(230a) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Delivery

As part of nanomaterial applications in biology and medicine, we are interested in developing single wall carbon nanotubes (SWCNTs) as intracellular biomaterials. Our research in this area has been directed to characterizing, developing, and utilizing SWCNT functionalization to control SWCNT sub-cellular localization and probe fundamental cellular processes utilizing SWCNTs. To produce biologically relevant solutions of individualized SWCNTs and overcome aggregation of SWCNTs, we use a surfactant dispersant. Specifically, we are focused on developing bio-inert, biological, and bioactive SWCNTs dispersions to understand and control how the SWCNT dispersions enter cells, where they go and what they do once inside cells, and do they leave cells.

The first step in targeted cell delivery is to determine how SWCNTs get into cells. To determine if SWCNTs dispersed in Pluronic F127 (PF127) could physically penetrate a cell membrane, we performed complementary studies with in vitro synthetic membranes, plasma membrane vesicles and live cell experiments. Our results demonstrated that, while P-F127 dispersed SWCNTs are membrane active and associate with cell membranes, they lack sufficient energy to physically penetrate a cell membrane bilayer. Further, our experiments suggested that SWCNTs are directly present in endosomes. Therefore, we experimentally validated that SWCNTs enter cells via endocytosis and not membrane penetration.

Once inside the cell, we were interested in the sub-cellular localization and cellular effects of SWCNTs. We showed that highly purified, length-selected, and SWCNTs well-dispersed with PF127 (bio-inert) are not acutely cytotoxic but reduce proliferation in a dose-dependent manner. Due to the similar mechanical and anisotropic properties of SWCNTs and F-actin, we investigated the interaction and effects of SWCNTs on F-actin. These SWCNTs reorganized F-actin structures, leading to more F-actin intensity in the apical cellular regions. Fluorescence lifetime imaging microscopy further suggested that SWCNTs interact with F-actin structures both in the cells and in purified actin filaments reconstituted in vitro. Functionally, SWCNTs reduce forces that can be generated by NIH-3T3.

Endocytosed SWCNTs coated with PF127 were able to enter cellular compartments and interact with actin, likely from their membrane activity which could destabilize endosomes. However, dispersion with bovine serum albumin (BSA) caused markedly different cellular effects. SWCNTs-BSA were not acutely cytotoxic and did not significantly reduce proliferation. Confocal Raman imaging co-registered to phase contrast showed different patterns of sub-cellular localization for these dispersions within perinuclear region of the cell. We also observed a significant increase in cellular uptake of SWCNTs-BSA into cells with 10’s of millions of SWCNTs entering human mesenchymal stem (hMSC) cells and HeLa and NIH-3T3 cells. Surprisingly, steady-state uptake per cell occurs in less than 1 minute, and there is a concentration threshold between 1 and 30 mg/mL as cell uptake machinery is saturated. Confocal Raman mapping of SWCNT radial breathing modes, fluorescence, and G-band demonstrated that SWCNTs primarily remain individually dispersed.

Finally, we determined that cells could recover from SWCNT exposure. Most cell lines exposed to SWCNTs-BSA were able to expel SWCNTs and recover cell phenotype, and after ~80 hr essentially no SWCNTs remained in cells. Conversely, cells exposed to SWCNTs-PF127 did not recover, maintaining substantial SWCNTs-PF127 throughout the cells, even after 80 hr. This differential ability to recover suggests different subcellular compartmentalization of SWCNTs as well as the possibility for cellular delivery of SWCNTs within the body.

In conclusion, we have dispersed SWCNTs – an important nanostructured material for biological applications – with different classes of biological dispersing agents to control uptake, sub-cellular processing, and recovery. Therefore, from these studies, we have shown that we can engineer and control SWCNT-cell interactions.

References

1. B. D. Holt, K. N. Dahl, and M. F. Islam, “Cells Uptake and Recover from Protein Stabilized Single Wall Carbon Nanotubes with Two Distinct Rates”, ACS Nano 6, 3481 (2012).
2. P. N. Yaron, B. D. Holt, P. A. Short, M. Lösche, M. F. Islam, and K. N. Dahl, “Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration”, Journal of Nanobiotechnology 9, 45 (2011).
3. B. D. Holt, K. N. Dahl, and M. F. Islam, “Quantification of Uptake and Localization of Bovine Serum Albumin-Stabilized Single-Wall Carbon Nanotubes in Different Human Cell Types”, Small 7, 2348 (2011).
4. B. D. Holt, P. A. Short, A. D. Rape, Y. Wang, M. F. Islam, and K. N. Dahl, “Carbon Nanotube Reorganize Actin Structures in Cells and ex vivo”, ACS Nano 4, 4872 (2010).