CO2 Delivery To Accelerate Incisional Wound Healing Following Single Irradiation of NearInfrared Lamp on the Coordinated Colloids Wei-Peng Li,†,# Chia-Hao Su,‡,# Sheng-Jung Wang,†,# Fu-Ju Tsai,† Chun-Ting Chang,† Min-Chiao Liao,‡ Chun-Chieh Yu,‡ Thi-Tuong Vi Tran,§ Chaw-Ning Lee,§ Wen-Tai Chiu,∥ Tak-Wah Wong,*,§,⊥ and Chen-Sheng Yeh*,† †
Department of Chemistry and Advanced Optoelectronic Technology Center, §Department of Biochemistry and Molecular Biology, College of Medicine, ∥Department of Biomedical Engineering, College of Engineering, and ⊥Department of Dermatology, National Cheng Kung University, Tainan 701, Taiwan ‡ Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan S Supporting Information *
ABSTRACT: Traditional wound care methods include wound infection control, adequate nutritional supplements, education of changing position every 2−3 h to avoid tissue hypoxia, vacuum assistant closure, debridement, skin graft, and tissue flap. Electric current stimulation, ultrasound, laser, and hydrotherapy have emerged as adjuvant therapies. However, most, if not all, of these therapies are expensive, and the treatment results are variable. The development of the active methods to improve wound healing is mandatory. CO2 administration has been known to improve microcirculation and local oxygen supply that are beneficial to wound healing. Here, the metal ion-ligand coordination nanoarchitecture was designed to reveal NIR light-induced CO2 generation for wound healing. The administration simply topically dropped the colloidal solution on the incisional wound, followed by exposure of near-infrared (NIR) lamp to yield CO2, resulting in the observation of the accelerated wound healing. KEYWORDS: wound healing, carbon dioxide, Bohr effect, near-infrared heating, copper sulfide of NPs alone such as Ag NPs against bacteria.5−8 Au NPs have also been used for their anti-inflammatory and antioxidant effects.9,10 At the proliferation stage, NP-growth factor nanoconjugates can be considered at the end of the inflammation phase.11,12 Gene therapy using NP−DNA has been used to target enzymatic activity in the last step of remodeling.13−15 Currently, most of the nanobased approaches have been limited to passive wound healing. Insufficient microcirculatory blood flow resulting in local hypoxia is often the first cause of nonhealing. Improvement of the microcirculation and oxygen supply has been one of the major goals of treatment. Dating back to 1932, carbon dioxide (CO2) therapy was initially used in spas to treat the patients with vascular impairment.16 Since then, clinical studies through transcutaneous or subcutaneous administration of CO2 have demonstrated the efficacy in improving microcirculation and the
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uccessful repair of wounds is a major challenge in the healthcare industry. The loss of functional ability and the increase in pain when wounds become chronic is a burden on the healthcare system. In the United States, chronic wounds affect around 6.5 million patients, and more than US$25 billion is spent annually for related treatments.1 The acute wound-healing process usually involves three phases: inflammation, proliferation, and scar-remodeling. Nanotechnology presents a host of promising materials and strategies to revolutionize medicine. Taking advantage of characteristic nanomaterials has given the advanced healing therapy which may accelerate wound repair with minimal scarring. Currently, different nanoapproaches based on nanocarrier and functional substrate have been proposed to target the specific phase of wound healing process. For example, thrombin is the product of the hemostatic response and was conjugated with iron oxide nanoparticle (NP) for the incisional wound on the rat skin to overcome the short half-life of thrombin.2 Antibiotics conjugated with NPs were formulated to avoid bacterial infection.3,4 Considering the overuse of the antibiotics possibly leading to the development of multidrugresistant organisms, antimicrobial therapy has introduced the use © 2017 American Chemical Society
Received: February 28, 2017 Accepted: May 18, 2017 Published: May 18, 2017 5826
DOI: 10.1021/acsnano.7b01442 ACS Nano 2017, 11, 5826−5835
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Figure 1. Preparation and characterization of hollow CuS nanoshells. (a) Schematic illustration of the preparation processes of the PEGylated hCuS/BC NPs for NIR irradiation. TEM images of (b) Cu2O NPs, (c) h-CuS NPs (insets: SEM image of h-CuS NPs), and (d) PEGylated h-CuS/ BC NPs. The scale bars were all set as 100 nm for TEM and SEM images. (e) UV−vis−NIR absorption spectra of the Cu2O and h-CuS colloidal solutions. (f) UV−vis−NIR absorption spectra of the h-CuS colloids as a function of (NH4)2S concentration. (g) HR-TEM image of a single hCuS NP. The magnified image showing the structure of the h-CuS NP with the (103) lattice plane and d spacing of 0.25 nm shown in the upper right inset. The electron diffraction pattern of the h-CuS NP giving a polycrystalline structure shown in the lower right inset. The elemental analysis of (h) S and Cu. (i) The EDS line scan profiles with the yellow line indicating the path of the electron beam displaying a concave profile to confirm a hollow structure with Cu and S mainly locating in the shell of h-CuS.
