Targeted silver nanoparticles for ratiometric cell phenotyping

Abstract

Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 ± 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 ± 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.

Fig. 1

Multiplexed ratiometric AgNP test system. The triplex system shown here is based on two targeting peptides: RPARPAR and SGKRK, and two biological targets: NRP-1 and p32. M21 cells express cell surface p32, whereas PPC-1 cells express both p32 and NRP-1 on the cell surface. Control biotin-AgNPs do not bind to either cell line and serve as a negative control. When an input of three different AgNPs is applied to the cells, their cell binding and uptake pattern will correlate with accessible cell surface receptor expression.

Fig. 2

Binding of peptide particles to receptor proteins. (A) R-AgNPs bind to immobilized NRP1-b1b2 domain but not to NRP1-b1b2 protein with mutated CendR binding pocket or to p32 protein; K-AgNPs bind to p32 and not to wt or mutant NRP1-b1b2. Data represent mean values ± SD (n=3); *** p < 0.001 by Student’s t-test. (B) Representative raw data from UV-vis spectroscopy for R-AgNPs. AgNPs were eluted from protein-loaded Ni-NTA magnetic beads with 0.4 M imidazole-containing buffer and the UV-vis spectrum of the eluate was measured. The absorbance spectrum seen for NRP-1 binding is due to R-AgNPs; the peak absorbance was taken for calculations.

Fig. 3

Receptor-dependent binding and uptake of nanoparticles by PPC-1 and M21 cells. (A) Flow cytometry of M21 and PPC-1 cells incubated for 1 h with R-, K-, or B-AgNPs, all labeled with CF555. Note that NRP-1-positive PPC-1 cells bind R-AgNPs (66 ± 1% positive), whereas M21 do not; both cell lines score positive for binding of K-AgNPs 7 ± 1% positive for M21 and 8 ± 1% for PPC-1 cells (n=3). B) Confocal microscopy pictures of M21 and PPC-1 cells co-cultured as attached cells. The cells can be distinguished by the high cell surface expression of NRP-1 by PPC-1 cells and absence of NRP-1 in M21 cells. AgNPs were labeled with CF555 fluorescent dye. Note cellular uptake of R-AgNPs in NRP-1-positive PPC-1 cells and binding of both PPC-1 and M21 cells by K-AgNPs. Scale bar = 50 μm.

Fig. 4

Isotopic multiplexing of AgNP binding and internalization into M21 and PPC-1 cells. A) Three isotope-tagged RPARPAR-coupled silver cores were incubated with PPC-1 or M21 cells for 1 h and total bound AgNPs were quantified by ICP-MS. Each of the three silver cores functionalized with RPARPAR peptide showed robust PPC-1 binding and low M21 binding. The Pd concentration is multiplied by 50 to visualize more clearly; the R-wt-Ag-Pd sample’s Pd content of 0.94 ± 0.04 ng/g is 100 times above the detection limit. Data represent mean values ± SD (n=3). B) Cells incubated with isotopically coded AgNPs (R-107AgNPs, K-109AgNPs, B-wtAg-PdNPs), followed by etching to remove the extracellular nanoparticles and quantification of the internalized nanoparticles by ICP-MS. PPC-1 cells demonstrated preferential uptake of R-AgNPs and M21 cells took up mainly K-AgNPs (n=3).