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K. L. Masselam, J. A. Young, J. Karen, C. Hunzeker, P.-J. Yu, P. Mignatti; Targeted Mouse Vascular Endothelial Cell Killing Using Gold Nanospheres. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1744.
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© ARVO (1962-2015); The Authors (2016-present)
To employ surface plasmon resonance to target and kill endothelial cells by means of gold nanospheres conjugated to IgG.
Mouse vascular endothelial cells were seeded at confluency in eight L-lysine coated LabTek wells. PCAM antibody was diluted to 0.1 mg/ml in 20 mM Hepes (pH 7.4) for a final concentration of 4 µg/ml antibody (80 µL used). Ten times the volume of gold (800 µL) was added to a 10:1 ratio of gold:antibody. BSA was added to a final concentration of 0.1 % and polyethylene glycol was added to a final concentration of 0.2 mg/ml in all wells. The suspension was then centrifuged at 5000 rpm in 4° C for 1 hour. The conjugate was then resuspended in PBS and 0.1% BSA. Endothelial monolayers were covered with 500 µl of media per well and 20 µL of conjugate was added to wells #1-4 (no conjugate was added to wells #5-8). The cells were incubated for 1 hour. The media was then replaced with PBS. Wells #1-3 and 5-7 were irradiated with 50 pulses per well of a 532-nm, 0.5 J/cm2, Q-switched Nd:YAG laser (6 mm spot). Media was removed and Trypan blue exclusion testing was performed to assay cell viability. Viable and non-viable cells were counted within the 6 mm irradiated spot for wells #1-4 and within a corresponding area for wells #5-8 by using an Axiovert 25 microscope (Carl Zeiss, Inc., Thornwood, NY) with a CWHK grid ocular (Olympus America Inc., Melville, NY) (ob 20×).
Irradiated wells with IgG-conjugated gold nanospheres (#1-3) demonstrated 45-65% cell death. Irradiated wells with unconjugated gold nanospheres (#5-7) showed 23-29% cell death. The conjugated (#4) and unconjugated (#8) non-irradiated wells yielded 24% and 22% cell death, respectively.
Wells containing IgG-conjugated gold nanospheres demonstrated a higher yield cell death compared to wells containing unconjugated gold nanospheres. Cell death in the non-irradiated wells was likely explained by cell preparation. Conjugated gold nanospheres could potentially be used in ophthalmology to treat neovascularization as well as in other areas of medicine for targeted cell killing.
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