Metallic nanoparticles to eradicate bacterial bone infection

NANO-01597; No of Pages 10

Nanomedicine: Nanotechnology, Biology, and Medicine xx (2017) xxx – xxx nanomedjournal.com

Metallic nanoparticles to eradicate bacterial bone infection

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College of Science and Engineering, Hamad Bin Khalifa University, Doha, Qatar Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE c Department of Medical Microbiology & Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE d Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE Received 26 November 2016; accepted 24 May 2017 b

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Shahnaz Qadri a , Yousef Haik a,⁎, Eric Mensah-Brown b , Ghada Bashir c , Maria J. Fernandez-Cabezudo d , Basel K. al-Ramadi c

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Abstract

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Treatment of osteomyelitis by conventional antibiotics has proven to be challenging due to limited accessibility to this unique location. Inorganic routes against bacterial infection have been reported for external and topical applications, however in vivo application of these antimicrobials has not been fully explored. Targeted delivery of metallic nanoparticles with inherent antimicrobial activity represents an alternative means of overcoming the challenges posed by multidrug-resistant bacteria and may potentially reduce overall morbidity. In this study we utilized silver–copper–boron composite nanoparticles in an attempt to eradicate S. aureus bone infection in mice. Our results demonstrate effective response when nanoparticles were administered via i.v. or i.m. route (1 mg/kg dose) where 99% of bacteria were eliminated in an induced osteomyelitis mouse model. The 1 mg/kg dose was neither toxic nor produced any adverse immune response, hence it is believed that metallic nanoparticles present an alternative to antibiotics for the treatment of bone infection. © 2017 Elsevier Inc. All rights reserved.

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Key words: Osteomyelitis; In vivo; Nanoparticles; Ag-cu-B; Animal model

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Osteomyelitis management involves long-term antibiotic therapy. Surgical intervention is highly recommended for chronic osteomyelitis. 1 Difficulties in treating osteomyelitis are believed to stem from the sheltered physiological environment offered to the bacteria and poor accessibility to the immune system and to therapeutic agents. 2,3 Several suggestions to control the amount of antibiotics by either novel delivery systems such as chewable tablets or localized delivery of the antimicrobials have been described. 4–7 Metal or polymeric implants with or without drugs have been studied to identify alternatives for osteomyelitis treatment. 8–10 Therapeutic modalities on animal models (e.g. sheep, 11 goat, 12 pig, 13 dog 14,15 and mouse 16–23) to manage chronic osteomyelitis have been developed, however many more studies are still needed to identify a modality with low recurrence rate and drug

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resistance. The recurrence rate of osteomyelitis is more than 50% following three months of antibiotic treatment. 24 The oligodynamic activity of metals provides a valuable alternative to the use of systemic antibiotics. Although silver seems to be the favorite antimicrobial metal, 25–28 there are several shortcomings associated with its use as a sole agent. Silver has a short-term antimicrobial activity that requires its continuous re-application and also the fact that silver requires an aqueous environment that produces the active ionic form to display its effect. 1,29 Most studies have examined the antimicrobial activity of Ag ions or nanoparticles in vitro, few have reported the toxicity of Ag using in vivo models. 30–34 Studies have shown that Ag-Cu is more effective as an antimicrobial agent compared to Ag lone or Cu alone. 35 The antimicrobial effectiveness of Ag-Cu complex is also however, limited by the

This study was in part supported by a grant from Qatar National Research Fund, grant number NPRP8-1744-3-357X (to YH) and in part by a UAEU-NRF grant number 20921 (to BKA). ⁎Corresponding author. E-mail address: [email protected] (Y. Haik). http://dx.doi.org/10.1016/j.nano.2017.05.013 1549-9634/© 2017 Elsevier Inc. All rights reserved. Please cite this article as: Qadri S., et al., Metallic nanoparticles to eradicate bacterial bone infection. Nanomedicine: NBM 2017;xx:1-10, http://dx.doi.org/ 10.1016/j.nano.2017.05.013

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Methods

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Synthesis and characterization of Ag-Cu-B nanoparticles

