DNA-Based Hydrogels Loaded with Au Nanoparticles or Au Nanorods


Mar 1, 2019 - Nonetheless, due to the soft nature of the healed hydrogels no “scars” or defective regions in the healed boundary could be detected...
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DNA-Based Hydrogels Loaded with Au Nanoparticles or Au Nanorods: Thermoresponsive Plasmonic Matrices for ShapeMemory, Self-Healing, Controlled Release and Mechanical Applications Chen Wang, Xia Liu, Verena Wulf, Margarita Vázquez-González, Michael Fadeev, and Itamar Willner ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09470 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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DNA-Based

Hydrogels

Loaded

with

Au

Nanoparticles or Au Nanorods: Thermoresponsive Plasmonic Healing,

Matrices Controlled

for

Shape-Memory,

Release

and

Self-

Mechanical

Applications Chen Wang,‡ Xia Liu,‡ Verena Wulf, Margarita Vázquez-González, Michael Fadeev, Itamar Willner*

Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

KEYWORDS: switchable hydrogels, photochemical stiffness control, nucleic acids, melting,

plasmon

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ABSTRACT: Gold nanoparticles (AuNPs) or gold nanorods (AuNRs) are loaded in polyacrylamide hydrogels cooperatively crosslinked by bis-acrylamide and nucleic acid duplexes

or

boronate

ester-glucosamine

and

nucleic

acid

duplexes.

The

thermoplasmonic properties of AuNPs and AuNRs are used to control the stiffness of the hydrogels. The irradiation of the AuNPs-loaded (λ = 532 nm) or the AuNRs-loaded (λ = 808 nm) hydrogels leads to the thermoplasmonic heating of the hydrogels, the dehybridization of the DNA duplexes, and the formation of hydrogels with lowerstiffness. By ON/OFF irradiation, the hydrogels are switched between low- and highstiffness states. The reversible control over the stiffness properties of the hydrogels is used to develop shape-memory hydrogels, self-healing soft materials, and to tailor thermoplasmonic switchable drug release. In addition, by designing bilayer composites of AuNPs- and AuNRs-loaded hydrogels, a reversible thermoplasmonic, light-induced bending is demonstrated where the bending direction is controlled by the stress generated in the respective bilayer composite.

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DNA-based stimuli-responsive hydrogels have attracted substantial recent research efforts.1–3 The signal-triggered reversible reconfiguration of nucleic acids that bridge the polymer chains, provides a versatile means to control the stiffness properties of the hydrogels. Different stimuli to trigger the reconfiguration of nucleic acids were reported, including pH,4–7 metal ions/ligand (e.g., Ag+-ions/cysteamine),8 the formation and separation of G-quadruplexes in the presence of K+-ions/crown ether,2,9 strand displacement

in

the

presence

of

appropriate

nucleic

acid

strands10

and

photoisomerization of trans/cis azobenzene tethered to the nucleic acids.2,11 The control over the stiffness properties of DNA-crosslinked hydrogels was applied to develop shape-memory12 and self-healing hydrogels,13,14 matrices for switchable catalysis,2,9 controlled release,4 and switchable transport through nanopores,15 as well as reversible, mechanical bending materials.16

In addition to the rapid advance in developing stimuli-responsive hydrogel materials, the size- and shape-controlled plasmonic properties of nanomaterials and the derived optical and electronic features17–22 have found broad application in diverse areas, such

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as sensors and biosensors,23–30 catalysis31–33 and photocatalysis,34 nanomedicine (nano-drug carriers, imaging),35,36 energy conversion,37,38 pollutant degradation and environmental safety control,38 and homeland security.39 One important property of plasmonic nanostructures is their photothermal effect.17,40–42 Photoirradiation and excitation of plasmonic nanostructures lead to local heating, and these photothermal properties have been widely used for imaging,43,44 sensing,21,45–47 and other nanomedical applications48,49 such as nanosurgery tools48,49 or phototherapy,48,50–52 water desalination53,54 or as heating source to drive the polymerization chain reaction (PCR) under light.55–58 In addition, plasmonic nanostructures were integrated with thermosensitive hydrogels,59 and light-induced phase transitions of the matrices were reported. In these systems poly isopropylacrylamide (p-NIPAM) thermoresponsive hydrogels loaded with plasmonic nanoparticles or nanorods were used to stimulate hydrogel-to-solid phase transitions and to control over the mechanical properties of the materials60. Also, the application of the hydrogel for dictated growth of cells61 was reported. Nonetheless, different functionalities of stimuli-responsive hydrogels such as

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self-healing, shape-memory or triggered mechanical functions using thermoplasmonic particles are rarely reported.

