Research Article www.acsami.org
Synthesis of Cu-Nanoparticle Hydrogel with Self-Healing and Photothermal Properties Shuai Chen,† Fu Tang,*,† Liangzhen Tang,† and Lidong Li*,†,‡ †
State Key Laboratory for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China ‡ State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P. R. China S Supporting Information *
ABSTRACT: Copper (Cu) nanoparticles possess unusual electrical, thermal, and optical properties. However, applications of these materials are often limited by their tendency to oxidize. We prepared Cu nanoparticles by a simple polyol method, with a good control over the particle size. The reaction required no inert atmosphere or surfactant agents. The as-prepared Cu nanoparticles showed good resistance to oxidation in solution. These Cu nanoparticles were then incorporated into a biocompatible polysaccharide hydrogel, which further stabilized the nanoparticles. The hybrid hydrogel exhibited a rapid self-healing ability. Because of the excellent photothermal conversion properties of the embedded Cu nanoparticles, the hybrid hydrogel showed rapid temperature elevation under laser irradiation. The hybrid hydrogel showed limited cytotoxicity; however, under laser irradiation the hydrogel displayed antibacterial properties owing to the heating effects. This study demonstrates that our hybrid hydrogel may have applications in biomedical fields and photothermal therapy. KEYWORDS: nanoparticles, hydrogel, self-healing, photothermal effect, antibacterial
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INTRODUCTION Metal nanoparticles have drawn considerable interest for their unique chemical and physical properties, which are different from those of their corresponding bulk materials owing to the effects of quantum confinement.1−4 Among these materials, copper (Cu) nanoparticles are of particular interest for their excellent electrical and thermal conductivity. Furthermore, Cu nanoparticles are lower in cost than related silver and gold nanoparticles and offer comparable optical and catalytic properties.5−9 Nevertheless, applications of Cu nanoparticles have been limited by their inherent instability under atmospheric conditions and easy oxidation.10 Thus, the synthesis of Cu nanoparticles is typically performed under an inert atmosphere or with surface-capping agents to minimize their exposure to oxygen.11,12 This involves more steps in particle preparation and purification, especially when further modification of the particles are needed. So more efficient methods would be desirable for more widespread applications. Hydrogels are soft materials that possess a high water content, similar to that of the human body tissue, which may allow for their application as biocompatible materials.13−15 Some hydrogels even show stimuli-responsive and self-healing behaviors.16−18 These properties have drawn attention to hydrogels for applications in pharmaceutical and medical science, such as tissue engineering and drug delivery.19−21 Moreover, metal nanoparticles can be incorporated into a three-dimensional network of hydrogels, to further modify the properties of these materials systems. It has been reported that © 2017 American Chemical Society
the network structure of hydrogels can facilitate the dispersion and stabilization of nanoparticles.22−24 However, there have been very few reports of Cu nanoparticles incorporated into hydrogels. Cu nanoparticles exhibit strong light absorption in the visible and near-infrared regions owing to localized surface plasmon resonance (LSPR), similar to that occurring in silver and gold nanoparticles.25−27 Resonance effects can dramatically increase the yield of conduction electrons, which are activated from their ground state to an excited state. Coupling between these electrons and the phonons of a metal lattice can elevate the temperature of the metal nanoparticles and that of the local environment over a short time period.28 Thus, it may be possible to realize highly efficient light-to-heat conversion processes. This LSPR-induced photothermal heating effect has drawn considerable research interests in the chemical and biological fields.29,30 However, studies of photothermal materials have been limited to gold or gold-based nanostructures. The photothermal properties of Cu nanostructures have not been widely studied; however, there have been a small number of reports on the photothermal effects of Cu nanostructures.31−34 Herein, we present the use of a polyol method to prepare Cu nanoparticles with controlled size. Unlike previously reported Received: April 8, 2017 Accepted: June 1, 2017 Published: June 1, 2017 20895
