UV Resonance Raman Study of Apoptosis, Platinum-Based Drugs, and Human Cell Lines.

As a noninvasive molecular analysis technique, ultraviolet resonance Raman (UVRR) spectroscopy represents a label-free method suitable for characterizing biomolecules. Using UVRR spectroscopy, we collected spectral fingerprints of UV absorbing cellular components, including proteins, nucleic acids, and unsaturated lipids. This knowledge was used to guide the assignment of spectra derived from intact human cell lines (i. e., HSC-3 and HaCaT) and from the apoptotic events induced by cisplatin. Notably, a jet-flow system was employed to generate flowing cell suspensions during UVRR measurements, minimizing UV-induced damage. A spectral marker is established based on the ratio of Raman intensities at 1488 and 1655 cm-1 ; this ratio correlates to the level of cell death due to apoptosis. Collectively, this work demonstrates that UVRR spectroscopy is a sensitive and informative probe of cellular physiology and molecular composition. The molecular insight obtained from UVRR measurements can be used to improve understanding of therapeutic treatment and to guide drug development and the choice of therapeutic agents.

An Ultraviolet Resonance Raman Spectroscopic Study of Cisplatin and Transplatin Interactions with Genomic DNA.

Ultraviolet resonance Raman (UVRR) spectroscopy is a label-free method to define biomacromolecular interactions with anticancer compounds. Using UVRR, we describe the binding interactions of two Pt(II) compounds, cisplatin (cis-diamminedichloroplatinum(II)) and its isomer, transplatin, with nucleotides and genomic DNA. Cisplatin binds to DNA and other cellular components and triggers apoptosis, whereas transplatin is clinically ineffective. Here, a 244 nm UVRR study shows that purine UVRR bands are altered in frequency and intensity when mononucleotides are treated with cisplatin. This result is consistent with previous suggestions that purine N7 provides the cisplatin-binding site. The addition of cisplatin to DNA also causes changes in the UVRR spectrum, consistent with binding of platinum to purine N7 and disruption of hydrogen-bonding interactions between base pairs. Equally important is that transplatin treatment of DNA generates similar UVRR spectral changes, when compared to cisplatin-treated samples. Kinetic analysis, performed by monitoring decreases of the 1492 cm-1 band, reveals biphasic kinetics and is consistent with a two-step binding mechanism for both platinum compounds. For cisplatin-DNA, the rate constants (6.8 × 10-5 and 6.5 × 10-6 s-1) are assigned to the formation of monofunctional adducts and to bifunctional, intrastrand cross-linking, respectively. In transplatin-DNA, there is a 3.4-fold decrease in the rate constant of the slow phase, compared with the cisplatin samples. This change is attributed to generation of interstrand, rather than intrastrand, adducts. This longer reaction time may result in increased competition in the cellular environment and account, at least in part, for the lower pharmacological efficacy of transplatin.

Platinum-Coated Gold Nanorods: Efficient Reactive Oxygen Scavengers That Prevent Oxidative Damage toward Healthy, Untreated Cells during Plasmonic Photothermal Therapy.

As a minimally invasive therapeutic strategy, gold nanorod (AuNR)-based plasmonic photothermal therapy (PPT) has shown significant promise for the selective ablation of cancer cells. However, the heat stress experienced by cells during the PPT treatment produces significant amounts of reactive oxygen species (ROS), which could harm healthy, untreated tissue near the point of care by inducing irreversible damage to DNA, lipids, and proteins, potentially causing cellular dysfunction or mutation. In this study, we utilized biocompatible Pt-coated AuNRs (PtAuNRs) with different platinum shell thicknesses as an alternative to AuNRs often used for the treatment. We show that the PtAuNRs maintain the efficacy of traditional AuNRs for inducing cell death while scavenging the ROS formed as a byproduct during PPT treatment, thereby protecting healthy, untreated cells from indirect death resulting from ROS formation. The synergistic effect of PtAuNRs in effectively killing cancer cells through hyperthermia with the simultaneous removal of heat stress induced ROS during PPT was validated in vitro using cell viability and fluorescence assays. Our results suggest that the high photothermal efficiency and ROS-scavenging activity of PtAuNRs makes them ideal candidates to improve the therapeutic efficacy of PPT treatment while reducing the risk of undesired side effects due to heat-stress-induced ROS formation.

