Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm

Abstract

Carbon-based nanomaterials such as single-walled carbon nanotubes and reduced graphene oxide are currently being evaluated for biomedical applications including in vivo drug delivery and tumor imaging. Several reports have studied the toxicity of carbon nanomaterials, but their effects on human male reproduction have not been fully examined. Additionally, it is not clear whether the nanomaterial exposure has any effect on sperm sorting procedures used in clinical settings. Here, we show that the presence of functionalized single walled carbon nanotubes (SWCNT-COOH) and reduced graphene oxide at concentrations of 1-25 μg/mL do not affect sperm viability. However, SWCNT-COOH generate significant reactive superoxide species at a higher concentration (25 μg/mL), while reduced graphene oxide does not initiate reactive species in human sperm. Further, we demonstrate that exposure to these nanomaterials does not hinder the sperm sorting process, and microfluidic sorting systems can select the sperm that show low oxidative stress post-exposure.

Figure 1. SWCNT-COOH/RGO concentration and time dependent sperm toxicity study.

(a) 1 mL semen vials were obtained from a cryobank. (b) Semen sample was thawed in a water bath at 37 °C for 15 min. (c) Sperm concentration and motility were quantified using a Makler hemocytometer. (d) Semen samples were mixed with HTF and 1% BSA and (e) centrifuged. (f) Sperm samples were placed in an incubator and motile sperm were allowed to swim up to the top media layer. (g) Supernatant containing motile sperm was collected and distributed in 8 wells. (h) Sorted semen were incubated with SWCNT-COOH and RGO at various concentrations; 1, 5, 25 μg/mL, and for 30 minutes or 3 hour time points. (i) Viability, sperm kinetic parameters, ROS, and NO were measured. (j) Sperm incubated with nanomaterials were processed using a microfluidic device to investigate the effects of nanomaterials on sperm sorting process.

Figure 2. Sperm viability analysis after incubation with nanomaterials.

Viable sperm population (control, 1, 5, and 25 μg/mL) after incubating with (a) SWCNT-COOH and (b) RGO for 30 minutes and 3 hours. (c) Fluorescent image of viable (green) and dead (red) sperm were used for viability quantification.

Figure 3. Sperm kinematic analysis at various SWCNT-COOH/RGO concentration and exposure time.

Linear (VSL) and curvilinear (VCL) sperm velocity for sperm samples (control, 1, 5, and 25 μg/mL) incubated with SWCNT-COOH for (a) 30 min and (b) 3 hours, as well as, incubated with RGO for (c) 30 minutes and (d) 3 hours. *p value < 0.05 between VSL of sperm incubated with RGO and control samples, N = 3. ^#p value < 0.05 between VCL of sperm incubated with RGO and control samples, N = 3. Error bars represent a standard error of the mean.

Figure 4. Superoxide generation at various SWCNT-COOH/RGO concentration and exposure time.

Sperm population generating reactive oxide stress (ROS) for sperm samples incubated with (a) SWCNT-COOH and (b) RGO at NP concentrations of 1, 5, and 25 μg/mL for two incubation periods of 30 minutes and 3 hours. *p value < 0.05 between sample treated with SWCNT-COOH and control, N = 3. Error bars represent a standard error of the mean.

Figure 5. Nitric oxide species generation due to SWCNT-COOH/RGO concentration and exposure time.

Sperm population generating nitric oxide species (NOS) for sperm samples incubated with (a) SWCNT-COOH and (b) RGO at concentrations of 1, 5, and 25 μg/mL for two incubation periods of 30 minutes and 3 hours.

Figure 6. ROS generation sperm population after sorting.

Sperm population generating reactive oxide species (ROS) for (a) sorted sperm collected from top chamber (n = 6, Modified Z-Scores method was used to detect outliers in the data. One data point out of 6 readings has Z-Score > 3.5, therefore it is neglected) and (b) sperm collected from bottom chamber of microfluidic device after samples are incubated with SWCNT-COOH (n = 6). Sperm population generating reactive oxide species (ROS) for (c) sorted sperm collected from top chamber (n = 6) and (d) sperm collected from bottom chamber of microfluidic device after samples are incubated with RGO (n = 6). *p value < 0.05 between sperm collected from top and bottom of the filter. ^#p value < 0.05 between sperm samples treated with SWCNT-COOH and untreated sample.