Polydopamine Nanoparticles Modulating Stimuli-Responsive PNIPAM

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Polydopamine nanoparticles modulating stimuliresponsive PNIPAM hydrogels with cell/tissue adhesiveness Lu Han, Yanning Zhang, Xiong Lu, Kefeng Wang, Zhenming Wang, and Hongping Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11043 • Publication Date (Web): 06 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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ACS Applied Materials & Interfaces

Polydopamine nanoparticles modulating stimuli-responsive PNIPAM hydrogels with cell/tissue adhesiveness Lu Han1 , Yanning Zhang1 , Xiong Lu1,2*, Kefeng Wang2, Zhenming Wang1, Hongping ★

★

Zhang3

1

Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, China 2

National Engineering Research Center for Biomaterials, Genome Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, China

3

Engineering Research Center of Biomass Materials, Ministry of Education, School of

Materials Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China

*Corresponding author: Xiong Lu Address: Chengdu, Sichuan Province, China Telephone: 86-28-87634023 Email: [email protected].

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Abstract Stimuli-responsive hydrogels can respond to stimuli by phase transformation or volume change and exhibit specific functions. Near-infrared (NIR) responsive hydrogel is a type of stimuli-responsive hydrogel, which can be precisely controlled by altering the radiation intensity, exposure time of the light source, and irradiation sites. Here, polydopamine nanoparticles (PDA-NPs) were introduced into a poly (N-isopropylacrylamide) (PNIPAM) network to fabricate a PDA-NPs/PNIPAM hydrogel with NIR responsibility, self-healability, and cell/tissue adhesiveness. After incorporation of PDA-NPs into the hydrogel, the PDA-NPs/PNIPAM hydrogel showed phase transitions and volume changes in response to NIR. Thus, the hydrogel can achieve triple response effects, including pulsatile drug release, NIR-driven actuation and NIR-assisted healing. After coating PDA-NPs onto hydrogel surfaces, the hydrogel showed improved cell affinity, good tissue adhesiveness, and growth factor/protein immobilization ability because of reactive catechol groups on PDA-NPs. The tissue adhesion strength to porcine skin was as high as 90 KPa. In vivo full-skin defect experiments demonstrated that PDA-NPs coating on the hydrogel and an immobilized growth factor had a synergistic effect on accelerating wound healing. In summary, we combined thermo-sensitive PNIPAM and mussel-inspired PDA-NPs to form a NIR-responsive hydrogel, which may have potential applications for chemical and physical therapies. Key word: NIR responsive, polydopamine nanoparticles, poly (N-isopropylacrylamide), adhesive, hydrogel

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1. Introduction Stimuli-responsive hydrogels can respond to external stimuli and alter their structural or properties depending upon environmental changes; thus, these materials have broadly promising applications, such as controlled drug delivery, sensors, and tissue engineering.1-2 Various stimuli, including temperature,3-4 pH,5-6 light,4,7and electrical8-9 or magnetic field,1, 6 have been employed to elicit changes in one or more properties of these hydrogels. Among these stimuli, near-infrared (NIR) light has been reported to be suitable for biological applications because it shows remarkably deep tissue penetration ability with low damage to biological specimens and living tissues.10 In addition, light can be applied instantaneously, making light-responsive hydrogels highly advantageous for various applications.

Poly(N-isopropylacrylamide) (PNIPAM)-based -responsive

hydrogels

because

of

their

hydrogels

show promise as

thermo-sensitivity,

stimuli

biocompatibility,

and

flexibility.11-12 Strategies have been developed to improve the poor response of PNIPAM hydrogels to NIR irradiation, such as introducing NIR-responsive nano-components, including gold nanorods,13-14 Fe3O4 nanoparticles,15 carbon nanotubes,16 graphene nanosheets,11 and graphene oxide nanosheets,17 into the hydrogel system. However, these nano-components are not easy to be uniformly dispersed in the hydrogels, which may decrease the responsive characteristics of hydrogels.18-19 In addition, these nanomaterials show dose-dependent toxicity to mammalian cells, and their long-term biocompatibility must be considered when applied for biomedical engineering. For instance, metallic nanoparticles are poorly biometabolized and the safety of the metal itself is a big concern.20-21

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Carbon-based nanomaterials have been demonstrated to induce toxic responses, such as oxidative stress and pulmonary inflammation.22-23 Especially, these light responsive hydrogels generally show poor cell-adhesive properties. However, for biomedical applications, the hydrogel should exhibit not only biocompatibility, but also cell/tissue adhesiveness for cell attachment and tissue integration.24

Based on the bio-adhesion mechanism of marine mussels, mussels-inspired materials shed a light on designing and synthesizing hydrogels with good cell affinity and tissue adhesiveness.25-26 Polydopamine (PDA), which has a similar structure to that of mussel-adhesive proteins,27 shows high adhesion to a wide range of substrates by forming covalent and/or non-covalent interactions with substrates. In addition, PDA also has good biocompatibility. Previous studies employed PDA coatings to modify material surfaces, and demonstrated that the PDA coatings can promote cell adhesion and proliferation.28-30 Recently, it is reported that PDA nanoparticles (PDA-NPs) have potential as photothermal agents with good biological functions.31 Thus, incorporating PDA-NPs in to a hydrogel system may produce hydrogel with both high cell affinity and NIR responsiveness.

In this study, a NIR-responsive PNIPAM hydrogel (PDA-NPs/PNIPAM hydrogel) with good cell/tissue adhesiveness was designed and modulated by PDA nanoparticles (PDA-NPs), as shown in Figure 1. First, PDA-NPs were prepared by oxidative self-polymerization. Next, PDA-NPs were dispersed in N-isopropylacrylamide (NIPAM) monomer solution to form PDA-NPs/PNIPAM hydrogel by free radical polymerization in the presence of an initiator

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and cross-linkers. Because of the excellent photo-thermal properties of PDA-NPs, the PDA-NPs/PNIPAM hydrogel showed phase transition and volume changes under NIR laser irradiation, thereby the hydrogel achieved triple response effects, including pulsatile drug release, NIR-driven actuation and NIR-assisted healing. Microstructure changes in the hydrogel before and after NIR irradiation were observed by scanning electron microscopy (SEM). The PDA-NPs also served as functional nanofillers to reinforce the hydrogel network, resulting a more elastic hydrogel, which was characterized by rheological test and swelling tests. Moreover, in order to enhance the cell/tissue adhesiveness of the hydrogels, PDA-NPs were further coated onto the PDA-NPs/PNIPAM hydrogel surfaces. The tissue adhesiveness and cell affinity of PDA-NPs on the hydrogels were investigated in vitro. The biocompatible, cell-affinitive, and tissue-adhesive hydrogel may be further employed as a wound dressing to accelerate tissue repair as demonstrated by our vivo full-skin wound defect experiment.

