Boron doped silver-copper alloy nanoparticle targeting intracellular S. aureus in bone cells

Abstract

Objectives: Alloyed metallic nanoparticles of silver and copper are effective against intracellular infection. However, systemic toxicity may arise due to the non-specific delivery of the nanoparticles. In addressing the issue, this study deals with the targeting of silver-copper-boron (ACB) nanoparticles to infected osteoblasts, which could decrease systemic toxicity and form the basis of targeting specific markers expressed in bone infections.

Methods: ACB nanoparticles were synthesized and conjugated to the Cadherin-11 antibody (OBAb). The effect of targeting nanoparticles against extracellular and intracellular S. aureus was determined by enumeration of bacterial growth. The binding of the targeting nanoparticles to infected osteoblasts as well as the visualization of live/dead bacteria due to treatment was carried out using fluorescence microscopy. MTT assay was used to determine the viability of osteoblasts with different concentrations of the nanoparticles.

Results: The ACB nanoparticles conjugated to OBAb (ACB-OBAb) were effective against extracellular S. aureus. The ACB-OBAb nanoparticles showed a 1.32 log reduction of intracellular S. aureus at a concentration of 1mg/L. The ACB-OBAb nanoparticles were able to bind to the infected osteoblast and showed toxicity to osteoblasts at levels ≥20mg/L. Also, the percentage of silver, copper, and boron in the nanoparticles determined the effectiveness of their antibacterial activity.

Conclusion: The ACB-OBAb nanoparticles were able to target the osteoblasts and demonstrated significant antibacterial activity against intracellular S. aureus. Targeting shows promise as a strategy to target specific markers expressed on infected osteoblasts for efficient nanoparticle delivery, and further animal studies are recommended to test its efficacy in vivo.

Fig 1. Characterization of nanoparticles.

i(a) Schematic representation of the synthesis of ACB nanoparticles, i(b) Plasmon resonance of ACB nanoparticles and i(c) Schematic representation of the conjugation of ACB nanoparticles to anti-osteoblast cadherin antibodies. ii(a) Line scan for the interatomic distance from high-resolution TEM, ii(b) TEM image of a single ACB nanoparticle, ii(c) High-resolution TEM of ACB nanoparticles, ii(d) Fast Fourier Transform (FFT) image of the ACB nanoparticles and ii(e) Size distribution of the ACB nanoparticles. iii(a) Size distribution by intensity measured using dynamic light scattering, iii(b) Zeta potential measurement of ACB and ACB-OBAb, iii(c) Average size of ACB nanoparticles measured by TEM and Zetasizer and iii(d) Silver and copper percentage in ACB nanoparticles determined using Energy Dispersive X-ray Spectroscopy (EDS). iv(a) TEM image of ACB nanoparticles and ii(b) STEM image of ACB nanoparticles. iv(c), iv(d), iv(e) and iv(f) are the elemental mapping for silver, copper, boron, and sulfur, respectively. iv(g) EDS spectrum of ACB nanoparticles.

Fig 2. Characterization of conjugation of OBAb to nanoparticles.

(a) Fourier Transform Infrared Spectroscopy (FTIR) of ACB and ACB-OBAb (Osteoblast cadherin antibody conjugated ACB nanoparticles), (b) Standard curve for IgG determined using Bradford assay. The blue marker represents the concentration of non-conjugated OBAb in the supernatant to determine the level of conjugation and (c) the fluorescent intensity of Alexa fluor 488 secondary antibody (goat anti-mouse IgG) bound to ACB-OBAb measured using spectrofluorimetry and the study was carried out in triplicates.

Fig 3. Antibacterial activity of nanoparticles and gentamicin against S. aureus.

(a) Screening for the extracellular antibacterial activity of ACB nanoparticles (with varying ratios of Ag, Cu, and B). Each nanoparticle was tested in triplicates (n = 3). Statistical analysis by 2-way ANOVA with Dunnett’s multiple comparison test. Each bar represents the mean ± S.D. The statistical significance was compared to the non-treated (0 mg/L) group. **** represents P ≤0.0001, *** represents P ≤0.001, ** represents P ≤0.01, * represents P ≤0.05 and ns represents >0.05. A t-test was conducted between the antibacterial data sets of ACB (6-3-1) and ACB (4.5–4.5–1) at 2.5mg/L, which showed a statistical significance of P ≤0.001 (***). The significance threshold was set at 0.05 for the 2way ANOVA and t-test. (b) The antibacterial activity of gentamicin against extracellular S. aureus. (c) The antibacterial activity of ACB nanoparticles against extracellular S. aureus. (d) The antibacterial activity of ACB-OBAb nanoparticles against S. aureus. For images (b), (c), and (d), each bar represents the mean ± S.D. The statistical significance was compared to the non-treated group using a t-test. The significance threshold was set at 0.05 for the t-test.

