Fig 1.
Gold nanoparticle characterization.
(A) TEM image of bare gold nanoparticles. (B) DLS plots of nanoparticle hydrodynamic diameter before after surface modification. (C) UV-VIS spectroscopy of gold nanoparticles, showing a shift in peak absorbance as a result of surface modification. (D) DLS measurements of average hydrodynamic diameter, polydispersity index, and zeta potential of nanoparticles with each surface modification. (E) Hydrodynamic diameter measurements of PEG, VHH, and C225-AuNPs before and after incubation with 10% fetal bovine serum for 72 hours.
Fig 2.
ELISA for EGFR binding to targeted AuNPs.
(A) shows the ELISA signal (reflecting EGFR binding) normalized by nanoparticle concentration. Both C225 and VHH-122 have high binding, while PEG has almost none. Differences in binding between all three nanoparticle types are statistically significant. (B) shows the ELISA signal normalized by antibody concentration. Here, C225 has much higher signal than VHH-122, consistent with its higher binding affinity and lower number of antibodies per nanoparticle.
Fig 3.
Darkfield images of AuNPs bound to A431 cells (high EGFR expression).
AuNPs appear as bright yellow-orange dots in the dark field images. PEG-AuNPs (A) showed little to no cell binding, VHH-AuNPs (B) showed a moderate level of cell binding, while C225-AuNPs (C) had a high number of nanoparticles bound to the cells in culture. This demonstrates effective EGFR-targeting, particularly for the C225-AuNPs. Scale bars are 30 μm.
Fig 4.
ICP-OES measurements of cell pellet gold concentrations.
C225-AuNPs showed the highest degree of A431 cell binding, followed by VHH-AuNPs. PEG-AuNPs showed only minimal cell binding. All three groups were statistically different from one another.
Fig 5.
CT enhancement in healthy mice after injection of AuNP contrast agents.
(A) shows a CT axial cross-section through the heart immediately after injection of VHH-AuNPs, demonstrating high CT contrast in the blood after injection. (B) shows the measured enhancement of each contrast agent in the blood over time, along with the exponential fit curve for each nanoparticle type. Enhancement at each time point is normalized to the initial enhancement immediately after injection. All nanoparticles showed an exponential decrease in concentration. C225 had a significantly shorter half-life than VHH-122 or PEG. (C) shows the organ enhancement at 36 hours (relative to the enhancement in the blood immediately after injection) for each contrast agent. C225 showed significantly more spleen accumulation than VHH-122 or PEG. All other differences were not statistically significant.
Fig 6.
Dual-energy CT slices of tumors 48 hours post-injection.
Axial CT slices of the mice through the center of the tumors are shown on the left, while a coronal maximum intensity projection (MIP) of an isolated tumor from each mouse is shown on the right. Each MIP includes 20 slices through the center of the tumor. Each image is an overlay of the single energy CT scan (in grayscale) and the dual-energy gold concentration map (in green). Gold nanoparticles were cleared from the bloodstream at this time point and accumulated predominately within the well-perfused tumor margins. The relatively poorly perfused tumor cores showed low nanoparticle uptake. All tumors had substantial nanoparticle accumulation, but C225-AuNPs showed the highest tumor enhancement. The single energy CT scans are windowed from -500 to 2000 HU for the axial slices and 0 to 3000 HU for the coronal MIPs while the dual-energy decomposition gold maps are windowed from 1.5 to 20 mg/mL for the axial slices and 0.5 to 20 for the coronal MIPs.
Fig 7.
CT quantification of gold nanoparticle accumulation in the tumor margins.
Enhancing regions of each tumor were segmented and the average gold concentration within each segmented tumor was calculated. C225-AuNPs have significantly higher accumulation in the enhancing portions of the tumor than either VHH-AuNPs or PEG-AuNPs. Targeting with VHH-122 did not provide any increase in accumulation relative to the PEG controls.