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Figure 1. Schematic of the PDA-NPs/PNIPAM hydrogel fabrication. (a) (i) PDA-NPs were prepared by oxidative self-polymerization; (ii) PDA-NPs/PNIPAM hydrogel was obtained by free radical polymerization, and PDA-NPs in the hydrogel served as functional cross-linkers to reinforce the hydrogels. (b) NIR-driven pulsatile drug release. (c) NIR-assisted healing. (d) PDA-NPs coated on hydrogel surfaces to enhance cell/tissue adhesiveness of the hydrogels. (e) Cells attached on the PDA-NPs coated PDA-NPs/PNIPAM hydrogels. (f) Adhesive hydrogel served as wound dressing in a full-skin wound defect model.

2. Experimental section 2.1. Materials. Polydopamine,

N-isopropylacrylamide

(NIPAM),

ammonium

6


persulfate

(APS),

N,

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N′-methylene bisacrylamide (BIS), and tetramethylethylenediamine (TMEDA) were purchased from Sigma-Aldrich (USA). Epidermal growth factor (EGF) was purchased from Shanghai Primegene Bio-Tech (Shanghai, China). Enzyme-linked immunosorption assay kits were purchased from R&D (USA). Fetal bovine serum (FBS), DMEM, and 1% penicillin-streptomycin solution were purchased from HyClone (USA). All other reagents and solvents were of reagent grade.

2.2 Synthesis of PDA-NPs PDA-NPs were synthesized via an oxidation and self-polymerization dispersion polymerization procedure as described in our previous study.28 Briefly, first, 2 mL NH4OH was mixed with 80 mL ethanol and 180 mL deionized water and stirred at 25 °C. Second, dopamine (DA) powder was dissolved in 20 mL deionized water and then poured into the above mixture. Finally, the mixture was stirred to allow DA polymerization at room temperature in the dark. After 30 h, the mixture was centrifuged (13000 g, 10 min) and purified by with ethanol. The PDA-NPs were obtained after drying. The morphology of PDA-NPs was observed by SEM (JSM 6390, JEOL, Japan). The particle size and polydispersity index (PDI) of the PDA-NPs was measured by dynamic light scattering (DLS) using a laser particle analyzer (ZETA-AIZER, Malvern, UK). The characterization results reveal that PDA-NPs are spherical in shape, with an average diameter of approximately 260 nm (Figure S1), and the PDI is 0.221, which demonstrates that PDA-NPs has uniform size distribution.

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2.3. Preparation of PDA-NPs/PNIPAM hydrogel The PDA-NPs/PNIPAM hydrogel was synthesized by in situ free radical polymerization of NIPAM in the PDA-NPs suspension. Briefly, PDA-NPs were first dispersed in 2 mL water by ultrasonic radiation for approximately 30 min. Subsequently, 0.226 g NIPAM monomers and BIS (0.002 g) were added to the PDA-NPs suspension, and then suspension was degassed and nitrogen-saturated under continuous and vigorous stirring in an ice-water bath for 20 min to remove dissolved oxygen. Finally, APS (1.1 mg) and catalyst TEMED (1 µL) were added while stirring. The solution was immediately transferred into a mold for polymerization at room temperature for 6 h to obtain the PDA-NPs/PNIPAM nanocomposite hydrogel. The compositions of various hydrogels were listed in Table S1.

2.4. Characterizations of PDA-NPs/PNIPAM hydrogel Rheological test Dynamic rheological tests of the hydrogels were conducted at room temperature using a Rheometric Scientific HAAKE (MARS, Germany) strain-controlled rheometer equipped with 20-mm of parallel plates. The hydrogels were loaded into a 0.5 mm gap between the plates and allowed to relax until the normal force was zero. Strain amplitude sweeps (0.01-100%) were first performed to determine the linear viscoelasticity region. Dynamic frequency sweeps were performed at angular velocities ranging from 0.01 to 10 Hz at 1.0% strain amplitude (liner region). All rheological measurements were performed in triplicate.

Swelling ratio

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To measure the swelling ratios of the hydrogels, samples (pure PNIPAM, 0.15 wt.% PDA-NPs/PNIPAM and 0.8 wt.% PDA-NPs/PNIPAM hydrogels) were first lyophilized to determinate the dry weight (Wd). Then the lyophilized samples were swollen in distilled water for 3 day at 25 °C until the equilibrium state was reached. Excess water was removed by filter paper and the weight of the swollen sample (Ws) was determined. Swelling ratio (%) = (Ws - Wd)/Wd ×100%

Characterization of NIR stimuli-responsive property To investigate the NIR stimuli-responsive properties of the PDA-NPs/PNIPAM hydrogels, the hydrogels were irradiated under NIR laser (808 nm) at a distance of 20 cm. The intensity of NIR laser was 2 W, and the diameter of the laser spot was 0.5 cm. Thus, the power density delivered to the sample was 10 W/cm2. To investigate thermal reversibility, the swelling and deswelling transitions of the PDA-NPs/PNIPAM hydrogels were repeated several times with the laser on (NIR irradiation for 1 min) and laser off (cooling in water at room temperature for 30 min) cycles. Surface temperature of the samples following irradiation was measured with a probe upon removal of the laser source. To investigate the change in volume of PDA-NPs/PNIPAM hydrogels, the dimensions of the hydrogel (pure PNIPAM hydrogel, 0.15 wt.% PDA-NPs/PNIPAM, and 0.8 wt.% PDA-NPs/PNIPAM hydrogels) were measured after the samples were irradiated with the NIR laser for 1 min. The volume change was quantified by measuring the dimension of cylindrical hydrogel samples before and after NIR irradiation. The initial hydrogel sample was a cylinder with the height of 10 mm and the diameter of 6.94 mm. The de-swelling rate of the hydrogels was expressed as the contraction volume ratio

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(Vt/V0), where Vt and V0 are the volume of hydrogel samples after and before irradiation, respectively.

