Multifunctional Magnetic Copper Ferrite Nanoparticles as Fenton-like

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Biological and Medical Applications of Materials and Interfaces

Multifunctional Magnetic Copper Ferrite Nanoparticles as Fenton-like Reaction and Near-Infrared Photothermal Agents for Synergetic Antibacterial Therapy Yingnan Liu, Zhirong Guo, Fan Li, Yaqing Xiao, Yalan Zhang, Tong Bu, Pei Jia, Taotao Zhe, and Li Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10096 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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ACS Applied Materials & Interfaces

Multifunctional Magnetic Copper Ferrite Nanoparticles as Fenton-like Reaction and Near-Infrared Photothermal Agents for Synergetic Antibacterial Therapy Yingnan Liu, Zhirong Guo, Fan Li, Yaqing Xiao, Yalan Zhang, Tong Bu, Pei Jia, Taotao Zhe, Li Wang* College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China *Corresponding

Author: [email protected]

Tel.: 029-87092486; Fax: 029-87092486.

1

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Abstract Synergistic therapeutic strategies for bacterial infection have attracted extensive attentions owing to their enhanced therapeutic effects and less adverse effects compared with monotherapy. Herein we report a novel synergistic antibacterial platform that integrates the nanocatalytic antibacterial therapy and photothermal therapy (PTT) by hemoglobin functionalized copper ferrite nanoparticles (Hb-CFNPs). In the presence of a low concentration of hydrogen peroxide (H2O2), the excellent Fenton and Fenton-like reaction activity of Hb-CFNPs can effectively catalyze the decomposition of H2O2 to produce hydroxyl radicals (·OH), rendering an increase in the permeability of the bacterial cell membrane and the sensitivity to heat. With the assistance of NIR irradiation, the hyperthermia induced by Hb-CFNPs can cause the death of the damaged bacteria. Additionally, owing to the outstanding magnetic property of Hb-CFNPs, it can improve the photothermal efficiency by about 20 times via magnetic enrichment, which facilitates to realize excellent bactericidal efficacy at a very low experimental dose (20 μg/mL). In vitro antibacterial experiment shows that this synergistic antibacterial strategy has a broad-spectrum antibacterial property against Gram-negative Escherichia coli (E.coli, 100%) and Gram-positive Staphylococcus

aureus

(S.aureus,

96.4%).

More

importantly,

in

vivo

S.aureus-infected abscess treatment studies indicate that Hb-CFNPs can serve as an antibacterial candidate with negligible toxicity to realize synergistic treatment of bacterial infections through catalytic and photothermal effects. Accordingly, this study proposes a novel, high-efficiency and multifunctional therapeutic system for the 2


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treatment of bacterial infection, which will open up a new avenue for the design of synergistic antibacterial systems in the future.

Keywords: copper ferrite nanoparticles, hemoglobin, Fenton or Fenton-like reaction, photothermal therapy, catalytic therapy, synergistic effect, bacterial infection, subcutaneous abscess

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1. Introduction Bacterial infection has turned into a fatal healthcare issue worldwide due to the high morbidity and mortality.1-3 The traditional method to combat bacterial infection is based on antibiotics therapy. However, following the indiscriminate use of antibiotics, the therapeutic effectiveness is inevitably weakened and the drug resistance continues to grow, even spurring the generation of superbacteria.4,5 Therefore, it is imperative to develop a novel therapeutic strategy that is able to combat bacterial infection in a more effective and safety way without developing resistance. In recent years, benefiting from the rapid development of nanotechnology, a variety of emerging therapies based nanomaterials have been developed, including chemotherapy, photothermal therapy (PTT), photocatalytic therapy and so on.6-8 Among them, PTT triggered by near-infrared (NIR) laser is recognized as an intriguing and effective alternative to conventional antibacterial strategies due to minimal invasive, high tissue-penetration, remote controllability, easy location and no resistance.9

During

the

photothermal

process,

the

photothermal-conversion

nanomaterials are able to convert light energy into heat energy, which can destroy bacteria through disruption the cell membrane and protein denaturation, resulting in cell death.10,11 Nevertheless, single modal PTT therapy usually suffers from the shortcoming that both high power density of NIR laser and long-term exposure to NIR laser may cause inflammation and thermal damage to nearby healthy tissue.12-15 PTT-based multimodal synergistic therapy is the most effective strategy to overcome this limitation, which integrates the advantages of single modality approaches to 4


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shorten irradiation time and reduce the dose of antibacterial agents as well as enhance antibacterial efficiency. On this ground, it is imperative to develop novel photothermal nanomaterials equipped with multiple antibacterial functions. Hydroxyl radical (·OH), the most toxic reactive oxygen species (ROS), has attracted extensive interest as active substance to eradicate bacteria.16-18 Compared with traditional antibiotics, it has high and broad-spectrum antibacterial activity and, whose antibacterial process is not dependent on the type of bacteria. Fenton reaction, as one of the main sources of ·OH, has been studied widely. The principle is to convert H2O2 into a highly toxic ·OH by using Fe2+/Fe3+ as catalysts.19,20 Then, the generated ·OH will induce initial oxidative damage to cell membrane, improving the permeability of the cell membrane and making it more sensitive to heat.21 Once combined with PTT, the damaged membrane will be destroyed in a shorter period of time, which greatly reduces the treatment time and minimizes harmful side effects of PTT. To date, various materials combining PTT and Fenton reaction have been developed. For example, Wu et al. design Fe3O4-based multifunctional nanospheres combining the Fenton reaction and photothermal effect for magnetic resonance imaging and tumor therapy.22 Liu and co-workers report ferrous phosphide (Fe2P) nanorods with photothermal effect and Fenton reaction activity for deep tumor synergetic theranostics.17 Hu et al. successfully prepare ultrasmall Cu2-xS nanodots as photothermal-enhanced Fenton nanocatalysts for synergistic tumor therapy at NIR-Ⅱ biowindow.23 Nevertheless, there are few studies investigating the potential of PTT/Fenton reaction system in antibacterial field. Therefore, the development of 5



