Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

Abstract

The carbon nanotubes (CNT) complexes (RNA-wrapped, oxidized DWNT) were endocytosed by capture in clathrin-coated vesicles, revealed by clear co-localization with both clathrin and transferrin. CNT complexes were also found in early endosomes, which was confirmed by co-localization with early endosome antigen (EEA). CNT complexes were not sorted for recycling back to the cell membrane (via fast recycling endosomes), as co-localization with Rab 4 was not observed.Instead, the overlapping of CNT complexes with both LAMP2 antibody and LysoTracker® marker indicated their sequestration in lysosomes.CNTs were also found within within vesicles of the secretory pathway (Rab11-positive vesicles), which for the first time suggests an exact route for their exocytosis. ABSTRACTCarbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT-RNA) is presented. Fixed cells, previously exposed to oxDWNT-RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT-RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT-RNA with the small GTP-binding protein (Rab11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network.

Design of double-walled carbon nanotubes for biomedical applications

“…A strong, locally confined Raman signal was detected within the whole cell, confirming successful DWNT uptake and possible localization within endosomes, as also reported in previous work by our group, which has looked at the intracellular distribution of functionalized DWNTs in greater detail [19,20]. An important factor for the design of DWNTs as a nano-vector is that they do not significantly modify the cell environment, so that perturbations due to the payload can be detected.…”

“…Furthermore, as we have shown in previous studies, they are released from the cells within 24 hours after having fulfilled their task and do not accumulate within cells [20]. In this study we investigated whether oxDWNT-RNA causes any changes in the cell biochemistry within this time frame, which they did not (figure 4).…”

Bioactive nanocarbon assemblies: Nanoarchitectonics and applications

“…Biocompatibility of CNTs with cardiac muscle cells has been tested revealing the lack of any observable cytotoxicity [71]. Uptake of CNTs was revealed to occur by a similar mechanism to that of nanoparticles [72]. Oxidized CNTs were encapsulated by RNA and the complex was internalized to cells by an endocytosis pathway.…”

Carbon for Sensing Devices

“…Heister [78,79,196] and Neves [197,198] have demonstrated that CNTs functionalized with polyethylene glycol (PEG) or ribonucleic acid (RNA) can be used to detect cancer cells within biological systems. Such carbon nanomaterial systems can be used for targeting tumor cells and delivering drugs for future cancer theranostics.…”