We report development of a nanoparticle-based, X-ray-activated anticancer "nanodrug" composed of yttrium oxide (Y(2)O(3)) nanoscintillators, a fragment of the HIV-1 TAT peptide, and psoralen. In this formulation, X-ray radiation is absorbed by the Y(2)O(3) nanoscintillators, which then emit UVA light. Absorption of UVA photons by nanoparticle-tethered psoralen has the potential to cross-link adenine and thymine residues in DNA. UVA-induced cross-linking by free psoralen upon activation with UVA light has previously been shown to cause apoptosis in vitro and an immunogenic response in vivo. Studies using the PC-3 human prostate cancer cell line demonstrate that X-ray excitation of these psoralen-functionalized Y(2)O(3) nanoscintillators yields concentration-dependent reductions in cell number when compared to control cultures containing psoralen-free Y(2)O(3) nanoscintillators.

A critical aspect for use of nanoprobes in biomedical research and clinical applications involves fundamental spatial and temporal characterization of their uptake and distribution in cells. Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate nanoprobes in single cells using surface-enhanced Raman scattering detection. To study the efficiency of cellular uptake, silver nanoparticles functionalized with three different positive-, negative-, and neutrally charged Raman labels were co-incubated with cell cultures and internalized via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake. These results indicate that the functionalized nanoparticle construct has potential for sensing and delivery in single living cells and that use of surface-enhanced Raman scattering for tracking and detection is a practical and advantageous alternative to traditional fluorescence methods.

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.

We report the first demonstration of surface-enhanced Raman spectroscopy (SERS) detection of para-mercapto benzoic acid (pMBA) and surface-enhanced resonance Raman spectroscopy (SERRS) detection of brilliant cresyl blue (BCB) and cresyl violet perchlorate (CVP) with continuous-wave excitation from a stand-off distance of 15 meters. We further report the first stand-off SERRS detection of BCB and CVP at that same distance in the presence of ambient fluorescent and incandescent/blackbody background light. These preliminary results suggest that it is possible to detect sub-nanomole amounts of material at reasonable distances with eye-safe laser powers using stand-off SERRS and serve as proof-of-concept highlighting the potential extension of stand-off Raman spectroscopy to include SERS and SERRS for remote, eye-safe chemical detection, analysis, and imaging in the presence of ambient background light.

L'invention porte sur une nanoparticule fonctionnalisee, ayant un cœur, ayant facultativement une enveloppe sur au moins une partie de celui-ci, le cœur contenant un materiau qui peut convertir de l'energie de rayons X appliques en une energie ultraviolette emise et l'enveloppe, lorsqu'elle est presente, contenant un materiau actif plasmonique; selon l'invention, la nanoparticule a, sur une surface de celle-ci, au moins un compose psoralene capable d'activation par l'energie ultraviolette emise. L'invention porte egalement sur l'utilisation de la nanoparticule fonctionnalisee dans un procede de traitement d'un trouble a proliferation cellulaire tel que le cancer.

In this paper, we describe the development and application of a pH-sensitive plasmonics-active fiber-optic nanoprobe suitable for intracellular bioanalysis in single living human cells using surface-enhanced Raman scattering (SERS) detection. The effectiveness and usefulness of SERS-based fiber-optic nanoprobes are illustrated by measurements of intracellular pH in HMEC-15/hTERT immortalized "normal" human mammary epithelial cells and PC-3 human prostate cancer cells. The results indicate that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor cellular microenvironments at the single cell level.

We describe the development of small molecule-sensitive plasmonics-active fiber-optic nanoprobes suitable for intracellular bioanalysis in single living human cells using surface-enhanced Raman scattering (SERS) detection. The practical utility of SERS-based fiber-optic nanoprobes is illustrated by measurements of intracellular pH in HMEC- 15/hTERT immortalized "normal" human mammary epithelial cells and PC-3 human prostate cancer cells. The results indicate that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically-relevant small molecules at the single cell level.

BACKGROUND:Fiber-optic nanosensors are fabricated by heating and pulling optical fibers to yield sub-micron diameter tips and have been used for in vitro analysis of individual living mammalian cells. Immobilization of bioreceptors (e.g., antibodies, peptides, DNA) selective to targeting analyte molecules of interest provides molecular specificity. Excitation light can be launched into the fiber, and the resulting evanescent field at the tip of the nanofiber can be used to excite target molecules bound to the bioreceptor molecules. The fluorescence or surface-enhanced Raman scattering produced by the analyte molecules is detected using an ultra-sensitive photodetector.OBJECTIVE:This article provides an overview of the development and application of fiber-optic nanosensors for drug discovery.CONCLUSIONS:The nanosensors provide minimally invasive tools to probe subcellular compartments inside single living cells for health effect studies (e.g., detection of benzopyrene adducts) and medical applications (e.g., monitoring of apoptosis in cells treated with anticancer drugs).

A methylene blue-encapsulated apoferritin complex shows cytotoxic effects on MCF-7 human breast adenocarcinoma cells when irradiated at the appropriate wavelength.

A critical aspect of the use of nanoprobes for intracellular studies in chemical and biological sensing involves a fundamental understanding of their uptake and trajectory in cells. In this study, we describe experiments using surface-enhanced Raman scattering (SERS) spectroscopy and mapping to track cellular uptake of plasmonics-active labeled nanoparticles. Three different Raman-active labels with positive, negative, and neutral charges were conjugated to silver colloidal nanoparticles with the aim of spatially and temporally profiling intracellular delivery and tracking of nanoprobes during uptake in single mammalian cells. 1-D Raman spectra and 2-D Raman mapping are used to identify and locate the probes via their SERS signal intensities. Because Raman spectroscopy is very specific for identification of chemical and molecular signatures, the development of functionalized plasmonics-active nanoprobes capable of exploring intracellular spaces and processes has the ability to provide specific information on the effects of biological and chemical pollutants in the intracellular environment. The results indicate that this technique will allow study of when, where, and how these substances affect cells and living organisms.