The hypoxic microenvironment present in rapidly proliferating solid tumors makes them among the most aggressive and difficult tumors to treat [1
]. Unlike the balance achieved in normal tissues, the consumption of oxygen in these tumors exceeds oxygen delivery from tumor blood vessels [3
]. Clinically, treatment failure results from the resistance of hypoxic cancer cells to both chemotherapy [4
] and radiotherapy [5
Hypoxia-inducible factor 1 alpha (HIF-1α) is a transcription factor whose activity is increased in human cancers in response to intratumoral hypoxia [7
]. HIF-1α plays important roles in many aspects of cancer biology, including cell proliferation, angiogenesis, invasion, and glycolysis, through the transcriptional regulation of a large number of genes [9
]. The significance of the contribution of HIF-1α to tumor growth is well documented [10
], and mouse xenograft studies have clearly demonstrated that the inhibition of HIF-1α activity suppresses tumor growth [6
]. HIF-1α is thus an attractive target for the development of anti-cancer agents. Several small-molecule inhibitors of HIF-1α have been identified, but their selectivity remains undefined. Selective inhibition of HIF-1α activity is an attractive approach to suppress tumor growth that may be achievable using siRNA as the therapeutic agent.
Exogenously synthesized, 19 to 22 base pair (bp) double-stranded small interfering RNA (siRNA) can activate the endogenous RNA interference (RNAi) pathway within mammalian cells and serve as a powerful tool for selective gene silencing. While RNAi holds great promise for targeted cancer therapy [15
], one of the key challenges is its delivery into tumor cells.
Single-walled carbon nanotubes (SWCNTs) are a new class of nanomaterials with significant biological potential. They are possible vehicles for delivering siRNA into mammalian cells, as they are capable of transporting various biological molecules into cancer cells through endocytosis, with negligible toxicity to the cells [16
]. However, before they can be used in biomedical applications, the hydrophobic pristine SWCNTs must be converted into a stable suspension in aqueous solution [21
]. This can be accomplished by noncovalently coating SWCNTs with surfactants or polymers, or by covalently functionalizing their side walls with suitable water-soluble groups [22
]. SWCNTs functionalized with covalently bound siRNA have been used to deliver siRNA into cancer cells in both in vitro
tissue culture and in tumor-bearing mice [17
]. In addition, SWCNTs have been noncovalently coated with phospholipids that were covalently linked to siRNA [27
]. These complexes were then delivered into cancer cells, T-cells, and human primary cells to knock down targeted genes. In both of these approaches, an intracellular chemical reaction is subsequently needed to break the covalent link and release the siRNA cargo [28
In the present study we have adopted an alternative approach in which unmodified siRNA serves both to noncovalently solubilize SWCNTS and also acts as the cargo. This method was suggested by the reported ability of single-stranded DNA (ssDNA) to coat pristine SWCNTs and give stable aqueous suspensions [29
]. The DNA in such complexes apparently forms a helical wrapping around the SWCNTs in which the bases interact closely with the hydrophobic surface of the nanotubes while the sugar-phosphate groups are exposed to water [29
]. Optimum binding was observed to be dependent on base composition [32
], with the best SWCNT solubilization obtained with a sequence containing alternating G and T and total lengths from 20 to 90 bp. Although the nanotube-coating interactions in such complexes must differ from those containing a double-stranded oligonucleotide such as siRNA, formation of our SWCNT/siRNA noncovalent complexes also required only simple ultrasonic agitation rather than chemical reactions. We found that the siRNA in these complexes retained its biological activity and readily entered cells—even in the presence of serum—as demonstrated by observation of the RNAi response in exposed cell culture. In addition, intratumoral administration of SWCNT/siRNA complexes targeting HIF-1α was found to significantly reduce HIF-1α activity in tumor-bearing mice. Our study demonstrates the potential for using pristine SWCNTs solubilized by siRNA as a therapeutic agent for cancer treatment.