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Particles were synthesized as reported in Ref. Briefly, 0.1 Molar salt of copper (II) sulfate, silver nitrate and boric acid were prepared in 100 ml deionized water in a ratio of 70:20:10 (Ag:Cu:B). Salt solution was heated to 90 °C in a triple neck flask with constant mixing by using Teflon rod fixed with homogenizer under the fume hood. Flask was purged with argon gas. Continuous stream of argon was used throughout the reaction. 8 M NaOH was added drop wise from the side neck of flask until the formation of black precipitate. The solution was heated for 20 min until the precipitate turned to grayish black. The precipitate formed was washed repeatedly three times or more in deionized water and centrifuged at 4000 rpm for 10 min for each wash. Furthermore 100 mM of lactic acid treatment for 10 min was used to break the nanoparticles in smaller size, particles were collected by centrifugation and washed 3 times or more with water to remove the lactic acid from samples. The final pellet was sonicated (Branson Sonifier–450) for 1 h on ice to prevent the rise in temperature associated with sonication. After sonication the sample was filtered through Whatman filter paper, freeze-dried and stored in 20 ml airtight glass vial with a screw cap. Prior to using the nanoparticles, frozen particles were weighed and suspended in deionized water, sonicated and used immediately. Ag-Cu-B nanoparticles were characterized by the XRD-technique (Agilent Technologies Oxford Gemini X-Ray Diffractometer). The XRD technique (Molybdenum source: Voltage 50 kV and with 30 mA current and λ Mo = 0.709 °A) was used to study the phase formation in and morphology of the Ag-Cu-B nanoparticle. Transmission electron microscopy was done with FEI Talos F200C, images were obtained at 200 KV. Atomic force microscopy (AFM) Bruker Model was employed to confirm the height of nanoparticles. Scanning was done at 1 Hz. The hydrodynamic size of nanoparticles was measured with a Nano-sizer NZS (Malvern). Scanning Electron Microscopy SEM JEOL6400 -Oxford EDS unit analyzed the surface morphology and elemental analysis.

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S. aureus XEN-36 strain which was derived from the parental strain S. aureus ATCC 49525 was obtained from Caliper Life Sciences, USA which possesses a stable copy of the modified photorhabdus luminescence lux ABCDE operon at a single integration site on a native plasmid, stock was stored in glycerol at −80 °C. This kanamycin resistant XEN-36 was streaked on tryptic soy agar plate with the recommended dose of kanamycin (200 μg/ml) as per guidelines of Caliperlife Sciences. Preparation of bacterial cultures was carried out essentially as previously described. 38 Bacterial colonies grown on T-soy agar plate were inoculated into 5 ml of T-soy broth and cultured stationary overnight with aeration. The overnight grown bacterial culture was then sub-cultured at 1:5 ratio and grown to mid-log phase for another 2 h with shaking at 200 rpm and was stopped when O.D. reached to 0.5 at 600 nm wave length. Colony forming units (CFU's) count was estimated at 0.5 O.D. by harvesting bacterial cells with centrifugation at 4000 rpm for 20 min and the pellet was re-suspended in 5 ml of PBS pH 7.4. Serial dilutions were performed in sterile PBS and 100 μl aliquots were plated on T-soy agar plates containing 200 μg/ml kanamycin. The number of bacterial colony forming units (CFUs) were enumerated after overnight incubation at 37 °C.

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S. aureus XEN-36 strain

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rapid Cu oxidation. We have found (data not reported here) that the Ag ion release is maintained for a longer period when boron is introduced to the Ag-Cu complex. The longevity of antimicrobial effectiveness for Ag-Cu-B is greater than that of Ag-Cu. This could be attributed to the B anticorrosive properties that delay the Cu oxidation. 36 The objective of our study is to report a silver–copper–boron (Ag-Cu-B) composite nanoparticles as an alternative for osteomyelitis management using in vivo model system. The Ag-Cu-B complexing overcomes the shortcomings associated with silver and silver copper. Antimicrobial function of Ag-Cu-B is reported for the first time in in vivo as therapeutic agent for bone infection. An osteotomy mouse model was developed. The development of infection was assessed by microbiological technique. The efficacy of the local application of Ag-Cu-B nanoparticles against an antibiotic was assessed.