In the present study we report on the integration of plasmonic gold nanoparticles (AuNPs, λmax= 530 nm) or gold nanorods (AuNRs, λmax= 790 nm) into two different types of DNA-based hydrogels. We demonstrate the wavelength-selective, light-induced and reversible thermoresponsive control over their stiffness properties, and highlight the use of AuNPs/AuNRs-loaded hydrogels as functional materials exhibiting shape-memory and self-healing properties, light-controlled drug release and light-regulated mechanical properties. In contrast to previous DNA-based stimuli-responsive hydrogels that revealed switchable stiffness transitions proceeding on long time-intervals, our plasmondriven, thermoresponsive hydrogels reveal stiffness transitions within short time-scales.

RESULTS AND DISCUSSION

Two types of plasmonic AuNPs/AuNRs-loaded hydrogels were prepared, Scheme 1. The hydrogels Ia and Ib consist of polyacrylamide chains crosslinked by bis-acrylamide and nucleic acid duplexes (1)/(1). AuNPs (λmax = 530 nm, Figure S1 (A-B)) and AuNRs

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(λmax = 790 nm, Figure S1 (C-D)) were integrated into Ia and Ib, respectively. The loading of (1) was evaluated by absorption spectroscopy to be 1:35 ((1):acrylamide), Figure S2. The hydrogels IIa and IIb were prepared by crosslinking of two polyacrylamide chains, PA and PB. PA was modified with boronic acid units and the self-complementary nucleic acid strand (2). PB was functionalized with glucosamine groups and the self-complementary nucleic acid strand (2). The loading of boronic acid units and (2) on PA was evaluated by NMR and absorption spectroscopy to be 1:1:50 (boronic acid:(2):acrylamide), Figure S3 and S4(A). Similarly, the loading of glucosamine units and (2) on PB was determined to be 5:1:64 (glucosamine:(2):acrylamide), Figure S3 and S4(B). The molecular weight of the polymers PA and PB was calculated by Diffusion Ordered Spectroscopy (DOSY) to be 170 KDa and 140 KDa, respectively (Figures S5-S6). Mixing PA and PB in the presence of AuNPs or AuNRs resulted in the hydrogel IIa or IIb, crosslinked by the boronate ester-glucosamine bridges and the duplexes (2)/(2) (For a detailed description of the synthesis and characterization of the hydrogels, see supporting information and Figures S7-S11). Bright-field scanning transmission electron microscopy (BF STEM), high-angle annular dark-field images (HAADF), scanning transmission electron microscopy

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(STEM) and scanning electron microscopy (SEM) images of the hydrogels Ia, Ib, IIa and IIb (Figures S7-S10) exhibit no nanoparticle aggregation inside the hydrogels. The nonaggregated states of the particles in the hydrogels is further supported by the absorption spectra of the AuNPs/AuNRs (Figure S11) and is attributed to electrostatic repulsion between the particles, due to the surface coating functionalities. The AuNPs are stabilized by citrate ligands and the AuNRs are stabilized by polyvinylpyrrolidone and sodium dodecyl sulfate.

Scheme 1. Preparation and light-induced, thermoresponsive stiffness control of AuNPs-loaded (Ia and IIa) or AuNRs-loaded (Ib and IIb) hydrogels crosslinked cooperatively by bis-acrylamide and nucleic acid duplexes (1)/(1) (A) or boronate ester-glucosamine and nucleic acid duplexes (2)/(2)

(B),

respectively.

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Figure 1 shows the characterization of the thermoresponsive hydrogels Ia and Ib by rheometry and SEM. The storage modulus (G’) and the loss modulus (G”) of Ia correspond to 55 Pa and 7 Pa, respectively. Photoirradiation of Ia at 532 nm for a timeinterval of 1 h resulted in a substantially lower stiffness, G’ = 22 Pa, G” = 4.5 Pa, Figure 1(A). By ON/OFF irradiation, the hydrogel was reversibly switched between high- and low-stiffness states, Figure 1(B). Control experiments revealed that the irradiation of Ia with an 808 nm laser source did not affect the stiffness (Figure S12A). In addition, irradiation (λ = 532 nm) of a hydrogel that lacks AuNPs did not yield any noticeable stiffness changes. These results indicate that the stiffness changes are selective to the 532 nm laser source that overlaps the plasmonic absorbance of AuNPs, and attributed to the thermo-induced dehybridization of the nucleic acid duplexes (1)/(1). After switching off the laser source, the hydrogel matrix was allowed to cool down, resulting in the re-hybridization of (1)/(1) and the formation of the stiffer hydrogel, cooperatively crosslinked by bis-acrylamide and the nucleic acid

duplexes (1)/(1). (For the

quantitative assessment of the temperature changes occurring in the hydrogels, vide

infra). The structural changes observed in the SEM images were consistent with the

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laser-induced stiffness changes. While the hydrogel crosslinked by bis-acrylamide and the duplexes (1)/(1) reveals a highly porous matrix, the irradiated hydrogel shows larger pores, consistent with a lower degree of crosslinking (only by bis-acrylamide), Figure 1(C).

