Beckman Coulter tools help quantify cellular toxicity caused by nanoparticles, reducing time taken to remove aggregated particles and to quantify cell toxicity in the presence of single walled carbon nanotubes.
Nanomedicine is an important field for materials scientists, chemists, biomedical engineers, biologists and medical scientists. This article will help researchers in this field to gain insight into the inherent shortcomings that are present in traditional cellular toxicity assays, and how these shortcomings are faced when the toxicity assay involves nanomaterials.
Researchers will also benefit from understanding how Beckman Coulter centrifugation and particle characterization instruments can considerably improve the accuracy and workflow in both preparing nanoparticles and assessing the nanoparticles in-vitro toxicity.
Nanotechnology biomedicine is an evolving field presently in its nascent phase. Nanoparticles, which include carbon nanotubes, quantum dots and graphene, have a number of unique spectral features that have specific applications for in vivo imaging and drug delivery.
Quantum dots, CNTs and graphene all fluoresce in the advantageous biological window enabling deeper imaging with better sensitivity. The strongly red-shifted Raman signal of CNTs is also used for in-vivo imaging.
The relatively large surface area and strong light absorption of CNTs and graphene also make them excellent drug delivery and photothermal therapy agents. Due to these unique properties, toxicity characterization of biologically focused nanomaterials poses a challenge to researchers.
Carbon Nanotube Preparation
The procedure followed for carbon nanotube preparation is provided below:
- Single-walled carbon nanotubes (Sigma-Aldrich) were mixed with 0.2% 1, 2-Distearoyl-phosphatidylethanolamine -methyl-polyethyleneglycol (DSPE-mPEG, 5kDa molecular weight, Laysan Bio) in 10 mL of water.
- Bath-sonication of the solution was done for 30mins to create well-dispersed carbon nanotubes following previously established procedures.
- Using a TLA-120.2 rotor in an Optima MAX-XP Ultracentrifuge, 5mL of SWCNT solution was centrifuged in open-top polycarbonate centrifuge tubes (Beckman Coulter P/N 343778) at 22°C, 55,000RPM (~131,000 x g) for two minutes.
- The top 650µL of supernatant was collected with care to ensure that the pelleted aggregates are not disturbed
- The ultracentrifuged SWCNT (referred to as UCF’d SWCNT) and the remaining, uncentrifuged SWCNT (referred to as As-Made SWCNT) were concentrated using 10kDa, Amicon Ultra 0.5mL Centrifugal Filters (Millipore) with a Beckman Coulter Microfuge 20microcentrifuge.
- The quantification of the concentration was done using a UV-Vis-NIR spectrophotometer (Paradigm, Molecular Devices) and the established mass extinction coefficient of SWCNTs at 808nm of 46.5 L/g*cm
- After concentration, UCF’d SWCNTs and As-Made SWCNTs were diluted using deionized water to concentrations of 0.6 mg/mL, 0.3mg/mL, and 0.06 mg/mL.
The toxicity assay was prepared as follows:
- Plating of the MCF-7 breast cancer cells was done at a density of 0.08 x 106 per well in a 24-well plate with 900 µL of RPMI/10% FBS (Invitrogen) 24h before the addition of nanotubes.
- Cell viability and growth were confirmed using one of the wells before the addition of nanotubes. 100 µL of SWCNT samples were added to wells on the second day.
- There were six SWCNT groups (n=2/group) in total: 0.06 mg/mL UCF’d SWCNTs; 0.06mg/mL As- Made SWCNTs; 0.03mg/mL UCF’d SWCNTs; 0.03 mg/mL As-Made SWCNTs; 0.006mg/mL UCF’d SWCNTs; and 0.006 mg/mL As-Made SWCNTs. 100µL of DSPE-mPEG only sample was added to wells serving as a control.
- There were three surfactant buffer control groups (n=2/group) in total: 0.2 mg/mL DSPE-mPEG; 0.02 mg/mL DSPE-mPEG; and 0.002 mg/mL DSPE-mPEG.
- Finally, control 1 (n=1) was a complete control, with cells left untouched, and control 2 (n=1) had 100 µL of sterilized water added.
- After 24 hours, all the wells were washed with PBS, trypsinized and mixed in 1mL of PBS for counting in the Vi-CELL XR. A new cell type was created in the Vi-CELL XR software to minimize the counting of aggregated nanotubes as cells.
- Percentages of viable cells were used to compare cell viability and difference in the two solutions.
Figures 1 and 2 show images of SWCNT and cell imaging respectively. In Figure 2, 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.
Black aggregates of SWNT can be seen in the image on the left and these aggregates are difficult to wash away without washing away the cells as well. The aggregates have absorption and fluorescence properties that will skew traditional toxicity assays.
Figure 1. Images of SWCNT Optical images of single-walled carbon nanotube (a) without centrifugation and (b) with ultracentrifugation for two minutes at 55,000RPM (~131,000 x g)
Figure 2. Cell Imaging. MCF-7 cells were imaged under an optical microscope after 24 hours of incubation with SWCNT.
Aggregated nanoparticles pose a challenge in nano-biomedicine. In this study, the toxicity of As-Made SWCNTs (which contained visible aggregates) was examined; however, this data is representative of most nanoparticles. The conclusions arrived at include the following:
- The SWCNTs were classified into two groups, one was As-Made without any purification step for aggregate removal and the second group underwent ultracentrifugation in the Beckman Coulter Optima MAX-XP ultracentrifuge.
- The new ultracentrifugation method shows that a two-minute, high-speed ultracentrifugation achieves the same biocompatibility and individual solubilized SWCNTs15 as the longer centrifugation time is a 180-fold time savings to researchers.
- Dynamic light scattering data and optical images taken during the DelsaMax PRO are evidence that all aggregated SWCNT have been removed by the rapid ultracentrifugation. Toxicity data was possible because of using Vi-CELL XR.
- The Vi-CELL XR was programmed to particularly look for spherical cells with defined outlines in a sharply delineated size range, making sure that counting of carbon nanotube aggregates as either viable or dead cells was minimized.
- Aggregates show increased toxicity over ultracentrifuged SWCNT, which can be attributed to poor surfactant coverage and larger size of aggregated SWCNT.
- At all concentrations, ultracentrifuged SWCNT (designated by UC) had minimal toxicity; 75% or more of the MCF-7 cells remained viable 24h after incubation.
- At a stock concentration of 0.6mg/mL, corresponding to a concentration in solution with cells at 0.06mg/mL, the aggregated SWCNTs had greater than 50% cell death.
Figure 3. Viability results
Figure 4. Size Distribution Data
This information has been sourced, reviewed and adapted from materials provided by Beckman Coulter, Inc.
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