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XEN-36- S. aureus growth on braided silk suture

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A bacterial suspension of XEN-36 containing 200 μg/ml of kanamycin was adjusted with Tryptic-Soy to O.D. 0.5 at 600 nm. A braided silk suture 5–0 size (SMI, REF.NO. 8151516, LOT NO. 110623) was tumbled in this bacterial suspension for 45 or 150 min, it was gently removed and air dried for 5 min by keeping on sterile Whatman filter paper and cut to a length of 2 cm. Further it was chopped into small pieces in 1 ml of PBS and followed by homogenization by tissue homogenizer. Serial dilutions of silk suture homogenate were performed in sterile PBS and 100 μl was plated on T-soy agar plates with kanamycin. Number of CFUs were enumerated after overnight growth at 37 °C in incubator.

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Mouse strain

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Female BALB/c mice were purchased from Harlan Olac (Biocester-UK). Mice were bred in the animal care facilities of the College of Medicine and Health Sciences United Arab Emirates University, and maintained in filter-topped isolator cages. Mice were housed under controlled dark and light cycle of 12 h each in groups of 5–6 mice per ventilated cages and post-surgery or treatment they were rehoused in same groups. They were fed standard diet with food and water ad libitum. All studies involving animals were conducted in accordance with and after approval of the animal research ethics committee of the College of Medicine and Health Sciences, United Arab Emirates University.

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Osteotomy

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8–12 week-old BALB/c female mice were anesthetized with a combination of xylazine (10 mg/kg of body wt.) and ketamine (100 mg/kg of body wt.). Xylazine and ketamine ratio were made

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in a final volume of 10 ml PBS. 0.3 ml of combined xylazine and ketamine was administered as single intra-peritoneal injection (i.p.). burpenorphine (0.1 mg/kg of body wt.) given subcutaneously (s.c.) as pre-operative pain medicine. After thorough cleaning of the lower limb with betadine and 70% ethanol, the shin was sectioned with a scalpel blade to expose the whole tibia. The tibia was exposed and the gastrocnemius and soleus muscles retracted gently to expose the posterior aspect of the tibia to avoid necrosis of bone that could occur as result of damage to vessel, especially the posterior tibial artery that supplies the posterior compartments of the leg and plantar surface of the foot. The tibia bone was drilled from the posterior to the anterior side to prevent purposely to avoid damaging the posterior tibial artery. Care was also taken not to fracture the fibula. A drilling machine (Cole palmer, Country), sterile steel drill bit of tip diameter 0.25 mm (size) was then used to drill a hole of a size that would allow a 5–0 silk suture to pass through immediately below the tibial tubercle. A braided silk suture with XEN-36 was passed through the drilled hole, stabilized by double knotting the

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Figure 1. Surgical steps: (A) shows the drilling of tibia with drilling bit of tip width 0.25 mm from posterior to anterior direction, (B) shows the drilled hole in tibia where silk suture can pass through. (C) and (D) show tibia bone after 7 days of infection with the silk suture and after silk suture is removed.

ends, and the sides cut close to knot. Prior to suturing of the skin, the exposed area of bone and surrounding tissue was wiped with 70% ethanol as a preventive measure against the development of a system infection or myositis. The mouse skin was sutured and anti-inflammatory caprofen (5 mg/ml) was given for two days post-surgery. Mice recovery from anesthesia was observed after 1 h and were returned to fresh cages. After surgery mice were fed normally for 6 days. Post 6th day of surgery, mice were given treatments, after 24 h of treatment mice were euthanized by carbon dioxide inhalation and were used for analysis. The steps of surgery are as shown in Figure 1.