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
Research Article
ACS Applied Materials & Interfaces
Figure 1. (a) Normalized UV−vis−NIR absorption spectrum, (b) SEM image, (c) size distribution, and (d) XRD pattern of the prepared Cu nanoparticles. synthesis, 6 mL of copper sulfate pentahydrate solution in ethylene glycol was first added to a glass vial. The vial was suspended in an oil bath, which was maintained at 85 °C. Then 6 mL of ascorbic acid solution in ethylene glycol was added under magnetic stirring, followed by a certain amount of aqueous ammonia solution (2.5% in weight). The concentration of ascorbic acid was maintained at 0.11 M. To tune the particle size, concentrations of the Cu ions and ammonia were varied from 3.5 to 30 mM, and 0 to 0.2 M, respectively. The reaction mixture was maintained at 85 °C for 30 min and then removed from the oil bath and cooled to room temperature. The particles were then centrifuged, washed with ethanol three times, and finally redispersed in 1 mL of distilled water. Synthesis of Cu Nanoparticles-Embedded Hydrogel. To synthesize the Cu-nanoparticle hydrogel, 20 mg of guar gum was first dissolved in 2 mL of distilled water under magnetic stirring to form a slightly viscous gel. Then, a 100 μL portion of the prepared Cu nanoparticles was added dropwise to the gel. The mixture was stirred for 15 min, followed by the addition of 200 μL of sodium tetraborate decahydrate aqueous solution (8 mM). The hydrogel gradually formed over 15 min. Measurements of Photothermal Performance. The Cunanoparticle hydrogel (1 cm × 1 cm × 1.5 cm) was added to a quartz cuvette cell (1 cm × 1 cm × 4.5 cm), and a laser beam (660 nm, power density 1.5 W/cm2, spot area 50 mm2) was directed through the cuvette. A guar-gum hydrogel without Cu nanoparticles was used as a control experiment. The temperature of the hydrogels was monitored with a digital thermometer, with the probe in contact with the hydrogel. For the in vivo photothermal elevation studies, the mice were subcutaneously injected with bare hydrogel and Cu-nanoparticle hydrogel, respectively. After irradiation by the 660 nm laser at a power density of 0.4 W/cm2 for 5 min, infrared thermal images were recorded by a Fluke Ti400EN IR Camera. Fluorescence Assay. First, a bacterial solution was prepared. A single colony of E. coli on a solid Luria−Bertani (LB) agar plate was transferred to 10 mL of liquid LB culture medium in the presence of 50 μg/mL ampicillin and grown at 37 °C for 6 h. The bacteria were collected by centrifugation at 4200g for 2 min and washed twice with phosphate-buffered saline (PBS). The supernatant was removed, and the remaining bacteria were resuspended in PBS and diluted to an optical density of 1.0 at 600 nm (OD600 = 1.0). A live/dead bacterial
methods, our reaction could be performed under mild atmospheric conditions, was surfactant-free, and was completed in 30 min. A natural polysaccharide, guar gum, was introduced to further stabilize the as-prepared Cu nanoparticles and form a Cu-nanoparticle hydrogel. Owing to the reversible linkages between the guar-gum hydroxyl groups and borate, hydrogel exhibited rapid self-healing properties upon external damage. The Cu nanoparticles embedded in the hydrogels showed a rapid thermal response to laser irradiation. A large increase in the local temperature was observed following 10 min of irradiation, revealing the efficient photothermal properties of the system. This sharp rise in temperature made the Cunanoparticle hydrogel effective for killing bacteria. The asprepared hydrogel shows a good promise as a material for applications in the biomedical fields such as photothermal therapy.
