Magnetic Nanoconjugated Teicoplanin: A Novel Tool for Bacterial Infection Site Targeting

Abstract

Nanoconjugated antibiotics can be regarded as next-generation drugs as they possess remarkable potential to overcome multidrug resistance in pathogenic bacteria. Iron oxide nanoparticles (IONPs) have been extensively used in the biomedical field because of their biocompatibility and magnetic properties. More recently, IONPs have been investigated as potential nanocarriers for antibiotics to be magnetically directed to/recovered from infection sites. Here, we conjugated the "last-resort" glycopeptide antibiotic teicoplanin to IONPs after surface functionalization with (3-aminopropyl) triethoxysilane (APTES). Classical microbiological methods and fluorescence and electron microscopy analysis were used to compare antimicrobial activity and surface interactions of naked IONPs, amino-functionalized NPs (NP-APTES), and nanoconjugated teicoplanin (NP-TEICO) with non-conjugated teicoplanin. As bacterial models, differently resistant strains of three Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis) and a Gram-negative representative (Escherichia coli) were used. The results indicated that teicoplanin conjugation conferred a valuable and prolonged antimicrobial activity to IONPs toward Gram-positive bacteria. No antimicrobial activity was detected using NP-TEICO toward the Gram-negative E. coli. Although IONPs and NP-APTES showed only insignificant antimicrobial activity in comparison to NP-TEICO, our data indicate that they might establish diverse interaction patterns at bacterial surfaces. Sensitivity of bacteria to NPs varied according to the surface provided by the bacteria and it was species specific. In addition, conjugation of teicoplanin improved the cytocompatibility of IONPs toward two human cell lines. Finally, NP-TEICO inhibited the formation of S. aureus biofilm, conserving the activity of non-conjugated teicoplanin versus planktonic cells and improving it toward adherent cells.

Keywords: Staphylococcus aureus biofilm; antibiotic resistance; antimicrobial activity; glycopeptide antibiotics; iron oxide nanoparticles; teicoplanin.

FIGURE 1

TEM images and size distribution of IONPs (A,D), NP-APTES (B,E), and NP-TEICO (C,F).

FIGURE 2

Synthetic route for teicoplanin conjugation to IONPs (not in scale). The first step is functionalization of the IONPs with APTES, followed by the conjugation of teicoplanin by covalent bonding of the terminal carboxylic groups of the antibiotic molecules with the amino groups of NP-APTES via EDC/NHS cross-linking.

FIGURE 3

Agar diffusion assay for measuring the antimicrobial activity of IONPs, NP-APTES, NP-TEICO, and non-conjugated teicoplanin versus the two Gram-positive bacteria S. aureus ATCC 6538P (A) and B. subtilis ATCC 6633 (B), and versus the Gram-negative E. coli ATCC 35218 (C).

FIGURE 4

Population growth kinetics of S. aureus ATCC 6538P (A), B. subtilis ATCC 6633 (B), and E. coli ATCC 35218 (C) exposed to teicoplanin (orange), IONPs (red), NP-APTES (green), and NP-TEICO (violet). Cultures without any addition (blue) were used as controls. Growth was recorded for 5 h. Black arrows indicate the addition (after 1 h of growth) of NP preparations and of teicoplanin to the bacterial populations. Triplicate experiments were conducted for each condition: standard errors were lower than 5%.

FIGURE 5

Bacterial cell viability of S. aureus ATCC 6538P, B. subtilis ATCC 6633, and E. coli ATCC 35218 measured as CFUs after 5-h growth (see Figure 4) in the presence of IONPs (black bar), NP-APTES (gray bar), NP-TEICO (light gray bar), and teicoplanin (white bar) compared to the untreated control populations (lined bar). Triplicate experiments were conducted for each condition, and the error bars represent the standard errors. One-way ANOVA analyses, ^∗p < 0.05 and ^∗∗∗p < 0.0001.

FIGURE 6

Fluorescence microscopy images of live and dead cells of S. aureus ATCC 6538P [first column on the left: (A,D,G,J,M)], B. subtilis ATCC 6633 [middle column: (B,E,H,K,N)], and E. coli ATCC 35218 [column on the right: (C,F,I,L,O)] in the absence and presence of different NP preparations and of teicoplanin. (A–C) untreated cells; (D–F) cells treated with IONPs; (G–I) cells treated with NP-APTES; (J–L) cells treated with NP-TEICO; (M–O) cells treated with teicoplanin. Scale bar: 12 μm.

FIGURE 7

TEM images of S. aureus ATCC 6538P [first column on the left: (A,D,G,J,M)], B. subtilis ATCC 6633 [middle column: (B,E,H,K,N)], and E. coli ATCC 35218 [column on the right: (C,F,I,L,O)] cells in the absence and presence of different NP preparations and of teicoplanin. (A–C) untreated cells; (D–F) cells treated with IONPs; (G–I) cells exposed to NP-APTES; (J–L) cells exposed to NP-TEICO; (M–O) cells treated with teicoplanin. Scale bars: 1 μm.

FIGURE 8

TEM images of S. aureus ATCC 6538P (A–C) exposed to NP-APTES (A), NP-TEICO (B), teicoplanin (C), and B. subtilis ATCC 6633 (D,E) exposed to NP-TEICO (D) and teicoplanin (E). Scale bar: 500 nm. indicates ghost cells; ^∗indicates lysed cells; white arrows indicate mesosome-like structures.

FIGURE 9

Effect of increasing concentrations of teicoplanin, NP-TEICO, IONPs, and NP-APTES on S. aureus ATCC 6538P biofilm formation. In the case of NP preparations, the amounts to be added were defined considering the teicoplanin loaded on IONPs under the conditions defined in the Materials and Methods. Effect on adherent biomass following crystal violet staining (A). Effect on planktonic (B) and adherent (C) cells exposed to teicoplanin (orange), IONPs (red), NP-APTES (green), and NP-TEICO (violet) on viability assay. The values are expressed as mean ± SD of three independent experiments. One-way ANOVA analyses, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.0001.

FIGURE 10

Cell viability of SKOV 3 (A,C) and hASC (B,D) after different times of exposure to IONPs (black), NP-TEICO (gray), and teicoplanin (white). Cell viability is expressed as a percentage of viable cells compared to the untreated sample, set as 100%. Here, 0.78 μg/mL of non-conjugated or nanoconjugated teicoplanin or 6.24 μg/mL of carrying NPs were added in (A,B); 6 μg/mL of non-conjugated or nanoconjugated teicoplanin or 48 μg/mL of carrying NPs were added in (C,D). In the case of NP preparations, the amounts to be added were defined considering the teicoplanin loaded on IONPs under the conditions defined in the Materials and Methods. The values are expressed as mean ± SD of three independent experiments. One-way ANOVA analyses, ^∗p < 0.05 and ^∗∗p < 0.01.