On demand drug release upon NIR stimulation To determine the NIR irradiation-induced behavior of the PDA-NPs/PNIPAM hydrogel, dexamethasone (Dex) was used as a model drug and pure PNIPAM hydrogel loaded with Dex was used as a control. Dex was loaded into the hydrogels by immersing the freeze-dry hydrogels in Dex solution (10 mg/mL) to allow the hydrogel take up drug solution for 3 days until the hydrogels reached equilibrium. After shaking for 24 h at 20°C, the percentage of loading drug was determined as the increased weight of the dried hydrogel after drug loading.

To assess the on-demand release capability of the hydrogels, Dex-loaded hydrogels were placed in 1mL PBS in 2 mL microtubes. NIR-stimulated on-demand release was conducted by exposing the hydrogel to the NIR laser with on/off cycles consisting 10 min laser-off and 1 min laser-on exposure. At predetermined time intervals, the supernatant was collected and replaced with an equal volume of fresh water. At defined time points, the release medium (1 mL) was collected and an equal amount of fresh PBS was added to the release medium. The amount of released Dex was monitored using a UV-Vis spectrophotometer (UV-2250) at 242 nm. All tests were performed in duplicate.

Hydrogel as actuator under NIR irradiation In order to evaluate the hydrogel as the NIR-driving soft actuator, we designed a bilayer

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hydrogel consisting of a pure PNIPAM hydrogel layer and a 0.8 wt.% PDA-NPs/PNIPAM hydrogel layer. The bilayer hydrogel was a slab with the width of 3 mm and length of 10 mm. The thickness of each layer was 1 mm. This bilayer structure is capable of bending, which is caused by asymmetric volume contraction of the upper PNIPAM layer and lower PDA-NPs/PNIPAM layer when irradiated by NIR. The NIR-driving actuation of the bilayer hydrogel was observed at the ambient condition.

Micromorphology of the hydrogel The scanning electron microscope (SEM, JSM 6390) was used to analyze the hydrogel morphology. For the sample before and after NIR irradiation, the hydrogel was quickly placed in -20 °C and then freeze-dried for 3 days. Before the SEM observation, the freeze-dried gel was carefully sliced and sputtered with gold. Through these procedures, the microstructure evolution of the hydrogels during NIR irradiation was well preserved.

2.5. NIR-assisted healing of PDA-NPs/PNIPAM hydrogels The hydrogel was cut into halves, and the two parts of gels were put into contact under NIR irradiation for 1 min at ambient temperature. The hydrogels, including PDA-NPs/PNIPAM hydrogels with different PDA-NPs contents, were tested, while the PNIPAM hydrogel was used as control. A tensile test with a 100 N load cell was employed to characterize the tensile strength of the original hydrogel and healed one at a speed of 120 mm/min. In addition, the healing behavior of the PDA-NPs/PNIPAM hydrogel without NIR irradiation was also performed as a control group (Figure S4).

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2.6. PDA-NPs coatings and tissue adhesiveness In order to enhance the cell affinity and tissue adhesiveness of hydrogels, PDA-NPs was coated on the surfaces of the PDA-NP incorporated light-responsive PDA-NPs/PNIPAM hydrogel. The PDA-NP coating process was described in our previous study.21 First, the cylindrical hydrogel samples (ϕ 10 mm× 1 mm; weight, 9 mg) were soaked in 1 mL of PDA-NP suspensions (1 mg/mL) for 2 days under gentle shaking to coat PDA-NPs on the hydrogel. Next, PDA-NPs-coated hydrogels were then treated by 10 min of ultrasonication and washed twice with distilled water to remove un-absorbed PDA-NPs. The residual PDA-NPs in the suspension were centrifuged, collected, lyophilized and weighted. Finally, the adsorbed amount of PDA-NPs on the hydrogels was quantified by subtracting the residual PDA-NPs from the initial mass of PDA-NPs in the suspension. The amount of absorbed PDA-NPs was 0.7 mg/sample.

The morphology of PDA-NPs/PNIPAM hydrogels with and without the PDA-NPs coating was observed by SEM (JSM 6390). The tissue adhesiveness of the hydrogels with and without the PDA-NPs coating was characterized by a tensile-adhesion test using porcine skin to mimic the natural tissue on a universal mechanical testing machine (Instron 5567, USA).

2.7. In vitro EGF loading and release EGF-loaded hydrogels were prepared using the same procedure that prepared Dex loaded hydrogels. Three kinds of hydrogels were used: PNIPAM hydrogel, PDA-NPs/PNIPAM

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hydrogels and PDA-NPs coated PDA-NPs/PNIPAM hydrogels. The in vitro release of EGF from the hydrogels was monitored in the phosphate buffer solution (PBS, pH 7.4) under shaking (100 rpm) at 37 °C. At predetermined intervals, the release medium was collected and equal amount of fresh PBS was added to the release medium. The amount of released EGF in the collected release medium was measured by an ELISA kit (Cloud-Clone Corp., USA) according to the manufacturer’s protocol.

2.8. In vitro cell culture and cell release Four types of hydrogels, including PNIPAM, PDA-NPs/PNPAM, PDA-NPs coated PDA-NPs/PNPAM hydrogels, and EGF-loaded PDA-NPs coated PDA-NPs/PNPAM hydrogels, were used to evaluate cell behaviors. Before cell culture, the hydrogels were purified in PBS for 3 days and sterilized in 75 wt.% alcohol. Fibroblasts (NIH-3T3, SCSP-515) were seeded onto the hydrogels with a density of 7 × 104 cells per sample and cultured in DMEM supplemented with 10 % FBS and 1 % penicillin-streptomycin solution at 37°C in a 5% CO2 incubator. The MTT assay was used to evaluate the proliferation of the cells after 3 day and 7 days of culture. Cell attachments on the hydrogels were observed using a confocal laser scanning microscope (CLSM TCSSP5, Leica, Germany) after staining with Calcein AM (A017, USA).

Cell release experiment: In order to realize NIR light triggered cell release, NIPAM was co-polymerized with acrylamide (AM) to form the PDA-NPs/PNPAM-PAM hydrogel and adjust LCST of the hydrogel above 37 °C.

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The hydrogels without PDA-NPs were used as

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the control groups. The procedures were described in Supporting Information (Figure S5). After the hydrogels were purified and sterilized, fibroblasts (2.0×105 cells per sample) were seeded on each hydrogels and incubated at 37 °C to allow cell adhesion. After 1 day, the hydrogels were taken out and placed in a new culture plate with fresh culture medium. Thereafter, the hydrogels were exposed to the 808 nm NIR laser for 1 min to trigger cell release. The released medium was centrifuged to collect the released cells, which were then imaged under the microscope with a digital camera. The released cells were re-incubated on the tissue culture plate and quantified by MTT assay after 1 day.

