of the CryoPNIPAM/CryoPAAm-DNA bilayer are substantially higher, as compared to the CryoPNIPAM/HydroPAAm-DNA system, consistent with convectional flow of the acidic solution, pH = 5.5, and enhanced pH-stimulated changes and accompanying i-motif reconfiguration in the pAAm cryogel matrix.The slower mechanical reconfiguration of the CryoPNIPAM/HydroPAAm-DNA bi-layer assembly as compared to the CryoPNIPAM/CryoPAAm-DNA system, is also reflected in the glucose-driven pH changes in the CryoPNIPAM/HydroPAAm-DNA-GOx system.Figure 5C, Panel I, depicts the temporal bending features of the CryoPNIPAM/HydroPAAm-DNA-GOx system, subjected to variable concentrations of glucose.In these systems, the GOxcatalyzed aerobic oxidation of glucose to gluconic acid and H 2 O 2 proceed in the hydrogel, leading to the acidification of the matrix.Within the time-interval of ≈20 min, no effect of glucose concentrations is observed, and the minute bending of the bilayer is very similar to the bending of the bilayer at pH = 7.4, Figure 5C, Panel II.Thus, within this time scale, the GOx-catalyzed aerobic oxidation of glucose and the accompanying pH changes are negligible, and the minute observed bending within this time scale is mainly ascribed to residual stiffness changes, introduced by phase transition of the pNIPAM layer.Nevertheless, on a substantially longer time scale, the CryoPNIPAM/HydroPAAm-DNA-GOx bilayer construct reveals glucose controlled bending differences and compacted coiled structure (curvature of ≈0.25 mm −1 ) are observed after time scale of 130 min, 10 mm, 120 min, 20 mm, and 108 min, 40 mm (cf. Figure S8, Supporting information), as compared to the time-intervals of 22 min, 10 mm, 18 min, 20 mm, 14 min, 40 mm, observed for the CryoPNIPAM/CryoPAAm-DNA-GOx system.(In all experiments, 19.3 μm, and 21.1 μm of GOx are integrated in the respective cryo/hydro gels).These results are consistent with convectional glucose mass-transport proceeding in the pAAm cryogel that result in enhanced GOxcatalyzed pH changes in the cryogel matrix and faster bending rates.
The CryoPNIPAM/CryoPAAm-DNA-GOx bilayer system and the CryoPNIPAM/HydroPAAm-DNA-GOx bilayer assemblies, revealing significant dynamic temporal bending features (different degrees of bending and different time-scale of bending) due to the stiffness and porosity of the cryogel/hydrogel frameworks, were characterized by SEM and fluorescence microscopy imaging.Figure 6A, Panel I depicts the SEM images of the interphase domain of the CryoPNIPAM/CryoPAAm. Figure 6A, Panel II, depicts the pore size distribution, revealing large pores of the CryoPNIPAM layer (average pore diameter 24 μm) and of the CryoPAAm layer (pore diameter 22 μm) of interconnected channels for both layers of the cryogel/cryogel system.In turn, Figure 6B, Panel I depicts the interphase domain of the CryoP-NIPAM/HydroPAAm bilayer system, and Figure 6B, Panel II, depicts the pore diameter distribution.While the CryoPNIPAM framework reveals large pores interconnected channels (26.7 μm pore diameter), the HydroPAAm layer reveals a highly-dense, small-pore matrix (6.1 μm pore diameter).For the fluorescence confocal microscopy images of the bilayer systems see Figure S6 (Supporting Information), and accompanying discussion.