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EXPERIMENTAL SECTION
Materials and Measurements. Copper sulfate pentahydrate, ethylene glycol, ascorbic acid, aqueous ammonia, guar gum, and sodium tetraborate decahydrate were purchased from Sigma-Aldrich and were used without further purification, unless otherwise noted. A live/dead bacterial viability kit (GMS60041) was purchased from GenMed Scientific Inc. Distilled water was used throughout the experiments. Escherichia coli (TOP10) were purchased from Beijing Bio-Med Technology Development Co., Ltd. Ultraviolet−visible−near-infrared (UV−vis−NIR) absorption spectra were collected on a Cary 5000 spectrophotometer. Transmission electron microscope (TEM) images were recorded with a Hitachi H7650B. Field-emission scanning electron microscopy (SEM) images were obtained with a Zeiss Supra 55. X-ray diffraction (XRD) patterns were measured with a Rigaku SmartLab X-ray diffractometer. The size distributions of the nanoparticles were measured by dynamic light scattering (DLS), with a Malvern Zetasizer Nano ZS90, at room temperature. The laser used for excitation was operated at 660 nm (MRL-III-660 nm; CNI Optoelectronics Tech. Co. Ltd., China). Synthesis of Cu Nanoparticles. Cu nanoparticles of various diameters were synthesized by a polyol method. Ethylene glycol was used as the dispersing medium instead of aqueous solution. In a typical 20896
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
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Figure 2. SEM images of the corresponding Cu particles prepared at copper precursor concentrations of (a) 11, (b) 15, and (c) 30 mM. Concentrations of ascorbic acid and ammonia were maintained at 0.11 and 0.1 M, respectively. viability kit was used to determine the bacterial cell viability.35,36 The bacterial suspensions (300 μL, 8.8 × 108 colony-forming unit (cfu)/ mL) and Cu nanoparticles-doped hydrogel (1 cm × 1 cm × 1.5 cm) were added into a quartz cuvette cell (1 cm × 1 cm × 4.5 cm). The laser beam (660 nm, power density 1.5 W/cm2, spot area 50 mm2) was directed through the cuvette for 10 min. The cuvette was then allowed to cool to room temperature. The bacterial suspensions were then mixed with 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) dyes according to the manufacturer’s instructions. After incubation in the dark at 37 °C for 10 min, the bacterial suspensions alongwith the dyes (1 μL) were dropped onto a glass coverslip (2.0 cm × 2.0 cm) and immediately observed with a confocal laser scanning microscope (CLSM; Olympus FV1000-IX81). The fluorescence of DAPI and PI from the specimens were observed on two different channels: a DM 405/488 dichroic mirror captured a bandwidth of 435−485 nm for the 405 nm scan and a DM 559/635 dichroic mirror captured a bandwidth of 590−690 nm for the 559 nm scan, respectively. Control experiments included the use of a bacterial solution, with a guar-gum hydrogel containing no Cu nanoparticles, and the use of the Cu-nanoparticle hydrogel without laser irradiation. For the colony formation assays, 100 μL of the bacteria suspensions were incubated on the Cu-nanoparticle hydrogel with and without irradiation (660 nm, power density 1.5 W/cm2, spot area 50 mm2). The hydrogels were washed twice with 100 μL of PBS and diluted 3 × 105-fold with PBS. The 100 μL portions of the bacterial solution incubated with and without Cu nanoparticles under the same conditions were used as control samples and then diluted 3 × 105fold with PBS. A 100 μL portion of the diluted bacteria solution was spread on the solid LB agar plate. Colonies were formed after 12 h of incubation at 37 °C. The number of cfus was counted. The survival fraction was determined by dividing the number of cfus of the different samples by the number of cfus of the sample incubated without Cu nanoparticles.