rise of oxygenation level because of the Bohr effect.17,18 The Bohr effect is a physiological phenomenon describing the release of oxygen from hemoglobin, allowing more oxygen within tissues, because of the decrease in blood pH. The currently available treatments have been limited to direct administration of CO2 gas through transdermal or subcutaneous delivery conducted by the
device equipped with CO2 gas, which may not be accessible in every clinic. For a course of treatment, multiple administrations were always needed to deliver CO2 on the wounds causing the disadvantages of being time-consuming,19,20 increasing the risk of injection site infection, and causing pain associated with multiple injections which may not be acceptable for elderly 5827
DOI: 10.1021/acsnano.7b01442 ACS Nano 2017, 11, 5826−5835
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Figure 2. Release amount of Fe3+ and CO2 of h-CuS/BC and PEGylated h-CuS/BC colloids. (a) Release amount of ferric ion (blue line) and CO2 (red line) from h-CuS/BC colloids as a function of NIR lamp exposure period at 0.58 W/cm2. (b) Stability of the coordinated ferric ion under different pH values at 37 °C for 7 days for h-CuS/BC colloids. (c) Stability of the PEGylated h-CuS/BC colloids for the coordinated ferric ion under PBS (pH 7) solution at 37 °C for 7 days. (d) Stability of the PEGylated h-CuS/BC colloids for the bicarbonate (BC) under PBS (pH 7) solution at 37 °C for 7 days. The detection of the CO2 release was conducted to monitor the release of the bicarbonate. The colloidal solutions were centrifuged to collect precipitates. Next, the precipitates were dispersed in 1 mM Ca(OH)2 and heated for 30 min at 50 °C to yield CO2 accompanied by the formation of CaCO3. Subsequently, CaCO3 was collected through centrifugation and subjected to ICP-AES measurements to derive the quantitation of CO2. All of the results were obtained triplicate at 300 ppm of Cu ion concentration.
feature of the h-CuS nanoshells with Fe3+ and BC to yield h-CuS/ BC NPs. Synthesized Cu2O NPs, which were used as the templates, were prepared as follows: First, hydrazine was used as a strong reducing agent to reduce Cu2+ ions to small Cu2O NPs (10−30 nm). Next, polyvyinylpyrrolidone (PVP) was employed to gather small Cu2O NPs into large Cu2O NPs through physical adsorption involving the electrostatic interaction between PVP and the small Cu2O NPs.29 The conversion of Cu2O to hollow CuS was conducted using (NH4)2S, followed by an ion-exchange process to substitute the oxygen ions in Cu2O with sulfide ions yielding the hollow structure of h-CuS. Transmission electron microscope (TEM) featured the Cu2O solid structures with a size of ∼162 nm (Figure 1b), whereas the sulfide substitution process resulted in the hollow-structured CuS (Figure 1c) with a particle size and shell thickness of ∼180 nm and ∼32 nm, respectively. The darkened shell relative to the core suggests the presence of the hollow interior. The scanning electron microscope (SEM) displayed the rough surface of the spherical h-CuS (inset in Figure 1c). To coordinate Fe3+ and BC, h-CuS was modified with cysteamines and dopamines (3,4-dihydroxybenzaldehyde). Specifically, the amine (−NH2) group of cysteamine and the aldehyde group of dopamine formed an imine bond, and the thiol (−SH) group of cysteamine bound on the surface of h-CuS, thereby linking the dopamines on h-CuS. The dopamine conjugation was completed in an acidic environment (pH 2.8− 3), indicating no oxidization process for the generation of polydopamine. The conjugated evidence can also be seen in the 1 H proton NMR spectra for cysteamine and dopamine (Figure S1). The PVP signals were also observed to reside on the particle surface. Because the two −OH groups on dopamine can coordinate with Fe3+ to form a double-chelation metal−ligand coordination bond, Fe3+ was then used as a bridge to form a single-chelation with BC. The hollow structure has offered more binding sites with outer and inner surface of the nanoshell