Conclusions
The present study has coupled the unique functions of cryogels and stimuli-responsive functions of gel matrices, specifically, pH-responsive DNA-based cryogels.The large-pore, interconnected, channels morphology of the cryogel matrices enable the convectional flow of the solute solution across the channels and the effective mass-transport of solute molecules, and functional ingredients loaded in the polymer framework comprising the cryogel.The convectional mass-transported dictated transport of chemicals within the cryogels, lead to enhanced chemical reactions and control over physical properties, as compared to analog hydrogel matrices dominated by smallpore structure and diffusional solute flow.The systems that were addressed in this study: (i) The enhanced operation of biocatalytic processes and biocatalytic cascades in polyacrylamide (pAAm) cryogels, as compared to pAAm hydrogels.(ii) The introduction of pH-responsive DNA-based pAAm cryogels (CryoPAAm-DNA) undergoing substantially enhanced pH-stimulated reconfiguration and separation of the DNA-bridges into the i-motif structures and accompanying stiffness changes, as compared to analog pH-responsive, DNA-based pAAm hydrogel matrices (HydroPAAm-DNA).(iii) The introduction of GOx-loaded pH-responsive DNA-based pAAm cryogels (CryoPAAm-DNA-GOx) demonstrating convectionally-controlled mass-transport of glucose and enhanced, metabolically-driven acidification of the cryogel framework through the aerobic, GOx-catalyzed, oxidation of glucose to gluconic acid.The superior mass-transport of glucose in the cryogel matrix led to enhanced acidification of the cryogel matrix, leading to fast reconfiguration of the DNA-based framework into i-motif constituents and accompanying fast metabolically stimulated changes, as compared to the analog GOx-loaded pH-responsive DNA-based pAAm hydrogel (HydroPAAm-DNA-GOx) matrices.The unique properties of the pH-responsive DNA-based cryogel matrices were, then, applied to develop bilayer mechanical bending device, stimulated by auxiliary pH-changes or by pHchanges, induced by the aerobic biocatalyzed oxidation of glucose by GOx integrated in the device.A bilayer device consisting of a thermo-responsive poly-N-isopropylacrylamide cryogel layer and GOx-loaded pH-responsive DNA-based pAAm cryogel layer (CryoPNIPAM/CryoPAAm-DNA-GOx) was fabricated.The pH-stimulated bending of the device was compared to a bilayer assembly, composed of the pNIPAM cryogel and the GOx-loaded pH-responsive DNA-based pAAm hydrogel (CryoPNIPAM/HydroPAAm-DNA-GOx).Significantly enhanced bending of cryogel/cryogel assemblies under auxiliary pH changes or upon pH changes induced by the GOx-catalyzed aerobic oxidation of glucose as compared to the cryogel/hydrogel bilayer systems were demonstrated.The temporal bending curvatures were controlled by the concentrations of glucose, and the bending processes were reversible.The enhanced bending of the cryogel/cryogel device was attributed to the convectional masstransport-stimulated acidification of the pH-responsive cryogel, and accompanying stiffness changes of the cryogel, through auxiliary pH changes or glucose transport into the GOx-loaded cryogel framework.In fact, the innovation of the present study is reflected by coupling of the bending of cryogel matrices, with stimuli-responsive reconfigurable gel matrices, and particularly, stimuli-responsive DNA-based gels.The convectional transport of other agents, leading to the formation of donor-acceptor complexes, metal-ligand complexes, formation of aptamer-ligand complexes, reversible reconfiguration of G-quadruplex, or triggered assembly and disassembly of triplex nucleic acid structures could be applied to control the properties of the cryogels.Beyond the use of cryogels for controlled robotic mechanical functions of the cryogel matrices, other broad applications of stiffnessswitchable DNA-based cryogels can be envisioned, including the use of these matrices as shape-memory, self-healing, controlled release of loads, and functional matrices for sensing, enhanced catalysis and biocatalysis.

Experimental Section
The nucleic acid sequences used in the study are: (1) /5Acryd/AAAAACCCAATCCCAATCCCAATCCCT-3′ (2) /5Acryd/AAAAATGATTGTGATTGTGACCG-3′ Further experimental details describing the methods to prepare the enzyme-loaded cryogels and hydrogels, the DNA-based cryogels and hydrogels, and the different bilayer constructs are described in Supporting Information.Also, methods to characterize the biocatalytic cascaded activity operating in the gel matrices, the gel matrices structural features and their corresponding stiffness properties with their respective triggering, and the bending curvature characterization are addressed.

Figure 1 .
Figure 1.A) Schematic biocatalytic cascade proceeding in the pAAm/bis-acrylamide crosslinked cryo/hydro gel matrices.B) Time-dependent absorbance changes of ABTS⋅ generated by: Panel I-The GOx-loaded cryogel, curve (a), the GOx-loaded hydrogel, curve (b).Panel II-The GOx/HRP cascade operating in the cryogel, curve (a), and the hydrogel, curve (b).Panel III-The GOx/HRP/-gal cascade operating in the cryogel, curve (a), and the hydrogel, curve (b), using lactose as the substrate.Error bars in the different experiments derived from N = 3 experiments.
, Panel I, curve (a) shows the time-dependent G' changes of the cryogel in state (i) (higher-stiffness) upon subjecting the cryogel to pH = 5.5.Time-dependent decrease in G' values from 120 Pa to ca. value of 55 Pa within 140 min is observed, consistent with the formation of a lower-stiffness cryogel, state (ii).On this timescale almost no temporal G' changes are observed for the hydrogel in state (i), G' ≈ 105 Pa, upon treatment with the buffer, pH = 5.5, curve (b).At substantially longer time-intervals the G' value of the hydrogel drops to 70-75 Pa after ≈7 h.Treatment of the lower-stiffness cryogel, state (ii), with a buffer solution at pH = 7.4 recovers the original state (i), G' value ≈ 120 Pa, and by cyclic treatment of the cryogel at pH = 5.5 and pH = 7.4, the G' value of the cryogel was switched between low and high values, Figure 2B, Panel II, curve a. Cyclic treatment of the hydrogel at pH = 5.5 and pH = 7.4 generated minute G' changes within this time-scale, Figure 2B, Panel II, curve (b).That is, the results demonstrate significant differences in the pH-stimulated stiffness changes of the cryogel versus hydrogel: (i) The ΔG' accompanying the stiffness changes of the cryogel corresponds to ≈65 Pa, whereas the ΔG' accompanying the stiffness changes of the hydrogel corresponds to ≈30 Pa.(ii) The response time for the stiffness changes in the cryo/hydro gels is significantly different.While the ΔG' differ-ences of the cryogel occur within 140 min, the ΔG′ differences observed in the hydrogel occur within ≈7 h!The enhanced stiffness changes of the pH-responsive cryogel, as compared to the hydrogel are attributed to the large interconnected porous channels of the cryogel that enable the convectional flow of the solute and the effective pH-stimulated reconfiguration of the polymer framework.Indeed, SEM images, Figure 2C, and confocal microscopy images (Figure S3, Supporting Information) of the bisacrylamide and (1)/(2) duplexes cooperatively crosslinked pAAm cryo/hydro gels reveal the different porous structures of the nucleic acids-based cryogel versus hydrogel.2.3.Glucose-Controlled, pH-Stimulated Dynamic Reversible Stiffness Changes in Glucose Oxidase (GOx)-Loaded DNA-based Cryogels (Cryo-PAAm-DNA-GOx) Versus Hydrogels (Hydro-PAAm-DNA-GOx) through the GOx-Catalyzed Aerobic Oxidation of Glucose to Gluconic Acid