The morphology of the prepared Cu nanoparticles was studied by SEM observations. Figure 1b shows an SEM image of the spherical nanoparticles, which had an average diameter of approximately 88 nm. And the DLS measurements (Figure 1c) showed that the average hydrodynamic diameter of the nanoparticles was about 110 nm, with a polydispersity index of about 0.15. The crystal structure of the prepared nanoparticles was confirmed by XRD (Figure 1d). Three diffraction peaks at 2θ = 43.4, 50.5, and 74.2° were clearly observed from the XRD pattern and indexed to the (111), (200), and (220) crystal planes, respectively. This result indicated that the Cu nanoparticles featured a face-centered cubic structure (JCPDS #04-0836). No diffraction peaks from the impurity phases, such as CuO and Cu2O, were detected in the XRD patterns, indicating the successful preparation of Cu nanoparticles without oxidation by our synthesis method. Moreover, the stability of Cu nanoparticles after irradiation has been confirmed. As shown in Figure S1, the morphology of Cu nanoparticles (Figure S1b) remained unchanged compared to that of the unirradiated Cu nanoparticles (Figure S1a). This result further demonstrated the good stability of the Cu nanoparticles prepared by our synthesis method. The sizes of the Cu nanoparticles could be controlled by changing the concentration of copper sulfate pentahydrate used during the synthesis. As the copper precursor pentahydrate concentration was increased, the size of the Cu nanoparticles increased. SEM images of the Cu nanoparticles prepared at precursor concentrations of 3.5, 11, 15, and 30 mM gave corresponding nanoparticle diameters of 88, 130, 220, and 530 nm, respectively, as shown in Figures 1b and 2. The UV−vis− NIR absorption spectra of the Cu nanoparticles, prepared with different concentrations of copper sulfate pentahydrate, are shown in Figure S2. With the increased concentration of copper sulfate pentahydrate, the characteristic peaks of Cu nanoparticles became broad, which related to the size effect of Cu nanoparticles. The broadness of the absorption peak probably stemmed from the wide size distribution and the formed large Cu nanoparticles. The increase in diameter can be understood from the effects arising from the higher copper salt concentration, while the reducing agent (ascorbic acid) concentration was maintained. Precipitation of copper atoms in the later period of the reaction tended to contribute to particle growth through collision with other nuclei rather than by the formation of new particles, thus leading to the formation of larger particles.37 Interestingly, the amount of ammonia used in the reaction also had an effect on synthesis of the Cu nanoparticles. We maintained the concentration of the copper precursor at 3.5 mM and that of ascorbic acid at 0.11 M and used SEM to study
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RESULTS AND DISCUSSION Synthesis and Characterization of the Cu Nanoparticles. The Cu nanoparticles were synthesized by a reaction of copper sulfate pentahydrate with ascorbic acid, which occurred upon addition of ammonia. Ethylene glycol was chosen as the reaction solvent because it has been reported that nonaqueous solvents can minimize the nanoparticle surface oxidation37 and may absorb onto the surface of metal nanoparticles to improve their dispersabilty.38,39 After the addition of ammonia (0.1 M) into the mixture of copper sulfate pentahydrate (3.5 mM) and ascorbic acid (0.11 M) in ethylene glycol, the color of the reaction solution turned from faint yellow to brown in 30 min. Figure 1a shows a UV−vis−NIR absorption spectrum of the prepared Cu nanoparticles. The nanoparticles exhibited a maximum absorption peak at about 595 nm, which was a characteristic surface plasmon resonance peak of the reported Cu nanoparticles.40 20897
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
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Figure 3. SEM images of Cu particles prepared with different concentrations of ammonia (a) 0, (b) 0.05, and (c) 0.2 M. The scale bar represents 500 nm.
Figure 4. (a) UV−vis absorption spectrum, (b) SEM image of the as-prepared Cu-nanoparticle hydrogel. Insets of (a) show the structure of guar gum and a photograph of the resulting Cu-nanoparticle hydrogel.
