Prostate-specific membrane antigen targeted gold nanoparticles for prostate cancer radiotherapy: does size matter for targeted particles?

Prostate-Specific Membrane Antigen (PSMA) targeted radiosensitizers are developed for prostate cancer CT imaging and radiotherapy based on gold nanoparticles and a high-affinity targeting peptide, PSMA-1, revealing a size-dependent pattern.

carboxyethyl)phosphine was removed by dialysis (MWCO=2kDa) against H 2 O. The final product was lyophilized and stored in -20 °C for future use. High-resolution matrix-assisted laser desorption/ionization−time-of-flight mass spectroscopy (MALDI-TOF MS, Bruker AutoFlex III) was used for characterization.

Synthesis and characterization of gold nanoparticles
Gold nanoparticles with three different sizes were synthesized. AuNPs with core size of 2 nm and 5 nm were synthesized in a two-phase toluene-H 2 O system. For 2 nm AuNPs, 80 mg of HAuCl 4 •3H 2 O and 260 mg of tetraoctylammonium bromide (TOAB) were dissolved in 5 ml H 2 O and 14 ml toluene respectively, and the two phases were mixed and stirred for 5 min. Then 18 mg DDA was added to the mixture and stirred for another 10 min. 5 ml of 13 mg/ml sodium borohydride (NaBH 4 ) ice-cold H 2 O solution was then added in very quickly (within 10s). The reaction mixture was stirred for more than 4 hours. For 5 nm AuNPs synthesis, 136.7 mg TOAB was dissolved in 5 ml toluene and 367 ul of gold(III) chloride solution (30 wt % in dilute HCl) was added and stirred for 5 min. 112 mg DDA was then added to the mixture. Following another 10 min of stirring, 1 ml of 75.6 mg/ml NaBH 4 ice-cold H 2 O solution was added dropwise. The reaction was then carried on for 2 hours. The produced AuNPs were then precipitated in pure ethanol and centrifuged at 4500 rpm for 10 min. The supernatants were decanted and AuNPs were washed again with pure ethanol. The precipitations were dried under N 2 gas stream and then dissolved in chloroform and centrifuged again at 4500 rpm for 10 min. The supernatants were collected and measured by UV-vis spectroscopy to determine the concentration.
AuNPs with core size of 19 nm were synthesized using the Turkevich method [1].
Generally, 100 ml of 0.01% HAuCl 4 •3H 2 O solution was refluxed and added with 2.5 ml of 1% sodium citrate solution was added under vigorous stirring. A color change from yellow to red would occur in 5 min. The heat source was removed after 30 min reacting and the solution was cooled down to room temperature.
The concentration of the AuNP samples was determined by UV−vis spectroscopy based on the plasmonic absorption band at 520 nm. To functionalize AuNPs with SH-PEG 2K -PSMA-1, a 1000 molar excess of SH-PEG 2K -PSMA-1 and SH-PEG 2K ligands at different molar ratio (1:8, 1:4, 1:1 to 2:1) were added to react with 1 equiv of AuNP-DDA or AuNPcitric acid for 2 days. Excess unreacted ligands were removed by extensive purification using centrifuge filters (MWCO = 30 kDa, GE Healthcare). As control, non-targeted AuNPs were also prepared in the same way with only SH-PEG 2K added and purified.
The hydrodynamic size of AuNPs was characterized with a dynamic light scattering system (DynoPro NanoStar). The zeta potential of AuNPs was measured with a Zetasizer Nano (Malvern). For absolute size determination, transmission electron microscopy (FEI Tecnai F300 kV). Samples were prepared by putting one drop of sample solution onto 400 mesh formvar/carbon supported copper grids (Ted Pella, Inc.), and dried naturally at room temperature. To visualize the polymer shells, the nanoparticles loaded copper grids were stained by one drop of 2% Phosphotungstic acid for 5 min and then the excess liquid was wicked off with filter paper. The grids after staining were dried again in air before TEM testing. Besides, gel electrophoresis for all the PSMA-targeted and untargeted nanoparticles with various sizes were performed on 1% agarose gel and 1× TAE running buffer at 120 kV. Each chamber was loaded with 10 μL of 2 uM AuNPs, 5 μL of glycerol, and 5 μL of 4× TAE.