Influence of anchoring ligands and particle size on the colloidal stability and in vivo biodistribution of polyethylene glycol-coated gold nanoparticles in tumor-xenografted mice

Abstract

Polyethylene glycol (PEG)-coated (pegylated) gold nanoparticles (AuNPs) have been proposed as drug carriers and diagnostic contrast agents. However, the impact of particle characteristics on the biodistribution and pharmacokinetics of pegylated AuNPs is not clear. We investigated the effects of PEG molecular weight, type of anchoring ligand, and particle size on the assembly properties and colloidal stability of PEG-coated AuNPs. The pharmacokinetics and biodistribution of the most stable PEG-coated AuNPs in nude mice bearing subcutaneous A431 squamous tumors were further studied using (111)In-labeled AuNPs. AuNPs coated with thioctic acid (TA)-anchored PEG exhibited higher colloidal stability in phosphate-buffered saline in the presence of dithiothreitol than did AuNPs coated with monothiol-anchored PEG. AuNPs coated with high-molecular-weight (5000 Da) PEG were more stable than AuNPs coated with low-molecular-weight (2000 Da) PEG. Of the 20-nm, 40-nm, and 80-nm AuNPs coated with TA-terminated PEG(5000), the 20-nm AuNPs exhibited the lowest uptake by reticuloendothelial cells and the slowest clearance from the body. Moreover, the 20-nm AuNPs coated with TA-terminated PEG(5000) showed significantly higher tumor uptake and extravasation from the tumor blood vessels than did the 40- and 80-nm AuNPs. Thus, 20-nm AuNPs coated with TA-terminated PEG(5000) are promising potential drug delivery vehicles and diagnostic imaging agents.

Fig. 1

(A) Schematic of the synthesis of indium-111 (¹¹¹In)-labeled, polyethylene glycol (PEG)-coated gold nanoparticles using thioctic acid (TA) as an anchoring ligand. (B) Synthesis of PEG-TA. (C) Synthesis of 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide (DTPA-TA). TEA, triethylamine; DIC, 1,3-diisopropylcarbodiimide; DMAP, 4-dimethylaminopyridine; TFA, trifluoroacetic acid; DCM, dichloromethane.

Fig. 2

Matrix-assisted laser desorption/ionization time-of-flight mass spectrum of O-(2-aminoethyl)-O′-methyl polyethylene glycol (molecular weight, 2000) terminated with thioctic acid (TA). The polydispersity index was 1.02. The intensity peak at 2180.64 corresponded to MeO-(CH₂CH₂O)₄₃-TA/Na⁺.

Fig. 3

Stability of pegylated gold nanoparticles (AuNPs) in phosphate-buffered saline containing 10 mM dithiothreitol (DTT) at 40°C. (A) Ultraviolet-visible spectra of 40- nm AuNPs coated with thiol-terminated polyethylene glycol with a molecular weight of 5000 (PEG₅₀₀₀-SH) acquired at various times after the addition of DTT. The concentration of AuNPs was 7.0 × 10¹¹ particles/ml. The arrow in the figure denotes absorbance at 650 nm. (B) Absorbance at 650 nm as a function of time. (C) Half-lives for 20-nm and 40-nm AuNPs coated with different types of PEG.

Fig. 4

Stability of 20-nm gold nanoparticles (AuNPs) in phosphate-buffered saline containing 10% fetal bovine serum at 37°C. The diameters of nanoparticles were measured using dynamic light scattering. Pegylated AuNPs did not show a significant change in particle size, whereas plain (uncoated) AuNPs aggregated within 10 min after incubation.

Fig. 5

Pharmacokinetics, expressed as the percentage of the injected dose per gram of tissue in mice (%ID/g), of 20-nm, 40-nm, and 80-nm gold nanoparticles (AuNPs) coated with thioctic acid-terminated polyethylene glycol. The solid lines are curves fitted to a two-compartment model.

Fig. 6

Gamma images of the in vivo distribution of 80-nm and 20-nm gold nanoparticles (AuNPs) in mice bearing A431 tumors 10 min and 2, 24, and 48 h after intravenous injection. Compared to 20-nm AuNPs, 80-nm AuNPs were cleared more rapidly from the blood and had higher concentrations in the liver and spleen. In contrast, 20-nm AuNPs had long blood pool activity and accumulated in the tumor 48 h after injection. The scales on the right side of each panel show the signal intensity range. Arrows indicate subcutaneous A431 tumors.

Fig. 7

Biodistribution of 20-nm and 80-nm gold nanoparticles (AuNPs) coated with thioctic acid-anchored polyethylene glycol with a molecular weight of 5000 in mice bearing subcutaneous A431 tumors.

Fig. 8

Fluorescence microscopic image (original magnification, 40×) of a 5-μm cryosectioned slice of an A431 tumor removed from a mouse 48 h after intravenous injection of 20-nm gold nanoparticles (AuNPs) coated with thioctic acid-anchored polyethylene glycol with a molecular weight of 5000. The slice was stained for platelet-endothelial cell adhesion molecule-1 (CD31, red) and epidermal growth factor receptor (blue). AuNPs were visualized using a dark field condenser (pseudocolored green). (A) Some nanoparticles remained in the blood vessel (arrows), while (B) others entered the extravascular fluid space (arrow heads).