Synergistic Antibacterial Effects of Amoxicillin and Gold Nanoparticles: A Therapeutic Option to Combat Antibiotic Resistance

Abstract

Compacted Au@16-mph-16/DNA-AMOX (NSi) nanosystems were prepared from amoxicillin (AMOX) and precursor Au@16-mph-16 gold nanoparticles (Ni) using a Deoxyribonucleic acid (DNA) biopolymer as a glue. The synthesized nanocarrier was tested on different bacterial strains of Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae to evaluate its effectiveness as an antibiotic as well as its internalization. Synthesis of the nanosystems required previous structural and thermodynamic studies using circular dichroism (CD) and UV-visible techniques to guarantee optimal complex formation and maximal DNA compaction, characteristics which facilitate the correct uptake of the nanocarrier. Two nanocomplexes with different compositions and structures, denoted NS₁ and NS₂, were prepared, the first involving external Au@16-mph-16 binding and the second partial intercalation. The Ni and NSi nanosystems obtained were characterized via transmission electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS) techniques to measure their charge, aggregation state and hydrodynamic size, and to verify their presence inside the bacteria. From these studies, it was concluded that the zeta potential values for gold nanoparticles, NS₁, and NS₂ nanosystems were 67.8, -36.7, and -45.1 mV. Moreover, the particle size distribution of the Au@16-mph-16 gold nanoparticles and NS₂ nanoformulation was found to be 2.6 nm and 69.0 nm, respectively. However, for NS₁ nanoformulation, a bimodal size distribution of 44 nm (95.5%) and 205 nm (4.5%) was found. Minimal inhibitory concentration (MIC) values were determined for the bacteria studied using a microdilution plates assay. The effect on Escherichia coli bacteria was notable, with MIC values of 17 µM for both the NS₁ and NS₂ nanosystems. The Staphylococcus aureus chart shows a greater inhibition effect of NS₂ and NP₂ in non-diluted wells, and clearly reveals a great effect on Streptococcus pneumoniae, reaching MIC values of 0.53 µM in more diluted wells. These results are in good agreement with TEM internalization studies of bacteria that reveal significant internalization and damage in Streptococcus pneumoniae. In all the treatments carried out, the antibiotic capacity of gold nanosystems as enhancers of amoxicillin was demonstrated, causing both the precursors and the nanosystems to act very quickly, and thus favoring microbial death with a small amount of antibiotic. Therefore, these gold nanosystems may constitute an effective therapy to combat resistance to antibiotics, in addition to avoiding the secondary effects derived from the administration of high doses of antibiotics.

Keywords: amoxicillin; antibiotic resistance; aureus nanosystem; gemini surfactant; gold nanoparticles.

Scheme 1

(A) Schematic representation of the synthesis of Au@16-mph-16 nanoparticles. (B) Schematic representation of the synthesis of Au@16-mph-16/DNA-AMOX nanosystem (for more details, see Graphic Abstract).

Figure 1

(A) Absorption spectra of the DNA/AMOX complex in the presence of different Au@16-mph-16 concentrations. Curves correspond from blue to red spectra to 0, 0.028, 0.075, 1.49, 2.44, 3.00, 3.86, 5.00, 5.70, 7.46, 9.30, 14.0, 18.0, 23.3, 28.7, 30.0, 31.6, 32.6, 33.6, 35.6, 37.6 and 40.9 nM of Au@16-mph-16. C_DNA = C_AMOX = 68 μM. (B) Analysis of the absorbance titration data for the DNA/AMOX-Au@16-mph-16 system in water. C_DNA = C_AMOX = 0.97 nM, λ = 518 nm, T = 298.2 K. Data are fitted according to a two-state model (Equation (3)).

Scheme 2

Schematic representation of the absorbance signal distribution according to the two-state model. A_f and A_b represent the absorbance of the free DNA/AMOX complex and the complex bound to the surface of AuNPs, respectively.

Figure 2

CD study for the interaction of Au@16-mph-16 with DNA/AMOX complex at different C_Au@16-mph-16 concentrations. C_DNA = 68 μM and C_AMOX = 68 μM. T = 298.2 K. (A) CD trend in molar ellipticity units ([θ]_280nm) at 280 nm. (B) Curves correspond to C_Au@16-mph-16 values of (B) (●) 0, (●) 1.49 nM, (●) 2.80 nM and (●) 3.4 nM. (C) (●) 3.4 nM, (●) 7.46 nM and (●) 9.30 nM. (D) (●) 9.30 nM, (●) 11.2 nM, (●) 14.0 nM, (●) 23.0 nM and (●) 33.3 nM. The arrows show the direction of CD change.