In analogy, the AuNRs-loaded hydrogel Ib reveals thermoresponsive, reversible stiffness changes, yet these proceed only upon the irradiation at 808 nm, Figure 1(D). The G’ and G” values of Ib correspond to 55 Pa and 3 Pa, respectively. Upon irradiation at 808 nm the G’ value dropped to 20 Pa (G” = 2 Pa), indicating lower stiffness and switching off the laser source led to the recovery of the stiffer state. This control over the stiffness features of the hydrogel was reversible, Figure 1(E). Furthermore, control experiment revealed that the irradiation of the hydrogel Ib (loaded with AuNRs) with the 532 nm laser did not affect the stiffness of the hydrogel, Figure S12(B), and resulted in a minute temperature change ≤ 5˚C. The absorption spectrum of the AuNRs-loaded hydrogel (Figure S11(B)) reveals a weak absorbance band (OD = 0.2) at λ = 508 nm after subtracting the background absorbance. This band corresponds to the transversal

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plasmon exciton of the NRs. This low absorbance at λ = 508 nm, compared to a high absorbance at λ = 530 nm for the AuNPs-loaded hydrogel (OD = 0.55), leads to the inefficient heating of the NRs-loaded hydrogel upon irradiation at 532 nm. In addition, the structural changes observed in the SEM images are consistent with the lightinduced control over the stiffness of Ib, Figure 1(F). Analogous results were observed with the AuNPs- and AuNRs-loaded boronate ester-glucosamine/(2)/(2) crosslinked hydrogels IIa and IIb (Figures S13-S14). It should be noted that the stiffness of the hydrogels can be further modulated by increasing the molar ratio of the acrylamide monomer units generating hydrogel Ia or by increasing the weight fraction of polymers comprising hydrogel IIa. The results are presented in Table S1.

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Figure 1. (A) Rheometric characterization of Ia: a and a’ correspond to G’ and G” before irradiation; b and b’ correspond to G’ and G” after irradiation at 532 nm. (B) Switchable stiffness control of Ia in dark (i) and upon irradiation at 532 nm (ii). (C) SEM images of Ia in dark (Panel I) and after irradiation at 532 nm (Panel II). (D) Rheometric characterization of Ib: a and a’ correspond to G’ and G” before irradiation; b and b’ correspond to G’ and G” after irradiation at 808 nm. (E) Switchable stiffness control of Ib in dark (i) and upon irradiation at 808 nm (ii). (F) SEM images of Ib in dark (Panel I) and after irradiation at 808 nm (Panel II).

One interesting facet of the thermoresponsive plasmonic AuNPs-loaded hydrogels, as compared to previously reported stimuli-responsive nucleic acid-base hydrogels, is the relatively fast plasmonic-induced transitions between the higher and lower stiffness

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states (ca. 30 minutes vs. several hours for other stimuli-responsive hydrogels). Figure 2 shows the stiffness changes of hydrogel Ia, Panel A, and hydrogel IIa, Panel B, upon irradiation of the hydrogels for different time intervals. As the irradiation time is prolonged the stiffness of the hydrogels decrease, consistent with the enhanced temperature induced separation of the duplex nucleic acids. After ca. 30 minutes the stiffness reaches a lower saturation value, suggesting complete separation of the duplex bridges. The control over the stiffness properties of Ia and IIa was further evaluated by irradiating for a fixed time-interval of 1 h with different laser powers, and as a function of AuNPs concentration. In all cases, an enhanced heating of the hydrogels increases the amount of dehybridized duplexes (1)/(1), resulting in the decrease of the stiffness, Figures S15-S16.

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Figure 2. (A) G’ and G’’ changes of the hydrogel Ia irradiated with 532 nm laser (80 mW) for different time-intervals; (B) G’ and G’’ changes of the hydrogel IIa irradiated with 532 nm laser (80

mW)

for

different

time-intervals.

