Multiphoton-Absorption-Induced-Luminescence (MAIL) Imaging of Tumor-Targeted Gold Nanoparticles

Abstract

We demonstrate that multiphoton-absorption-induced luminescence (MAIL) is an effective means of monitoring the uptake of targeted nanoparticles into cells. Gold nanoparticles (AuNPs) with diameters of 4.5 and 16 nm were surface-functionalized with monocyclic RGDfK, an RGD peptide analogue that specifically targets the α(v)β₃ integrin, a membrane protein that is highly overexpressed in activated endothelial cells during tumor angiogenesis. To determine whether cyclic RGD can enhance the uptake of the functionalized AuNPs into activated endothelium, human umbilical vein endothelial cells (HUVECs) were used as a model system. MAIL imaging of HUVECs incubated with AuNPs demonstrates differential uptake of AuNPs functionalized with RGD analogues: RGDfK-modified nanoparticles are taken up by the HUVECs preferentially compared to AuNPs modified with linear RGD (GRGDSP) conjugates or with no surface conjugates. The luminescence counts observed for the AuNP-RGDfK conjugates are an order of magnitude greater than for AuNP-GRGDSP conjugates. Transmission electron microscopy shows that, once internalized, the AuNP-RGDfK conjugates remain primarily within endosomal and lysosomal vesicles in the cytoplasm of the cells. Significant aggregation of these particles was observed within the cells. MAIL imaging studies in the presence of specific uptake inhibitors indicate that AuNP-RGDfK conjugate uptake involves a specific binding event, with α(v)β₃ integrin-mediated endocytosis being an important uptake mechanism.

Setup for investigating gold nanoparticle penetration through reconstructed skin and comparison to published human skin data

“…[23][24][25] Based on this, we developed a method to detect AuNP in skin. 8,26,28 Though multiphoton microscopy is a nondestructive optical technique, skin sectioning was required to avoid the possible limitations when imaging from the top view in case of nonsectioned skin specimens in which AuNP could be detected up to variable depths from the skin surface ranging from 14 to 100 μm, depending on skin pre-treatment (tissue fixation) and area of examination (presence or absence of wrinkles).…”

“…This technique is referred to as multiphoton-absorption-induced luminescence (MAIL), where the absorption of multiple photons from a near-infrared pulsed femtosecond laser can lead to robust luminescence of metal nanoparticles. 8,[23][24][25][26] Multiphoton microscopy was therefore employed as a useful optical technique to track particle penetration in skin 8,22,[26][27][28] and was the imaging tool in our study to track model AuNP.…”

Organic–inorganic nanohybrids for fluorescence, photoacoustic and Raman bioimaging

“…There are two major classes of nanomaterials that have a good multiphoton absorption cross-section: QDs and metallic nanoparticles. For example, different sizes of gold nanoparticles conjugated with RGD peptide can generate distinct multiphoton absorption-induced luminescence (MAIL) signals under 800 nm [72]. This surfacefunctionalized nanostructure was found to have an ability to target tumor cells and to give transmission images via MAIL.…”

Spectroscopic Properties of Gold Nanoparticles at the Single‐Particle Level in Biological Environments

“…[1, 12, 13, 14, 15, 16] It has been demonstrated that Multiphoton-Absorption-Induced Luminescence from gold NPs is efficiently generated using a 2-photon excitation source at 800 nm with excitation intensities that are lower than those typically used for 2-photon imaging of living cells. [1] 25 years ago, Boyd and co-workers showed that gold surfaces exhibit ~100 times more luminescence when irradiated with a 2-photon excitation source compared to 1-photon excitation.…”