Synthesis and Characterization of Size- and Charge-Tunable Silver Nanoparticles for Selective Anticancer and Antibacterial Treatment

Abstract

Advances in the research of nanoparticles (NPs) with controlled charge and size are driven by their potential application in the development of novel technologies and innovative therapeutics. This work reports the synthesis, characterization, and comprehensive biological evaluation of AgNPs functionalized by N,N,N-trimethyl-(11-mercaptoundecyl) ammonium chloride (TMA) and trisodium citrate (TSC). The prepared AgNPs were well characterized in terms of their morphological, spectroscopic and functional properties and biological activities. The implementation of several complementary techniques allowed not only the estimation of the average particle size (from 3 to 40 nm depending on the synthesis procedure used) but also the confirmation of the crystalline nature of the NPs and their round shape. To prove the usefulness of these materials in biological systems, cellular uptake and cytotoxicity in microbial and mammalian cells were determined. Positively charged 10 nm Ag@TMA2 revealed antimicrobial activity against Gram-negative bacteria with a minimum inhibitory concentration (MIC) value of 0.17 μg/mL and complete eradication of Escherichia coli (7 logs) for Ag@TMA2 at a concentration of 0.50 μg/mL, whereas negatively charged 10 nm Ag@TSC1 was effective against Gram-positive bacteria (MIC = 0.05 μg/mL), leading to inactivation of Staphylococcus aureus at relatively low concentrations. In addition, the largest 40 nm Ag@TSC2 was shown to exhibit pronounced anticancer activity against murine colon carcinoma (CT26) and murine mammary gland carcinoma (4T1) cells cultured as 2D and 3D tumor models and reduced toxicity against human HaCaT keratinocytes. Among the possible mechanisms of AgNPs are their ability to generate reactive oxygen species, which was further evaluated in vitro and correlates well with cellular accumulation and overall activity of AgNPs. Furthermore, we confirmed the anticancer efficacy of the most potent Ag@TSC2 in hiPSC-derived colonic organoids and demonstrated that the NPs are biocompatible and applicable in vivo. A pilot study in BALB/c mice evidenced that the treatment with Ag@TSC2 resulted in temporary (>60 days) remission of CT26 tumors.

Keywords: advanced cellular models; antibacterial activity; anticancer activity; organoids; silver nanoparticles.

Scheme 1. Schematic Illustration of AgNPs Synthesis, Characterization, and Biological Activity

Figure 1

(A) Schematic illustration of the Ag@TMA and Ag@TSC NPs preparation procedures: synthesis, growth stage, and ligand exchange. (B) Electronic absorption spectra of Ag@TMA1 and Ag@TSC2 were recorded in water at room temperature (RT) and the corresponding photographs of AgNPs solution (inset).

Figure 2

Characterization of AgNPs by detailed SEM imaging at 20 k and 100 k magnification (A), TEM images with SAED insets (B), with their corresponding bimodal size distribution (C), DLS measurements results (D), and XPS spectra (E).

Figure 3

Stability of AgNPs in DMEM cell media monitored by UV–vis absorption measurements (A) and melting curves of DNA (50 mM) in the presence of Ag@TMA2 (0.50 μg/mL) (B). The results are expressed as mean ± SEM.

Figure 4

Cellular uptake of AgNPs in (A, B) microbial and (C, D) mammalian cells after 2 and 24 h incubation, determined by ICP. Data are expressed as mean ± SEM (*** P-value < 0.001, ** P-value < 0.01, and * P-value < 0.5).

Figure 5

Antibacterial efficacy of investigated AgNPs against (A)E. coli and (B)S. aureus and ROS detection in vitro in (C)E. coli and (D)S. aureus. Data are expressed as mean ± SEM (*** P-value < 0.001, ** P-value < 0.01, and * P-value < 0.5).

Figure 6

Laser scanning confocal microscopy imaging of live/dead staining of bacteria after 2 h of incubation with AgNPs: (A)E. coli and (B) S. aureus. The live cells were stained with Hoechst33342 (blue fluorescence), and dead cells were stained with propidium iodide (PI, red fluorescence). Scale bar—5 μm.

Figure 7

(A) Morphological changes induced by AgNPs treatment in E. coli and S. aureus determined by SEM imaging. (B) EDX spectra for the E. coli treated with Ag@TMA2 and (C) SEM images of Ag@TMA2-treated colonies of E. coli with corresponding spatially resolved EDX elemental maps: Ag, C, and Cl.

Figure 8

Cytotoxicity of AgNPs against (A) HaCaT, (B) CT26, and (C) 4T1 cells; in vitro ROS generation determined by APF fluorescence signals (D), determination of the mechanism of AgNPs-induced cell death determined by flow cytometry analysis of cells stained with Annexin V-FITC for apoptosis detection and PI for necrosis, respectively (E), and AgNPs efficacy against the 3D tumor spheroid model. Spheroids were treated with AgNPs and stained with Hoechst33342 (nuclei, blue fluorescence), Calcein AM (live cells, green fluorescence), and PI (dead cells, red fluorescence); scale bar—100 μm (F). Data are expressed as mean ± SEM (*** P-value < 0.001, ** P-value < 0.01, and * P-value < 0.5).

Figure 9

(A) Scheme illustrating the differentiation of iPSC to iPSC-derived colonic organoids, (B) pluripotency and colonic tissue markers staining: phalloidin—green, OCT-4, SOX17, Nanog, FOXA2, CDX2 and cyclin D1—red, and Hoechst 33342—blue; scale bars—50 μm, and (C) live/dead staining of colonic organoids after treatments with AgNPs: Calcein AM—green, PI—red, and Hoechst 33342—blue, scale bars—50 μm.

Figure 10

In vivo efficacy of Ag@TSC2. The kinetics of CT26 tumor growth in BALB/c mice after injection of Ag@TSC2 (left) and photographs of representative tumors from the control and treated group.