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Experiment plan

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The number of surgeries per day was limited to 15–18 mice as a result each experiment has only 2 to 3 groups of mice with 4–6 mice in each group. All mice received similar infected silk suture incubated from one common bacterial culture each day except groups of mice in experiment-5. Mice used in

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All experimental mouse groups were euthanized after 24 h of treatment except for experiment-5, in which animals were followed up for survival test for up to 12 weeks. Mice processing and homogenization of tissues

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After treatment, mice were sacrificed and osteotomized tibia isolated from the tissue; care was taken not to break the bone. After weighing, bone was immersed in 1 ml of sterile cold PBS (100 mM) buffer pH 7.4 in a 35 mm petri-dish, placed on ice and was chopped into tiny pieces with the help of a pair sterile scissor. 1 ml of bone extract containing the chopped pieces of bone was transferred into 15 ml pyrex tube and homogenized in a final volume of 10 ml PBS buffer. 100 μl homogenization samples were serially diluted by 10 fold in sterile PBS and 100 μl aliquot was placed on a T-soy agar plate containing 200 μg/ml of kanamycin. The remainder of the homogenized tissue was stored at −80 °C and utilized for other biochemical analysis. Similarly, a piece of liver and whole spleen were collected and weighed, and tissues were homogenized and plated on an agar plate to enumerate bacterial growth, as detailed previously. 40

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Histology

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Groups of BALB/c mice treated with Ag-Cu-B NP at a dose of 1 mg/kg of bw. and a control group treated with saline only were euthanized after 24 h of treatment. The tibia bone was resected as mentioned earlier; bone was decalcified by treating with 0.5 M EDTA in a 100 mM PBS buffer and was kept overnight. The bone was embedded in paraffin and processed for histological analysis as previously described. 41 Paraffin embedded bone was sectioned with 5 μm thickness using rotary microtome (Shandon AS 325, USA). Sections were stained with hematoxylin and eosin stain (H&E). Olympus BX53 microscope equipped with digital camera DP26 (Tokyo, Japan) was used to capture the images.

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GSH was measured from homogenized tissues of spleen, liver and bone of mice treated or non-treated with nanoparticles or antibiotics. Sample homogenates were diluted in cold lysis buffer pH 8.8 (50 mM of Tris and 1 mM of EDTA) with protease inhibitor (Sigma Cat. No. P8340) and were lysed by cell disruptor in cold conditions. Cell lysate was collected in an Eppendorf tube and centrifuged at 10,000 g for 10 min at 4 °C and supernatant was collected for glutathione assay. A GSH standard curve was obtained and done as described in Coleman et al. 42 Briefly 2.5 mM of GSH (L-Glutathione, Sigma cat# G6013) was made in Tris-EDTA buffer (50 mM of Tris and 1 mM of EDTA) pH 8.8, and was serially diluted in Tris-EDTA buffer (50 mM of Tris and 1 mM of EDTA) in a 96 well plate. To each well was added with 100 μl of 2.5 mM of 5,5′-Dithiobis (2-nitroobenzoic acid) DTNB (Sigma, Cat# D8130), which was prepared in Tris-EDTA buffer. Plate was incubated for 10 min at room temperature and absorbance was measured at 405 nm. A standard curve was generated from GSH in nanomole verses absorbance. 100 μl of supernatant of different samples were added to solution in the 96 well plate followed by addition of 100 μl of 2.5 mM DTNB. The mixture was incubated for 10 min at room temperature, absorbance was measured at 405 nm and concentration of GSH was calculated from the standard curve. Concentration of GSH for each sample was normalized with total protein of each sample and GSH concentration was expressed in μmoles/gram of total protein.

