Nanomedicine is an area common to chemists, materials scientists, biologists, biomedical engineers and medical scientists. This article aims to provide researchers with an insight into the shortcomings of conventional cellular toxicity assays, and how these are magnified when nanoparticles are involved in the assays.
Researchers will also gain an insight into how the accuracy and workflow can be significantly improved in both the preparation and assessment of nanoparticles in vitro toxicity by using Beckman Coulter centrifugation and particle characterization instruments.
This article shows how the Beckman Coulter Optima MAX-XP ultracentrifuge effectively minimized the time taken to remove the aggregated nanotubes, while the Vi-CELL XR from Beckman Coulter enabled quantification of cell toxicity in the presence of single-walled carbon nanotubes (SWCNT).
Optima XPN Centrifuge. Image credit: Beckman Coulter
The field of nanotechnology biomedicine is an evolving area that can still be considered as being in its nascent phase. Nanoparticles such as carbon nanotubes (CNTs), quantum dots (QDs) and graphene have several unique spectral features that have specific applications in drug delivery and in vivo imaging. As QDs, graphene and CNTs all fluoresce in the advantageous biological window, deeper imaging with increased sensitivity is achievable. In vivo imaging also exploits the strongly red-shifted Raman signal of CNTs. Graphene and CNTs are also excellent photothermal therapy and drug delivery agents due to their strong light absorption and high surface area. These unique properties, however, mean that characterizing the toxicity of biologically focused nanomaterial can be challenging for researchers.
In vitro cell toxicity assays typically use an absorption and fluorescence region that is overlapped by the intrinsic fluorescence and absorption of each of these nanomaterials, which can lead to misleading and inaccurate results.
Furthermore, the fluorescence, absorption and toxicity of these materials are all significantly altered when they are aggregated together rather than individually solubilized, meaning that systemic bias occurs in toxicity studies that compare aggregated versus non-aggregated. Long centrifugation processes are typically used to remove aggregated nanoparticles. For example, CNTs are subjected to 6 h centrifugations at 22,000 x g to pellet aggregates.
Carbon Nanotube Preparation
Single-walled carbon nanotubes (Sigma-Aldrich) and 0.2% 1, 2-distearoyl-phosphatidylethanolamine -methyl-polyethyleneglycol (DSPE-mPEG, 5 kDa molecular weight, Laysan Bio) were mixed in 10mL of water. Well-dispersed carbon nanotubes were created by bath sonicating the solution for 30 minutes, as per previously established procedures.
5 mL of SWCNT was centrifuged in open-top polycarbonate centrifuge tubes (Beckman Coulter P/N 343778) at 22°C, 55,000 RPM (~I31,000 x g) for two minutes using a TLA-120.2 rotor in an Optima MAX-XP Ultracentrifuge. The top 650µL of supernatant was collected carefully to avoid disrupting the pelleted aggregates.
Using 10 kDa, Amicon Ultra 0.5 mL Centrifugal Filters (Millipore) with a Beckman Coulter Microfuge 20 microcentrifuge, concentration of the ultracentrifuged SWCNT(UCF’d SWCNT) and the uncentrifuged SWCNTs (As made SWCNT) was performed.
Measurement of the concentration was carried out using a UV-Vis-NIR spectrophotometer (Paradigm, Molecular Devices) and the established mass extinction coefficient of SWCNTs at 808nm of 46.5L/g*cm2 Deionized water was used to dilute the concentrated UCF'd SWCNTs and As-Made SWCNTs to 0.6mg/mL, 0.3mg/mL, and 0.06mg/mL concentrations.
Figure 1. Images of SWCNT. Optical images of single-walled carbon nanotube (a) without centrifugation and (b) with ultracentrifugation for two minutes at 55,000 RPM (~131,000 x g). Note the presence of black, aggregated SWCNT in the sample that was not ultracentrifuged. Image credit: Beckman Coulter
24 hours prior to adding nanotubes, MCF-7 breast cancer cells were plated at a density of 0.08 x 106 per well in a 24-well plate with 900µL of RPMI/10% FBS (Invitrogen). Using one of the wells before nanotubes were added, cell growth and viability were confirmed. On the next day, 100µL of SWCNT samples were added to the wells.
In total, there were six SWCNT groups (n=2/group):
- 0.06 mg/mL UCF'd SWCNTs
- 0.06 mg/mL As-Made SWCNTs
- 0.03 mg/mL UCF'd SWCNTs
- 0.03 mg/mL As-Made SWCNTs
- 0.006 mg/mL UCF'd SWCNTs
- and 0.006 mg/mL As-Made SWCNTs.
