By properly conjugating gold nanoparticles with specific peptides, we were successful in selectively transporting them to the nuclei of cancer cells. Confocal microscopy images of DNA double-strand breaks showed that localization of gold nanoparticles at the nucleus of a cancer cell damages the DNA. Gold nanoparticle dark-field imaging of live cells in real time revealed that the nuclear targeting of gold nanoparticles specifically induces cytokinesis arrest in cancer cells, where binucleate cell formation occurs after mitosis takes place. Flow cytometry results indicated that the failure to complete cell division led to programmed cell death (apoptosis) in cancer cells. These results show that gold nanoparticles localized at the nuclei of cancer cells have important implications in understanding the interaction between nanomaterials and living systems.

Gold nanoparticles possess a unique combination of properties which allow them to act as highly multifunctional anti-cancer agents (X. H. Huang, P. K. Jain, I. H. El-Sayed and M. A. El-Sayed, Nanomedicine, 2007, 2, 681–693; P. Ghosh, G. Han, M. De, C. K. Kim and V. M. Rotello, Adv. Drug Delivery Rev., 2008, 60, 1307–1315; S. Lal, S. E. Clare and N. J. Halas, Acc. Chem. Res., 2008, 41, 1842–1851; D. A. Giljohann, D. S. Seferos, W. L. Daniel, M. D. Massich, P. C. Patel and C. A. Mirkin, Angew. Chem., Int. Ed., 2010, 49, 3280–3294). Not only can they be used as targeted contrast agents for photothermal cancer therapy, they can serve as scaffolds for increasingly potent cancer drug delivery, as transfection agents for selective gene therapy, and as intrinsic antineoplastic agents. This tutorial review will highlight some of the many forms and recent applications of these gold nanoparticle conjugates by our lab and others, as well as their rational design and physiologic interactions.

Silver nanoparticles (AgNPs) are increasingly being used as antimicrobial agents and drug carriers in biomedical fields. However, toxicological information on their effects on red blood cells (RBCs) and the mechanisms involved remain sparse. In this article, we examined the size dependent nanotoxicity of AgNPs using three different characteristic sizes of 15 nm (AgNPs15), 50 nm (AgNPs50), and 100 nm (AgNPs100) against fish RBCs. Optical microscopy and transmission electron microscopy observations showed that AgNPs exhibited a size effect on their adsorption and uptake by RBCs. The middle sized AgNPs50, compared with the smaller or bigger ones, showed the highest level of adsorption and uptake by the RBCs, suggesting an optimal size of ∼50 nm for passive uptake by RBCs. The toxic effects determined based on the hemolysis, membrane injury, lipid peroxidation, and antioxidant enzyme production were fairly size and dose dependent. In particular, the smallest sized AgNPs15 displayed a greater ability to induce hemolysis and membrane damage than AgNPs50 and AgNPs100. Such cytotoxicity induced by AgNPs should be attributed to the direct interaction of the nanoparticle with the RBCs, resulting in the production of oxidative stress, membrane injury, and subsequently hemolysis. Overall, the results suggest that particle size is a critical factor influencing the interaction between AgNPs and the RBCs.

Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.

Drug delivery systems (DDSs) offer efficient and localized drug transportation as well as reduce associated side effects. In order to better understand DDSs, precise observation of drug release and delivery is required. Here, we present a strategy, plasmonic-tunable Raman/fluorescence imaging spectroscopy, to track the release and delivery of an anticancer drug (doxorubicin) from gold nanoparticle carriers in real time at a single living cell level. A pH-responsive drug release profile was attained through the conjugation of doxorubicin (DOX) to the nanoparticle surface via a pH-sensitive hydrazone linkage. When DOX is bound to the surface of the gold nanoparticle, its surface-enhanced Raman spectrum can be seen, but its fluorescence is quenched. When released, due to the lysosomes' acidic pH, its Raman enhancement is greatly reduced, changing the acquired Raman spectrum and in turn allowing for the visualization of its fluorescence signal. The plasmonic-tunable Raman/fluorescence properties enabled the tracking of the DOX release and delivery process from the gold nanoparticle surface to the lysosomes of single living cells under the acidic pH change of their microenvironments. This technique offers great potential to follow the molecular mechanisms of drug delivery and release in living cells, as well as the cellular response to drug action.

A unique approach using the large photoacoustic effect of single-walled carbon nanotubes (SWNTs) for targeting and selective destruction of cancer cells is demonstrated. SWNTs exhibit a large photoacoustic effect in suspension under the irradiation of a 1064-nm Q-switched millisecond pulsed laser and trigger a firecracker-like explosion at the nanoscale. By using such an explosion, a photoacoustic agent is developed by functionalizing the SWNTs with folate acid (FA) that can selectively bind to cancer cells overexpressing folate receptor on the surface of the cell membrane and kill them through SWNT explosion inside the cells under the excitation of millisecond pulsed laser. The uptake pathway of folate-conjugated SWNTs into cancer cells is investigated via fluorescence imaging and it is found that the FA-SWNTs can enter into cancer cells selectively with a high targeting capability of 17-28. Under the treatment of 1064-nm millisecond pulsed laser, 85% of cancer cells with SWNT uptake die within 20 s, while 90% of the normal cells remain alive due to the lack of SWNTs inside cells. Temperature changes during laser treatment are monitored and no temperature increases of more than +/- 3 degrees C are observed. With this approach, the laser power used for cancer killing is reduced 150-1500 times and the therapy efficiency is improved. The death mechanism of cancer cells caused by the photoacoustic explosion of SWNTs is also studied and discussed in detail. These discoveries provide a new way to use the photoacoustic properties of SWNTs for therapeutic applications.

Due to their strong enhancement of scattered light, plasmonic nanoparticles have been utilized for various biological and medical applications. Here, we describe a new technique, Targeted Plasmonic-Enhanced Single-Cell Rayleigh/Raman Spectroscopy, to monitor the molecular changes of any cell-component, such as the nucleus, during the different phases of its full cell cycle by simultaneously recording its Rayleigh images and Raman vibration spectra in real-time. The analysis of the observed Raman DNA and protein peaks allowed the different phases of the cell cycle to be identified. This technique could be used for disease diagnostics and potentially improve our understanding of the molecular mechanisms of cellular functions such as division, death, signaling, and drug action.

Plasmonic nanoparticles (NPs) have become a useful platform in the biomedical field due to their potential use in disease diagnosis and treatment. Recently, it has been reported that plasmonic NPs conjugated to nuclear-targeting peptides cause DNA damage and apoptotic populations in cancer cells. In the present work, we utilized the plasmonic scattering property and the ability of nuclear-targeted silver nanoparticles (NLS/RGD-AgNPs) to induce programmed cell death in order to image in real-time the behavior of human oral squamous carcinoma (HSC-3) cell communities during and after the induction of apoptosis. Plasmonic live-cell imaging (movie) revealed that HSC-3 cells behave as non-professional phagocytes. The induction of apoptosis in some cells led to the attraction and their subsequent engulfment by the neighboring cells. Attraction to apoptotic cells resulted in clustering of cellular community. The live-cell imaging movies also revealed that as the initial concentration of NLS/RGD-AgNPs increases, the rate of self-killing increases and the degree of attraction and clustering decreases. These results are discussed in terms of the proposed mechanism of cells undergoing programmed cell death.