Physico-Chemical Characterization and Assessment of Cytotoxic and Genotoxic Effects of Poly-Ethylene-Glycol Coated and Uncoated Gold Nanoparticles on Human Kidney (HK-2) Cells

Research Article

Austin J Environ Toxicol. 2021; 7(2): 1042.

Physico-Chemical Characterization and Assessment of Cytotoxic and Genotoxic Effects of Poly-Ethylene-Glycol Coated and Uncoated Gold Nanoparticles on Human Kidney (HK-2) Cells

Rogers CR1,2, Dasari S1,2, Patlolla AK1,2 and Tchounwou PB1*

¹RCMI Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, USA

²Department of Biology, CSET, Jackson State University, USA

³Jackson Public School, USA

*Corresponding author: Paul B Tchounwou, Presidential Distinguished Professor & Director, RCMI Center for Environmental Health, Jackson State University, Jackson, MS, USA

Received: November 12, 2021; Accepted: December 06, 2021; Published: December 13, 2021


Although gold Nanoparticles (Au-NPs) have been widely used in medicine for the diagnosis and treatment of patients due to their unique physicochemical properties, chemical stability and biocompatibility, recent reports have also highlighted their potential to induce toxicity to humans. In the present study, we investigated the toxic effects of uncoated and Polyethylene Glycol (PEG)-coated AuNPs on human kidney (HK-2) cells. Both forms of AuNP were synthesized and characterized using standard protocols. Dynamic Light Scattering (DLS), Zeta Sizer Nano ZS analyzer, Transmission Electron Microscopy (TEM), and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) were used to measure their distribution, zeta potential/surface charge, morphological size, and Au concentrations, respectively. Cytotoxicity was measured by Cyto- Tox assay and trypan blue exclusion test. Oxidative Stress (OS) was assessed by quantifying the levels of Glutathione (GSH), and Mitochondria Membrane Potential (MMP). Genotoxicity was assessed by single cell gel electrophoresis (Comet assay) and Chromosomal Aberration (CA) assay. Uncoated AuNPs significantly reduced cell viability, increased ROS, decreased GSH, depolarized the MMP, and induced significant DNA damage and chromosomal alterations including chromosome gaps, centric rings, breaks, deletions, and intra and interchromosome exchanges, in a concentration-dependent manner. PEG-coated AuNPs displayed lower cytotoxic and genotoxic effects, and did not produce any significant increase in ROS or significant decrease in GSH along with negligible polarization of the MMP. Hence, PEG-coated AuNPs are relatively less toxic than uncoated AuNPs and therefore, may have potential applications in nanomedicine.

Keywords: AuNP polymer formulations; Cell viability; Oxidative stress; Cytotoxicity; Genotoxicity; HK-2 cells


Gold Nanoparticles (AuNPs) are reported to have many biomedical and biotechnological applications including their incorporation into several diagnostic and therapeutic strategies in biology and medicine. AuNPS have been very useful not only in diagnosis of certain chronic diseases like cancer but also beneficial in treatment [1-6]. AuNPs have been extensively used due to their dynamic physical and chemical properties; however, this activity has been a public health concern [7]. Published research has demonstrated that nanoparticles can be absorbed and exhibit a long residence time with a big half-life because of the physico-chemical characteristics associated with nanosizes, shapes, and surface charges that increase their resistance to attack by the body’s immune system. Therefore, toxicological implications of AuNPs appears to be a major disquietude, limiting their utilization for diagnosis and treatment of chronic health conditions.

It is generally believed that AuNPs are relatively noncytotoxic, however, there are differing reports alluding to the potential toxicity associated with their physico-chemical characteristics. Primarily, several in vitro studies of NPs have confirmed that their toxic potential is associated with their specific particle sizes [8-11]. Size of a nanoparticle is directly linked to its function. In addition, studies on surface chemistry of AuNPs reveal novel issues on their cytotoxic potential. Small AuNPs have larger surface area exhibiting high reactivity compared to larger size AuNPs with lower adsorption and reactivity. Such feature is the fundamental factor for AuNP application in nanotechnology. Because of their size, smaller AuNPs enter the cells more rapidly and once internalized, they may interact with many cellular components to cause biological effects. The molecular mechanism of their cytotoxicity is via triggering of apoptosis signaling pathway by inducing oxidative stress (ROS) [12,13]. Studies confirm that AuNPs induce oxidative stress leading to ROS production, lipid peroxidation and apoptotic mediated cell death [14,15]. Next, surface charge constitutes one of the key factors that modulate AuNPs toxicity. It is commonly determined by measuring the zeta potentials which help in assessing the stability of nanomaterials based on whether or not their charges are positive or negative. According to Platel et al. [16] information on surface charge is important to explain uptake mechanisms that would assist in predicting likely biological interactions that would be either protective or harmful.

