Engineered metal nanoparticles in the sub-nanomolar levels kill cancer cells

Abstract

Background

Small metal nanoparticles obtained from animal blood were observed to be toxic to cultured cancer cells, whereas noncancerous cells were much less affected. In this work, engineered zinc and copper metal nanoparticles were produced from bulk metal rods by an underwater high-voltage discharge method. The metal nanoparticles were characterized by atomic force microscopy and X-ray photoelectron spectroscopy. The metal nanoparticles, with estimated diameters of 1 nm–2 nm, were determined to be more than 85% nonoxidized. A cell viability assay and high-resolution light microscopy showed that exposure of RG2, cultured rat brain glioma cancer cells, to the zinc and copper nanoparticles resulted in cell morphological changes, including decreased cell adherence, shrinking/rounding, nuclear condensation, and budding from cell bodies. The metal-induced cell injuries were similar to the effects of staurosporine, an active apoptotic reagent. The viability experiments conducted for zinc and copper yielded values of dissociation constants of 0.22±0.08 nmol/L (standard error [SE]) and 0.12±0.02 nmol/L (SE), respectively. The noncancerous astrocytes were not affected at the same conditions. Because metal nanoparticles were lethal to the cancer cells at sub-nanomolar concentrations, they are potentially important as nanomedicine.

Purpose

Lethal concentrations of synthetic metal nanoparticles reported in the literature are a few orders of magnitude higher than the natural, blood-isolated metal nanoparticles; therefore, in this work, engineered metal nanoparticles were examined to mimic the properties of endogenous metal nanoparticles.

Materials and methods

RG2, rat brain glioma cells CTX TNA2 brain rat astrocytes, obtained from the American Type Culture Collection, high-voltage discharge, atomic force microscope, X-ray photoelectron spectroscopy, high-resolution light microscopy, zeta potential measurements, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay were used in this work.

Results

Engineered zinc and copper metal nanoparticles of size 1 nm–2 nm were lethal to cultured RG2 glioma cancer cells. Cell death was confirmed by MTT assay, showing that the relative viability of RG2 glioma cells is reduced in a dose-dependent manner at sub-nanomolar concentrations of the nanoparticles. The noncancerous astrocytes were not affected at the same conditions.

Conclusion

The engineered and characterized zinc and copper nanoparticles are potentially significant as biomedicine.

Keywords:

• nanoparticles
• XPS
• atomic force
• glioma cancer cell
• zinc
• copper

Supplementary materials

Metal nanoparticles

Metal nanoparticles were produced by the modification as per the method described.^1,2 The system consists of a water container and a high-voltage generator connected with two metal electrodes submerged in water. By controlling the voltage and distance between the electrodes, the plasma created under water produces a very fine dispersion of metal into nanoparticles (Figure S1).

Two metal electrodes (99.9999%; Alfa Aesar, Haverhill, MA, USA) of 2 mm diameter are positioned in a large Pyrex jar 1 mm below the gas–water interface at a distance of ≈0.5 cm. A total of 750 mL of liquid chromatography - mass spectrometry (LC-MS) grade water (Omnisolv, Charlotte, NC, USA) is used in this procedure (Figure S1). The jar filled with nitrogen is placed in the water bath with running water to prevent overheating. An AC voltage of 15,000 volts is applied to electrodes and the electric discharge is sustained for 1 hour. The water suspension is collected in 1 L glass beaker and placed in the refrigerator for 12 hours to allow large metal particles to sediment. Then the suspended particles are separated from the sediment and subjected to centrifugation at 15,000× g for 2 hours at 25°C. After centrifugation, the pellet is discarded and the supernatant is subjected to further centrifugations to produce fractions of nanoparticles enriched in particles of particular sizes. The centrifuge speed and time to separate nanoparticles by size are estimated with Stock’s equation. The particle suspensions are analyzed similarly to that described in the work by Samoylov et al.³ The total concentration of metal in suspension was measured by atomic absorption spectra (GTW Analytical Services, Memphis, TN, USA), and the size and the number of particles are determined by atomic force microscopy.