2.9. In-vivo wound healing The wound healing characteristics of hydrogels were evaluated in a rat model. Briefly, 5 male Sprague Dawley (SD) rats weighing 250-300 g were used. After anesthetization with pentobarbital (2 wt.%, 2 mL/kg), the dorsal area of the rats was totally depilated and 4 full-thickness circular wounds (8 mm in diameter) were created on the upper back of each mouse by a biopsy punch. On each rat, a blank wound without hydrogel was used as a control. The hydrogels with EGF (EGF, 30 µg/sample, Shanghai Primegene Bio-Tech Co., Ltd. China) were implanted on other wound sites of the rats. A total of 5 parallel specimens of each type of hydrogels were tested. The wounds were additionally covered with a 4 × 10 cm piece of Tegaderm TM dressing (3 M, St. Paul, MN, USA) to keep the hydrogel in place and to protect the rats from infection. In the following 3 days, rats were injected with penicillin to reduce the risk of infection. The experiments were performed in accordance with protocols approved by the local ethical committee and laboratory animal administration rules of China.

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Wound healing was grossly evaluated by wound size reduction measurement. After 15 days of healing, skins including the entire wound with adjacent normal skin were harvested and fixed in 4% buffered paraformaldehyde. Then, the samples were embedded in paraffin, and sections of 3-5 µm were stained with hematoxylin and eosin (HE) and Masson's trichrom for histological analysis.

3. Results 3.1. Characterizations of the hydrogel The rheological test showed that PDA-NP incorporation resulted in high elasticity of the hydrogel. As shown in Figure 2a, all hydrogels showed elastic behavior with storages modulus (G′) ≫ loss modulus (G′′) over the whole frequency range. The values of G′ and G′′ of the PDA-NPs/PNIPAM hydrogels were higher than those of pure PNIPAM hydrogel, indicating that the PDA-NPs/PNIPAM hydrogel was more elastically than pure PNIPAM hydrogel. Storage modulus increased with PDA-NPs content, which is comparable with the mechanical properties of the PNIPAM hydrogels with different PDA-NPs contents. This can be explained by the influence of PDA-NPs crosslinking in the hydrogels. PDA-NPs have abundant of functional groups on their surfaces and can interact with the PNIPAM network, serving as a crosslinking site to weaken the mobility of chain and increase the elastic modulus. In addition, the storage modulus (G′) of the PDA-NPs/PNIPAM hydrogel decreased when the temperature increased to 32 °C, indicating that the critical phase transition temperature (LCST) of PNIPAM was not affected by PDA-NPs. The PDA-NPs/PNIPAM hydrogels showed a smaller swelling ratio than PNIPAM hydrogels, and the swelling ratio

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decreased with the PDA-NPs content in the hydrogels (Figure 2b). The decreased swelling ratio also revealed that PDA-NPs could serve as nano-reinforcements to generate a highly entangled and cross-linked network structure.

Figure 2. (a) Frequency-sweeping rheological behavior of the PDA-NPs/PNIPAM hydrogels different contents of PDA-NPs. (b) Thermal rheological behavior of the PDA-NPs/PNIPAM hydrogels different contents of PDA NPs. (c) Swelling behavior of the PDA-NPs/PNIPAM hydrogels with different contents of PDA-NPs. (d) Equilibrium swelling weight ratio of PDA-NPs/PNIPAM hydrogels with different contents of PDA-NPs.

3.2. NIR responsiveness Volume contraction: The NIR responsiveness of the PDA-NPs/PNIPAM hydrogel was 16


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investigated by irradiation using the NIR laser. Figure 3a shows photographs of the volume change of PNIPAM hydrogels with different PDA-NPs contents before and after NIR laser irradiation for 1 min and cooling in water for 4 h. The PDA-NPs/PNIPAM hydrogels showed distinct contraction after NIR laser irradiation for 1 min (Figure 3a-ii). After cooling in water, the hydrogels recovered their original shape (Figure 3a-iii), indicating a notable photo-thermal phase transition. In contrast, pure PNIPAM hydrogel remained unchanged under the same irradiations, showing no photo-thermal responsiveness (Figure 3a). The shrinking volume ratios of 0.15 wt.% PDA-NPs/PNIPAM and 0.8 wt.% PDA-NPs/PNIPAM hydrogels under NIR irradiation were 10% and 77%, respectively (Figure 3b), indicating that the volume contraction significantly increased with PDA-NPs content. Moreover, similarly to other NIR-responsive hydrogels,15,

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the PDA-NPs/PNIPAM hydrogel also showed a

reversible swelling-shrinking transition during the NIR laser on and off cycles (Figure 3c). The temperature of 0.8 wt.% PDA-NPs/PNIPAM hydrogel increased form 25 °C to 37°C after NIR irradiation for 1 min, and 0.15 wt.% PDA-NPs/PNIPAM hydrogel increased form 25 °C to 33.5 °C, whereas pure PNIPAM hydrogels showed negligible temperature change under the same irradiations. These results indicate that PDA-NPs could absorb NIR laser and convert the energy into heat.31 Higher PDA-NP contents can absorb more NIR energy and therefore result in a larger temperature increase (Figure S1). Thus, increasing the weight percentage of PDA-NPs in the hydrogel also leads to high temperature increase and large volume contraction.

Pulsatile drug release under NIR irradiation: The PDA-NPs/PNIPAM hydrogel exhibits

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an excellent NIR controllability for Dex release. As shown in Figure 3d, during the initial period, Dex was automatically released from two of the hydrogels by diffusion. After 10 min, the released amount did not increase. However, if the NIR laser was switched on, the release of Dex from PDA-NPs/PNIPAM hydrogel was initiated; and if the NIR light was switched off, the release completely stopped, and therefore a pulsatile release profile was observed during laser on-off cycles, indicating an on demand drug release mode controlled by switching the NIR laser on/off. In contrast, no Dex released from pure PNIPAM hydrogel under NIR irradiation. The temperature of the hydrogel was around 45 °C after 2 min NIR irradiation

during

drug

release.

The

NIR-responsive

of

drug-release

from

the

PDA-NPs/PNIPAM hydrogel was attributed to the structure contraction caused by increased temperature under NIR irradiation.

