摘要：

IntroductionProtein toxins have played an important role in cancer therapy.1,2Ricin is one kind of protein toxin that can kill mammalian cells by undergoing retrograde transport from the Golgi apparatus to the endoplasmic reticulum, and then to the cytosol. It is naturally composed of an A chain (RTA) and a B chain (RTB), connected by a dithiol bond. RTA is a protein of 263 amino acids, which acts as a cytotoxin by catalytically inactivating ribosomes; however, it is generally non-toxic outside the cell due to the low efficiency with which it enters cells by itself. It needs the ricin B chain (RTB) to bind to the cell surface and transport it to an intracellular compartment to exert the action. Thus, RTB, as a galactose domain responsible for the translocation of RTA across the cell membrane,3is critical for binding, internalization and intracellular trafficking of the ricin toxin.Natural ricin is known to be very efficient and one molecule inactivates several thousand ribosomes per minute, leading to rapid inhibition of protein synthesis and cell death. However, its extremely high cytotoxicity is non-specific, which seriously limits its application in cancer therapy.4Immunization against ricin exposure using inactivated ricin toxoid or a genetically engineered ricin A chain are realistic future therapeutic options. A recent advance in this field has involved the use of antibodies that can target tumour cellsviacarrying specific antigenic markers5–8to replace the binding moiety. However, most of the binding moieties that replaced the B chain greatly lost the ability to transport the A chain to the Golgi apparatus, and then to endoplasmic reticulum.9Thus, it would be particularly valuable to develop a simple, routine and efficient way to specifically deliver toxin, such as RTA, into tumor cells with no or little loss of their biological activity, as performed naturally by RTB.Recently, there have been several investigations concerning the ability of single-walled or multi-walled carbon nanotubes (SWNTs or MWNTs) to shuttle various molecular cargos inside living cells without cytotoxicity.10–18In the original work of Pantarottoet al.10and Daiet al.,11fluorescently labelled nanotubes were internalized into cells with no apparent toxic effects. They also found that an active peptide, when covalently linked to SWNTs, penetrated into the cells. Subsequently, SWNTs were found to mediate the intracellular transport of proteins11and nucleic acids.12–15The mechanism of nanotube–protein uptake into cells has been suggested to be eitherviaa hydrophobic/hydrophilic effect or by a self-uptaking behavior.12Recently, an energy-dependent endocytosis process has been proposed by Daiet al.19–21These findings reveal that the uniqueness of SWNTs as molecular transporters is beginning to emerge and needs to be fully developed for variousin vitroandin vivodelivery applications.In this study, recombined RTA protein was delivered into cells by MWNTs. Delivered in this way, RTA was able to function and to induce cell death. The MWNT–RTA conjugates (Fig. 1a) produced three times higher cell death rates for L-929 cells compared to those achieved by RTA alone (Fig. 1b, FACS). The intracellular transport of protein toxinviaMWNTs and similar subsequent destruction for cells were also observed for various cell lines, including HL7702, MCF-7, HeLa and COS-7 cells. The selective destruction of tumor cells was also achieved by coupling MWNTs with anti-HER2, which targets breast cancer cells that over-expressed HER2. These results provide the first proof for MWNTs as molecular transporters to deliver recombined protein toxins into tumour cells. Furthermore, a full toxic effect of MWNT–RTA conjugates was investigated.Schematic representation of (a) MWNT–RTA conjugates, (b) the intracellular protein toxin transportedviaMWNT carriers.

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