form. When the amount of ammonia is in great excess, competition between copper-ion complexation by ammonia and their reduction by ascorbic acid prevents the formation of copper particles. In addition, TEM images are provided in Figure S4. These results further demonstrated the growth of Cu nanoparticles. Therefore, the diameter and size distribution of Cu nanoparticles can be controlled by changing the concentration of copper sulfate pentahydrate and the amount of ammonia. Self-Healing Properties of Cu-Nanoparticle Hydrogel. To prepare Cu-nanoparticle hydrogel, guar gum, a biocompatible and environment-friendly polysaccharide, was used to form a composite with the Cu nanoparticles. Guar gum is a galactomannan extracted from the seed of the leguminous shrub Cyamopsis tetragonoloba and consists of (1,4)-linked β-Dmannopyranose main chain, with branched α-D-galactopyranose units at the 6-position.43 The structure of guar gum is shown in the inset of Figure 4a. The cis-diol groups on the guar-gum chains may be cross-linked with borate ions to form an intermolecular cross-linking hydrogel.44 The Cu-nanoparticle hydrogel was realized by adding an aqueous solution of sodium tetraborate decahydrate into the mixture of Cu nanoparticles and guar gum. The hydrogel formed quickly as borate ions were generated from hydrolysis of sodium tetraborate decahydrate. The resulting Cu-nanoparticle hydrogel had a brown appearance similar to that of the Cunanoparticle solution. A UV−vis absorption spectrum of a hydrogel, which contained Cu nanoparticles with diameters of approximately 88 nm, is shown in Figure 4a. The maximum surface plasmon absorption of the Cu nanoparticles featured at 617 nm, which represents a 22 nm redshift, compared with that of the Cunanoparticle solutions. This redshift could be attributed to an increase of the refractive index around the Cu nanoparticles
the morphology of the Cu particles, which resulted at various ammonia concentrations, as shown in Figures 3 and 1b. As shown in Figure 3a, in the absence of ammonia only bulk Cu powders with micron-sized particles could be obtained and these rapidly precipitated from the reaction medium. When the concentration of ammonia was increased to 0.05 M, Cu particles with diameters of about 165 nm could be obtained (Figure 3b). As the concentration of ammonia was increased further to 0.1 M, much smaller Cu nanoparticles, with diameters of approximately 88 nm, were observed (Figure 1b). Thus, higher ammonia concentrations gave smaller Cu particles. However, at an ammonia concentration of 0.2 M, Cu particles with diameters of 340 nm (Figure 3c) were observed. No other obvious particles or precipitates were formed when the concentration of ammonia was increased further. The UV− vis−NIR absorption spectra of the Cu particles prepared with different amounts of ammonia are shown in Figure S3. The results coincided with the SEM images. The role of ammonia in this reaction may be explained, as follows. Alkaline conditions can deprotonate ascorbic acid, and the resulting anions can accelerate the rate of reduction of copper ions.41 Thus, at low ammonia concentrations, the reducibility of ascorbic acid is weak and the rate of reduction of the copper precursor is sluggish. Consequently, only a few nuclei form during the nucleation step, and the copper atoms precipitated in the later period of the reaction contribute mainly to particle growth such that large particles are formed. As the ammonia concentration is increased, the rate of reduction of the copper precursor by ascorbic acid is increased and more copper nuclei form leading to smaller nanoparticles.37 However, as the ammonia concentration was increased further, complexation of copper ions by ammonia decreased the concentration of free copper ions in the reaction mixture.42 Thus, nucleation was disrupted and larger particles tended to 20898
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
Research Article
ACS Applied Materials & Interfaces after becoming embedded in the hydrogel.45,46 The microstructure of the resulting hydrogel was studied by SEM observations, as shown in Figure 4b. The Cu nanoparticles were uniformly distributed in the network of the hydrogel, with no visible aggregation. These results demonstrate the successful preparation of the Cu-nanoparticle hydrogel. The combination and dissociation of borates and cis-diols is reversible suggesting that the resultant hydrogel may have the ability to self-heal through dynamic cross-linking of hydroxyl groups of the guar gum and those of the borate ions. Thus, we selected a piece of the Cu-nanoparticle hydrogel to study its self-healing ability. A piece of bare guar-gum hydrogel without nanoparticles was also prepared and joined to a piece of the Cunanoparticle hydrogel. As shown in Figure 5, after combining
on the photothermal conversion efficiency. The concentration of the Cu nanoparticles was maintained at 4 mM (by controlling the concentration of Cu ions). The guar-gum hydrogel without the Cu nanoparticles was used in a control experiment. The laser irradiation temperature elevation profile, shown in Figure 6a, indicates the photothermal efficiency of our
Figure 5. Self-healing test of the prepared Cu-nanoparticle hydrogel. For clarity, we used pieces of hydrogels with and without Cu nanoparticles. (a) Two pieces of hydrogels were prepared for selfhealing. (b) Two hydrogels are spliced and healed in 5 min. (c) The hydrogels were stretched.