photothermal/Fenton agents for antimicrobial treatment is of great importance. Magnetic spinel structured copper ferrite, possessing outstanding chemical stability, high catalytic activity, has aroused wide attention in the degradation of various organic hazards and the purification of wastewater.24-27 However, its good photothermal conversion capability and its potential in biomedical application have been neglected. In this study, we prepare hemoglobin (Hb) functionalized copper ferrite nanoparticles (abbreviated to Hb-CFNPs) to construct a new therapeutic platform based on PTT and Fenton reaction for the eradication of pathogenic bacteria. As illustrated in Figure 1, the coupling between the two redox pairs of CFNPs (Fe2+/Fe3+ and Cu+/Cu2+) can catalyze the decomposition of H2O2 at a low concentration to generate ·OH through Fenton and Fenton-like reactions, resulting in the oxidative damage on the cell membrane. The introduction of Hb not only improves the biocompatibility and dispersion of CFNPs, but also further improves the overall catalytic efficiency of the nanocomposites through the Fenton reaction between hemoglobin and H2O2. Under the action of an external magnetic field, Hb-CFNPs can be enriched on the target position, and the abscess is heated in situ by an 808 nm laser irradiation. The considerable photothermal conversion ability of Hb-CFNPs can induce the death of the damaged cells in a short treatment time so as to avoid damage to normal tissues. On the basis of this system, a novel dual-modal synergistic antibacterial platform is developed for the ablation of bacteria, and the potential of CFNPs for biomedical use is revealed.

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Figure 1. Schematic illustration of the synthesis Hb-CFNPs and the corresponding antibacterial application through the combination of Fenton reaction and photothermal effect.

2. Materials and Methods 2.1 Chemical and Reagents Hemoglobin (Hb), glutathione (GSH), 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and phenanthroline were purchased from Solarbio Sci-Tech Co (Beijing, China). Sodium acetate anhydrous (NaAc), ethylene glycol and dimethyl sulfoxide (DMSO) were supplied by Kelong Chemical Reagent Co., Ltd (Chengdu, China). Copper (II) chloride dihydrate (CuCl2·2H2O), iron (III) chloride hexahydrate (FeCl3·6H2O) and methylene blue (MB) were obtained from Guanghua Technology Co., Ltd. (Guangdong, China). H2O2 (30%) and 3,3’,5,5’-tetramethy benzidine (TMB) were purchased from Aladdin Industrial Corporation (Shanghai, China). All 7



chemicals were used as received without any purification. Twice-distilled water was used in all experiments. 2.2 Synthesis of Hb-CFNPs The Hb-CFNPs were synthesized by a hydrothermal synthesis method according to a literature protocol with a minor modification.28 In detail, 25 mL of ethylene glycol was added into 5 mL of Hb aqueous solution (100 mg/mL). After mixing evenly, FeCl3·6H2O (0.54 g), CuCl2·2H2O (0.17 g) and NaAc (0.75 g) were introduced and sufficiently stirred for 2 h. Then the mixture solution was transferred into a Teflon-sealed autoclave and heated under 180℃ for 24 h. The precipitate was separated from the solution by a magnet and washed three times with deionized water by centrifugation (12000 rpm, 20 min). Finally, the products were obtained by vacuum freeze-drying and stored at 4℃ for further application. 2.3 Characterization The morphology of the prepared Hb-CFNPs was characterized by a Hitachi S4800 scanning electron microscopy (SEM, Japan) and a Tecnai G2 F30 transmission electron microscopy (TEM). The crystalline structure was determined by a D8 Rigaku 9000 X-ray diffraction (XRD) system. The chemical composition and the chemical states were analyzed by the X-ray photoelectron spectrum (XPS, Thermo ESCALAB 250, USA) and the Fourier transform infrared spectrum (FT-IR, Bruker Vetex 70, Germany). The content of Hb on Hb-CFNPs was acquired using thermogravimetric analysis (DTG-60A, Shimadzu, Japan) in a N2 environment. The magnetic performance of the as-prepared Hb-CFNPs was examined through an MPMS XL-7 8


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vibrating sample magnetometer (VSM) at ambient temperature. UV-vis absorption spectra were recorded on a Shimadzu UV-2550 spectrophotometer (Japan). The morphology changes of bacteria were characterized by a Nova Nano SEM-450 scanning electron microscopy. The fluorescent-based cell live/dead tests and microscopic fluorescence images were harvested using a Nikon AR laser scanning confocal microscopy (Japan). A BX51 biological microscope (Olympus, Japan) was used to obtain the light microscopy images. 2.4 Photothermal Effect of Hb-CFNPs The photothermal conversion property of Hb-CFNPs was studied by monitoring the

temperature

changes

of

Hb-CFNPs

aqueous

dispersions

of

different

concentrations exposed to an 808 nm laser (2 W/cm2, Changchun Optoelectronics MDL-Ⅲ-808-2W). The real-time temperature and thermal images of dispersions were recorded using a Fotric 226S infrared thermal camera. The thermal stability of the Hb-CFNPs suspension was evaluated by irradiating for 10 min each time and then naturally cooling to room temperature. The cycle was repeated for three times. The photothermal conversion efficiency of Hb-CFNPs was calculated according to Xu’s report.1 The photothermal performance of CFNPs was analyzed by the same procedure. 2.5 Analysis of Hydroxyl Radical (·OH) Generation ·OH was analyzed according to the principle that ·OH can degrade MB and weaken the absorption of MB at 663 nm. Briefly, different mass concentrations of Hb-CFNPs solutions (0.3 mL) were separately added into MB solution (8 mg/L, 2.7 9