We further evaluated the temperature changes induced by the irradiation of Ia and Ib. Towards this goal, we made use of the fact that the fluorescence of CdSe/ZnS quantum dots (QDs) is dependent on the temperature and thus the QDs may act as nanothermometers.62 Accordingly, the QDs were incorporated into the hydrogels and the fluorescence intensity was followed at variable temperatures to derive the appropriate calibration curves, Figure S17(A-B). Subsequently, Ia and Ib that include the QDs were irradiated with the 532 nm and 808 nm laser sources (80 mW, 1 h), respectively. From the fluorescence intensities of the QDs and using the calibration curves, we found that the temperature rose to 45˚C in Ia and 50˚C in Ib (Figure S17(CD)). The temperature-induced control over the stiffness of the hydrogels by means of the NPs/NRs was further supported by control experiments, characterizing the stiffness properties of the AuNPs/NRs-loaded hydrogels in an aqueous solution at 45˚C and

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50˚C, Figure S18. The rheometric experiments demonstrated that the externally-heated hydrogels also reveal lower stiffness values, comparable to the stiffness features observed upon plasmonic irradiation of the particles.

The control over the stiffness of the thermoresponsive hydrogels was then used to develop shape-memory hydrogels. Shape-memory materials have attracted substantial interest due to their abilities to encrypt information,63–66 their use as sensors,67–69 and the development of shape-driven drug release matrices.4,70 Shape-memory polymers are functional polymers that undergo triggered transitions between a permanent shape into a metastable state of a perturbed shape, which includes an internally embedded code to regenerate the original state in the presence of a counter trigger.71–74 The development of DNA-based hydrogels, and particularly the coupling of permanent nucleic acid linkers and signal-triggered reconfigurable nucleic acids, provides a versatile paradigm to design shape-memory hydrogels.1 In these systems, the two coupled crosslinking motifs generate stiff, shaped hydrogels. The triggered separation of the stimuli-responsive bridges lead to the lower-stiffness and shapeless state, where

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the remaining permanent linkers provide a memory code (appropriate entanglement of the polymer chains) to restore the original stiff and shaped state, stabilized by the two cooperative crosslinkers. Different switchable stimuli, such as K+-stabilized Gquadruplexes,13,14 pH-controlled formation and dissociation of i-motif6,7 or DNA triplexes4 were reported to yield shape-memory hydrogels. The thermo-controlled stiffness properties of Ia, IIa and Ib, IIb by plasmonic AuNPs or AuNRs allowed us to develop plasmonic-induced shape-memory hydrogels, Figure 3(A). In Figure 3(B), the shape-memory properties of the 532 nm-responsive Ia is demonstrated. The cooperative crosslinking of the hydrogel by bis-acrylamide and the duplexes (1)/(1) allowed the generation of AuNPs-loaded hydrogel in a Teflon mold, that was extruded as a triangle-shaped hydrogel. Irradiation of the triangle-shaped Ia with the 532 nm laser source led to the heating of the hydrogel and the transition of the triangle-shaped state into a shapeless, quasi-liquid state, that recovers the triangle-shaped state after switching off the light and cooling the sample (the recovery of the shaped hydrogel proceeds within 1 h). The transitions between the shaped and shapeless states were reversible (the shaped hydrogel was recovered for at least four ON/OFF irradiation

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steps). Similarly, Figure 3(C-E) depicts the shape-memory properties of the hydrogels Ib, IIa and IIb, respectively. By ON/OFF irradiation, the hydrogels underwent reversible and switchable transitions between the shapeless, low-stiffness states and the stiff, triangle-shaped states. It should be noted that irradiation of Ia and IIa with the 808 nm laser source did not lead to any shape transitions, implying that the shape transitions are, indeed, stimulated by the excitation of the plasmonic AuNPs. Also, irradiation of the triangle-shaped Ib and IIb with the 532 nm laser source did not induce any structural changes. That is, the thermo-induced separation of the nucleic acid duplexes resulted in the shapeless states of the hydrogel matrices, where bis-acrylamide or boronate esterglucosamine provides an internal memory code. In the low-stiffness state, the entanglement of the polymer chains retains the tethers (1) or (2) in confined orientations, enabling the memory-guided hybridization to reform the triangle-shaped hydrogels upon cooling.

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Figure 3. (A) Schematic representation of the reversible shape-memory properties of the hydrogels. Reversible light-induced shape-memory transitions of Ia (B), Ib (C), IIa (D) and IIb (E), respectively.