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Figure 3. (a) Photographs of volume change of PDA-NPs/PNIPAM hydrogels with different PDA-NPs contents. (i) Original shape; (ii) Volume contraction after 1 min of NIR irradiation; (iii) Recovery to original shape after 4 h of cooling in water. (b) Volume contraction ratio of PDA-NPs/PNIPAM hydrogels with different PDA-NP contents. (c) Temperature changes of PDA-NPs/PNIPAM hydrogels as a function of irradiation-cooling cycles (irradiation time: 1 min). (d) Cumulative release profile of dexamethasone from PNIPAM hydrogels with PDA-NPs/PNIPAM hydrogels in response to NIR laser irradiation for 2 min (yellow arrows) and without laser irradiation.

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Actuation movements of bilayer NC gels: The actuation movement of the bilayer hydrogel containing a PDA-NPs/PNIPAM layer and a pure PNIPAM layer induced under NIR irradiation was demonstrated in Figure 4. After NIR irradiation for 30 s, the hydrogels were clearly bent, and a water drop was observed. During NIR irradiation, PDA-NPs absorbed and transformed the NIR energy to thermal energy, and therefore caused the PDA-NPs/PNIPAM layer to be heated above its LCST faster than the PNIPAM layer. Thus, the PDA-NPs/PNIPAM layer contracted prior to the PNIPAM layer, causing the bending of the bilayer hydrogel.

We also prepared another bilayer hydrogel with pure polyacrylamide (PAM) layer and PDA-NPs/PNIPAM layer as a control (Figure S3). The bilayer hydrogel also bent after NIR irradiation for 30 s, and the two layers had the tendency to separate during the large bending. This is because the PAM layer does not have ability to absorb heat and contract, whereas the PDA-NPs/PNIPAM layer has large contraction. Thus, the large discrepancy of the asymmetry deformation between the PAM and PDA-NPs/PNIPAM layers caused the separation of the two layers. However, for the bilayer hydrogel consisting of the PNIPAM layer and PDA-NPs/PNIPAM layer (Figure 4), the PNIPAM layer also had certain contraction due to the heat diffusion, which buffered the large deformation discrepancy between the two layers. Consequently, the integration of the bilayers was well preserved during NIR light irradiation. These results indicate that the thermo-responsive hydrogels with a bilayer structure is able to convert the isotropic volume contraction to anisotropic bending.33

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Figure 4. (a) Schematics of NIR responsive actuation of the bilayer hydrogel consisting of PDA-NPs/PNIPAM layer (left) and pure PNIPAM layer (right); (b) Photographs of the bilayer hydrogel before and after NIR irradiation; the blue oval indicates water was squeezed out from the hydrogel after NIR irradiation.

Microstructure evolution: The microstructures change in the PDA-NPs/PNIPAM hydrogel in response to NIR irradiation was investigated to understand the mechanism of volume changes. Cross-sections of hydrogels before and after exposure to the NIR laser are shown in Figure 5. Before NIR irradiation, the PDA-NPs/PNIPAM hydrogel exhibited an interconnected microporous network (Figure 5a). PDA-NPs were uniformly distributed in the composite hydrogel by the high-magnification of SEM observation (inset of Figure 5a, Figure S2). After NIR irradiation, the internal macro-pores structure PDA-NPs/PNIPAM hydrogel collapsed (Figure 5b), resulting in the large volume contraction and drug release. After the laser was turned off and the hydrogel was cooled in water, the hydrogel returned to its original state and the pores expanded again by absorbing the surrounding water (Figure 5c). 21



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This microstructure evolution was attributed to the phase transition of PNIPAM under NIR irradiation. When the laser was off and the temperature was below the LCST of PNIPAM, water remained in the polymer network, resulting in the formation of a homogeneous internal microstructure after freeze-drying. However, when the laser was on and the temperature was above the LCST of PNIPAM, water was squeezed out from the polymer network due to the phase transition of PNIPAM, resulting in micropore collapse after freeze-drying.

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Figure 5. (a) Microstructure of PDA-NPs/PNIPAM hydrogel, the inset shows the PDA-NPs embedded in the hydrogel matrix. (b) After NIR irradiation, pores in hydrogel collapsed. (c) Hydrogel after cooling in water and the pores opened again.

3.3. NIR-assisted healing ability The PDA-NPs/PNIPAM hydrogel has NIR-assisted healing ability. As shown in Figure 6a, the hydrogel was firstly cut into halves, and then the two halves were placed in to contact. After NIR irradiation for 1 min, the PDA-NPs/PINPAM hydrogels tended to heal, and the 0.8 wt.% PDA-NPs/PNIPAM hydrogel could withstand its own gravity weight upon hanging. In contrast, the control PNIPAM hydrogel never healed under NIR irradiation. A tensile test was conducted to characterize the healing effect caused by NIR irradiation (Figure 6b). The healed hydrogel withstood stretching stress of 16 KPa. Notably, the healed hydrogel was stiffer than the original one, likely because a slight loss of water occurred during irradiation process. In addition, we also demonstrated that the PDA-NPs /PNIPAM hydrogels could not heal without the assistance of NIR irradiation (Figure S4). NIR-assisted healing of the hydrogel can be explained in two ways. First, laser irradiation increased the activity of the PDA-NPs, facilitating the interaction of PNIPAM chains through covalent/non-covalent bonds. Secondly, the laser irradiation-induced temperature increase enhanced the mobility of PNIPAM chains,34-36 allowing the PNIPAM chains at the broken surfaces to diffuse into each other and interact with neighboring PDA-NPs, leading damage repair.37-38

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Figure 6. NIR-assisted healing test of hydrogels prepared with four groups. (a) The slices of pure PNIPAM, 0.15 wt.% PDA-NPs/PNIPAM, and 0.8 wt.% PDA-NPs/PNIPAM hydrogels were cut in half to expose fresh surfaces, put into contact, and irradiated by NIR for 1 s and 10 s. The healed hydrogels were hung under gravity. (b) Tensile stress of original and healed 0.8 wt.% PDA-NPs/PNIPAM hydrogels.

