Quantifying the Cellular Toxicity Caused by Nanoparticles

Nanoparticles such as carbon nanotubes (CNTs), graphene and quantum dots (QDs) have unique spectral features that have precise applications in both drug delivery and in vivo imaging. These particles can fluoresce in the biological window which allow for better imaging with enhanced sensitivity.

Graphene and CNTs have high surface area and good light absorption properties, which make them as excellent photothermal therapy and drug delivery agents. However, these properties make it difficult for researchers to characterize the toxicity of biologically-focused nanomaterials.

This is because the intrinsic absorption and fluorescence of these nanomaterials overlap with the fluorescence and absorption region utilized in vitro cell toxicity assays. These aspects result in ambiguous and incorrect results. Moreover, when nanoparticles are combined together, their toxicity, fluorescence, and absorption properties change significantly. These changes, however, are less obvious in materials that are individually solubilized.

Hence, toxicity studies that compare aggregated against non-aggregated are known to have a systemic bias. This article demonstrates how Beckman Coulter’s Optima MAX-XP ultracentrifuge can be used to reduce the time taken to remove the combined nanotubes. It also describes how the Vi-CELL XR cell viability analyzer from Beckman Coulter can be used to determine cell toxicity in the presence of single-walled carbon nanotubes (SWCNT).

Carbon Nanotubes Preparation

SWCNTs were combined with 0.2% 1, 2-Distearoyl- hosphatidylethanolamine -methyl-polyethyleneglycol (DSPE-mPEG, 5 kDa molecular weight) in 10mL water. The solution thus obtained was bath-sonicated for half an hour, and then using established procedures well-dispersed carbon nanotubes were produced.

Next, by means of a TLA-120.2 rotor in an Optima MAX-XP Ultracentrifuge, 5mL SWCNT solution was centrifuged in polycarbonate centrifuge tubes, i.e., Beckman Coulter P/N 343778, at 22°C temperature and 55,000 RPM for a couple of minutes (Figure 1). Then, 650µL of supernatant was collected, and the ultracentrifuged SWCNT (UCF’d SWCNT) and uncentrifuged SWCNT (As-Made SWCNT) were concentrated with 10 kDa, Amicon Ultra 0.5mL centrifugal filters and Beckman Coulter’s Microfuge 20 microcentrifuge.

Using the mass extinction coefficient of SWCNTs at 808nm of 46.5 L/g*cm and a UV-Vis-NIR spectrophotometer, the concentration was determined. Subsequently, both As-Made SWCNTs and UCF’d SWCNTs were diluted with deionized water to 0.06mg/mL, 0.3mg/mL, and 0.6mg/mL.

Figure 1. Optical images of SWCNT (a) without centrifugation and (b) with ultracentrifugation for two minutes at 55,000 RPM (~131,000 x g). Image credit: Beckman Coulter

Toxicity Assay

MCF-7 breast cancer cells were utilized for toxicity assay (Figure 2). These cells were plated at 0.08 x 106 for each well in a 24-well plate using 900µL of RPMI/10% FBS (Invitrogen) for a period of 24 hours. Nanotubes were later added to the well plate. One of the wells was used to confirm the viability and growth of cells.

Figure 2. MCF-7 cells were imaged under an optical microscope after 24 hours of incubation with SWCNT. The cells, incubated with either 0.06mg/mL As-Made SWNT (left image) or 0.06mg/mL ultracentrifuged SWNT (right image), have not yet reached confluence. Image credit: Beckman Coulter

On the second day, 100µL of SWCNT samples were introduced into the wells. Overall, there were six SWCNT groups (n=2/group) and three surfactant buffer control groups (n=2/group) as follows:

• 0.06 mg/mL UCF’d SWCNTs
• 0.06 mg/mL ASMAde SWCNTs
• 0.03 mg/mL UCF’d SWCNTs
• 0.03 mg/mL As-Made SWCNTs
• 0.006 mg/mL UCF’d SWCNTs
• 0.006 mg/mL As-Made SWCNTs
• 0.2 mg/mL DSPE-mPEG
• 0.02 mg/mL DSPE-mPEG
• 0.002 mg/mL DSPE-mPEG

Next, 100µL of DSPE-mPEG only sample was introduced into the wells, which served as a control. Here, control 1 (n=1) had untouched cells and control 2 (n=1) had 100µL of uncontaminated water. After a period of 24 hours, the wells were cleaned with PBS, trypsinized and then combined in 1mL of PBS for counting in the Vi-CELL XR cell viability analyzer.

Subsequently, a fresh cell type was produced in the Vi-CELL XR software so as to reduce the counting of collective nanotubes as cells. Finally, percentages of feasible cells were utilized to compare the difference and viability of cells in both solutions (Figure 3).

Figure 3. At all concentrations, ultracentrifuged SWCNT (designated by UC) had minimal toxicity; 75% or more of the MCF-7 cells remained viable 24 hours after incubation. Contrastingly, SWCNT that were not centrifuged and contained aggregated species (designated by AG) had increasing toxicity toward MCF-7 cells that scaled with increasing concentration. Image credit: Beckman Coulter

Results and Discussion

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In this analysis, the toxicity of As Made SWCNTs containing visible aggregates was studied in detail; this information represents most nanoparticles. The SWCNTs were divided into two groups: one group was As-Made in which no purification step was preformed to remove aggregates, and in the second group ultracentrifugation was carried out in Beckman Coulter’s Optima MAX-XP ultracentrifuge.

Although centrifugation procedures were effective in removing aggregated nanoparticles, the lengthy centrifugation times can impede research workflow. In contrast, the new ultracentrifugation technique shows how rapid ultracentrifugation was effective in achieving the same individual solubilized SWCNTs and biocompatibility as the longer centrifugation time, but at a fraction of time (Figure 4).

Figure 4. SWCNTs, after sonication in surfactant, still have a number of aggregated species. Size distribution, determined by dynamic light scattering on the DelsaMax PRO, showed two broad species (red line). The first size range represents individually solubilized carbon nanotubes. The second species, containing mostly aggregated carbon nanotubes, has a diameter peak closer to one micron in size. Image credit: Beckman Coulter

Data relating to dynamic light scattering and optical images show that all aggregated SWCNTs were removed by high-speed ultracentrifugation. Thanks to the Vi-CELL XR cell viability analyzer, it was possible to obtain the toxicity data which otherwise would not have been obtained using standard MMP and MTT toxicity assays.

In this study, aggregates display a high level of toxicity when compared to ultracentrifuged SWCNTs. This can be due to larger size of aggregated SWCNT and poor surfactant coverage. SWCNTs that are aggregated have more surfactant-free surface, and this high surface availability plays a key role in increased reactive oxygen species.

In addition, As-Made or aggregated SWCNTs are much larger and this increased size can interrupt cellular action and thus prevent cell growth. To overcome this issue, aggregated nanoparticles should be removed before being utilized in vivo or in vitro studies.

Conclusion

Beckman Coulter offers particle characterization and centrifugation tools that can be effectively used for quantifying cellular toxicity induced by nanoparticles. These instruments reduce the time taken to remove aggregated particles and enhance nanotechnology workflow.

This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc. - Particle Characterization.

For more information on this source, please visit Beckman Coulter, Inc. - Particle Size Characterization.