Besides the shape-memory functions of the hydrogels, the related phenomenon of self-healing was explored, Figure 4(A). Self-healing of hydrogels has attracted research efforts directed to biomedical tissue engineering applications.75–78 Figure 4(B-C) shows the self-healing properties of Ia and IIa, respectively. The stiff hydrogels were irradiated with the 532 nm laser source, resulting in the transition of the stiff states to the lowstiffness states. The resulting low-stiffness hydrogels were cut into two pieces that were physically connected under 532 nm laser source. Switching off the light source and

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allowing the hydrogels to cool down resulted in integrated, intact, self-healed matrices. Similar self-healing features were demonstrated for Ib and IIb, Figure 4(D-E). Control experiments revealed that the physically connected pieces exhibiting high-stiffness properties did not lead to integrated and self-healed hydrogels, implying that free nucleic acids included in the low-stiffness hydrogel pieces are essential to heal the hydrogel matrix (Figures S19-S20). That is, the hybridization of the free nucleic acids provides the self-healing mechanism. It should be noted that tensile stress values of the hydrogels after the self-healing process confirm the formation of intact healed hydrogels. Nonetheless, due to the soft nature of the healed hydrogels no “scars” or defective regions in the healed boundary could be detected. Table S2 summarize the tensile stress values of the as prepared and the healed hydrogels Ia and IIa in the presence of different concentrations AuNPs. The results demonstrate that the healed hydrogels reveal slightly lower tensile stress values as compared to the original tensile stress values of the hydrogels. The tensile stress values of the healed hydrogels confirm, however, the formation of intact matrices, and the lower tensile stress values might be attributed to defective sites of incomplete duplex bridging at the healed

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boundaries. Furthermore, the results demonstrate that the tensile stress values, before and after self-healing are dependent on the loading degree of the AuNPs. As the concentration of the NPs increases, the tensile-stress values are higher, consistent with the higher stiffness of the hydrogels (cf. Figure S15B and S16B). We note, however, that the self-healing process involves a primary irradiation of the hydrogel pieces. This is essential to yield soft hydrogel pieces that can be physically connected and selfhealed by the formation of duplexes at the inter-connected boundary.

Figure 4. (A) Schematic representation of the self-healing of hydrogels Ia, IIa, Ib and IIb. The thermoplasmonic activation of the respective hydrogels leads to the generation of low-stiffness

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hydrogels that after being cut into two pieces, rejoin into the healed hydrogels upon switching off the respective light sources. Self-healing of Ia (B), IIa (C), Ib (D) and IIb (E), respectively.

The control over the stiffness properties of the hydrogels was further used to stimulate light-induced drug release from the different hydrogels. The doxorubicin anticancer drug was loaded into the respective hydrogel matrices during the formation of the hydrogels. Figure 5(A) shows the drug release from Ia upon the switchable ON/OFF irradiation with the 532 nm laser source, curve a. The irradiation of the hydrogel stimulated a fast drug release and switching off the light source retarded the release process. For comparison, Figure 5(A), curve b, shows the drug release from the same hydrogel without irradiation. Very inefficient ON/OFF release from the hydrogel that lacks AuNPs was observed. It should be noted that irradiation of Ia with the 808 nm light source yielded a similar inefficient drug release pattern. Thus, the thermo-induced stiffness changes in Ia stimulate the effective drug release and the ON/OFF irradiation of Ia provides a means to control the dose of the drug released from the hydrogel. In addition, Figure 5(B), curve a, depicts the switchable drug release from Ib upon ON/OFF

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irradiation with the 808 nm laser source. Effective release of doxorubicin proceeded under irradiation, consistent with the formation of a low-stiffness state (due to the thermal plasmon-induced separation of the duplexes (1)/(1)). Similar switchable drug release from Ib and IIb and time dependent drug release functions from Ia, IIa, Ib and IIb were demonstrated (Figure S21).

Figure 5. (A) Switchable release of doxorubicin from Ia upon switching ON and OFF the light source, λ= 532 nm (curve a) and without irradiation (curve b). (B) Switchable release of doxorubicin from Ib upon switching ON and OFF the light source, λ= 808 nm (curve a) and without irradiation (curve b).

Finally, the control over the stiffness properties of the hydrogels was used to construct stimuli-responsive hydrogel composites exhibiting triggered, mechanical bending properties. In a previous study,16 we demonstrated that the guided-bending of bilayered, stimuli-responsive, macroscopic hydrogels can be controlled by the stiffness

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of one of the hydrogel layers. That is, the increase of the stiffness of one of the layers resulted in the stress-induced bending of the bilayer composite. Different triggers, such as pH or K+-ion-assisted formation of G-quadruplexes and their separation by crown ether were used to stimulate the switchable and reversible bending of bilayered hydrogels, controlled by the respective hydrogel in which the trigger-stimulated stress proceeds. In addition, knowing the effective Young's moduli of the respective bilayer composites in different states, a quantitative model for the evaluation of the bending curvature was developed.

