Surface-Enhanced Raman Scattering Detection and Tracking of Nanoprobes: Enhanced Uptake and Nuclear Targeting in Single Cells

Abstract

We describe the development and application of a co-functionalized nanoprobe and biodelivery platform combining a nuclear targeting peptide (NTP) for improved cellular uptake and intracellular targeting with p-mercaptobenzoic acid (pMBA) as a surface-enhanced Raman scattering (SERS) reporter for tracking and imaging. The nuclear targeting peptide, an HIV-1 protein-derived TAT sequence, has been previously shown to aid entry of cargo through the cell membrane via normal cellular processes, and furthermore, to localize small cargo to the nucleus of the cell. Previous work in our lab has verified cell uptake and distribution of the nanoprobes in clinically relevant mouse and human cell lines. In this work, two-dimensional SERS mapping was used to track the spatial and temporal progress of nanoparticle uptake in PC-3 human prostate cells and to characterize localization at various time points, demonstrating the potential for an intracellularly targeted multiplexed nanobiosensing system with excellent sensitivity and specificity. Silver nanoparticles co-functionalized with the TAT peptide showed greatly enhanced cellular uptake over the control nanoparticles lacking the targeting moiety. The ability to detect and monitor nanoprobe trafficking using SERS spectroscopy offers an improved alternative over previous tracking and detection methods such as light microscopy and fluorescence methods. The development of multifunctional nanoconstructs for intracellular delivery has potential clinical applications in early detection and selective treatment of disease in affected cells. Other applications include use in basic research aimed at understanding the inner workings of living cells and how they respond to chemical and biological stimuli.

SERS Tags: Novel Optical Nanoprobes for Bioanalysis

“…249 The intensity of a particular Raman band in the SERS tag spectrum can be plotted by using a color gradient or a series of contours, resulting in an "image map" showing the location of the tags over a select physical area as a function of their SERS signal intensity at that particular band. 250 SERS signal intensity depends upon the number of SERS tags within small excitation laser spots (usually less than 200 μm 2 ), and the biological samples, such as cells and tissues, are intrinsically inhomogeneous.…”

Raman spectroscopy: techniques and applications in the life sciences

“…SERS particles functionalized with immunolabels have also been used to measure the external surface of endothelial cell membranes [292]. Specific nucleus-targeting peptides were used to functionalize NPs and also enhanced NP uptake into cells [293]. Modulating the surface charge of the functionalized NP was also shown to give some control over uptake as well [294].…”

Peptide-Guided Surface-Enhanced Raman Scattering Probes for Localized Cell Composition Analysis

“…This approach results in convoluted SERS spectra composed of contributions from biochemicals of diverse intracellular environments simultaneously. Therefore, the ability to control the localization of SERS probes inside the cell is critical to the production of minimal SERS spectra capable of being deconvoluted to resolve chemical composition and phenotype data for localized intracellular environments independently.Metal nanoparticles, conjugated with a variety of ligands, have been used as intracellular SERS probes with exceptional selectivity for the purposes of imaging and detection, but these have not been designed to determine cell composition or phenotype (4,14,17,20). Zeiri et al (24) developed protocols to direct the production of silver nanoparticles (SNPs) to either the cytosol of a bacterium or to the cell wall.…”

“…Metal nanoparticles, conjugated with a variety of ligands, have been used as intracellular SERS probes with exceptional selectivity for the purposes of imaging and detection, but these have not been designed to determine cell composition or phenotype (4,14,17,20). Zeiri et al (24) developed protocols to direct the production of silver nanoparticles (SNPs) to either the cytosol of a bacterium or to the cell wall.…”