Microscopy

The illumination optical system⁴ with a high-aperture cardioid annular condenser has been used in this work. The system produces a highly oblique hollow cone of light (numerical aperture [NA]=1.2–1.4). The illumination system is positioned in an Olympus BX51 microscope by replacing a regular condenser. The illumination system is connected with a light source (EXFO120; Photonic Solution Ltd, Edinburgh, UK) by a liquid light guide. The objective used for this work is an infinity-corrected objective HCX PL APO 100/1.40–0.70, oil, iris from Leica. The image is magnified by a zoom intermediate lens (2×-U-CA, Olympus Corporation, Shinjuku, Tokyo City, Tokyo, Japan), a homebuilt 40× relay lens, and captured by a Sony MCC-500MD video and a Dimension 8200 Dell computer. The microscope is placed on a vibration-isolated platform (manufactured by TMC, Peabody, MA, USA).

Atomic force microscopic images

The images of metal nanoparticles were taken by Bruker MultiMode 8 (Santa Barbara, CA, USA) atomic force microscope (AFM) in tapping^® (intermittent-contact) mode, using PointProbe^® Plus SEIKO microscopes – Non-Contact/Tapping Mode High Force Constant (PPP-SEIH) made by Nanosensors™ (Neuchatel, Switzerland) AFM probes; the nominal values specified by the vendor for the force constant and resonance frequency of these probes are 15 N/m and 130 kHz, respectively. The AFM imaging was used to measure the size distribution of particles. Monolayers of zinc and copper nanoparticles were prepared on a mica substrate for all measurements by adding small amount of 0.01% nanoparticles solution on freshly cleaved mica surfaces.

XPS spectra

X-ray photoelectron spectroscopy (XPS) was used to make quantitative spectroscopic measurements of the elemental composition of the nanoparticles’ surfaces. The Kratos Axis Ultra delay-line detector instrument in hybrid mode using a monochromatic Al Kα1,2 X-ray source (hυ=1,486.6 eV) was used. High-resolution spectra of Zn 2p (1,017–1,057 eV) and Cu 2p (925–965 eV) were acquired using a pass energy of 40 eV with an energy resolution of 0.1 eV. Zinc and copper nanoparticles were analyzed in a water suspension on a silicon wafer.

Cell viability assay

A cell viability assay⁵ quantitatively determined the effect of metal nanoparticles on cell viability. Cells were plated in D5648 media (Sigma 96-well polystyrene plates) at a density of 3×10³ cells/well. At 24 hours after plating, the medium was replaced with Dulbecco’s Modified Eagle’s Medium (100 μL/well) containing either 1 μmol/L staurosporine or zinc or copper nanoparticles with various concentrations (0.05–0.3 nm). At 20 hours after treatment, a 20 μL aliquot of tetrazolium salt (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide; MTT, 5 mg/mL in phosphate buffer solution [PBS]) was added to each well, and the cells were incubated for 4 hours at 37°C. MTT was reduced in metabolically active cells to form purple formazan crystals that were subsequently dissolved in dimethyl sulfoxide and quantified by a plate reader (Bio-Rad, Hercules, CA, USA). The dye is cleared to a colored product by the activity of NAD(P) H-dependent dehydrogenase enzymes, and this indicates the level of energy metabolism in cells. The color development (yellow to blue) is proportional to the number of metabolically active cells. The analysis was carried out with Origin: Data Analysis and Graphing Software (OriginLab).

Binding equations

The purpose of this section is to present a quantitative description of binding by using known binding equations and to describe the interaction of metal nanoparticles (M) and target molecules in cells (T) and resulting complexes in terms of binding parameters. The applicability of the binding equations to the M–T system is not trivial and special considerations are required. The reaction between M and T can be schematically presented as follows:

$\text{nM}+\mathrm{T}\leftrightarrows {\text{TM}}_{\mathrm{n}}$(S1)

where n is the number of metal nanoparticles bound to a single binding site on the target molecule. We accept that one, two, or more binding sites on the target molecule can be involved in the binding of one metal nanoparticle depending on the mono-, bi-, or multivalency of binding. Then n might be equal to 1, 0.5, or 1/k, where k is the valence of binding.

