Visualizing systemic clearance and cellular level biodistribution of gold nanorods by intrinsic two-photon luminescence

Abstract

Characterization of systemic performance of gold nanostructures is critical to the advancement of biomedical applications of these nanomaterials as imaging or therapeutic agents. The accuracy of current in vitro methods, however, is limited by interanimal variability. We present a novel method capable of monitoring the pharmacokinetics of PEGylated gold nanorods (GNRs) in the same animal by using intravital two-photon luminescence (TPL) imaging of GNRs flowing through a surface blood vessel. The TPL imaging with high speed and submicrometer resolution allowed for studying the clearance of GNRs as a function of surface coating. PEGylated-GNRs (PEG-NRs) were found to exhibit a biphasic clearance mode, with a significantly prolonged blood residence time for branched poly(ethylene glycol) (PEG) as compared to the linear PEG. With spectral detection to distinguish GNR TPL from tissue autofluorescence, we also mapped the cellular distribution of GNRs in the explanted organs, and found most GNRs resided in the macrophages in liver and spleen.

Figure 1.

Surface modification of GNRs and characterization of TPL from GNRs. (a) Structure of PEG-NRs (linear PEG: 2k and 5k; branched PEG: 7k). (b) TPL emission spectra from CTAB-NR and PEG-NR solution. (c) TPL emission spectra from PEG-NRs in water, PBS, and serum. For b and c, 6 μL of each solution was dropped into a small chamber on a coverslip. TPL was excited at the wavelength of the longitudinal plasmon peak of each sample with the same laser power and photomultiplier tube voltage, and spectra were recorded from the solution with the laser focused at 20 μm above the coverslip. (d) TPL intensity vs PEG-NR concentration in aqueous solution. A linear dependence was observed from 4.7 nM to 0.11 nM.

Figure 2.

Intravital TPL imaging of PEG-NRs circulating in peripheral blood vessels. (a) A single-frame TPL image of individual flowing GNRs (yellow) marked by red circles. The excitation wavelength was 774 nm. TPL intensity profile from the two GNRs was shown under the figure, with a signal-to-noise ratio of 4:1. The background (~400 au) was due to the autofluorescence from the blood vessel. (b–g) TPL images of PEG-NRs flowing in blood vessels of the same mouse at p.i. time of (b) 10 min, (c) 1 h, (d) 2 h, (e) 4 h, (f) 8 h, and (g) 24 h. Each image was compiled as a stack of 60 frames collected continuously at a rate of 0.4 s per frame. The excitation wavelength was 774 nm. Scale bar = 20 μm.

Figure 3.

Quantitative analysis of clearance of PEG-NRs. Blood circulation kinetics of mPEG_2k-NRs (a), mPEG_5k-NRs (b), and branched PEG_7k-NRs (c) probed by intravital TPL imaging method. The blood concentration of GNRs was represented by TPL contrast, I_NR/I_blood. Each data point was an average from 3 mice (n = 3). The data were fitted with one-compartment model (red, y = Ae^{−(x − x₀)/t}₁ + y₀) and two-compartment model (green, A₁e^{−(x − x₀)/t}₁ + A₂e^{−(x − x₀)/t}₂ + y₀) (d) ICP-MS analysis of gold concentration in blood at different p.i. times, normalized by the gold concentration at 0.5 h after injection. n = 3.

Figure 4.

Serum binding test. mPEG_2k-NRs (a, 0.52 nM), mPEG_5k-NRs (b, 0.52 nM), and branched PEG_7k-NRs (c, 0.52 nM) were incubated in mouse serum, and the extinction spectra were monitored for 24 h. The spectrum was first measured in PBS and then in serum after incubation for 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. A gradual widening of the longitudinal plasmon resonance band was observed for mPEG_2k-NRs and mPEG_5k-NRs, but not for branched PEG_7k-NRs.

Figure 5.

TPL imaging of PEG-NRs in explanted organs. (a) Emission spectra from liver autofluorescence (black) and TPL from PEG-NRs in liver (red), excited by an fs laser at 774 nm. (b) Autofluoresecence from liver and TPL from PEG-NRs excited by 774 nm laser and detected with spectral detector within 460–560 nm. PEG-NRs (bright dots) were observed between adjacent hepatocytes. (c) Image acquired with spectral detector within 650–700 nm in the same area with (b). Only TPL from PEG-NRs was detected, while no autofluorescence was observed in this range. (d) Enlarged image of PEG-NRs in liver, detected within 460–560 nm. GNRs were accumulated in the intercellular space between hepatocytes, which were assigned as Kupffer cells. (e) PEG-NRs observed in spleen. The green circle indicated the uptake of GNRs by a macrophage. (f) No TPL signal from GNRs was detected in kidney. Scale bar = 10 μm.