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3.4. PDA-NPs-coated hydrogels with tissue adhesiveness PDA-NPs were coated on the hydrogels to enhance cell affinity and tissue adhesiveness of the hydrogels. As shown in Figure 7, pure PNIPAM hydrogel only showed smooth surfaces. In the PDA-NPs/PNIPAM hydrogel, which was obtained by adding PDA-NPs to the hydrogel precursor, PDA-NPs were wrapped in polymer matrix. After the PDA-NPs/PNIPAM hydrogel was incubated in the dispersion of PDA-NPs for 3 days, the PDA-NPs fully covered the whole surface of the hydrogels. These results demonstrated that PDA-NPs were easily immobilized on the surfaces of hydrogels through the intrinsic adhesiveness of PDA.39

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Figure 7. Micrographs of various hydrogels. (a) Pure PNIPAM hydrogel. (b) 0.8 wt.% PDA-NPs/PNIPAM hydrogels. (c) PDA-NPs coated on PDA-NPs/PNIPAM hydrogels.

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Figure 8. (a) Schematics of adhesion test. (b) Adhesiveness of pure PNIPAM hydrogel, 0.8 wt.% PDA-NPs/PNIPAM hydrogel, and PDA-NPs coated 0.8 wt.% PDA-NPs/PNIPAM hydrogel.

The PDA-NPs-coated PDA-NPs/PNIPAM hydrogel exhibited high adhesive strength to porcine skin, as demonstrated in a tensile-adhesion test (Figure 8a). The quantitated adhesive

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strength of various hydrogels is shown in Figure 8b. Without PDA-NPs coating, the PDA-NPs/PNIPAM hydrogel showed low adhesive strength of approximately 30 KPa, which was because PDA-NPs were wrapped into the hydrogel and could not adhere. After the hydrogel was coated by PDA-NPs, the adhesive strength of the hydrogel sharply increased to 90 KPa, which was comparable with previously reported mussel-inspired PEG-based adhesive hydrogels.40 The high adhesiveness of the hydrogel occurred because PDA-NPs were exposed on the hydrogel surfaces, and therefore the functional groups on PDA-NPs, including catechol groups and quinone groups, could form covalent/non-covalent interactions with adjacent tissue surfaces.41-42 The high tissue adhesiveness to porcine skin of PDA-NPs/PNIPAM hydrogels after PDA-NPs coating would facilitate the integration of the hydrogel and surrounding tissue during in vivo implantation.

3.5. In vitro EGF release The PDA-NPs-coated PDA-NPs/PNIPAM hydrogel showed sustained release of EGF in vitro up to 20 day, as shown in Figure 9. During the initial 14 days, the amount of EGF released from PNIPAM and uncoated PDA-NPs/PNIPAM was 45.8% and 32%, respectively. After PDA-NPs were coated on the hydrogels, the amount of EGF released further decreased to 23%. The release rates gradually decreased in the latter periods. The results indicated that incorporating PDA-NPs in the hydrogel and coating PDA-NPs on the surfaces of the hydrogel helps to immobilize and sustain the release of EGF. The EGF sustained release properties of PDA-NPs coated hydrogels was ascribed to the high bonding ability of PDA to proteins via Schiff’s base reactions or Michael-type addition.42 In addition, the

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nano-architectures on hydrogel surfaces created by PDA-NPs also contributed to the good immobilization and sustained release of EGF.19

Figure 9. In vitro EGF cumulative release from pure PNIPAM hydrogel, 0.8 wt.% PDA-NPs/PNIPAM hydrogel, and 0.8 wt.% PDA-NPs/PNIPAM hydrogel with PDA-NPs coating.

3.6. In vitro cell culture Cell affinity. The PDA-NPs/PNIPAM hydrogel showed good biocompatibility and favored cell adhesion and attachment. CLSM observation revealed that the fibroblasts adhered well on all hydrogels (Figure 10a-d), which is consistent with the results of previous study showing that PINPAM hydrogels have good cytocompatibility.43-45 The MTT assay further quantitatively evaluated cell proliferation on hydrogels after 1 and 3 days of culture (Figure 10e). After 1 day of culture, the number of cells on PDA-NP incorporated hydrogels was higher than that of cells on pure PNIPAM hydrogels, indicating that PDA-NP facilitates cell adhesion at initial stage. After 3 days of culture, fibroblasts proliferated on all hydrogels, and

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the number of cells on the EGF-loaded PDA-NPs coated hydrogels was the highest among all the hydrogels. These results indicated that PDA-NPs intrinsically enhanced the focal adhesion of fibroblasts and PDA-NPs and EGF synergistically promoted cell proliferation. The excellent cell affinity of hydrogels can be attributed to the unique chemistry and nano-structure PDA-NPs in the hydrogel.

NIR light triggered cell release. The PDA-NPs coated PDA-NPs/PNIPAM hydrogel realized NIR light triggered cell release (Figure S5a). After NIR irradiation for 1 min, the hydrogel started to contract and release cells. The just released cells were clearly observed under microscope (Figure S5b). However, nearly no cell was released from the PNIPAM hydrogel (Figure S5c). MTT assay illustrated that around 50% of the seeded cells were released after NIR light irradiation (Figure S5d). The cell release results indicated that the coated PDA-NPs could provide adhesive sites to promote cell immobilization and adhesion at initial stage, while the incorporated PDA-NPs could absorb NIR energy and convert into heat leading to PNIPAM network contraction to release the cells on demand under an NIR irradiation. Once the NIR light was switched on, PDA-NPs could generate heat locally by a photo-thermal effect. When the temperature was increased above its LCST, the hydrogel underwent a transition from a hydrophilic swollen state to a hydrophobic collapsed state, leading to an obvious volume shrinking and the release of cells from hydrogel. These results demonstrated the advantage of combining the NIR responsiveness from incorporated PDA-NPs and cell affinity from PDA-NPs coating, suggesting that the currently light responsive and cell adhesive hydrogel has great promise to realize better dynamic control on

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cells as well as other biological agents for therapy.

Figure 10. CLSM image of fibroblasts adhered on (a) PNIPAM hydrogel, (b) 0.8 wt.% PDA-NPs/PNIPAM hydrogel, (c) PDA-NPs coated PDA-NPs/PNIPAM hydrogel, (d) EGF-loaded hydrogel PDA-NPs coated PDA-NPs/PNIPAM hydrogel. Scale bar: 100 µm. (e) Cell growth on different hydrogels after 1 and 3 days of culture. 31



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3.7. In vivo wound healing In vivo full-skin defect experiments supported the enhanced wound healing ability of PDA-NPs-coated PDA-NPs/PNIPAM hydrogels. The photographs of the wound healing process showed that the defect areas were closed in all groups after 15 days of healing (Figure 11a). The EGF-loaded hydrogels showed better healing after 9-day treatment than other groups. Quantitative evaluation of the defect area confirmed that the PDA-NPs-coated hydrogel had a higher healing ratio of 50.5% than the control group (45.2%) and the hydrogel without PDA-NPs coating (40.1%) after 9-day treatment, while EGF-loaded hydrogels further accelerated wound healing with the highest healing ratio of 68.5% (Figure 11b).

