Integrated molecular targeting of IGR1R and HER2 surface receptors and destruction of breast cancer cells using single wall carbon nanotubes

Ning Shao, Shaoxin Lu, Eric Wickstrom, Balaji Panchapakesan, Nanotechnology 18 (2007) 315101 (9pp).

Purpose of the Study:   

Demonstrate the in vitro ability of single walled carbon nanotubes (SWCNT) functionalized with anti-IGF1R and anti-HER2 monoclonal antibodies to destroy breast cancer cells upon excitation with near infrared light (808nm). 

Methods:

 

•  SWCNTs having an average diameter of 1.4nm and lengths ranging from 500nm to 1mm were fabricated with a self-assembled monolayer of polyethylene glycol (PEG) to prevent undesirable binding with other biomolecules.
• Transmission electron micrography was used to characterize the SWCNTs.
• SWCNTs were divided into three groups and functionalized with a non-specific antibody (control), anti-IGF1R antibody, or anti HER2 antibody.
• Cytotoxicity was minimized, consistent with published research, by chemically modifying SWCNT surface with –OH and –COOH groups in addition to the PEG conjugates.
• SWCNTs were mixed with fluorescent dyes (phalloidin 488 & 555) to form fluorescent SWCNT-antibody hybrids.
• SWCNTs were incubated with two different human breast cancer cell lines:  (a) BT474, which exhibit high expression of HER2 receptors and lower expression of IGF1R receptors, and (b) MCF7, which exhibit high expression of IGF1R receptors and lower expression of HER2 receptors.  Each cell line was incubated with both anti-HER2 SWCNTs and anti-IGF1R SWCNTs.
• Confocal fluorescent microscopy was used to characterize the fluorescent SWCNT-antibody hybrids and the cellular uptake thereof.
• An 808nm laser at 800mW cm^-2 and 3 min exposure time was used for the photo-thermal therapy regimen. 
• Trypan blue assay was used to verify and quantify cell death (i.e. cell viability).  

Key Findings:

 

1. SWCNT-receptor specific antibody conjugates were readily internalized into the cells over large areas while the SWCNTs with the non-specific antibody were not.  The authors hypothesize the following mechanism (consistent w/ previous research):  upon receptor-specific attachment, stresses are generated due to free energy release which generates a pressure differential across membrane pores, thereby facilitating endocytosis.
2. Cells receiving only the NIR light without internalized SWCNT survived the photo-thermal therapy whereas those incubated with the SWCNT anti-HER2 and anti-IGF1R conjugates did not survive (100% kill rate).  Those cells to which SWCNT-non specific antibody conjugates were attached, 50% survived (most likely due to fewer attached).
3. Cells were killed due to the localized increase in temperature resulting from high absorption by the SWCNT that occurs with NIR light while normal cells remain transparent to NIR.   
4. The energy used to destroy the cancer cells was estimated to be ~200nW per cell, too low to create any damage to the normal cells.  Due to the high specificity, antibody-directed targeting, the authors were able to use half the laser power of past nanotube based cell killing techniques. 

Definitions:

Her2:  human epidermal growth factor receptor (often over expressed by breast cancer cells)

IGF1R:  insulin-like growth factor receptor (often over expressed by breast cancer cells)