The kidneys function primarily to remove metabolic waste such as urea and ammonia from the body. In addition, it has been reported that the kidneys may play a role in the excretion of other waste products including toxic metals, and Nanomaterials (NMs) [17]. The kidneys are highly vascularized and receive about 20% of blood coming from the heart. Kidneys are highly sensitive to nanoparticlemediated toxicity due to their physiological activity in excretion of waste materials from human body. Hence, the major anatomical components of the kidneys including glomerular structures and tubular epithelial cells may be adversely affected by NMs. Because of their key role in the excretory system and their vulnerability to toxic chemicals, the kidneys represent important targets and test models for studying the deleterious effects of potential nephrotoxicants including nanomaterials. Hence, scientific reports show that kidneys act as target for nanomaterial toxicity [18].

However, AuNPs toxicity can be highly modulated by functionalized surface agents. Polymeric ligands improve the longterm stability of AuNPs and increase hydrophilicity of the surface. These nanoparticle-polymer formulations not only help in target site specificity but also enhance the circulation time in the blood [19]. In order to reduce the toxic side effects of AuNPs, coating agents like Polyethylene glycol (PEG) have been used [20-23]. Nanoparticle coating will increase the biocompatibility by modification of their surface to water solubility and other factors. To improve the stability and solubility of nanoparticle by conjugation with a polymer like PEG has been successful as it prevents aggregation, opsonization and enzyme degradation [24].

Therefore, the specific aims of this research were to synthesize and characterize PEG-coated and uncoated AuNPs using analytical methods such as TEM, zeta potential, dynamic light scattering and ICP-OES, and to assess their cytotoxicity and genotoxicity to human kidney (HK-2) cells.

Materials and Methods

Cells and culture media

Human Kidney (HK-2) epithelial cells, streptomycin (10000U/ ml) and penicillin (10000U/ml) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells grown in DMEM/F12 (Dulbecco’s Modified Eagles Medium) supplemented with fetal bovine serum (FBS) were purchased from ThermoFisher (Life Technologies) (Suwanee, GA, USA).

Synthesis of gold nanoparticles (AuNPs)

For the synthesis of uncoated AuNPs 3.75mL of Au chloride was added to boiling nanopure water for one hour. A reducing agent, (1% Trisodium Citrate) was added and allowed to cool for 10 min then centrifuge for three hours at 5,000 rpm. For the synthesis of PEGcoated nanospheres, AuNPs were conditioned with 0.5% vol ethanol and then immersed in the dispersion media. They were then mixed by sonication for 20 min and 8% of emulsifying solution contain 4% each of Polyoxyethylene Glycerol Trioleate and Tween 20 was used to create a more stable and biocompatible structure.

Size determination of gold nanoparticles

The size and distribution of AuNPs in ultrapure water were measured by dynamic light scattering (DLS), using Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., Worcestershire, UK). Transmission electron microscopy (TEM) was performed to characterize the studied nanoparticles. The preparation and examination of AuNPs on TEM were conducted as previously described by Patlolla et al. [25].

Elemental analysis of gold nanoparticles composition

HK-2 cells treated with AuNPs were analyzed by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) using a Perkin Elmer ICP-OES Optima 8000 DV available in our Environmental Toxicology Research Laboratory at Jackson state University. Sample preparation, and spectral collection and analysis of gold content were performed following the protocol described by Schmidt et al. [26].

Physicochemical properties of AuNPs

Zeta potential (ζ-potential) measurements were performed on both coated and uncoated AuNP using Malvern Instruments’ Zetasizer Nano ZS. Sample preparation and instrumental analysis were carried out following the analytical protocol previously described in our laboratory [27].

Cell treatment with gold nanoparticles

The experiments were conducted in 96-well plates with three replicates for each positive control, negative control, non-cell control (serum free growth media without cells) and treatment sample of PEG coated and uncoated AuNP. 1x106 cells/mL was used for both controls and AuNP-treated cells. In order to determine AuNPs toxicity and their LC50 values, 6.25, 12.5, 25, 50, 100 and 200 μM NP were prepared in serum free DMEM//F12 media. All samples were kept for 24 hours at in a humidified 5% CO2 incubator at 37°C.