Histological analysis was further used to assess the quality of the regenerated skin tissue. Compared with the untreated defect (blank), defects treated with hydrogels were covered with an intact and complete layer of epidermis (Figure 11c). We also investigated the granulation tissue in each defect to evaluate the quality of newly formed skin tissue because granulation tissue formation with an epidermis from the wound edge is a common phenomenon during the healing of full-thickness wounds.46 For the defect treated by hydrogels, more granulation tissue was replaced by collagen fibers than that of the untreated defects (Figure 11c-1). EGF loaded hydrogel showed the least granulation tissue (Figure 11c-7). The amplified sections revealed that EGF-loaded hydrogel induced the regeneration of mature skin tissue that closely mimicked natural skin tissue with well-arranged collagen fibers and hair follicles (Figure 11c-8). Masson staining showed that EGF-loaded hydrogel

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largely improved collagen deposition and alignment, and resulted in more organized collagen fibrils in the defects, compared with the pure hydrogel or blank groups (Figure 11d). In summary, the high functionality of PDA-NPs and their nano-structures was beneficial for immobilizing of extracellular matrix (ECM) proteins.25,

28

The hydrogels with PDA-NPs

showed high tissue adhesiveness facilitating wound suture and repair.

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Figure 11. (a) Representative photos of gross appearance of defects treated with PNIPAM, 0.8 wt.% PDA-NPs/PNPAM, and PDA-NPs-coated-PDA-NPs/PNIPAM hydrogels, and EGF loaded PDA-NPs-coated-PDA-NPs/PNIPAM hydrogels at days 0, 5, 9, and 14. (b) Percent wound area at 5, 9, and 14 days of post-wounding. (c) HE staining of wound sections after 15 days of treatment. (d) Masson staining of collagen deposited in the wound sites. GT: granulation tissue; HT: host tissue.

Discussion This study combined thermo-sensitive PNIPAM and mussel-inspired PDA-NPs to form a NIR-responsive hydrogel. PDA-NPs have a high photothermal conversion efficiency that is comparable and even higher than those of previously reported agents, such as gold nanorods or carbon based nanomaterials.

31

Compared with traditional NIR photothermal materials,

PDA-NPs displays extra unique properties that are suitable for fabricating light responsive hydrogel to meet the requirements of biomedical applications. First, PDA-NPs have good biocompatibility and biodegradability, and do not exhibit long-term toxicity during their retention in vivo.42 Secondly, PDA-NPs have intrinsic adhesiveness to various substrates regardless of surface chemistry, and modify the surface properties of the substrates.27 Thirdly, PDA-NPs can provide multiple react sites to bind biomolecules, such as drugs, peptides and growth factors, and enhance cell attachment and growth.47-49 Fourthly, PDA-NPs are easily and cost-effectively prepared by the oxidation and self-polymerization of dopamine in a mixture containing water, ethanol, and ammonia at room temperature.28 Fifthly, PDA-NPs have a better dispersibility and stability in water and biological media, which is critical for fabricating NC hydrogels with homogeneous distribution of NPs.31, 50

Due to these unique

properties of PDA-NPs, the resulted PDA-NPs/PNIPAM hydrogel achieved multiple properties

superior

than

previous

reported

light

responsive

34


hydrogels.

The

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PDA-NPs/PNIPAM hydrogel not only shows triple NIR response effects, including pulsatile drug release, NIR driven actuation and NIR assisted healing, but also exhibits good cell affinity and tissue adhesiveness.

Note that PDA-NPs are not only incorporated into hydrogel to introduce photothermal properties, but also coated on the hydrogel surface to improve cell affinity and tissue adhesiveness for the first time. Previous reported light responsive hydrogels have poor cell affinity, and the adhesive RGD oligopeptide was introduced into hydrogel to improve cell adhesion.32 Recently, mussel inspired PDA films have been widely used in surface modification of biomaterials due to the intrinsic adhesiveness of PDA.27, 39, 51 However, there is nearly no report on the PDA coated hydrogels, to the author’s knowledge. Our previous studies have employed PDA-NPs to modify porous scaffolds with enhanced biological properties for bone tissue regeneration.28 Compared with previous reported dense and continuous PDA films, using PDA-NPs to modify substrates have several prominent advantages. First, PDA-NPs create micro/nanostructures on the hydrogel surfaces, which more closely mimics natural ECM structures that play a positive role in regulating cell behaviors.52-54. Secondly, PDA-NPs possess higher speciﬁc surface area than PDA films, which provides more multiple react sites to bind ECM proteins. Thirdly, PDA-NPs enhance cell attachment and biomolecule immobilization without trading off the intrinsic physicochemical properties of substrates.28 Finally, PDA-NPs coated hydrogels show high tissue adhesiveness to skin tissue, which facilitates the integration of the hydrogel and surrounding tissue during in vivo implantation.

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Conclusions A NIR-responsive hydrogel with high cell/tissue adhesiveness and fast NIR-assisted healing ability was synthesized by incorporating PDA-NPs into PNIPAM networks. The PDA-NPs acted as highly effective photo-thermal agents, and nanoreinforcements as well as provided bioactive bonding sites for cell attachment, growth factor immobilization and tissue adhesion. The hydrogel showed rapid and repeatable NIR-responsive changes in volume, which is beneficial for pulsatile release of drugs. The hydrogel can quickly heal (1 min) after unfavorable damage with the assistance of NIR laser-irradiation. In addition, the hydrogel incorporated with PDA-NPs showed enhanced the elasticity of the hydrogels, compared with pure PNIPAM hydrogels. Interestingly, after coating PDA-NPs onto the hydrogel surfaces, the tissue adhesiveness significantly increased compared with pristine hydrogel. The PDA-NPs-coated hydrogel also exhibited good cell affinity and was successfully applied for skin wound dressing, demonstrating that this PDA-coated hydrogel can be used for skin tissue repair. In summary, the hydrogel combined thermo-sensitive PNIPAM and mussel-inspired PDA-NPs to form a cell affinitive and NIR-responsive hydrogel, which might have potential applications for chemical and physical therapy.