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Experiment-1: 6th day post-operation, group 1 was injected with saline; groups 2 and 3 were injected with Ag-Cu-B nanoparticles (NP) at 1 mg/kg bw, via i.p. or i.m. (near infection site), respectively. Experiment-2: 6th day post-operation group 4 and group 5 were given 0.5 mg/kg of NP and saline respectively. Experiment-3: 6th day post-operation, group 6 was injected with saline (i.v. via tail vein). Group 7 was injected with 0.1 mg/kg of NP. Experiment-4: 6th day post-operation, group 8 was injected with saline (i.v.). Group 9 was injected negative control made out of iron oxide nanoparticles 1 mg/kg (i.v.). Group 10 was fed orally with antibiotics co-trimoxazole at a dose of 95 mg/kg/24 h in drinking water. 39 Experiment-5: To test for nanoparticle toxicity, mice were given either saline (group 11) a single dose of NP i.v. (1 mg/kg; group 12) or multiple doses of NP, once per week for four weeks (group 13).

Reduced glutathione (GSH) measurement for oxidative stress

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experiment-5 did not receive osteotomy and were used for survival assay.

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Total protein assay

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Total protein content in the supernatant was measured by BCA assay (Thermo Scientific Cat. No. 23227) according to the manufacturer's instructions and was used as directed by manufacturer. Reagents A and B were mixed in a 50:1 ratio and 180 μl of the mixture of A and B added to 20 μl of sample or dilutions of used for obtaining standard curve in a 96 well flat bottom plate. Plate was covered and incubated for 30 min. Absorbance was measured at 562 nm by spectrophotometer Plate Reader (Biotek-USA).

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Statistics

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Data were expressed as the mean ± standard error. Statistical differences were analyzed using the Mann–Whitney test. P b 0.05 was considered statistically significant.

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Results

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Nanoparticle characterizations

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High magnification TEM images as shown in Figure 2, A showed nanoparticles to be spherical with diameters ranging from 18 to 27 nm. AFM image of sensor height is shown in Figure 2, B. The average height of nanoparticles was 26.6 ± 3 nm. Figure 2, C shows the major diffraction peaks of silver are clearly seen and indexed. Considerable amount of the Cu and the Cu2 phases are also formed in the microstructure of the complex. From the calculations of the peak intensities and the d-values of atomic spacing, a relatively high degree of crystallinity is observed.

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However, no traces of boron and its phases have been observed in this pattern due to the low reactivity of boron with silver at relatively low temperature. To confirm the composition of the Ag-Cu-B NPs ICP-OES 700 was used. 1 ml of 10 mg/ml of nanoparticles was digested in 5% nitric acid to a final concentration of 10 ppm in a volumetric flask and a standard addition method was used to detect the concentration of silver, copper and boron. The ratio of Ag:Cu:B was found to be 66.6:19.9:13.5. The hydrodynamic diameter of nanoparticles as shown in Figure 2,D, the maximum peak height of nanoparticles were at 30 nm. The SEM images showed spherical morphology of Ag-Cu-B nanoparticles with peaks indexed to Ag and Cu and O2, the semi-quantitative analysis of nanoparticles compositions showed for Ag:Cu 60:16, the B does not show due to the low atomic number. The morphology and size of particles was characterized by Hitachi cold field emission scanning electron microscope (CFe-SEM S4800) and the average size of the particles was determined as 100 nm before lactic acid treatment. After lactic acid treatment the particle sizes were 20 to 30 nm. 37

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Figure 2. Characterization of Ag-Cu-B nanoparticle treated with lactic acid (A) TEM micrograph low magnification (insert) and high magnification. (B) AFM micrograph (C) powder graph, lyophilized powder sample of nanoparticles was used to analyze X-ray diffraction pattern (insert). (D) Dynamic light scattering for size distribution. (E) EDS of the particle and SEM micrograph (insert). (F) MIC of different compositions of nanoparticles containing Ag, Cu and B.