To provide a control, 100µL of DSPE-mPEG only sample was added to the wells.
In total, there were three surfactant buffer control groups (n=2/group):
- 0.2 mg/mL DSPE-mPEG;
- 0.02 mg/mL DSPE-mPEG;
- 0.002 mg/mL DSPE-mPEG.
Lastly, control 1 (n=1) was a complete control, with cells left untouched and 100µl of sterilized water was added to control 2(n=1). After 24 hours, in order to enable counting in the Vi-CELL XR, all wells were rinsed using PBS and then trypsinized and mixed in 1mL of PBS. To minimize aggregated nanotubes being counted as cells a novel cell type was developed using the Vi-CELL XR software. Comparison of cell viability and difference in the two solutions was carried out using the percentages of viable cells.
Figure 2. Cell Imaging. MCF-7 cells were imaged under an optical microscope after 24 hours of incubation with SWCNT. The cells, incubated with either 0.06 mg/mL As-Made SWNT (left image) or 0.06 mg/mL ultracentrifuged SWNT (right image), have not yet reached confluence. Black aggregates of SWNT can be seen in the image on the left; 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. Image credt: Beckman Coulter
Figure 3. Viability Results. 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. At a stock concentration of 0.6 mg/mL, corresponding to a concentration in solution with cells at 0.06 mg/mL, the aggregated SWCNTs had greater than 50% cell death. Image credt: Beckman Coulter
Results and Discussion
One of the key challenges of nano-biomedicine is aggregated nanoparticles. This article examined the toxicity of As-Made SWCNTs (which included visible aggregates); however, this information is representative of most nanoparticles. The SWCNTs were separated into the As-Made group and the ultracentrifuged group. The former group did not undergo any purification for aggregate removal while the latter underwent ultracentrifugation in the Beckman Coulter Optima MAX-XP ultracentrifuge.
Although centrifugation is effective for the removal of aggregated nanoparticles, the research workflow is somewhat hindered by long centrifugation times, of at least 6 h at low speeds (5,000 x g to 22,000 x g). This new ultracentrifugation technique shows that a high-speed, two-minute ultracentrifugation can achieve the same biocompatibility and individual solubilized SWCNTs as the longer centrifugation time can, offering researchers a 180-fold time saving.
Dynamic light scattering data and optical images captured with the DelsaMax PRO provide proof that all aggregated SWCNT has been eliminated using the rapid ultracentrifugation. It was possible to collect the toxicity data in this study because Vi-CELL XR was used; aggregates have a strong absorption that would confound typical MMP and MTT toxicity assays.
The Vi-CELL XR was programmed to specifically look for spherical cells having defined outlines in a sharply delineated size range, to minimize the counting of carbon nanotube aggregates as either dead or viable. Due to the large number of aggregates present even after cell washing, the Vi-CELL XR optimization is very important.
Ultracentrifuged nanotubes were run without any cells, as a control, and in this trial, the Vi-CELL XR did not count a single cell as dead or live.
Figure 4. Size Distribution Data. Single-walled carbon nanotubes, 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, roughly 100 nm in diameter, represents individually solubilized carbon nanotubes. The second species, containing mostly aggregated carbon nanotubes, has a diameter peak closer to one micron in size. After a two-minute ultracentrifugation, the SWNT demonstrate only a single broad species at 100 nm, indicating that virtually all aggregates have been removed. This is further indicated by a 59% decrease in polydispersity. Interestingly, the zeta potential remains unchanged between aggregated and centrifuged carbon nanotubes. This is most likely due to the fact that steric repulsion, from the Poly(ethylene glycol) surfactant, provides most of the stability to the carbon nanotubes, while electrostatic repulsion does not play a major role. Image credt: Beckman Coulter
Figure 5. Flowchart. Image credt: Beckman Coulter
Compared with ultracentrifuged SWCNT, aggregates show higher toxicity, something that can be attributed to poor surfactant coverage and the larger size of aggregated SWCNT. The surfactant-free surface is more exposed in aggregated SWCNT and this increased surface availability of SWCNT contributes directly to increasing the reactive oxygen species (ROS). Also, on the whole, As-Made or aggregated SWCNTs are much larger, as shown by the dynamic light scattering data. The larger size of SWCNT can block cell-signaling pathways or hinder cellular action and thereby inhibit cell growth. It is therefore essential that removal of aggregated nanoparticles is performed before being used in vitro or in vivo.
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