Synthesis of monodispersed GNPs
GNPs were prepared according to the method outlined by Anshup et al.24 Briefly, in a 250 mL round-bottomed flask, 100 mL of 0.05% HAuCl4 was added and heated. Under prompt stirring, 3.5 mL of sodium citrate (1%) was supplemented and further rapidly stirred for 15 minutes at 120°C.
After 30 minutes of stirring, the solution was then steadily cooled to room temperature. The citrate concentration was adjusted to attain different particle sizes.
After 15 minutes, the liquid was removed and cooled to room temperature and filtered. The prepared GNPs were resuspended and purified by centrifugation and double precipitation from distilled water.
Preparation of hydrophilic GNPs
MSA (0.16 M) was dissolved in 70 mL of water in a 250 mL three-necked flask containing GNPs. The solution was stirred for 3 hours in nitrogen atmosphere. After the reaction was complete, the water was separated by vacuum, and the residue was transferred into a centrifuge tube and mixed with 4 mL of water.
Subsequently, 20 mL of water was added to the mixed solution, and the precipitated product was separated and subjected to centrifugation at 3,000 rpm (revolutions per minute) for 15 minutes and washed with water.
The prepared MSA-conjugated GNPs were dissolved and purified by centrifugation and double precipitation from water.
Immobilization of herceptin on GNPs
The immobilization of herceptin on GNPs (GNP–Her) was carried out by the reaction of water-soluble GNPs with herceptin (Figure 1).
Water-soluble GNPs (1.2 mmol) and herceptin (7 mmol) were added to a two-necked flask and dissolved in 10 mL of PBS buffer. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 2 mmol) and N-hydroxysuccinimide (NHS; 2 mmol) were supplemented to the reaction solution, followed by stirring for nearly 5 hours at room temperature.
The reaction product was filtered to remove the precipitate and added to a 100,000 molecular weight cutoff dialysis membrane in deionized water for 24 hours to discard the unreacted EDC, herceptin, and NHS. Lastly, the solution was filtered through a 0.45 μm membrane and dried under vacuum for 24 hours.
Preparation of cyanine dye–GNP–Her conjugates
Cyanine dye 5 (Cy5)-conjugated GNP–Her was obtained by reacting 1.5×10−3 mol/L GNP–Her with 4.8×10−5 mol/L fluorescent Cy5 in a carbonate buffer solution.
The concentration of Cy5 dye was examined by UV–vis spectroscopy to calculate the number of NPs internalized into the cytoplasm of SK-BR3.25