ASSOCIATED CONTENT Supporting Information SEM micrographs of PDA-NPs. change

of

PDA-NPs

solution

Plots of the NIR irradiation time versus the temperature with

different

concentrations.

Microstructure

of

PDA-NPs/PNIPAM hydrogel with different concentrations of PDA-NPs. Photographs of the

36


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bilayer hydrogel consisting of PAM layer and PDA-NPs/PNIPAM layer before and after NIR irradiation. The healing test of PDA-NPs/PNIPAM hydrogels without NIR irradiation. Synthesis process for PDA-NPs/PNIPAM-PAM hydrogels for NIR light triggered cell release.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing ﬁnancial interest. ★

These two authors contributed equally to this work.

Acknowledgements The work was financially supported by the National key research and development program of China (2016YFB0700802), NSFC (81671824), Open fund of Key Lab of Advanced Technologies of Materials (MOE), and Fundamental Research Funds for the Central Universities.

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Figure 1. Schematic of the PDA-NPs/PNIPAM hydrogel fabrication. (a) (i) PDA-NPs were prepared by oxidative self-polymerization; (ii) PDA-NPs/PNIPAM hydrogel was obtained by free radical polymerization, and PDA-NPs in the hydrogel served as functional cross-linkers to reinforce the hydrogels. (b) NIR-driven pulsatile drug release. (c) NIR-assisted healing. (d) PDA-NPs coated on hydrogel surfaces to enhance cell/tissue adhesiveness of the hydrogels. (e) Cells attached on the PDA-NPs coated PDA-NPs/PNIPAM hydrogels. (f) Adhesive hydrogel served as wound dressing in a full-skin wound defect model. 153x147mm (300 x 300 DPI)



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Figure 2. (a) Frequency-sweeping rheological behavior of the PDA-NPs/PNIPAM hydrogels different contents of PDA-NPs. (b) Thermal rheological behavior of the PDA-NPs/PNIPAM hydrogels different contents of PDA NPs. (c) Swelling behavior of the PDA-NPs/PNIPAM hydrogels with different contents of PDA-NPs. (d) Equilibrium swelling weight ratio of PDA-NPs/PNIPAM hydrogels with different contents of PDA-NPs. 145x131mm (300 x 300 DPI)


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Figure 3. (a) Photographs of volume change of PDA-NPs/PNIPAM hydrogels with different PDA-NPs contents. (i) Original shape; (ii) Volume contraction after 1 min of NIR irradiation; (iii) Recovery to original shape after 4 h of cooling in water. (b) Volume contraction ratio of PDA-NPs/PNIPAM hydrogels with different PDA-NP contents. (c) Temperature changes of PDA-NPs/PNIPAM hydrogels as a function of irradiation-cooling cycles (irradiation time: 1 min). (d) Cumulative release profile of dexamethasone from PNIPAM hydrogels with PDA-NPs/PNIPAM hydrogels in response to NIR laser irradiation for 2 min (yellow arrows) and without laser irradiation. 164x169mm (300 x 300 DPI)



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Figure 4. (a) Schematics of NIR responsive actuation of the bilayer hydrogel consisting of PDA-NPs/PNIPAM layer (left) and pure PNIPAM layer (right); (b) Photographs of the bilayer hydrogel before and after NIR irradiation; the blue oval indicates water was squeezed out from the hydrogel after NIR irradiation. 127x107mm (300 x 300 DPI)


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Figure 5. (a) Microstructure of PDA-NPs/PNIPAM hydrogel, the inset shows the PDA-NPs embedded in the hydrogel matrix. (b) After NIR irradiation, pores in hydrogel collapsed. (c) Hydrogel after cooling in water and the pores opened again. 176x387mm (300 x 300 DPI)



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Figure 6. NIR-assisted healing test of hydrogels prepared with four groups. (a) The slices of pure PNIPAM, 0.15 wt.% PDA-NPs/PNIPAM, and 0.8 wt.% PDA-NPs/PNIPAM hydrogels were cut in half to expose fresh surfaces, put into contact, and irradiated by NIR for 1 s and 10 s. The healed hydrogels were hung under gravity. (b) Tensile stress of original and healed 0.8 wt.% PDA-NPs/PNIPAM hydrogels. 155x200mm (300 x 300 DPI)


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Figure 7. Micrographs of various hydrogels. (a) Pure PNIPAM hydrogel. (b) 0.8 wt.% PDA-NPs/PNIPAM hydrogels. (c) PDA-NPs coated on PDA-NPs/PNIPAM hydrogels. 80x187mm (300 x 300 DPI)



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Figure 8. (a) Schematics of adhesion test. (b) Adhesiveness of pure PNIPAM hydrogel, 0.8 wt.% PDANPs/PNIPAM hydrogel, and PDA-NPs coated 0.8 wt.% PDA-NPs/PNIPAM hydrogel. 111x156mm (300 x 300 DPI)


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Figure 9. In vitro EGF cumulative release from pure PNIPAM hydrogel, 0.8 wt.% PDA-NPs/PNIPAM hydrogel, and 0.8 wt.% PDA-NPs/PNIPAM hydrogel with PDA-NPs coating. 59x44mm (300 x 300 DPI)



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Figure 10. CLSM image of fibroblasts adhered on (a) PNIPAM hydrogel, (b) PDA-NPs/PNIPAM hydrogel, (c) PDA-NPs coated 0.8 wt.% PDA-NPs/PNIPAM hydrogel, (d) EGF-loaded hydrogel. Scale bar: 100 µm. (e) Cell growth on different hydrogels after 1 and 3 days of culture. 224x419mm (300 x 300 DPI)


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Figure 11. (a) Representative photos of gross appearance of defects treated with PNIPAM, 0.8 wt.% PDANPs/PNPAM, and PDA-NPs-coated-PDA-NPs/PNIPAM hydrogels, and EGF loaded PDA-NPs-coated-PDANPs/PNIPAM hydrogels at days 0, 5, 9, and 14. (b) Percent wound area at 5, 9, and 14 days of postwounding. (c) HE staining of wound sections after 15 days of treatment. (d) Masson staining of collagen deposited in the wound sites. GT: granulation tissue; HT: host tissue. 184x211mm (300 x 300 DPI)


Polydopamine Nanoparticles Modulating Stimuli-Responsive PNIPAM

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