Figure 6. Reversible thermoplasmonic light-induced bending of the Ia/Ib (A) and the IIa/IIb (B) bilayer composites. (C) Schematic representation of the geometrical parameter, and effective

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Young’s moduli, Yn, used to calculate the curvatures of the respective irradiated bilayer composites (see supporting information the model for the evaluation of the bending curvature).

The ability to control the stiffness by light suggested that, by designing bilayered hydrogels loaded individually with AuNPs or AuNRs, the directional light-induced bending of the hybrid hydrogels could be accomplished. Figure 6(A) shows the reversible, light-induced bending of the Ia/Ib bilayer composite. Irradiation of the bilayer composite, λ = 532 nm, resulted in the decrease of the stiffness of the lower layer Ia and the upward bending of the composite. Switching off the light yielded the recovery of the linear bilayer with original stiffness. Further irradiation of the bilayer composite, λ = 808 nm, resulted in the thermoplasmonic decrease of the stiffness of the upper layer Ib and the stress-induced downward bending of the bilayer composite. Again, switching off the light recovered the linear composite. Similar selective bending features were observed for the bilayer composite IIa/IIb, Figure 6(B). Knowing lengths (Ln), width (b) and thickness (cn) of the bilayer composites and the effective Young's moduli (Yn), Figure 6(C) and Table S3, we are able to calculate the curvatures and compare them to the

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experimentally obtained curvatures of the bilayer composites, Table S4. A very good agreement between the experimental and the calculated values was obtained.

In addition to the different applications of the plasmonic thermoresponsive hydrogels discussed above, one could add the potential use of the drug-loaded hydrogels in nanomedicine. The soft properties of the hydrogels and their injectability into target tissues turn the thermoresponsive hydrogels into ideal materials for the targeted lightinduced

drug

release.

As

a

first

step

toward

this

goal,

we

applied

the

doxorubicin/AuNPs-loaded or doxorubicin/AuNRs-loaded thermoresponsive carrier for the light-induced release of the anti-cancer drug doxorubicin and the evaluation of the cytotoxicity of the released drug on MDA-MB-231 breast cancer cells, Figure 7. In these experiments, the MDA-MB-231 cells in the growth medium were treated with the hydrogel matrices loaded with the doxorubicin/AuNPs or the doxorubicin/AuNRs. Prior to the introduction of the cells, the respective hydrogels were irradiated for 10 minutes, 20 minutes and 30 minutes to release the drug from the hydrogel matrices. The cytotoxic effects of the resulting hydrogels on the MDA-MB-231 cells were monitored

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after a time-interval of three days. Appropriate control experiments were conducted to probe the plasmonic thermoresponsive release of the drug on the cytotoxicity towards the

MDA-MB-231

cells.

Figure

7(A)

shows

the

cytotoxic

effect

of

the

doxorubicin/AuNPs-loaded hydrogel Ia on the MDA-MB-231 cells after three days. The cell viability after three days corresponded to 15%, 10% and 5% for the hydrogels irradiated at 532 nm for 10, 20 and 30 minutes, respectively. These results are consistent with the enhanced release of the drug into the hydrogel matrix as the initial irradiation was prolonged. Control experiments revealed that the doxorubicin/AuNPsloaded hydrogel in dark or upon irradiation at 808 nm had minute cytotoxicity toward the cells (80% and 85% cell viability, respectively). These results are consistent with the selective release of the doxorubicin from the drug-loaded AuNPs hydrogel Ia, upon irradiation at λ = 532 nm. The minute cytotoxicity of the non-irradiated, or 808 nmirradiated hydrogels are attributed to leakage of doxorubicin from the stiff hydrogel matrix into the growth medium. Similarly, Figure 7(B) shows the cytotoxic effect of the doxorubicin/AuNRs-loaded hydrogel Ib on the MDA-MB-231 cells after three days. The cell viability after three days corresponded to 35%, 17% and 8% for the hydrogels

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irradiated at 808 nm for 10, 20 and 30 minutes, respectively. Control experiments revealed that the hydrogel Ib in dark or upon irradiation at 532 nm had minute cytotoxicity toward the cells (80% and 76% cell viability, respectively). These results are consistent with the selective release of the drug from the AuNRs-loaded hydrogel Ib, upon irradiation at λ= 808 nm.