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In vivo enumeration of S. aureus XEN-36 strain

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To confirm the number of CFUs corresponding to O.D. values in mid log phase, CFUs were enumerated from T-soy broth when O.D. was to 0.5 or more. We found 6 × 10 7 CFUs corresponded to O.D. of 0.5 at 600 nm wavelength as shown in Figure 3, A. Bacteria from mid logarithmic phase are highly dividing cells 47 and are ideal for developing infection. It was found that 150 min of incubation of silk suture in T-Soy broth provided a 0.5 O.D., 5 × 10 6CFUs/cm of silk suture as shown in Figure 3, B. The development of localized infection and post treatment progress was observed by counting number of CFUs as shown in Figure 4, which depicts a significant decrease in CFUs count after i.v. or i.m. administration of NP compared to non-treated control mice. The CFUs were also counted in liver, spleen and blood of the same experimental groups. We observed less than 100 CFUs (per ml of blood and per mg tissue for liver and spleen) in all

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Figure 3. Bacterial load on silk suture, (A) enumeration of bacterial growth at (O.D) measured at 600 nm, (B) Number of CFU's obtained from silk suture after 45 or 150 min of incubation time.

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liver, spleen or blood, which clearly indicates that bacterial infection remained largely localized and was locally progressive. Furthermore, no reduction in animal body weight or fluid intake was observed in any of the experimental groups (data not shown). To confirm the antimicrobial activity of nanoparticles with lower than 1 mg/kg concentration, we observed a reduction in CFUs as shown in Figure 5. In graph 5A, the CFUs were reduced from ~50 million in control to ~5 million in NP treated group, representing a 10-fold reduction. In contrast, in Figure 5, B, the CFU counts were 5 million for control and ~2 million in NP treated mice, which represents ~2.5 fold reduction. Thus, the extent of reduction in CFUs is directly correlated with the dose of NP used for the treatment. To confirm that the observed antimicrobial activity is solely from Ag-Cu-B nanoparticles we tested the effect of injecting iron oxide metallic nanoparticles (1 mg/kg, i.v.) on bacterial CFUs (Figure 6). Our results showed that iron oxide nanoparticles failed to give any reduction in CFUs compared to non-treated group (Figure 6). In contrast, a second group of animals given co-trimoxazole antibiotics (as a positive control) showed a significant reduction (~10-fold) in bacterial CFUs. To investigate the long-term effects of systemic Ag-Cu-B nanoparticle we did survival tests for single dose and multiple doses and monitored the mice for 12 weeks. All the mice treated

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Figure 4. Reduction in bone infection following NP administration. Infected mice were injected with 1 mg/kg bw of NP, either i.v. (via tail vein) or i.m. bacterial CFUs of mice via tail vein or intramuscular in tibia near the infection site, all bars represent the mean values ±S.E. P value b0.05** or b0.5*.

either with a single dose or multiple doses survived as well as those not treated at all. These data suggest that Ag-Cu-B nanoparticles at the dose of 1 mg/kg concentration is not lethal. Moreover, animal body weights measured twice per week over the entire observation period revealed no significant changes (data not shown). These findings suggest that systemic administration of Ag-Cu-B NPs at the 1 mg/kg dose are not associated with any overt signs of morbidity or mortality. Post treatment, the tibia was disarticulated gently and cleaned of all soft tissues. We observed that the size of the drilled hole in tibia had enlarged which could have arisen as result of loss of necrotic bone tissue due to the localized S. aureus infection or necrosis as a result of drilling.

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Oxidative stress

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Silver and copper nanoparticles are prone to produce metal ions, which could cause oxidative stress. To evaluate their toxicity GSH level was determined for mice groups used in Experiments 1 and 3. The reduced glutathione levels in bone, spleen, and liver were non-significant for all treated groups as shown in Figure 7. GSH levels in bone (treated or non-treated) was 2–3 fold higher than that in the liver or spleen. There was no decrease in the level of reduced glutathione, and therefore there

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Figure 5. Ag-Cu-B NP administered via iv, Figure 5, A and B a dose of 0.5 and 0.1 mg/kg of bw, respectively. Each bar represents mean values with ± S.E.M.

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Figure 6. Group of mice administered via IV with iron oxide nanoparticles at a dose of 1 mg/kg bw as negative control and group of mice treated with co-trimoxazole at a dose of 95 mg/kg of bw/24 h via drinking water. Each bar represent mean value ±S.E.M, *P-value b0.5.