the two pieces of hydrogels, we observed merging of the hydrogels within 5 min as they became a single piece. The combination was irreversible, and only elongation of the hydrogels was observed under a stretching force. The rapid selfhealing property of the Cu-nanoparticle hydrogel suggests a good potential for applications of this material in biomedical fields. Photothermal Conversion Performance of Cu-Nanoparticle Hydrogel. The photothermal effect of Cu nanoparticles is due to LSPR.29 Described by the two-temperature model, the Cu nanoparticles absorb a certain wavelength of the incident light in the visible and near-infrared region. LSPR excitations significantly increase the yield of “hot” electrons (excited electrons). These excited electrons rapidly dephase and equilibrate via electron−electron scattering, forming a “hot electron gas.” Through the coupling between the hot electrons and phonons of the metal lattice, the lattice temperature increases. Finally, the energy of lattice transfers to the local environment. The process generates a large amount of thermal energy around the Cu nanoparticles. To investigate the LSPRinduced photothermal conversion performance, the Cu-nanoparticle hydrogel was cut into a small piece (1 cm × 1 cm × 1.5 cm) and placed in a quartz cuvette cell (1 cm × 1 cm × 4.5 cm). The cuvette was then irradiated by a 660 nm laser beam, with an energy density of 1.5 W/cm2. Hydrogels containing different sizes of Cu nanoparticles were also prepared and applied in this experiment to study possible particle-size effects
Figure 6. Temperature change curves of Cu-nanoparticle hydrogel vs laser irradiation time for (a) different particle sizes and (b) different concentrations. (c) Temperature change of Cu-nanoparticle hydrogel over three cycles of laser irradiation on/off. The laser was operated at 660 nm and 1.5 W/cm2.
hydrogels containing different sizes of Cu nanoparticles. As shown in Figure 6a, the control hydrogel showed a mild temperature increase (about 3 °C) after 10 min of laser irradiation, whereas the temperature of Cu-nanoparticle hydrogels with nanoparticle diameters of 88, 130, 220, 340, and 530 nm, showed temperature increases of approximately 29, 28, 23, 18, and 10 °C after 10 min, respectively. These results indicated that Cu nanoparticles with smaller sizes (such as 88 nm) showed more efficient photothermal conversion in our system. The photothermal efficiency of the Cu-nanoparticle hydrogel was calculated to be 22.3% under irradiation by a 660 nm laser (see Figure 4a, as well as Figure S5 in the Supporting Information). This laser heat conversion efficiency is higher than that of the other Cu nanostructures previously reported (12.5%).33 Thus, when the concentration of Cu ions was 20899
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
Research Article
ACS Applied Materials & Interfaces
Figure 7. Colocalization CLSM images showing the photothermal cytotoxicity in E. coli, with PI (red) to show dead bacteria and DAPI (blue) to show all bacteria. E. coli incubated without hydrogel or Cu nanoparticles (a, e, i) was used as the control experiment. E. coli incubated on the Cunanoparticle hydrogel (b, f, j), hydrogel with irradiation (c, g, k), and Cu-nanoparticle hydrogel with irradiation (d, h, l) were used as the experimental groups. The scale bar indicates 20 μm.