mL) to establish an adsorption/desorption in a dark environment for 1 h at room temperature. Then, H2O2 solutions (1 M, 6 μL) were added into the above mixture. After another 20 min of incubation, the mixture was centrifuged (12000 rpm, 20 min) to remove Hb-CFNPs and the absorbance at λ=663 nm of supernatant was recorded on a UV-vis spectrophotometer. Additionally, TMB was employed as an indicator to visualize and monitor the ·OH production. In detail, 1 mL aqueous solution containing TMB (1 mM), Hb-CFNPs (50 μg/mL) and a series H2O2 concentrations (0.5, 1.0, 1.5, 2.0 and 4.0 mM) was incubated for 5 min at room temperature. The colors of the solutions were photographed and the UV-vis spectra were recorded. 2.6 In Vitro Antibacterial Experiment The in vitro antibacterial efficiency of Hb-CFNPs was evaluated using a spread plate method. Gram-negative Escherichia coli (E.coli) and Gram-positive Staphylococcus aureus (S.aureus) were employed as model bacteria. First, the bacteria dispersion (OD600=1.6) was diluted 100 times with phosphate buffered solution (PBS, pH 7.4). Then 0.8 mL of the diluted bacterial suspension was incubated for 20 min with 0.2 mL of PBS, H2O2, Hb-CFNPs and Hb-CFNPs+H2O2, respectively. Another four groups were processed under the same condition except that after 15 min of co-incubation, they were exposed to an 808 nm laser (2.0 W/cm2) for 5 min. The final concentrations of Hb-CFNPs and H2O2 were 20 μg/mL and 1 mM, respectively. The total volume of sample solution in each treatment group was 1 mL. Finally, 100 μL of treated bacterial suspension was spread on a solid medium and 10


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cultured at 37℃ for 18 h. The bacterial colonies were counted and the survival rate was calculated according to the following formula: CFUeg

survival rate (%) = CFUcg × 100% (1) where CFUeg refers to the number of colonies formed in the experimental group and CFUcg represents the number of colonies formed in the PBS treatment group. Afterwards, the above bacterial suspension after various treatments was collected via centrifugation and washed with PBS. Ten microliter of propidium iodide (PI, 10 μg/mL, Solarbio) and 10 μL of calcein acetoxymethyl ester (calcein-AM, 10 μg/mL, Solarbio) were simultaneously incubated with 20 μL of the obtained bacteria suspension in the dark for 15 min at room temperature. The viable bacteria were stained green fluorescence by calcein-AM and the dead bacteria were labeled red fluorescence by PI. All samples were placed on the surfaces of slides and the images were captured by a Nikon AIR laser scanning confocal microscopy to reflect the membrane integrity of bacteria. 2.7 Morphological Characterization of Bacteria For the morphological characterization of bacteria, the bacterial suspensions of the above eight treatment groups were fixed overnight with 2.5% glutaraldehyde solution after the assessment of antibacterial properties. Then they were dehydrated by sequential treatments with 30, 50, 70, 80, 90 and 100% of ethanol successively for 10 min. After drying with a critical point dryer (Leica, EMCPD300, Germany), all samples were sputter-coated with platinum for SEM observation. 2.8 In Vitro Cytotoxicity Assay 11



The in vitro cytotoxicity of Hb-CFNPs was evaluated by a standard 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on mouse embryonic fibroblast cells (NIH-3T3 cells). NIH-3T3 cells were first seeded into a 96-well plate (about 5000 cells per well, six wells for each concentration). After incubation for 24 h, the culture was discarded and all cells were washed with PBS (0.01 M, pH=7.4). Then, the fresh cultures containing different concentrations (10-100 μg/mL) of Hb-CFNPs were added and co-cultured for another 6 h. The cells without Hb-CFNPs were used as a control. After washing with PBS, MTT solution (0.5 mg/mL) was added into each well and co-cultured with NIH-3T3 cells for another 4 h. Afterwards, the MTT solution was removed and DMSO (150 μL per well) was introduced to dissolve the formazan crystals. The absorbance at 492 nm of each well was measured using a microplate reader (MultiskanFC, ThermoFisher Scientific, USA) after jiggling for 15 min. The cell viability was calculated according to the formula: Aex

cell viability = Acon × 100% (2) where Aex and Acon represent the absorbances of the experimental group and the control group, respectively. 2.9 Hemolytic Assay of Hb-CFNPs Hemolysis assay was performed using fresh mouse blood. First, the red blood cells (RBCs) were collected by centrifugation (10000 rpm, 5 min) and rinsed with PBS (0.02 M, pH=7.4) five times. Next, the obtained RBCs (0.4 mL) were added into 7.6 mL of PBS to prepare the stock dispersion. Different concentrations of Hb-CFNPs 12


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suspension (in PBS, 0.7 mL) were gently mixed with 0.3 mL of the RBCs stock dispersion. After incubated at room temperature for 4 h, the mixed dispersions were centrifuged and the supernatant absorbance at 492 nm was measured via a MultiskanFC microplate reader. The deionized water treatment group was used as a positive control group, and the PBS treatment group acted as a negative control group. Hemolysis ratio was calculated according to the following equation: AS ― AN

hemolysis ratio (%) = AP ― AN × 100% (3) where AS, AN and AP represent to the absorbances of Hb-CFNPs treatment group, the negative control and the positive control, respectively. 2.10 Animal Model To further evaluate the in vivo antibacterial activity of Hb-CFNPs, subcutaneous abscess was created on Kunming mice (4 weeks, 16-20 g, Dashuo Experimental Animal Co., Ltd). Briefly, the mice were firstly shaved and disinfected with 75% ethyl alcohol. Then, 100 μL of S.aureus (107 CFU/mL) was inoculated on the back of the mouse by a subcutaneous injection. After 48 h, an infected abscess was formed subcutaneously in each mouse. According to the size of the abscess, the mice were divided into six groups with five mice in each group: (Ⅰ) PBS, (Ⅱ) H2O2, (Ⅲ) Hb-CFNPs,