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Histology

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H & E staining of tibial bone tissue showed normal structure with no obvious infiltration of inflammatory cells, no hemorrhage or bone necrosis. Experiment was not designed for osteoclasts or bacterial staining. Figure 8 shows the histology of treated bone.

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Discussion

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Osteomyelitis due to S. aureus is being seen with increasing frequency in patients with chronic diseases such as diabetes and peripheral vascular disease and in patients with poor dental hygiene. 48 Osteomyelitis is a progressive infection that could result in limb amputation or patient death. The objective of this

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was no apparent evidence of toxicity. There was however an increase in the level of GSH in bones treated with 1 mg/kg (i.m.), 1 mg/kg (i.v.) nanoparticles, and co-trimoxazole. The increase in GSH level was 18%, 7% and 44%, respectively but these were not significant. Liver and spleen did not show any differences in GSH level mice treated or not-treated with nanoparticles. However, mice treated with co-trimoxazole antibiotic showed an increase in the level of GSH in the liver compared to controls. The increase was however, not statistically significant.

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study was to deploy a potential inorganic route (metalo-antibiotic) to investigate therapeutic effects in an animal model for osteomyelitis treatment that may introduce a new intervention strategy for osteomyelitis management. To our knowledge this is the first in vivo study showing antimicrobial activity of AgCuB nanoparticles in osteomyelitis induced in mice and presents an antibiotic free and effective alternative that could be utilized for therapeutic management of this distressing condition. Investigators have demonstrated effectiveness of antibiotics using polymeric delivery systems, 8–10 however to our knowledge delivery of metallic particles to the site of infection has not been done or tested. S. aureus deep bone infection treatments with antibiotics has a rate of failure more than 50%. 24 There may be several reasons for failure of antibiotic therapy. It is well known that antibiotic susceptible bacteria escape therapy because of the inability of antibiotics to cross the cell membrane. 7,48 This sheltered bacteria inside the cell is then able to cause cellular damage. To address the failure of antibiotics to kill intracellular bacteria, we developed a tri element Ag, Cu, and B antimicrobial nanoparticle with the ability to effectively limit intracellular infection of bone. The antimicrobial activity of Ag +, 25,27,28 Cu + 49,50 or Ag-Cu nanoparticle 51 have been reported against S. aureus, E.coli and several other bacterial strains in vitro. In this study, we present

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Figure 7. Effect on GSH level. Each bar represents mean ± S.E.M. (A) shows reduced glutathione level in group of mice administered iron oxide nanoparticles via IV or co-trimoxazole 95 mg/kg of bw/24 h orally in their drinking water, (B) Ag-Cu-B nanoparticles administered I.V. or I.M.

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evidence demonstrating the efficacy of AgCuB nanoparticles against an established S. aureus infection deep inside the tibial bone of mice. It has been shown that silver and copper nanoparticles may exhibit strain specific antimicrobial activity, and claimed that silver has potent antimicrobial activity for E. coli and S. aureus, while as copper has shown higher antimicrobial activity for B. subtillis. 46 Silver–Copper nanoparticles were reported as one of the most efficient antimicrobial nanoparticles against E. coli with MIC of 0.5 μg/ml 46,51 in an in vitro study. The tri-element AgCuB antimicrobial nanoparticle was found to prolong the release of its components ions (Ag +, Cu +) when placed in solution compared to the single element or dual element (AgCu). In our studies (data not shown here) we found that tri element nanoparticles of the composition we used (Ag70:Cu20:B10) has a greater longevity by several folds and was found to be the most effective composition against S. aureus. Metallic nanoparticles are prone to produce free radicals, which cause cellular damage by oxidative stress. 52 Besides oxidative stress, immune response induced by AgCuB nanoparticles exposure via NOD like receptor containing pyrin domain 3 (NLRP3) has been recently reported. 37 Earlier we showed that exposure of AgCuB nanoparticles with 2 mg/kg exposure in