,
Panel I, curve (a), depicts the temporal G' changes of the CryoPAAm-DNA-GOx matrix treated with glucose, 20 mm.A temporal decrease of the G'-values is observed, and within a time-scale of 180 min, the G'-values drops from 120 Pa to a leveled-off value of 50 Pa.In turn, subjecting the HydroPAAm-DNA-GOx matrix, cooperatively crosslinked by bis-acrylamide and (1)/(2)-bridges to glucose, 20 mm, leads to very slow G' changes within this time-scale, curve (b), and after a time-scale of 180 min, the G' values decrease from G' = 100 Pa to G' = 85 Pa.(Nonetheless, the G' values decreased to the leveled-off value of ≈60 Pa within a substantially longer time-scale of 7 h!). Figure 3B, Panel II, depicts the

Figure 2 .
Figure 2. A) Schematic representation of pH-responsive cryo/hydro-DNA gel matrices and the reversible stiffness-controlled properties of the gels.The left and right schemes represent the state of the bulk gels, according to the pH values, and respectively, the nucleic acids state of the different states are depicted in the enlarged schemes.B) Panel I-Time dependent G' changes of: (a) The high-stiffness, bis-acrylamide and (1)/(2) duplex cooperativelybridged cryogel, treated at pH = 5.5.(b) The high-stiffness, bis-acrylamide and (1)/(2) duplex cooperatively-bridged hydrogel, treated at pH = 5.5.Panel II-Cyclic G' changes upon treatment of: (a) The cryogel at (i) pH = 7.4, (ii) pH = 5.5.(b) The hydrogel at (i) pH = 7.4, (ii) pH = 5.5.C) SEM images and the respective histograms corresponding to the pore analysis of: Panel I-The cryogel.Panel II-The hydrogel.Error bars in the different experiments derived from N = 3 experiments.

Figure 3 .
Figure 3. A) Schematic control over the stiffness properties of the GOx-loaded pH-responsive pAAm cryo/hydro gels cooperatievly crosslinked by bisacrylamide and (1)/(2) bridges.The pH changes controlling the stiffness of the cryo/hydro gels are guided by the GOx-catalyzed aerobic oxidation of glucose to gluconic acid and H 2 O 2 .B) Panel I-Temporal G' changes upon treatment of: (a) The GOx-loaded cryogel with glucose 20 mm, (b) The GOxloaded hydrogel with glucose 20 mm.Panel II-Cyclic G' values of: (a) The GOx-loaded cryogel treated reversibly with glucose 20 mm for 180 min (ii), followed by rinsing with a buffer solution at pH = 7.4 (i).(b) The GOx-loaded hydrogel treated reversibly with glucose 20 mm for 180 min (ii), followed by rinsing with a buffer solution at pH = 7.4 (i).C) Panel I-Time dependent G'-values of the GOx-loaded cryogel in the presence of different concentrations of glucose: (a) 5 mm, (b) 10 mm, (c) 20 mm.Panel II-Time dependent G' values of the GOx-loaded hydrogel in the presence of different concentrations of glucose: (a) 5 mm, (b) 10 mm, (c) 20 mm.Error bars in the different experiments derived from N = 3 experiments.
, Panel II, (a), while only minute time-dependent structural changes are observed at pH = 7.4, Figure 4C, Panel II, (b), indicating that the relatively slow reconfiguration of the (1)/(2) duplexes in the DNA responsive cryogel induce stiffness changes reflected by the dynamic bending of the bilayer construct.The resulting compact coiled structure recovers into the linear bilayer configuration, upon rinsing the system at pH = 7.4, and concomitant cooling to 25 °C.(The bending of the bilayer system within the first two minutes is very similar at pH = 7.4 or pH = 5.5).This phenomenon is further demonstrated by following the temporal bending features of the CryoPNIPAM/CryoPAAm upon operation of the GOx-catalyzed acidification of the pAAm cryogel by the aerobic oxidation of glucose, in the presence of different concentrations of glucose, Figure 4D, Panel I.