Figure 7. Cytotoxicity of the doxorubicin/AuNPs- and doxorubicin/AuNRs-loaded hydrogels toward MDA-MB-231 breast cancer cells. The bars represent the cell viability after the treatment of the cells with doxorubicin-loaded hydrogels for three days. (A) Entry I: cells treated with the doxorubicin/AuNPs-loaded hydrogel Ia irradiated with the 532 nm light source for 10, 20 and 30 minutes. Entry II: untreated cells in growth media; untreated cells in growth media and HEPES buffer. Entry III: control experiments, where the cells were treated with doxorubicin/AuNPsloaded hydrogel Ia in dark or upon irradiation with 808 nm light source for 30 minutes. (B) Entry I: cells treated with the doxorubicin/AuNRs-loaded hydrogel Ib irradiated with the 808 nm light

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source for 10, 20 and 30 minutes. Entry II: untreated cells in growth media; untreated cells in growth media and HEPES buffer. Entry III: control experiments, where the cells were treated with doxorubicin/AuNRs-loaded hydrogel Ib in dark or upon irradiation with 532 nm light source for 30 minutes.

CONCLUSION

In conclusion, the present study has introduced AuNPs- or AuNRs-loaded DNAbased matrices as functional, thermoresponsive, plasmonic materials that reveal shapememory, self-healing, controlled release and mechanical properties. Two different acrylamide-based hydrogels crosslinked by bis-acrylamide and nucleic acid duplexes or boronate ester-glucosamine and nucleic acid duplexes were prepared, and the hydrogels were loaded either with AuNPs or AuNRs. Photoirradiation of the AuNPs- or AuNRs-loaded hydrogels led to the wavelength-selective, plasmonic heating of the hydrogels, resulting in the dehybridization of the DNA duplexes and the formation of the hydrogels in low-stiffness states. Upon switching off the light source, the hydrogels

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recovered the stiff states that are stabilized by two crosslinking motifs, e.g., bisacrylamide/duplexes or boronate ester-glucosamine/duplexes. The significant results demonstrated in the study include: (i) The plasmonic heating of the hydrogels provides a versatile means to control the stiffness properties of hydrogels; (ii) The use of different shaped plasmonic nanostructures (AuNPs or AuNRs) enables the tunable, wavelengthselective and switchable control over the stiffness of the hydrogels; (iii) The response time of the thermo-sensitive transitions is significantly shorter as compared to the timeintervals of DNA-based hydrogels triggered by chemical means, e.g., strand displacement, pH or the formation of G-quadruplexes and their separation in the presence of K+-ions/crown ether.

The present study has demonstrated versatile applications of the thermoresponsive, plasmonic nanostructure-loaded hydrogels, including the development of reversible shape-memory materials, the use of these hydrogels as light-guided self-healing matrices, the use of these matrices for controlled drug release (e.g., the anti-cancer drug, doxorubicin) and the light-induced, spatially-dictated bending of bilayered

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hydrogels. These results provide a rich arsenal of applications in nanomedicine (e.g., dose-controlled drug release, tissue-engineering), functional actuators (e.g., in microfluidic devices) and light-induced pumps (e.g., through pores). In addition, the shape-memory hydrogels might act as sensors or information storage devices. Finally, the light-stimulated, stress-induced deformation of the hydrogels might be extended to design moveable structures of enhanced complexities for robotic applications. We note, however, that the results spark numerous future capabilities, such as the light-induced thermal activation of plasmonic hydrogels on surfaces for solar-light induced switchable wettability or switchable catalytic functions.

EXPERIMENTAL SECTION

Chemicals. 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), sodium phosphate dibasic, sodium phosphate monobasic, acrylamide (AAm) solution (40%), bisacrylamide (BAA), acrylic

acid,

3-(acrylamido)phenylboronic

acid

(AAPBA),

D-(+)-glucosamine

hydrochloride, ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine

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(TEMED), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (irgacure D-2959), 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfoNHS), gold chloride trihydrate (HAuCl4∙3H2O), sodium citrate, silver nitrate (AgNO3), cetrimonium bromide (CTAB), sodium borohydride (NaBH4), hydrochloride (HCl), ascorbic acid, sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP, Mw = 40000) and CdSe/ZnS auantum dots (QDs) were purchased from Sigma-Aldrich. Desalted 5′end acrydite-modified nucleic acid strands were purchased from Integrated DNA Technologies Inc. (Coralville, IA). Doxrubicin (DOX) was purchased from Life Technologies Corporation. Ultrapure water purified by a NANOpure Diamond instrument (Barnstead International, Dubuque, IA, USA).