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Figure 8. Hematoxylin and eosin (H&E) staining of tibia bone in experimental group. Following tibial bone surgery, mice received suture impregnated with S. aureus. On day 6 post-surgery, mice were administered NP i.v. (1 mg/kg). After 24 h, all mice were sacrificed and bone tissue was processed for H&E staining. (A) 10× (B) 40×. Cortical bone (cb), bone marrow (bm), osteocytes (red arrows), megakaryocytes (black arrows).

mice produce no lethal effects. 37 In this study we utilized a single dose or multiple doses of 1 mg/kg AgCuB nanoparticles and we observed 100% survival over a period of 12 weeks after administering AgCuB nanoparticles. The dose utilized in this study was 50% less than the toxicity tolerable dose that we reported earlier. 37 The osteomyelitis mouse model developed for this study followed similar features of infection rate as was reported earlier. 6,19,53 We showed that 10 5 to 10 7 CFU'S were utilized to develop bone infection as shown in Figure 3, A. In this study the silk suture contained 6.5 × 10 6 CFU′s/cm which developed a significant local infection within the bone. In the post induction of bone infection with silk suture containing S. aureus, it was observed that infection peaks in the bones on the 6th day post-surgery, while no presence of S. aureus was observed in blood, liver or spleen tissues, suggesting that there had been no spread of systemic infections. Administration of nanoparticles 1 mg/ml (i.v.) or (i.m.) showed more than 90% reduction of CFUs at infection site. Both administration techniques are similar to other common dose administration procedures. 54 Other administration techniques such as oral or subcutaneous were not investigated in this study. Eradicating more than 90% of infection with a single dose of

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The authors would like to acknowledge Dr. Said Mansour, Dr. Sergey Suslov and Dr. Daniel Johnson at Qatar Energy and

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nanoparticles inside the bone is a major significant achievement of this study, and presents an effective alternative to address the challenge facing patients with bone infections treatment of difficult bone infections. Our study showed that the antimicrobial activity of nanoparticles applied in induced osteomyelitis is proportional to dose of the particles (Figures 4 and 5), however the negative control (iron oxide) did not show any antimicrobial activity and was similar to sham group. Co-trimoxazole was selected as a positive control for osteomyelitis therapy because of its common use in rodents and also because it was available in the animal house. 55 It is preferred to administer antibiotics to rodents by the oral route in their drinking water to avoid stress with antibiotic concentrations at or above the therapeutic limit. 56 Co-trimoxazole was therefore administered orally in the drinking water at the recommended dosage 95 mg/kg/24 h. A significant reduction of 50% in CFUs in the bone was observed after oral antibiotic administration. It was observed that when antibiotics were administered in drinking water the antibiotic concentration in the plasma was below the effective therapeutic concentration. 6 Silver or copper ions are prone to cause oxidative stress by the Fenton reaction, and it has been evaluated in vitro that up to 30 μM of silver ions exhibited no oxidative stress. 57 There is a possibility that the accumulation of nanoparticles or ions in liver or spleen may occur, and so to assess the risks associated with nanoparticle accumulation, the reduced GSH levels in the liver and spleen were measured. The data show no depletion in GSH level in the nanoparticles and antibiotic treated groups of mice (Figure 7), however a slight increase in GSH level was observed. Increase in GSH level has been reported when supplemented with N-Acetylcysteine (NAC), that spiked serum GSH level, 58 in this study antioxidant supplements were not supplied. It is possible that the rise in GSH level may be due to bone healing. The histology of treated bones did not show inflammatory infiltrate in the tibia bone, however a higher number of neutrophils for both treated and control group (as shown in Figure 8). No significant changes were observed in tibia treated with nanoparticles. The histological analysis supports results obtained by the biochemical tests as well as tests for oxidative stress markers that no-significant changes occur in bones treated with 1 mg/kg bw of AgCuB nanoparticles. We conclude that AgCuB nanoparticles present an alternative to antibiotics for treating osteomyelitis. The study also demonstrated that the administration of a single dose of AgCuB nanoparticles of 1 mg/kg significantly eradicated bacteria in the infected area of tibia bone. The administration route, (i.v. or i.m.) did not show significant difference in the bacterial reduction. The particles did not show toxic or immune responses.

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