min, a piece of the Cu-nanoparticle hydrogel and a piece of the bare guar-gum hydrogel became a single piece and also showed a self-healing property (Figure S6). The result can further demonstrate that the Cu-nanoparticle hydrogel has reusable capability. For preliminary assessment of the hydrogel application value in vivo, mice were injected with bare hydrogel or Cunanoparticle hydrogel (88 nm, 8 mM), with exposure to a 660 nm laser for 5 min (Figure S7a). The local temperature variation was recorded by a Fluke Ti400EN IR camera. After laser irradiation of 5 min, a higher temperature increase from 29.2 to 32.6 °C was monitored on the Cu-nanoparticle hydrogel injected, but only a mild increase of temperature from 29.1 to 30.4 °C was detected on the controlled hydrogel (Figure S7b). The difference of the two increased temperatures was attributed to the photothermal effect of Cu nanoparticles. Therefore, Cu-nanoparticle hydrogel showed good photothermal effect in vivo. Photothermal Antibacterial Assay. The excellent photothermal conversion of the Cu-nanoparticle hydrogel motivated us to investigate potential antibacterial properties. It has been reported that elevated temperatures may cause irreversible cell damage via disruption of metabolic signals, denaturation of proteins, and rupture of cell walls.48−50 We first studied the photothermal antibacterial performance of the Cu-nanoparticle hydrogel through a fluorescence-based bacterial labeling assay, in which DAPI (blue) was used to indicate all bacterial cells and PI (red) indicated dead bacterial cells only. Using fluorescence
maintained, the surface area of the Cu nanoparticles increased and more effective photoheat conversion resulted. It has also been reported that photothermal conversion efficiency decreases with decreasing nanoparticle volume because of increased scattering.31,47 We also studied the concentration-dependent photothermal elevation studies of hydrogels containing Cu nanoparticles, with diameters of 88 nm (Figure 6b). A higher concentration of Cu nanoparticles gave a greater temperature increase. The hydrogel containing 8 mM of Cu nanoparticles exhibited a considerable temperature increase of 42 °C over 10 min from 25 to 67 °C. These results indicated that the Cu-nanoparticle hydrogels can rapidly and efficiently convert laser energy into thermal energy. No obvious changes of the Cu nanoparticles in the hydrogels were observed during the laser irradiation experiment; however, irradiation of the Cu nanoparticles in an aqueous suspension induced irreversible aggregation owing to increased collision probabilities of particles as the temperature was increased. This phenomenon further demonstrated that our hydrogel system improves the dispersion and stabilization of Cu nanoparticles, thus opening a range of potential applications for these systems. The Cu-nanoparticle hydrogel was subjected to three cycles of laser irradiation at 660 nm. The results (Figure 6c) indicated good repeatability, and only a slight difference of the temperature elevation was observed after the cycle, which demonstrates that the Cu nanoparticles-embedded hydrogel can withstand repeated laser irradiation over long time periods. More importantly, after irradiation by a 660 nm laser for 20 20900
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
Research Article
ACS Applied Materials & Interfaces
with the fluorescence assay. After laser irradiation, the E. coli bacteria were almost unaffected, as the percentage of viability was nearly 100%. Interestingly, there seemed to be a small increase in the number of E. coli colony units for E. coli incubated with the blank hydrogel, which indicates excellent biocompatibility of the guar gum. The Cu-nanoparticle hydrogel exhibited limited cytotoxicity before irradiation, and there was only about a 20% decrease in the bacterial viability. Moreover, after laser irradiation for 10 min with the Cunanoparticle hydrogel, the bacterial viability reduced sharply; nearly 99% of the bacteria were killed. However, the Cu nanoparticles without hydrogel protection showed high toxicity to E. coli under light irradiation or in the dark (Figure S8). The bactericidal ability of the Cu nanoparticles could not be controlled by laser irradiation. Therefore, we conclude that laser irradiation alone or guar-gum hydrogel in itself had no specific antibacterial properties, but the temperature increase caused by the photothermal conversion of Cu nanoparticles in the hydrogel induced the antibacterial behavior. The prepared Cu-nanoparticle hydrogel possessed light-induced bactericidal ability.