(Ⅳ)

Hb-CFNPs+H2O2,

(Ⅶ)

Hb-CFNPs+NIR,

(Ⅷ)

Hb-CFNPs+H2O2+NIR. For the Hb-CFNPs+H2O2+NIR treatment group, 30 μL of Hb-CFNPs solution (20 μg/mL) and 20 μL of H2O2 solution (1 mM) were directly injected into the infected abscess. After 15 min, the injected Hb-CFNPs was enriched in the center of the abscess with a magnet, and then the site was irradiated with an 808 13



nm NIR laser (power density: 2 W/cm2) for 2 min. The other groups were also administered using the same procedure under the same condition. The abscesses were photographed every day and the mouse weights were measured by an electronic balance. After 10 days of treatment, all the mice were scratched, and skin tissues and major organs (heart, liver, spleen, lung and kidney) were excised. Then they was fixed with 4% paraformaldehyde solution and stained with hematoxylin and eosin (H&E) for histology analysis. All mice were treated in accordance with the guidelines of the Institutional Animal Care and Use Committee. The mice were discarded according to the standard approved protocol after we finished the experiment. 2.11 In Vivo Biodistribution of Hb-CFNPs The biodistribution of Hb-CFNPs was evaluated by measuring the content of Cu element in the mice from the Hb-CFNPs treated group. First, the major organs and tissues excised from mice’s body were lyophilized and weighted. Then they were digested via a microwave disgestion system (ETHOS UP, Milestone, Italy) in nitric acid at 150℃. The obtained tissue solutions were analyzed using an atomic absorption spectrometer (Hitachi ZA3000, Japan).

3. Results and Discussion 3.1 Synthesis and Characterization of Hb-CFNPs

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Figure 2. Characterization of Hb-CFNPs. (a, d) SEM images of Hb-CFNPs. (b, e) TEM images of Hb-CFNPs. The inset of (b) is the size distribution of Hb-CFNPs. (c, f) Selected area electron diffraction pattern, STEM image and elemental mapping of Cu, Fe, C, N, S, and O of Hb-CFNPs. (g) XRD pattern of Hb-CFNPs. (h) and (i) FT-IR spectra of Hb, CFNPs and Hb-CFNPs. (j) TGA curves of Hb, CFNPs and Hb-CFNPs. (k) Magnetic hysteresis loop of the Hb-CFNPs. (l) UV-vis spectra of Hb, CFNPs and Hb-CFNPs.

15



Hb-CFNPs were synthesized by a one-step hydrothermal reaction. Their morphologies and microstructures were first characterized by SEM images (Figure 2a, d). It could be seen that the as-made Hb-CFNPs exhibited a granular structure and they accumulated to form a continuous ordered cluster-like structure. TEM images (Figure 2b, e) were consistent with the SEM images, reflecting the uniform spherical structure of Hb-CFNPs with an average diameter of 12.8 nm. The selected area electron diffraction pattern (SAEDT) (Figure 2c) showed (220), (311), (400), (511), (440) and (533) metallic planes, which corresponded well to cubic spinel structure data of CuFe2O4 (JCPDS No. 77-0010).24,29,30 The energy dispersive spectrometer (EDS)-elemental mappings in Figure 2c and f displayed the homogenous distribution of Cu, Fe, C, N, S and O in Hb-CFNPs. In addition, the crystalline structure of Hb-CFNPs was further manifested via wide-angle X-ray diffraction (XRD) test. As shown in Figure 2g, the pattern presented seven typical diffraction peaks at 30.2° (220), 35.5° (311), 43.4° (400), 50.5° (200), 57.2° (511), 62.8° (440) and 74.2° (533), again verified the cubic spinel structure. The chemical states of Hb-CFNPs were studied by XPS. The high resolution spectra in Figure S1 reflected the coexistence of Fe2+/Fe3+, Cu+/Cu2+ in Hb-CFNPs, which provided the possibility for the generation of ·OH through Fenton and Fenton-like reactions. In addition, FT-IR spectra were also measured to further validate the successful modification of Hb on the Hb-CFNPs. As indicated in Figure 2h and i, the spectrum of CFNPs showed two characteristic peaks at 588 cm-1 and 864 cm-1, which corresponded to the stretching vibration of metal oxygen (Cu-O and Fe-O) bonds.30 In contrast, in addition to exhibiting Cu/Fe-O 16


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characteristic peaks at the same position, the FT-IR spectrum of Hb-CFNPs also showed a series of new bands belonging to Hb. The peak at 3375 cm-1 was attributed to the N-H stretching vibration. The peak at 1128 cm-1 was derived from the in-plane skeleton vibration of benzene ring.31 And the characteristic amide band Ⅰ (1654 cm-1) arose from the C=O stretching vibration of peptide linkages.29,30 Such results confirmed that Hb was present on the surface of Hb-CFNPs. The thermal gravity analysis (TGA) result in Figure 2j indicated that the content of Hb in Hb-CFNPs was about 22%. In addition, the magnetic behavior of the as-obtained Hb-CFNPs was also explored by the VSM curve. As displayed in Figure 2k, the measured magnetization saturation (Ms) value of Hb-CFNPs was 43.8 emu/g, which was high enough for magnetic enrichment. The optical property of Hb-CFNPs aqueous dispersion was also characterized via the UV-vis-NIR spectrometer (Figure 2l). Compared to CFNPs, Hb-CFNPs exhibited a characteristic absorption peak at 412 nm, which was considered to be the characteristic peak of Hb with acceptable deviation.31,33 In addition, a broad and intense absorption band extending from the visible to the NIR region was observed, indicating that Hb-CFNPs had a photothermal potential to convert NIR laser into heat for PTT.

