AgCuB nanoparticle eradicates intracellular S. aureus infection in bone cells: in vitro

Emergent Materials (2019) 2:219–231 https://doi.org/10.1007/s42247-019-00035-7 ORIGINAL ARTICLE AgCuB nanoparticle eradicates intracellular S. aureus infection in bone cells: in vitro Shahnaz Qadri 1 & Tahir Abdulrehman 2 & Jamil Azzi 3 & Said Mansour 4 & Yousef Haik 1 Received: 18 May 2019 / Accepted: 19 June 2019 / Published online: 24 July 2019 # The Author(s) 2019 Abstract Staphylococcus aureus is the leading cause of internalized bone infection. Internalized bacteria are shielded from the immune system and antibiotics causing complications of conventional antibiotic treatment. In this study, we investigate silver-copperboron (AgCuB) nanoparticles (NPs) as a potential alternative to eradicate internalized bacterial infection without causing a harming effect on the host cells. The antimicrobial property, as well as the toxicity of the AgCuB NP’s, is reported as dosedependent between 0 and 20 μg/ml. Our results showed that 1–5 μg/ml of AgCuB NPs significantly reduced internalized infection in osteoblast cells with a single dose of treatment. The host cell toxicity observed at 20 μg/ml is ten times higher than the effective antimicrobial dose. 1 Introduction Osteomyelitis by S. aureus is a damaging bone infection and if left untreated could cause patient death [1, 2]. The bacterial infection is mostly a result of hematogenous spread from a skin infection, direct exposure to open wounds, and direct contamination during surgical procedures [3]. Prognosis of direct osteomyelitis is factored due to systemic factors such as obesity and diabetes. S. aureus is well known for its affinity to bind to the extracellular matrix (BEM) of osteoblast cells and direct interaction of the intracellular space following its internalization of the osteoblasts membrane [4]. Internalization of S. aureus leads to apoptosis of osteoblast cells [5]. S. aureus which colonizes inside the osteoblast cells is responsible for the spreading and relapse of infection in the presence of antibiotics [6]. Internalized S. aureus remains protected from the immune * Yousef Haik 1 College of Science and Engineering, Hamad Bin Khalifa University, Doha, Qatar 2 College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar 3 Brigham and Women’s Hospital, Harvard Medical School, Boston, USA 4 Qatar Environment and Energy Research Institute, Hamad Bin Khalifa University, Doha, Qatar system as a result of the sheltered environment [7] leading to acute or chronic infection [8]. Recent studies have reported that the most commonly used antibiotic (vancomycin, daptomycin, and linezolid) failed to eradicate intracellular S. aureus infections [9]. Studies reported that S. aureus developed resistance to many types of antibiotics, e.g., all beta-lactams, linezolid, daptomycin, and vancomycin [10]. Hence, it is clear that therapy to eradicate intracellular infection is crucial for clinical management of osteomyelitis. Recently, several methods are employed to deliver antimicrobials at the intracellular environment [11]. For example, a planktonic S. aureus bacterium was used as a vesicle to deliver antibiotic-conjugated antibody at the intracellular environment [9]. Ultrasound-mediated delivery of antibiotic at the infection site was employed due to the enhancement of drug penetration in the cells due to the induced vibrations [12]. However, such techniques have not demonstrated effective intracellular delivery of antibiotics to eradicate intracellular infection significantly. Metallic nanoparticles (NPs) and metal ions have shown a great promise of bactericidal activity in in vitro studies and wound infection therapy [13]. Among all metallic NPs, silver NP, copper NP, and silver-copper alloys have shown the most potent antimicrobial activity against a wide range of Grampositive or Gram-negative bacterial infections [14–16]. The silver-copper alloy NPs have been reported as the most effective antimicrobial by the release of dual ions (Ag+, Cu+) compared with silver alone or copper alone nanoparticles; this ion release process is believed to cause DNA damage of the bacteria [17, 18]. Silver-copper-boron (AgCuB) have been 220 studied as antimicrobial therapy in osteomyelitis animal model [19]. Exospore of AgCuB nanoparticles related immune response and its role in inflammation has been reported which depicted that an overdose can cause hepatotoxicity [20]. The in vitro study of AgCuB NP’s toxicity in osteoblast cells and efficiency of antimicrobial activity at the intracellular level has not been reported. This study reports AgCuB NP’s ability to eradicate the intracellular infection of S. aureus invaded into osteoblast cells and AgCuB NP’s toxicity to the osteoblast cells. We have added boron element with Ag–Cu because boron is a known anticorrosive agent that delays the oxidation of copper [21, 22]. Copper oxide has less antimicrobial activity than Cu0, Cu+; hence, it is essential to minimize copper oxidation for retaining sufficient antimicrobial property of copper nanoparticles or silver-copper nanoparticles. A massive work of antimicrobial property for silver or copper nanoparticles appears in the open literature; however, the antimicrobial activity of AgCuB NPs in osteoblast cells has not been studied. We believe this is the first report that addresses the effectiveness of AgCuB nanoparticle as an antimicrobial agent for eradicating the internalized bacterial infection in osteoblasts and this study identifies a therapeutic tolerance dose of AgCuB NPs. emergent mater. (2019) 2:219–231 transmission electron microscope (TEM) holey carbon-coated copper grids and was kept under desiccation. The TEM (TalosF200X) was used to analyze the size and shape of nanoparticles. The energy-dispersive X-ray (EDX) detector installed on TalosF200X was used to identify the distribution of elements in nanoparticles at high magnification. The hydrodynamic size and zeta potential were measured after dispersing nanoparticles in deionized water, and 1 ml of nanoparticles using 5-ml syringe was loaded in the disposable zeta potential cuvette (DTS1070, Malvern). This cuvette was used for measuring zeta potential and size in the Zetasizer (ZSP–Malvern). The percentage of silver, copper, and boron was confirmed by inductive coupled optical emission spectroscopy (ICP-OES) that was used to characterize the elemental analysis. Briefly, 10 mg/ml of AgCuB nanoparticles was digested in 5% nitric acid in a volumetric flask covered with aluminum foil and was kept for 2 h at room temperature. Standard addition method was used to identify the concentration of silver, copper, and boron elements. The final working concentration of digested nanoparticles was made to 10 ppm. The standard curve was generated by using a multielement standard solution containing silver, copper, and boron, and nanoparticle-digested solution was spiked with 1, 5, and 10 ppm of standard solutions before the machine was calibrated for silver, copper, (...truncated)