A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles.

Plasmonic nanoparticles are increasingly utilized in biomedical applications including imaging, diagnostics, drug delivery, and plasmonic photothermal therapy (PPT). PPT involves the rapid conversion of light into heat by plasmonic nanoparticles targeted to a tumor, causing hyperthermia-induced cell death. These nanoparticles can be passively targeted utilizing the enhanced permeability and retention effect, or actively targeted using proteins, peptides, or other small molecules. Here, we report the use of actively targeted spherical gold nanoparticles (AuNPs), both to induce PPT cell death, and to monitor the associated molecular changes through time-dependent surface enhanced Raman spectroscopy within a single cell. We monitored these changes in real-time and found that heat generated from the aggregated nanoparticles absorbing near-infrared (NIR) laser light of sufficient powers caused modifications in the protein and lipid structures within the cell and ultimately led to cell death. The same molecular changes were observed using different nanoparticle sizes and laser intensities, indicating the consistency of the molecular changes throughout PPT-induced cell death from actively targeted AuNPs. We also confirmed these observations by comparing them to reference spectra obtained by cell death induced by oven heating at 100 °C. The ability to monitor PPT-induced cell death in real-time will help understand the changes on a molecular level and offers us a basis to understand the molecular mechanisms involved in photothermal cancer cell death.

Determining Drug Efficacy Using Plasmonically Enhanced Imaging of the Morphological Changes of Cells upon Death.

Recently, we utilized the optical properties of gold nanoparticles (AuNPs) for plasmonically enhanced Rayleigh scattering imaging spectroscopy (PERSIS), a new technique that enabled the direct observation of AuNP localization. In this study, we employ PERSIS by using AuNPs as light-scattering probes to compare the relative efficacy of three chemotherapeutic drugs on human oral squamous carcinoma cells. Although the drugs induced apoptotic cell death through differing mechanisms, morphological changes including cell membrane blebbing and shrinkage, accompanied by an increase in white light scattering, were visually evident. By utilizing the AuNPs to increase the cells' inherent Rayleigh scattering, we have obtained the time profile of cell death from the anticancer drugs using a single sample of cells in real time, using inexpensive equipment available in any lab. From this time profile, we calculated cell death enhancement factors to compare the relative efficacies of the different drugs using our technique, which corresponded to those calculated from the commonly used XTT cell viability assay. Although this technique does not impart molecular insights into cell death, the ability to quantitatively correlate cell death to morphological changes suggests the potential use of this technique for the rapid screening of drug analogues to determine the most effective structure against a disease or cell line.

Biological Targeting of Plasmonic Nanoparticles Improves Cellular Imaging via the Enhanced Scattering in the Aggregates Formed.

Gold nanoparticles (AuNPs) demonstrate great promise in biomedical applications due to their plasmonically enhanced imaging properties. When in close proximity, AuNPs plasmonic fields couple together, increasing their scattering cross-section due to the formation of hot spots, improving their imaging utility. In the present study, we modified the AuNPs surface with different peptides to target the nucleus and/or the cell as a whole, resulting in similar cellular uptake but different scattering intensities. Nuclear-targeted AuNPs showed the greatest scattering due to the formation of denser nanoparticle clusters (i.e., increased localization). We also obtained a dynamic profile of AuNP localization in living cells, indicating that nuclear localization is directly related to the number of nuclear-targeting peptides on the AuNP surface. Increased localization led to increased plasmonic field coupling, resulting in significantly higher scattering intensity. Thus, biochemical targeting of plasmonic nanoparticles to subcellular components is expected to lead to more resolved imaging of cellular processes.