3.2 Photothermal Performance of Hb-CFNPs

17



Figure 3. Photothermal conversion property of Hb-CFNPs. (a) Thermal images of different concentrations of Hb-CFNPs aqueous dispersions. (b) The temperature elevation corresponding to (a). (c) The temperature profile of Hb-CFNPs solution (400 μg/mL) after exposure to 808 nm laser irradiation for one laser on/off cycle. (d) Linear time data versus –Ln(θ) obtained from the cooling period of (c). Time constant for heat transfer from Hb-CFNPs was determined to be τs=644.17 s by applying the linear time data from the cooling period (after 600 s) versus negative natural logarithm of driving force temperature. (e) Recycling-heating profiles of Hb-CFNPs dispersion (400 μg/mL) irradiated for three on/off cycles. (f) Photothermal images of Hb-CFNPs aqueous dispersion (20 μg/mL) with and without magnetic enrichment in 5 min. The laser used in all above measurements is an 808 nm near-infrared laser with 18


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a power intensity of 2W/cm2.

The photothermal effect of Hb-CFNPs was investigated under an 808 nm laser irradiation at a power density of 2 W/cm2. As shown in Figure 3a and b, the temperature of Hb-CFNPs suspensions displayed an irradiation time- and concentration-dependent rise, while the temperature of pure water hardly increased under the same condition. This result implied that Hb-CFNPs possessed the competence of rapid and efficient conversion of 808 nm NIR light energy into thermal energy. The photothermal conversion efficiency was calculated to be ~28.6% (Figure 3c, d), which is higher than that of traditional Au nanorods (21%), Pt nanoparticles (22.99%), prussian blue nanocages (26%) and WO3-X nanoparticles (25.8%).34-37 Additionally, the photothermal stability of Hb-CFNPs was also assessed by photothermal performance cycle tests. As shown in Figure 3e, there was no significant attenuation of the temperature elevation observed after three cycles of laser on/off, revealing that Hb-CFNPs could withstand repeated laser irradiation over long time periods and they possessed high photothermal stability. The photothermal performance of CFNPs was also measured under the same condition. As shown in Figure S2, the solution temperature increased with the increase of illumination time and the concentration of CFNPs, which was in accordance with the trend of Hb-CFNPs. However, when the concentration of CFNPs was 400 μg/mL, there was only a 15℃ increase within 10 min, which was much lower than a 25.5℃ increase of Hb-CFNPs. Additionally, a slight decrease in temperature elevation was observed 19



after three cycles of laser on/off, indicating that CFNPs couldn’t be repeatedly irradiated multiple times. Thus, the introduction of Hb could improve the photothermal efficacy and photothermal stability of CFNPs. More importantly, Hb-CFNPs had great potential as an excellent photothermal therapeutic agent. Considering the outstanding magnetic property, we further explored the photothermal effect of Hb-CFNPs under the magnetic enrichment. As depicted in Figure 3f, in the absence of magnetic enrichment, the temperature of Hb-CFNPs dispersion at a concentration of 20 μg/mL increased only 2.9℃ after 5 min of NIR irradiation. However, once combined with magnet enrichment, the temperature of Hb-CFNPs agglomerate rapidly increased from 22℃ to 43℃ within 1 min, following reached 52℃ after 5 min of irradiation. The phenomenon reflected that magnetic enrichment significantly enhanced the photothermal effect of Hb-CFNPs, thereby reducing the concentration of Hb-CFNPs served as a photothermal agent and improving the biosafety. Additionally, it was worth mentioning that Hb-CFNPs could capture bacteria via the electrostatic force and the affinity of Hb for lipopolysaccharides of bacterial cell wall.28,38 Under magnetic mediated, bacteria was enriched (Figure S3), resulting in a significant increase in photothermal sterilization efficiency. Thus, Hb-CFNPs could be exploited as an ideal photothermal agent for ablation of bacteria. 3.3 Catalytic Activity of Hb-CFNPs

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Figure 4. Catalytic activity of Hb-CFNPs. (a), (b) and (c) represent the results of degraded MB experiments at different Hb-CFNPs concentrations, different H2O2 concentrations and different incubation time, respectively. Concentration of MB: 8 mg/L. (d), (e) and (f) correspond to the results of the chromogenic reaction of TMB under different Hb-CFNPs concentrations, different H2O2 concentrations, and different incubation time. The concentrations of Hb-CFNPs and H2O2 are 50 μg/mL and 4 mM, respectively. The concentration of TMB in all of the above measurements is 1 mM and the incubation time is 5 min.

The Fenton reaction catalytic activity of Hb-CFNPs was examined by the MB bleaching experiment where MB was degraded by ·OH radicals. As shown in Figure 4a-c, the absorption peak of MB was dependent on the Hb-CFNPs concentration, H2O2 concentration and incubation time, suggesting that Hb-CFNPs could effectively catalyze the generation of ·OH by H2O2 to degrade MB. Owing to the fact that NIR irradiation caused an increase in temperature, we further explored the influence of 21



