Antitumor Activity and Prolonged Survival by Carbon‐Nanotube‐Mediated Therapeutic siRNA Silencing in a Human Lung Xenograft Model

Abstract

Carbon nanotubes are novel nanomaterials that are thought to offer potential benefits to a variety of biomedical and clinical applications. In this study, the treatment of a human lung carcinoma model in vivo using siRNA sequences leading to cytotoxicity and cell death is carried out using either cationic liposomes (DOTAP:cholesterol) or amino-functionalized multi-walled carbon nanotubes (MWNT - NH(+)(3)). Validation for the most cytotoxic siRNA sequence using a panel of human carcinoma and murine cells reveals that the proprietary siTOX sequence is human specific and can lead to significant cytotoxic activities delivered both by liposome or MWNT - NH(+)(3) in vitro. A comparative study using both types of vector indicates that only MWNT - NH(+)(3):siRNA complexes administered intratumorally can elicit delayed tumor growth and increased survival of xenograft-bearing animals. siTOX delivery via the cationic MWNT - NH(+)(3) is biologically active in vivo by triggering an apoptotic cascade, leading to extensive necrosis of the human tumor mass. This suggests that carbon-nanotube-mediated delivery of siRNA by intratumoral administration leads to successful and statistically significant suppression of tumor volume, followed by a concomitant prolongation of survival of human lung tumor-bearing animals. The direct comparison between carbon nanotubes and liposomes demonstrates the potential advantages offered by carbon nanotubes for the intracellular delivery of therapeutic agents in vivo. The present work may act as the impetus for further studies to explore the therapeutic capacity of chemically functionalized carbon nanotubes to deliver siRNA directly into the cytoplasm of target cells and achieve effective therapeutic silencing in various disease indications where local delivery is feasible or desirable.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing

“…We have recently reported the ability of this type of chemically functionalized CNT to complex and transfer siRNA into mammalian cells (27,28,32). To confirm such results in neurons, noncoding siRNA fluorescently labeled with AlexaFluor 546 (siNEG-AF546) was complexed with f -CNT at 1∶8 mass ratio and incubated for 24 h with primary neurons isolated from the mouse motor cortex.…”

“…These results further confirmed the neuroprotective effect achieved by carbon nanotube-mediated siCas 3 treatments and further illustrated that this could translate to functional preservation of motor skills after local ischemic damage in the rat motor cortex. The current work was motivated by previous findings that f -CNTs are able to translocate into the cell cytoplasm (17) and act as transporters of nucleic acids, including siRNA, in vitro (27,32) and in vivo (28). The delivered siRNA has proven to be functional with the ability to silence specific genes.…”

“…The delivered siRNA has proven to be functional with the ability to silence specific genes. Furthermore, we have previously reported that intratumoral delivery of an siRNA sequence mediating apoptotic responses by ammonium functionalized multiwalled carbon nanotubes into human lung (Calu 6) xenografts resulted in biological effects and a therapeutic outcome, evidenced by significant tumor growth suppression and improved animal survival (28). In the present study, ammonium functionalized CNTs were also shown to be capable of effectively transporting siRNA into primary neurons in the absence of any cell morphology alterations (e.g., dendrite contraction) or unwanted cytotoxic responses at therapeutically relevant doses, confirming previous in vitro studies showing axonal growth in primary neuronal cultures treated with positively charged CNTs (38).…”

“…Most importantly, f -CNT have the ability to interact with biological components and be internalized efficiently in different cell types by different mechanisms (20)(21)(22). Several studies have shown promising results, exploiting f -CNT characteristics for pDNA and siRNA delivery in vitro and in vivo (17,18,20,(23)(24)(25)(26)(27)(28). Chemically functionalized single-walled CNT have been able to deliver siRNA to knockdown the expression of telomerase reverse transcriptase in an Lewis Lung Carcinoma mouse tumor model (26).…”

Synthesis of mesitylene-based polyamine dendrimer for functionalisation of single-walled carbon nanotubes

“…[39,40] Prato and co-workers have designed simple ammonium-f-CNTs and more sophisticated dendron-based nanotubes. [41][42] The different conjugates were used for gene-silencing experiments in vitro and in vivo demonstrating the capacity of such systems to deliver and eventually knock-down different genes efficiently. Recently, we reported the synthesis of cationic nanotubes through reaction with a series of linear polyamines, such as putrescine, spermidine and spermine.…”