Effect of Gold Nanoparticle Size and Coating on Labeling Monocytes for CT Tracking

Abstract

With advances in cell therapies, interest in cell tracking techniques to monitor the migration, localization, and viability of these cells continues to grow. X-ray computed tomography (CT) is a cornerstone of medical imaging but has been limited in cell tracking applications due to its low sensitivity toward contrast media. In this study, we investigate the role of size and surface functionality of gold nanoparticles for monocyte uptake to optimize the labeling of these cells for tracking in CT. We synthesized gold nanoparticles (AuNP) that range from 15 to 150 nm in diameter and examined several capping ligands, generating 44 distinct AuNP formulations. In vitro cytotoxicity and uptake experiments were performed with the RAW 264.7 monocyte cell line. The majority of formulations at each size were found to be biocompatible, with only certain 150 nm PEG functionalized particles reducing viability at high concentrations. High uptake of AuNP was found using small capping ligands with distal carboxylic acids (11-MUA and 16-MHA). Similar uptake values were found with intermediate sizes (50 and 75 nm) of AuNP when coated with 2000 MW poly(ethylene-glycol) carboxylic acid ligands (PCOOH). Low uptake values were observed with 15, 25, 100, and 150 nm PCOOH AuNP, revealing interplay between size and surface functionality. Transmission electron microscopy (TEM) and CT performed on cells revealed similar patterns of high gold uptake for 50 nm PCOOH and 75 nm PCOOH AuNP. These results demonstrate that highly negatively charged carboxylic acid coatings for AuNP provide the greatest internalization of AuNP in monocytes, with a complex dependency on size.

Figure 1

TEM images of spherical gold nanoparticle of increasing size from 15 to 150 nm. 15 and 25 nm AuNP were synthesized through the Turkevich method. 50, 75, 100, and 150 nm AuNP were synthesized through a seeded growth method.

Figure 2

Schematic depiction of the range of AuNP sizes used in this study and the chemical structures of the ligands used as coatings. PEG-amine coatings were mixed in 1:4 ratios with MPEG and PCOOH to provide particle stability. Ligands examined represent different functionalities and charges.

Figure 3

Cell viability of monocytes incubated with AuNP of a range of diameters and coatings. Cells were incubated with AuNP for 24 hours at 0.1, 0.5, 1.0 mg Au/ml. Viability was assessed with the LIVE/DEAD assay. Values are normalized to those for untreated monocytes. Values represent the mean of 3 replicates (n=3) and error bars represent standard error of mean. Significance for 150 nm AuNP was examined with one-way ANOVA and post hoc Tukey pairwise comparisons. * indicates significance of treated groups compared to non-treated group (p<0.05).

Figure 4

Evaluation of gold content in monocytes incubated with 15 nm 11-MUA AuNP (0.5 mg Au/ml) for up to 48 hours. In addition, the total number cells at each time point is displayed. Values represent mean of 3 replicates (n=3) and error bars are standard error of mean. A one-way ANOVA was used to determine significance for both uptake and number of cells. Post-hoc Tukey pairwise comparison was used to compare significance between time points (p<0.05). Results for statistical comparisons between 24 and 48 hours shown only.

Figure 5

Gold uptake in monocytes incubated with AuNP of a variety of sizes and coatings. Formulations exhibiting particle instability (significant aggregation and sedimentation) or cytotoxicity were excluded from the study. The cells were treated for 24 hours at 0.5 mg Au/ml and evaluated for gold content with ICP-OES. Values represent mean of typically six replicates (n=6) and error bars are standard error of mean. A two-way ANOVA was used to determine significance of AuNP uptake for size and coating. A Tukey pairwise mean comparison was used to determine significance between coatings at each size. * indicates significance of PCOOH compared to all other coatings at that size.

Figure 6

TEM of monocytes treated with 15, 75, 100, and 150 nm PCOOH AuNP. Cells were treated at 0.5 mg/ml for 24 hours before being harvested and fixed for TEM.

Figure 7

(A) CT scans of monocytes settled into pellets after treatment with 75 and 150 nm PCOOH AuNP for 24 hours at 0, 0.1, 0.25 and 0.5 mg/ml. Cells were collected in 4% PFA solution after treatment. Scans performed on small animal microCT scanner with 100 μm spatial resolution at 80 kVP and 500 μA. (B) Quantification of attenuation in Fig. 7A of monocytes incubated with PCOOH AuNP of various sizes. Values normalized to untreated cells. Error bars are standard error of the mean with (n=3 for each data point). A one-way ANOVA and post-hoc Tukey pairwise mean comparison used to compare different sizes at each concentration group. * indicates significance between all sizes at that concentration (p<0.05).