temperature on the catalytic performance of Hb-CFNPs. As shown in Figure S4a, under NIR irradiation, the absorption peak of MB gradually decreased with the increase of temperature, indicating that high temperature was benefit for enhancing the catalytic activity of Hb-CFNPs. This phenomenon was due to that the ionization process could be promoted as the temperature rises, gradually enhancing the efficiency of the Fenton reaction.17 Furthermore, we also incubated the reaction solution in water baths at different temperatures to further verify the above speculation. The absorption peak of MB in Figure S4b exhibited the same trend of change as that in Figure S4a. Thus, the catalytic activity of Hb-CFNPs could synergistically sterilize bacteria with the photothermal effect. We also compared the catalytic activity of Hb-CFNPs with CFNPs and BSA-CFNPs. As exhibited in Figure S5a and Figure S6, the absorption peaks of MB in Hb-CFNPs treatment group were significantly lower than those in BSA-CFNPs treatment group and CFNPs treatment groups, indicating that Hb played an important role in enhancing catalytic activity of Hb-CFNPs. According to the previous reports, the mechanism was a Fenton-like reaction between Hb and H2O2, wherein H2O2 was decomposed into ·OH and hydroxide anions (-OH), and HbFe2+ was oxidized to metHb (HbFe3+).39-41 The reaction equation was as follows: HbFe2+ + H2O2 → HbFe3+ + ·OH + -OH

(4)

Additionally, the catalytic performance as triggered by Hb-CFNPs in the presence of a low concentration of H2O2 was further characterized by the chromogenic reaction of TMB. As shown in the insets of Figure 4d, a bright blue-green was 22


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observed when Hb-CFNPs were added to the TMB solution. It confirmed that Hb-CFNPs catalyzed the generation of ·OH, triggering the oxidation of colorless TMB into chromogenic TMB. The absorbance intensities of the reaction solutions are presented in Hb-CFNPs-dependent, H2O2-dependent and time-dependent manners (Figure 4d-f). The result in Figure S5b reflected a higher catalytic activity of Hb-CFNPs compared to BSA-CFNPs, again verifying the Fenton reactivity of Hb and the successful preparation of Hb-CFNPs. 3.4 Consumption of GSH GSH, as a tripeptide molecule, widely presents in bacteria. It plays an important role in the bacterial antioxidant defense system and it can prevent damage to cellular components induced by oxidative stress. Thus, the GSH level can serve as an oxidative stress indicator in cells. The Ellman’s assay was performed to evaluate the GSH consumption by Hb-CFNPs. As shown in Figure S7, the GSH level decreased with the increase of Hb-CFNPs, which was mainly due to the reduction of Fe3+ and Cu2+ on the surface of Hb-CFNPs, resulting in an increase in the GSH consumption. Additionally, the amount of Fe2+ was also determined to further verify the above result. It could be seen in Figure S8 that the amount of Fe2+ increased sharply with the increase of GSH concentration, indicating that Fe3+ on the surface of Hb-CFNPs was reduced to Fe2+ by GSH. Therefore, Hb-CFNPs could break the self-defense of bacteria by consuming GSH, thereby enhancing the microbicidal efficacy and inducing bacterial death. 3.5 In Vitro Antibacterial Test 23



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Figure 5. Photographs of the bacterial colonies formed by (a) S.aureus and (b) E.coli after the exposure to (Ⅰ) PBS, (Ⅱ) H2O2, (Ⅲ) Hb-CFNPs, (Ⅳ) Hb-CFNPs+H2O2, (Ⅴ)

PBS+NIR,

(Ⅵ)

H2O2+NIR,

(Ⅶ)

Hb-CFNPs+NIR

and

(Ⅷ)

Hb-CFNPs+H2O2+NIR. (c) and (d) are the survival rates corresponding to (a) and (b). The concentrations of Hb-CFNPs and H2O2 are 20 μg/mL and 1 mM, respectively. (e) Fluorescence staining images of different treatment groups, where viable bacteria are labeled green by calcein-AM and dead bacteria are labeled red by PI.

24


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Motivated by the above results, we concluded that Hb-CFNPs were a kind of potential therapeutic agent that were capable to be used in catalytic and photothermal therapy for bacterial infections. Thus we evaluated the in vitro synergetic antibacterial effect of Hb-CFNPs against Gram-positive S.aureus and Gram-negative E.coli. As presented in Figure 5a, a large number of viable colonies formed on LB agar plates in (Ⅰ) PBS group and (Ⅴ) PBS+NIR group, indicating that NIR irradiation alone didn’t affect S.aureus growth. Weak antibacterial effects were observed in (Ⅱ) H2O2 group and (Ⅵ) H2O2+NIR group, which were attributed to the poor bactericidal efficacy of H2O2 at a low concentration. With the treatment of (Ⅲ) Hb-CFNPs, the number of colonies was reduced by ~29%. A reasonable explanation for this phenomenon was that Hb-CFNPs adsorbed bacteria on their surface, thereby resulting in a decrease of the bacteria number in the bacterial suspension after removal of Hb-CFNPs. When the bacteria were incubated with Hb-CFNPs for 15 min and then exposure to the 808 nm laser for 5 min, the number of bacterial colonies was significantly reduced and the bacterial survival rate decreased from ~71% to ~36%. Obviously, photothermal effect itself could kill S.aureus, but it was not enough. The (Ⅳ) Hb-CFNPs+H2O2 group showed an obvious antibacterial efficiency compared with (Ⅱ) H2O2 group and (Ⅲ) Hb-CFNPs group, with a bacterial survival rate of 54.4%. This data confirmed that Hb-CFNPs catalyzed the decomposition of H2O2 into ·OH via Fenton reaction, causing the bacterial death. When NIR irradiation was introduced into the Hb-CFNPs+H2O2 treatment system, there were only a few colonies observed. The antibacterial rate reached 96.4%, indicating a Fenton reaction/PTT synergetic 25