patients. The potential risk of overdose by cause carbon dioxide poisoning, such as suffering anoxia or asphyxiation, should also be considered accordingly. Therefore, we anticipate designing the carrier in a manner of on-demand release of CO2 with benefits associated with a noninvasive, simple, effective, and active method for both physicians and patients. Unfortunately, its gaseous nature has made CO2 more challenging for controlled delivery. Because CO2 gas can dissolve in water, it would be meritorious to ponder and deliberate the design of water-dispersed NPs, which are simply topically administrated on the wound in the form of colloidal solution, upon exposure of near-infrared (NIR) lamp to yield CO2. For this reason, a NIR-driven coordinated CuS NPs was constructed to generate CO2. Covellite CuS NP was selected because of its appreciable NIR absorption covering the first NIR biological window (650−950 nm) to the second NIR window (1000−1350 nm), showing effective photothermal transduction. The design started with the synthesis of solid Cu2O nanoparticles followed by a modified Kirkendall process to hollow and convert Cu2O, yielding hollow porous CuS. Subsequently, the ferric ion (Fe3+) was used as the coordination center to bridge both dopamine and bicarbonate (BC).21−23 BC can decompose into CO2 at the elevated temperature roughly at 42 °C.24,25 The present coordinating architecture has conjugated dopamine on the surface of the hollow CuS nanoshells (h-CuS). The ene−diol-containing compounds such as dopamine can serve as a bidentate enediol ligand to coordinate Fe3+.26,27 On the other hand, BC coordinated to Fe3+ in a monodentate manner.28 These metal ion−ligand coordination features would cover the inner and outer h-CuS.
RESULTS Coordinating h-CuS Nanoshells and the Corresponding Characterization. Figure 1a illustrates the coordinating 5828
DOI: 10.1021/acsnano.7b01442 ACS Nano 2017, 11, 5826−5835
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Figure 3. NIR lamp setup to irradiate PEGylated h-CuS/BC colloids for the measurements of the temperature elevation curves and pH change. (a) NIR lamp irradiation was displayed showing the relative positions for the lamp, fan, and irradiated area (red dashed frame). (b) Temperature elevation curves of the colloids (at 300 ppm of Cu ion concentration) and H2O upon NIR lamp exposure as a function of period. (c) pH value of PEGylated h-CuS/BC NPs colloids at different dosages (Cu ion concentration) for 5 min of NIR lamp exposure. The NIR lamp was set at 0.58 W/ cm2. All data were obtained in triplicate.
temperature elevation of the h-CuS colloidal solutions was dependent on the dosage of the NPs and the power density of NIR lamp (Figure S7). Using 0.58 W/cm2 irradiation of the NIR lamp showed that the amount of CO2 increased as a function of the period of light exposure (Figure 2a). The CO2 release reached 100% after a short period of 10 min irradiation. On the contrary, iron ions were liberated less than 10% upon NIR light irradiation. This indicates that the bidentate enediol ligand (dopamine) to coordinate Fe3+ is slightly affected, while the monodentate bicarbonate to Fe3+ can be easily broken accompanied by CO2 production by heat. Because of the creation of the weak acidic environment when CO2 dissolves in water, the liberation of Fe3+ was conducted under different pH values (Figure 2b). Specifically, when the material was settled under pH 5, 6, or 7 for 7 days, the Fe3+ release rate was