The association binding constant (K_b) for this reaction can be defined using the mass action law.^6,7

${\mathrm{K}}_{\mathrm{b}}=\frac{{\text{TM}}_{\mathrm{n}}}{([\mathrm{T}]{[\mathrm{M}]}^{\mathrm{n}})}$(S2)

If we neglect the number of target molecules bound to nonspecific molecules, then the total number of the target-molecule binding sites (C_T) is composed of the free binding sites and the sites bound to the metal nanoparticles:

${\mathrm{C}}_{\mathrm{T}}=\mathrm{T}+{\text{TM}}_{\mathrm{n}}$(S3)

Combining Equations S2$\text{nM}+\mathrm{T}\leftrightarrows {\text{TM}}_{\mathrm{n}}$(S1) and S3${\mathrm{K}}_{\mathrm{b}}=\frac{{\text{TM}}_{\mathrm{n}}}{([\mathrm{T}]{[\mathrm{M}]}^{\mathrm{n}})}$(S2) , we can determine the fraction of the target-molecule binding sites occupied by the metal nanoparticles:

$\mathrm{Y}=\frac{{\text{TM}}_{\mathrm{n}}}{{\mathrm{C}}_{\mathrm{T}}}=\frac{{\mathrm{K}}_{\mathrm{b}}{[\mathrm{M}]}^{\mathrm{n}}}{1+{\mathrm{K}}_{\mathrm{b}}{[\mathrm{M}]}^{\mathrm{n}}}$(S4)

The ratio of occupied and free target-molecule binding sites can be defined as follows:

$\frac{\mathrm{Y}}{1-\mathrm{Y}}={\mathrm{K}}_{\mathrm{b}}{[\mathrm{M}]}^{\mathrm{n}}$(S5)

Taking the logarithm of both sides, we get

$\log \frac{\mathrm{Y}}{1-\mathrm{Y}}=\log {\mathrm{K}}_{\mathrm{b}}+\text{nlog}[\mathrm{M}]$(S6)

A plot of the left-hand side of Equation S6$\frac{\mathrm{Y}}{1-\mathrm{Y}}={\mathrm{K}}_{\mathrm{b}}{[\mathrm{M}]}^{\mathrm{n}}$(S5) versus log[M] is known as a Hill plot. It gives an estimate of n from the slope and K_b from the ordinate intercept.

We can speculate that the binding of the metal nanoparticles and the target molecules in cells results in cell death. Then the concentration of the TM_n complexes is directly proportional to the relative cell mortality. If we denote the number of cells at no exposure to metal nanoparticles as N and the number of dead cells at the metal nanoparticles of concentration [M] as N_d, then the relative cell mortality (D) is equal to the following:

$D=\frac{N}{N_d}$(S7)

Similarly, the relative cell viability (V) is equal to:

$V=\frac{N}{N_v},$(S8)

where N_v is the number of the viable cells at the metal nano-particles of concentration [M].

From Equations S4${\mathrm{C}}_{\mathrm{T}}=\mathrm{T}+{\text{TM}}_{\mathrm{n}}$(S3) , S7$\log \frac{\mathrm{Y}}{1-\mathrm{Y}}=\log {\mathrm{K}}_{\mathrm{b}}+\text{nlog}[\mathrm{M}]$(S6) , and S8$D=\frac{N}{N_d}$(S7) , it is inferred that Equation S6$\frac{\mathrm{Y}}{1-\mathrm{Y}}={\mathrm{K}}_{\mathrm{b}}{[\mathrm{M}]}^{\mathrm{n}}$(S5) can be presented as follows:

$\log \frac{\mathrm{Y}}{1-\mathrm{Y}}=\log \frac{1-V}{V}=\log {\mathrm{K}}_{\mathrm{b}}+\text{nlog}[\mathrm{M}]$(1)

where V is the relative cell viability measured by the cell viability assay.

Thus, the measurements of the cell viability as a function of the concentration of metal nanoparticles yield values of the association constant and the valence of the binding of metal nanoparticles and the cell target molecules.

Zeta potential

Zeta potentials were calculated with Henry’s equation:

$\zeta =3\mathrm{\eta }\mathrm{\mu }/2\epsilon \mathrm{F}(k\alpha )$(S9)

Disclaimer

Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Figure S1 Setup for the production of metal nanoparticles by the underwater spark at high voltage.

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Acknowledgments

The work was funded by National Institute of Standards and Technology grant number 70 NANB14H324. The authors thank M Mansour and A David for useful discussion.

Disclosure

The authors report no conflicts of interest in this work. Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.