antibacterial system was successfully constructed. As for killing E.coli (Figure 5b), the corresponding treatment groups exhibited the same antibacterial trends with those of S.aureus. However, it was worth mentioning that the response of E.coli showed more sensitive toward various antibacterial treatments and the sterilization efficiency of (Ⅷ) Hb-CFNPs+H2O2+NIR group reached 100% (Figure 5d). This may be related to the difference in membrane structure that the cytoplasmic membrane of Gram-positive bacteria consists of a multilayered peptidoglycan polymer, while the plasma membrane of Gram-negative bacteria is composed of a thin layer of peptidoglycan.42-44 To further decipher the in vitro antibacterial effect described above, a live/dead bacterial cell staining assay was performed to characterize the membrane integrity of bacteria. The viable bacteria with intact cell membrane were stained green fluorescence by calcein-AM, and the dead bacteria with damaged membranes were stained red fluorescence by PI. As shown in Figure 5e, the bacteria in the PBS and PBS+NIR treatment groups emitted intense green fluorescence and almost no red fluorescence was observed. It revealed that treatment with laser alone negligibly affected the viability of bacteria. In the presence of H2O2, small red fluorescence signals were observed, demonstrating a weak antibacterial efficiency of H2O2 at a low concentration. In Hb-CFNPs treatment group, most of the bacteria were labeled green and only a few of red spots appeared. However, when the bacteria were treated with Hb-CFNPs+H2O2, the red spots apparently increased, indicating that the Fenton reaction significantly enhanced the bactericidal effect. After introducing of NIR 26


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irradiation, no green spots were observed and all bacteria were stained red fluorescence. This result proved that PTT and catalytic effect could synergistically eradicate bacteria.

Figure 6. Morphologies of S.aureus and E.coli incubated with (Ⅰ) PBS, (Ⅱ) H2O2 (1 mM), (Ⅲ) Hb-CFNPs (20 μg/mL) (Ⅳ) Hb-CFNPs+H2O2, (Ⅴ) PBS+NIR, (Ⅵ) H2O2+NIR, (Ⅶ) Hb-CFNPs+NIR and (Ⅷ) Hb-CFNPs+H2O2+NIR. Irradiation time: 5 min.

Field emission scanning electron microscopy (FE-SEM) was also used to illuminate the antibacterial effect by characterizing the change in the morphologies of bacteria. As shown in Figure 6, smooth and intact bacteria surfaces were observed in 27



PBS treatment groups (either no NIR irradiation or under NIR irradiation). When co-incubated with H2O2 or Hb-CFNPs for 20 min, slight distorted and wrinkled appeared at the cell membranes, indicating that H2O2 or Hb-CFNPs alone only had a minor impact on the integrity of bacterial cell membranes. In contrast, significant cellular deformation and content leakage were observed in the Hb-CFNPs+NIR treatment group, suggesting that the photothermal effect of Hb-CFNPs caused damage to the cells. When the bacteria were treated with Hb-CFNPs+H2O2, the bacterial surfaces became rougher and more wrinkled compared with those in H2O2 and Hb-CFNPs treatment groups. This phenomenon was owing to that ·OH generated by Hb-CFNPs catalyzing H2O2 oxidized lipids and damaged the cell membrane. After 15 min of treatment with Hb-CFNPs+H2O2 followed by 5 min of NIR irradiation, the bacteria completely lost the cellular integrity and the intracellular matrix flowed out, implying a stronger antibacterial ability of the synergistic strategy. Consequently, this constructed antibacterial platform based on the PTT effect and catalysis effect was able to kill E.coli and S.aureus rapidly and effectively. 3.5 In Vitro Cytotoxicity Assays and Hemolysis Assay Biocompatibility was a prerequisite for the use of Hb-CFNPs for in vivo applications. Thus, prior to using Hb-CFNPs for bacterial infection therapeutics in vivo, the biocompatibility including cytotoxicity and hemolysis ratio was evaluated. As shown in Figure 7a, Hb-CFNPs showed ignorable cytotoxicity to NIH-3T3 cells, with cell viability remaining greater than 90% in the concentration range from 10 to 100 μg/mL. It indicated the excellent biocompatibility of Hb-CFNPs, which might be 28


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attributed to the presence of Hb on the surface of Hb-CFNPs. Additionally, a hemolysis assay was performed to explore the impact of Hb-CFNPs on RBCs. As shown in Figure 7b, the supernatant of the water treatment group was bright red, while the supernatants of the PBS treatment group and Hb-CFNPs treatment groups were colorless and transparent, demonstrating the negligible hemolysis ratio of Hb-CFNPs. Even if the concentration of Hb-CFNPs reached 30 μg/mL, the hemolysis ratio was only 4.6%, which was lower than the permissible limit (5%).37 Thus, Hb-CFNPs possessed good biocompatibility and were able to be applied in the bacterial infection treatment in vivo.

29



Figure 7. (a) Cell viability of NIH-3T3 cells after incubation with Hb-CFNPs at different concentrations for 6 h. (b) Relative Hemolysis ratios of water, PBS and different concentrations of Hb-CFNPs. The Insets are the corresponding photos. (c) Thermal images of a dosed mouse treated with Hb-CFNPs before and after NIR irradiation for 2 min (808 nm, 2 W/cm2). (d) Photographs of S.aureus infected abscesses treated with (Ⅰ) PBS, (Ⅱ) H2O2 (1 mM), (Ⅲ) Hb-CFNPs (20 μg/mL), (Ⅳ) Hb-CFNPs+H2O2, (Ⅶ) Hb-CFNPs+NIR, (Ⅷ) Hb-CFNPs+H2O2+NIR and the corresponding histologic sections. 30