microscopy, we were able to simultaneously distinguish the cytotoxic effects. A suspension of the bacterial strains (E. coli) was incubated with the Cu-nanoparticle hydrogel under irradiation by a 660 nm laser beam for 10 min. Next, the E. coli suspensions were extracted, mixed with the dyes, and then observed with a CLSM. In the case of E. coli incubated with the Cu-nanoparticle hydrogel, after laser irradiation, a strong red fluorescence was observed from the dead bacteria in the CLSM images, as shown in Figure 7d. Most of the red fluorescent spots clearly overlapped with emission from the blue stain, which indicated all bacteria. Numerous violet spots were found in the merged images (Figure 7l) demonstrating the bacterial killing efficiency. The group of E. coli incubated with the hydrogel containing no Cu nanoparticles (Figure 7c) showed no particular antibacterial properties through laser irradiation. The CLSM images from this sample were very similar to those of the control (E. coli bacterial only) (Figure 7a), indicating that the presence of the Cu nanoparticles was the main cause of cytotoxicity. Furthermore, there were few red fluorescent spots from the incubated sample from the Cu-nanoparticle hydrogel that was not laser irradiated (Figure 7b), showing the limited innate cytotoxicity of the hybrid hydrogel. These studies indicated that the photothermal properties of the Cu nanoparticles were responsible for the antibacterial effects observed in this assay. In addition, the colony formation assays were performed accordingly. The results shown in Figure 8 were in accordance
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CONCLUSIONS We present a simple and rapid strategy for synthesizing Cu nanoparticles under mild conditions. No inert atmosphere or surfactant agents were needed for this reaction. The sizes of Cu nanoparticles produced could be efficiently tuned by controlling the concentration of copper ions and ammonia in the reaction. The nanoparticles were successfully blended into a biocompatible guar-gum hydrogel, which further stabilized the nanoparticles to form Cu-nanoparticle hydrogels. Owing to the reversible interaction of borates and cis-diol groups of the guar gum, the resultant hydrogel showed rapid self-healing abilities upon damage by an external force. The Cu nanoparticles embedded in the hydrogel showed excellent photothermal conversion, and a temperature elevation of 67 °C was observed after the hydrogel was irradiated with laser light for 10 min. This sharp rise of temperature contributed to efficient antibacterial properties of the irradiated Cu-nanoparticle hydrogels. These properties demonstrate that Cu-nanoparticle hydrogels show potential as materials for photothermal therapy.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04956. SEM images of the Cu nanoparticles before and after laser irradiation; UV−vis−NIR absorption spectra and TEM images of the Cu nanoparticles; monitored temperature profiles and linear time data from the cooling period; calculation of the photothermal conversion efficiency; self-healing test of the Cu-nanoparticle hydrogel after irradiation; photograph of the setup of in vivo photothermal performance measurement and thermal images of mouse exposed to the laser; bactericidal activity of Cu nanoparticles (PDF)
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AUTHOR INFORMATION
Corresponding Authors
Figure 8. (a) Photographs and (b) bacteria viability of E. coli colonies grown on agar plates. Viability ratio data were obtained by counting the cfu’s in (a). Error bars represent the standard deviation.
*E-mail:
[email protected] (F.T.). *E-mail:
[email protected] (L.L.). 20901
DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
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ACS Applied Materials & Interfaces ORCID
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Lidong Li: 0000-0003-0797-2518 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51673022 and 51403018), the State Key Laboratory for Advanced Metals and Materials (2016Z-08), and the State Key Laboratory of Fine Chemicals (KF1613).
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DOI: 10.1021/acsami.7b04956 ACS Appl. Mater. Interfaces 2017, 9, 20895−20903