Sequences of the DNA strands. (1)5’-Acrydite-AAATTCGCGCGCGAA-3’, (2) 5’Acrydite-AAATTCGCGCGAA-3’.

Synthesis of the hydrogel Ia and Ib. A 120 L HEPES buffer (HEPES, 10 mM, MgCl2, 100 mM, pH = 7.0) that contains AAm (5 M, 12 L), BAA (0.01 M, 3 L), irgacure (0.4 M, 2.4 L) and DNA (1) (5 mM, 8 L) was prepared and AuNPs (80 nM) or AuNRs (50 nM) were added

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into the solution. The solution was bubbled for 10 min with nitrogen and then irradiated under UV light for 10 min to form the hydrogels Ia and Ib. Preparation of the hydrogel Ia and Ib in the triangle-shaped mold and the switchable laser-induced transitions between different states

A 120 L HEPES buffer (HEPES, 10 mM, MgCl2, 100 mM, pH = 7.0) that contains AAm (5 M, 12 L), BAA (0.01 M, 3 L), irgacure (0.4 M, 2.4 L) and DNA (1) (5 mM, 8 L) was prepared and AuNPs (80 nM) or AuNRs (50 nM) were added into the solution. Then the solution and the mold covered by parafilm were bubbled for 10 min with nitrogen. Immediately after bubbling, the solution was added into the mold and bubbled for another 10 min with nitrogen. Then the solution in the mold was irradiated under UV light for 10 min to form the shaped hydrogels Ia and Ib. To trigger the switchable transitions between low-stiffness and high-stiffness states, the hydrogels Ia and Ib were extruded from the mold and irradiated with 532 nm and 808 nm laser light source for 1h, respectively, and cooled down for 1 h at room temperature.

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Preparation of the hydrogel IIa and IIb in the triangle-shaped mold and the switchable laser-induced transitions between different states

The dried copolymer samples were dissolved in a 100 µL HEPES buffer (10 mM HEPES, 100 mM MgCl2, pH = 8.0) that contains AuNPs (50 nM) or AuNRs (20 nM). The resulting solution was heated to 90 °C and then poured into the triangle-shaped mold. After incubation overnight, the hydrogels IIa and IIb were extruded from the mold. To trigger the switchable transitions between low-stiffness and high-stiffness states the hydrogels were irradiated with 532 nm and 808 nm laser light source for 1 h, respectively, and cooled down for 1 h at room temperature.

Preparation of the bilayer hydrogel and laser-induced transitions between linear shape and curved shape

AAm (5 M, 15 L), BAA (0.01 M, 15 L), irgacure (0.4 M, 3.75 L) and DNA (1) (5 mM, 8 L) were mixed in 53.25 L HEPES buffer (HEPES, 10 mM, MgCl2, 100 mM, pH = 7.0), and AuNPs (80 nM) or AuNRs (50 nM) were added into the solution. Then the solution and the mold, covered by parafilm, were bubbled for 10 min with nitrogen.

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Immediately after bubbling, the first solution that contains AuNPs (100 L) was added into the mold and bubbled for another 10 min with nitrogen. Then the solution in the mold was irradiated under UV light for 10 min to form the first layer of hydrogel Ia. After the first layer was formed, the solution that contains AuNRs (100 L) was added onto the top of the first layer and bubbled for 10 min with nitrogen. Subsequently, the solution in the mold was irradiated for 10 min to form the bilayered hydrogel. To trigger the switchable transitions between linear shape and curved shape the bilayer composite was extruded from the mold and irradiated with 532 nm or 808 nm laser light source for 1 h, respectively, and then cooled down for 1 h at room temperature.

For additional details on the synthesis of AuNPs and AuNRs, the preparation of the triangle-shaped hydrogels IIa and IIb, the loading of the hydrogels with doxorubicin (DOX), and the model for the evaluation of the bent curvature, see supporting information.

ASSOCIATED CONTENT

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Supporting Information. Experimental section; TEM images and absorption spectra of AuNPs and AuNRs; 1H NMR and DOSY of polymers; drug release from the hydrogels and evaluation of the bent curvature.

AUTHOR INFORMATION

Corresponding Author E-mail: [email protected]

Author Contributions ‡Chen Wang and Xia Liu contributed equally to this work.

ACKNOWLEDGMENT The authors acknowledge financial support from the Israel Science Foundation.

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