Fig 4. S. aureus internalization and cadherin expression.

Upper panel (scale 25μm)—Noninfected osteoblasts. (a) Osteoblasts exhibiting autofluorescence. (b) Osteoblasts stained with DAPI (blue) and (c) Merged image of the non-infected osteoblasts. Middle panel (scale 20μm)–S. aureus infected osteoblasts. The S. aureus bacteria were labeled with FITC (green). (d) Osteoblasts with internalized S. aureus appear green, indicating internalization. (e) The infected osteoblasts are stained with DAPI (blue). Osteoblasts nucleus as well as the S. aureus (small blue spots) were stained with DAPI, which appear blue (f) Merged image of the infected osteoblasts. Lower Panel (scale 25μm)–Cadherin-11 expression by infected osteoblasts. Osteoblasts were treated with primary mouse anti-osteoblast cadherin antibodies and counterstained using Alexa fluor 555 (AF555) labeled secondary goat anti-mouse IgG. (g) Osteoblast expressing cadherin-11 labeled with AF555 secondary antibody (green). (h) Osteoblasts labeled with DAPI (blue). (i) Merged image of g and h.

Fig 5. Internalization of ACB-OBAb by osteoblasts.

Upper Panel (scale 20μm)—The binding of ACB-OBAb to osteoblasts. (a) DIC image of osteoblast treated with ACB-OBAb. (b) ACB-OBAb stained (red) with AF555 secondary goat anti-mouse IgG. (c) The ACB-OBAb treated osteoblasts treated with DAPI (blue). (d) Merged image of (b) and (c) showing the binding of ACB-OBAb (red) binding to osteoblast. Lower Panel (scale 25μm)—The binding of ACB-OBAb to infected osteoblasts. (e) DIC image of osteoblast treated with ACB-OBAb. (f) ACB-OBAb stained (red) with AF555 secondary goat anti-mouse IgG. (g) The internalized S. aureus stained using FITC labeled anti-S. aureus mouse IgG. (h) The infected osteoblasts treated with DAPI (blue). The S. aureus was also stained with DAPI besides the osteoblast nucleus. (i) Merged image of f, g, and h, showing the binding of ACB-OBAb to infected osteoblasts.

Fig 6. The antibacterial activity of ACB and ACB-OBAb against internalized S. aureus.

Each bar represents the mean ± S.D. Each nanoparticle treatment concentration against internalized bacteria was tested in a minimum of triplicates. The statistical significance was compared to non-treated (0μg/mL) group) using a t-test. **** represents P-value ≤0.0001. The significance threshold was set at 0.05 for the t-test.

Fig 7. Live/dead bacterial staining of infected osteoblasts.

Propidium iodide (PI) (Ex 535/ Em 617) Syto 9 (Ex 485/ Em 498) Staining. Upper panel (Scale 20μm): S. aureus infected osteoblast treated with 5mg/L of ACB-OBAb. (a) PI (red) staining showed dead intracellular S. aureus (small red spots). PI is not permeable to live cells. (b) Syto 9 (green) staining which is taken up by both live and dead bacteria. (c) Merged image in which the orange-red spots indicate dead bacteria. Middle Panel (Scale 25μm): Infected osteoblasts not treated with ACB-OBAb. (d) PI staining—small red spots indicate dead bacteria, (e) Syto 9 staining in which the green spots indicate live bacteria. (f) Merged image of (d) and (e). A higher number of live bacteria was seen in non-treated infected osteoblasts. Lower Panel (Scale 25μm)–Control: Non-infected and not treated with ACB-OBAb. (h) PI stained osteoblasts. (i) Syto 9 stained osteoblasts. (h) Merged image of (h) and (i), which does not show green or red spots of dead or live bacteria.

Fig 8. MTT assay.

The cytotoxicity of (a) ACB to osteoblasts (b) ACB-OBAb to osteoblasts. Each bar represents the mean ± S.D. The cytotoxicity of each nanoparticle concentration to osteoblast was carried out in a minimum of triplicates. Each treatment concentration was compared to the control (0 mg/L) using a t-test with Welch’s correction. **** represents P-value ≤0.0001, *** represents P-value ≤0.001, ** represents P-value ≤0.01 and “ns” represents non-significant. The significance threshold was set at 0.05 for the t-test.