Figure 4 .
Figure 4. A) Schematic preparation of a thermoresponsive CryoPNIPAM/CryoPAAm-DNA (or CryoPNIPAM/HydroPAAm-DNA) bilayer construct where the pH-responsive pAAm layer is unloaded or loaded with GOx.The thermally-induced cyclic and reversible bending of the bilayer construct is schematically depicted.B) Panel I-Images corresponding to the reversible, thermally-induced, bending of the CryoPNIPAM/CryoPAAm-DNA system, and quantification of the bending curvature, 1/r, as a function of geometrical parameters associated with the bent structure.Panel II-Cyclic and reversible bending curvature, upon thermal cycling of the system between 25 °C (i), and 35 °C (ii).C) Panel I-Images, at time-intervals, of the CryoPNIPAM/CryoPAAm-DNA bilayer system subjected to an auxiliary buffer solution, pH = 5.5 at 35 °C, and the reversible temporal transition of the compacted coiled bilayer, treated with an auxiliary buffer solution, pH = 7.4 at 25 °C.Panel II-Time-dependent curvature changes of: curve (a) The CryoPNIPAM/CryoPAAm-DNA bilayer treated with the auxiliary buffer solution, pH = 5.5 at 35 °C.curve (b) The CryoPNIPAM/CryoPAAm-DNA bilayer treated with the auxiliary buffer solution, pH = 7.4 at 35 °C.D) Panel I-Images, at time-intervals, of the CryoPNIPAM/CryoPAAm-DNA-GOx bilayer, treated at 35 °C with variable concentrations of glucose.Panel II-Time-dependent bending curvatures of the CryoPNIPAM/CryoPAAm-DNA-GOx bilayer system, treated at 35 °C with variable concentrations of glucose: curve (a) 10 mm, curve (b) 20 mm, curve (c) 40 mm.Error bars derived from N = 3 experiments.

Figure 5 .
Figure 5. A) Panel I-Images corresponding to the reversible, thermally-induced bending of the CryoPNIPAM/HydroPAAm-DNA bilayer system.Panel II-Reversible and cyclic bending curvature corresponding to the thermally-induced reconfiguration of the CryoPNIPAM/HydroPAAm-DNA system.B) Panel I-Images, at time-intervals corresponding to the CryoPNIPAM/HydroPAAm-DNA bilayer subjected to an auxiliary buffer solution, pH = 5.5 at 35 °C.Panel II-Time-dependent bending curvature of: curve (b) The CryoPNIPAM/HydroPAAm-DNA to buffer solution, pH = 5.5 at 35 °C.For comparison, the time-dependent bending curvature of the CryoPNIPAM/CryoPAAm-DNA bilayer system is depicted in curve (a).C) Panel I-Images, at time-intervals of the CryoPNIPAM/HydroPAAm-DNA-GOx bilayer system subjected at 35 °C to variable concentrations of glucose.Panel II-Curves (i), (ii), (iii) correspond to time-dependent bending curvature of the bilayer CryoPNIPAM/HydroPAAm-DNA-GOx in the presence of variable glucose concentrations corresponding to 10 mm, 20 mm, and 40 mm, respectively.For comparison, time-dependent bending curvatures of the CryoPNIPAM/CryoPAAm-DNA-GOx in the presence of variable concentrations of glucose: curve (i)' 10 mm, curve (ii)' 20 mm, and curve (iii)' 40 mm are provided.For the time-dependent curvature changes of the CryoPNIPAM/HydroPAAm-DNA-GOx bilayer construct in the presence of variable concentrations of glucose, at substantially longer timescale, see Figure S8 (Supporting Information), and accompanying discussion.Error bars derived from N = 3 experiments.

Figure 6 .
Figure 6.A) Panel I-SEM images of the CryoPNIPAM/CryoAAm bilayer interphase domain and the images of the respective layers.Panel II-Pore size distribution of the respective layers.B) Panel I-SEM images of the CryoPNIPAM/HydroAAm bilayer interphase domain and the images of the respective layers.Panel II-Pore size distribution of the respective layers.