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3.6 In Vivo Antibacterial Activity Assessment Inspired by the outstanding bactericidal efficacy and biocompatibility of Hb-CFNPs in vitro, we further investigated the in vivo therapeutic effectiveness of Hb-CFNPs by using Kunming mice with S.aureus-infected subcutaneous abscess as a model. As shown in Figure 7c, after 2 min of irradiation, the abscess region temperature of the mouse in Hb-CFNPs treatment group increased from 38.2℃ to 49.7℃, providing intuitive proof that Hb-CFNPs could act as a photothermal therapeutic agent. Figure 7d showed the optical images of abscesses in different treatment groups. On day 1, the scars appeared in all treatment groups except (Ⅰ) PBS and (Ⅱ) H2O2 treatment groups. As time went on, the color of all scars gradually became darker and the sizes gradually decreased, indicating that subcutaneous bacterial infection was inhibited and the wound was healing. The scar rates of the (Ⅲ) Hb-CFNPs,

(Ⅳ)

Hb-CFNPs+H2O2,

(Ⅶ)

Hb-CFNPs+NIR

and

(Ⅷ)

Hb-CFNPs+H2O2+NIR treatment groups were significantly better than other two treatment groups. After treating for 10 days, the scar vanished for (Ⅷ) Hb-CFNPs+H2O2+NIR group and the wound basically healed, demonstrating that rapid and effective sterilization by the synergistic action of Fenton reaction and PTT played an important role in wound healing. Although the scar in (Ⅰ) PBS treatment group also disappeared, the wound was not closed and ichor was still visible inside the wound. Furthermore, a new subcutaneous abscess appeared around the wound, which may be related to the spread of bacterial infection. The healing of the infected wounds was further evaluated by H&E staining. As 31



shown in Figure 7d, an intact epidermis layer of the wound tissues and less inflammatory cells were observed in the Hb-CFNPs+H2O2+NIR group, while disordered skin epidermal layers and obvious infiltrations of inflammatory cells were observed in other groups. Therefore, the combination of Fenton reaction and PTT provided the potential for Hb-CFNPs serving as an effective therapeutic agent for treatment of infected wounds in vivo. 3.7 In Vivo Biosafety Evaluation

32


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Figure 8. In vivo biosafety experiments. (a) Body weight change curves of mice in different groups as a function of time. (b) H&E-stained tissue slices from major organs (heart, liver, spleen, lung and kidney) of different treatment groups (Ⅰ: PBS group, Ⅱ: H2O2 (1 mM) group, Ⅲ: Hb-CFNPs (20 μg/mL) group, Ⅳ: Hb-CFNPs +H2O2 group, Ⅶ: Hb-CFNPs+NIR group, Ⅷ: Hb-CFNPs+H2O2+NIR group). Scale bar: 200 μm. (c) In vivo accumulation of Hb-CFNPs in mice after various treatments. 33



The in vivo biosafety of this therapy was systematically assessed by the body weight, H&E staining of key organs and the biodistribution of Hb-CFNPs. In Figure 8a, the weight gain of the mice in Hb-CFNPs+H2O2+NIR treatment group was consistent with that of healthy mice, indicating the negligible in vivo toxicity of this therapy. The histological analysis results of key organs in Figure 8b showed that there was no appreciable inflammatory lesion, injury or necrosis observed. It confirmed the insignificant long-term toxicity at the dose we used. The biodistribution experiment of Hb-CFNPs was also carried out to further verify the above results. Following in vivo focal infection therapy, the key organs and tissues (including heart, liver, spleen, lung, kidney, skin, blood and scar) were harvested, lyophilized and weighed. The content of Cu element was measured by an atomic absorption spectrometer. As shown in Figure 8c, in the treatment groups without NIR irradiation, Hb-CFNPs were mainly accumulated in scar and some metabolic organs like kidney owing to the reticuloendothelial system.45 In contrast, after NIR irradiation treatment, the accumulation of Hb-CFNPs in the body disappeared, indicating that Hb-CFNPs were excreted through metabolism. A reasonable explanation for this was that NIR accelerated blood circulation and promoted the transport of Hb-CFNPs to metabolic organs for excretion. Therefore, as a therapeutic agent for bacterial infection by subcutaneous injection, Hb-CFNPs exhibited excellent therapeutic effect accompanied by low in vivo toxicity.

4. Conclusions 34


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In summary, we have constructed a novel, biocompatible, synergistic antibacterial platform based on Hb-CFNPs for rapidly and effectively eliminating bacteria and accelerating the abscess ablation. In the presence of a low concentration of H2O2, Hb-CFNPs catalyze H2O2 into highly reactive and toxic ·OH via Fenton and Fenton-like catalytic reaction to induce cell membrane damage. Combined with NIR illumination, hyperthermia generated by Hb-CFNPs induces severe deformation of the damaged cell membrane and leakage of contents, resulting in bacterial death. Both in vitro and in vivo experiments demonstrate that the impressive synergistic antibacterial effect of Hb-CFNPs and excellent biosafety. This nanomaterial successfully incorporates the catalytic therapy with photothermal therapy for combating the bacterial infection, paving a new way for developing antibacterial agents with high synergistic therapeutic outcome. In addition, it is worth mentioning that the remarkable magnetic property of Hb-CFNPs can increase the photothermal effect by 20 times via magnetic enrichment, which facilitates to realize a satisfactory bactericidal effect at a very low dose, thereby enhancing the biosafety. It provides a new idea for the design of antibacterial agents with high photothermal effect.

Conflicts of interest The authors declare that there is no conflict of interest.

Acknowledgements The authors would like to thank the financial supports of the Fundamental Research Funds for the Northwest A&F University (Nos. Z111021601) and Talented Program (A279021724). 35



ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: High-resolution XPS spectra of Hb-CFNPs, photothermal performance of CFNPs, photograph of magnetic separation of E.coli, the degraded MB experiments at different temperatures, comparison of catalytic activity between Hb-CFNPs and BSA-CFNPs, comparison of catalytic activity between Hb-CFNPs and CFNPs at different temperatures, GSH consumption assay, and the determination of Fe2+.

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Multifunctional Magnetic Copper Ferrite Nanoparticles as Fenton-like

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