Measurement of cell viability and cytotoxicity

The cell viability was assessed by the trypan blue exclusion test using Nexcelom cellometer (Lawrence, MA, USA). This assay reflects all treatment-related effects (necrosis, cell-cycle delay, and apoptosis) that reduce the number of living cells, as described by Bumah et al. [28]. Results were expressed as the percentages of cell viability in treated cells compared to the negative control.

Cyto Tox-Glo assay

Cyto tox-Glo assay describe by Niles et al [29] determines cell mortality by measuring the amount of stable protease activity being release into the cell culture medium. The luminescence reflecting the number of viable cells was assessed as previously described [30].

ROS analysis using 2’,7’-dichlorofluorescein (H2DCF)

ROS levels were determined using 2’, 7’-dichlorofluorescein (H2DCF) obtained from Sigma Company. After incubation the cells were treated with H2DCF and spectrophotometric measurements of ROS were taken at 530nm following the procedure previously described by Skalska et al. [31].

Measurement of glutathione (GSH)

GSH-Glo Glutathione luminescence-based assay (Promega, Madison, WI, USA) was conducted to detect and quantify glutathione in controls and AuNP-treated cells. This process involved seeding a 96-well tissue culture flask with 100μL aliquots containing 104 cells per well and incubating for 24 hours. A standard curve with a GSH range of 0-5 μM was generated by diluting 5mM Glutathione stock solution (1:100) in distilled water. The complete assay was performed according to the protocol instructions (Technical Manual TM344 - GSH/GSSG-Glo assay V6611) from Promega Corporation (Madison, Wisconsin, USA).

Measurement of mitochondrial membrane potential (Δѱm)

5x 104 cells were placed in each well of a 96 well black-sided plate, and kept overnight to allow attachment, and subsequently washed one time with the dilution buffer. The fluorescence of each sample was read using an Omega Polarstar platform-reader with an excitation wavelength of 535±17.5 nm and an emission wavelength of 590±17.5 nm. The calculations and data analysis were conducted following the manufacturer’s protocol [32].

Assessment of genotoxicity

Comet assay: Genotoxicity in AuNP-treated and untreated HK-2 cells was assessed by alkaline gel electrophoresis using Comet assay kit for single cell gel electrophoresis from Trevigen (Gaithersburg, MD, USA). Both controls and treated cells were analyzed for DNA damage following the Comet Assay protocol by Kermanizadeh [33]. The data were evaluated using the DNA damage analysis software (Loats Associates Inc., USA).

Chromosome aberration assay: Chromosome aberration was assessed using a previously described standard protocol [34]. A maximum of 200 cells of best quality metaphase spread of each treatment were captured and 100 cells of each treatment were scored manually using an Olympus microscope with a 10x magnification. Various types of chromosomal aberrations such as breaks, dicentrics, rings, and chromatid breaks were scored and analyzed for each treatment group.

Statistical analysis

Triplicate experiments were performed, and the data were expressed as means ± SDs. Using SAS 9.1 software, assessment of differences in mean values between controls and AuNP-treated cells was done by one-way analysis of variance (ANOVA) for multiple comparisons or Student’s t-test for paired groups. Statistically significant differences were noted for p-value less than 0.05.


Characterization of AuNPs

The nanoparticles that were characterized included Polyethylene Glycol (PEG) coated and uncoated and gold nanoparticles. Their size distribution and morphology were respectively characterized by DLS and TEM analysis (Figure 1). Data generated from this analysis indicated that the size of both forms of gold nanoparticles ranged from 20-25 nm (Table 1) and their shape was spherical. Dynamic Light Scattering (DLS) was used to determine average aggregate hydrodynamic diameters. Since light scattering is exponentially proportional to NP size, we have reported our DLS data as percentages of total volume and not percentages of measured intensity. Using this technique, we observe a 5nm increase in the hydrodynamic diameter of 25nm AuNPs after addition PEG increasing aggregation [35]. TEM images were obtained with a JOEL 1011 TEM system calibrated at 100keV and 300kX magnification. Among the two structures analyzed, PEG-coated AuNPs (Type 1) agglomerated in the 25-30 nm size range, while uncoated AuNPs (Type 2) were mainly spherical in the same range of 20-25 nm.