Figure 3

Microphotograph made with TEM TALOS, showing the gold core of the nanoparticles and the corresponding histogram of size distribution on 350 gold cores of the nanoparticles, produced using ImageJ software.

Figure 4

EDS-MET graph showing the chemical composition of Au@16-mph-16 gold core nanoparticles. Inorganic compounds such as Copper (Cu) and Gold (Au) were observed.

Figure 5

Stability study over time, performed using UV-visible spectrophotometry, for Au@16-mph-16 nanoparticles.

Figure 6

Absorbance spectra and evolution over time of different nanosystems. (A,B) Absorbance spectra of N₁ and N₂ (Au@16-mph-16 precursors). (C,D) Absorbance spectra of NS₁ and NS₂ (Au@16-mph-16/ADN/AMOX) compacted nanosystems. (E) Evolution over time of absorbance at fixed 518 nm wavelength (SPR location) for Au@16-mpPh-16 precursors at different concentrations; blue triangles correspond to [Au@16-mph-16] = N₁, and red diamonds correspond to [Au@16-mph-16] = N₂. (F) Evolution over time of absorbance at a fixed 520 nm wavelength for compacted nanocomplexes at different concentrations; blue triangles correspond to [Au@16-mph-16/DNA/AMOX] = NS₁ and red diamonds correspond to [Au@16-mph-16/DNA–AMOX] = NS₂.

Figure 7

SEM images of different populations of bacteria: (A) E. coli, (C) S. aureus and (E) S. pneumoniae. SEM-EDS analysis of the chemical analysis of the sample showing the presence of Au from the nanoparticles: (B) E. coli, (D) S. aureus and (F) S. pneumoniae.

Figure 8

Spectrum produced to verify the presence of gold inside the microorganisms (A) E. coli, (B) S. aureus, and (C) S. pneumoniae. Note that elements such as the constituents of microorganisms appear alongside some used in the preparations.

Figure 9

Unreleased amounts of AMOX (%) from the nanocomplexes vs. time in Mueller Hinton medium at 37 °C. The continuous line corresponds to the best fit obtained with the bi-exponential first-order kinetic model described in Equation (4). (A) NS₁ formulation, (B) NS₂ formulation.

Figure 10

Microdilution assay. Spectrophotometry results for distinct bacterial agents. Dots correspond to experimental data, and solid lines to trend lines. AMOX control is shown in red, N₁ and N₂ AuNPs in orange and in blue, respectively, and NS₁ and NS₂ nanosystems in black and in cyan, respectively. OD_CORR corresponds to the corrected optical density of the systems (see Section 2.2.8 for more details).

Scheme 3

Illustration of the possible mechanisms of action of Ni precursors (Au@16-mph-16) and NSi nanosystems (Au@16-mph-16/DNA-AMOX) on Gram-negative (A) and Gram-positive bacteria (B). On the left of the image, the differences between the walls of Gram-negative and Gram-positive bacteria are illustrated.

Figure 11

TEM microphotograph of E. coli after 24 h of treatment. (A) Control bacteria without treatment. (B) Bacteria treated with AMOX. (C,D) Bacteria treated with the N₂ precursor. (E,F) Bacteria treated with the NS₂ nanosystem. Arrows indicate the presence of metallic nuclei compatible with the gold cores of the nanoparticles. Asterisks (*) indicate the remains of nanoparticles surrounded by biological material because of the action of the nanosystems.

Figure 12

TEM photomicrograph of S. aureus after 24 h of treatment. In (A), the control is shown, corresponding to a population of bacteria without treatment. In (B), populations of bacteria treated only with the antibiotic (AMOX) are observed. The treatment with the precursors is shown in (C,D). The population of bacteria treated with NS₂ is shown in (E,F). The arrow indicates the presence of metallic nuclei compatible with the gold nuclei of the nanoparticles.

Figure 13

TEM microphotograph corresponding to a population of S. pneumoniae after 24 h of treatment. (A) Control bacteria without treatment. (B) Bacteria treated with the AMOX antibiotic. (C,D) Bacteria treated with the N₂ precursor. (E,F) Bacteria treated with the NS₂ nanosystem. Arrows indicate the presence of metallic nuclei compatible with the gold cores of the nanoparticles. Asterisks (*) indicate the remains of nanoparticles surrounded by biological material because of the action of the nanosystems.