Vibrational Spectroscopy as a Tool for Studying Drug-Cell Interaction: Could High Throughput Vibrational Spectroscopic Screening Improve Drug Development

Vibrational spectroscopy is currently widely explored as a tool in biomedical applications. An area at the forefront of this field is the use of vibrational spectroscopy for disease diagnosis, ultimately aiming towards spectral pathology. However, while this field shows promising results, moving this technique into the clinic faces the challenges of widespread clinical trials and legislative approval. While spectral pathology has received a lot of attention, there are many other biomedical applications of vibrational spectroscopy, which could potentially be translated to applications with greater ease. A particularly promising application is the use of vibrational spectroscopic techniques to study the interaction of drugs with cells. Many studies have demonstrated the ability to detect biochemical changes in cells in response to drug application, using both infrared and Raman spectroscopy. This has shown potential for use in high throughput screening (HTS) applications, for screening of efficacy and mode of action of potential drug candidates, to speed up the drug discovery process. HTS is still a relatively new and growing area of research and, therefore, there is more potential for new techniques to move into and shape this field. Vibrational spectroscopic techniques come with many benefits over the techniques used currently in HTS, primarily based on fluorescence assays to detect specific binding interactions or phenotypes. They are label free, and an infrared or Raman spectrum provides a wealth of biochemical information, and therefore could reveal not only information about a specific interaction, but about how the overall biochemistry of a cell changes in response to application of a drug candidate. Therefore, drug mode of action could be elucidated. This review will investigate the potential for vibrational spectroscopy, particularly FTIR and Raman spectroscopy, to benefit the field of HTS and improve the drug development process. In addition to FTIR and Raman spectroscopy, surface enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS) and stimulated Raman spectroscopy (SRS), will be investigated as an alternative tool in the HTS process

Raman spectroscopy investigation of biochemical changes in tumour spheroids with ageing and after treatment with staurosporine

There has been increasing use of in vitro cell culture models that more realistically replicate the three dimensional (3D) environment found in vivo. Multicellular tumour spheroids (MTS) using cell lines or patient-derived organoids have become an important in vitro drug development tool, where cells are grown in a 3D 'sphere' that exhibits many of the characteristics found in vivo. Significantly, MTS develop gradients in nutrients and oxygen, commonly found in tumours, that contribute to therapy resistance. While MTS show promise as a more realistic in vitro culture model, there is a massive need to improve imaging technologies for assessing biochemical characteristics and drug response in such models to maximize their translation into useful applications such as high throughput screening (HTS). In this study we investigate the potential for Raman spectroscopy to unveil biochemical information in MTS and have investigated how spheroid age influences drug response, shedding light on increased therapy resistance in developing tumours. The wealth of molecular level information delivered by Raman spectroscopy in a noninvasive manner, could aid translation of these 3D models into HTS applications.PostprintPeer reviewe

Bioanalytical measurements enabled by surface-enhanced Raman scattering (SERS) probes

Since its discovery in 1974, surface-enhanced Raman scattering (SERS) has gained momentum as an important tool in analytical chemistry. SERS is used widely for analysis of biological samples, ranging from in vitro cell culture models, ex vivo tissue and blood samples, and direct in vivo application. New insights have been gained into biochemistry, with an emphasis on biomolecule detection, from small molecules such as glucose and amino acids to larger biomolecules such as DNA, proteins, and lipids. These measurements have increased our understanding of biological systems, and significantly, they have improved diagnostic capabilities. SERS probes display unique advantages in their detection sensitivity and multiplexing capability. We highlight key considerations that are required when performing bioanalytical SERS measurements, including sample preparation, probe selection, instrumental configuration, and data analysis. Some of the key bioanalytical measurements enabled by SERS probes with application to in vitro, ex vivo, and in vivo biological environments are discussed

Surface-enhanced Raman spectroscopy for in vivo biosensing

Surface-enhanced Raman scattering (SERS) is of interest for biomedical analysis and imaging because of its sensitivity, specificity and multiplexing capabilities. The successful application of SERS for in vivo biosensing requires probes to be biocompatible and procedures to be minimally invasive, challenges that have respectively been met by developing new nanoprobes and instrumentation. This Review presents recent developments in these areas, describing case studies in which sensors have been implemented, as well as outlining shortcomings that must be addressed before SERS sees clinical use

Ratiometric analysis using Raman spectroscopy as a powerful predictor of structural properties of fatty acids

Raman spectroscopy has been used extensively for analysis of biological samples in vitro, ex vivo and in vivo. While important progress has been made towards using this analytical technique in clinical applications, there is a limit to how much chemically specific information can be extracted from a spectrum of a biological sample, which consists of multiple overlapping peaks from a large number of species in any particular sample. In an attempt to elucidate more specific information regarding individual biochemical species, as opposed to very broad assignments by species class, we propose a bottom up approach beginning with detailed analysis of pure biochemical components. Here we demonstrate a simple ratiometric approach applied to fatty acids, a subsection of the lipid class, to allow the key structural features, in particular degree of saturation and chain length, to be predicted. This is proposed as a starting point for allowing more chemically and species specific information to be elucidated from the highly multiplexed spectrum of multiple overlapping signals found in a real biological sample. The power of simple ratiometric analysis is also demonstrated by comparing the prediction of degree of unsaturation in food oil samples using ratiometric and multivariate analysis techniques which could be used for food oil authentication

Through tissue imaging of a live breast cancer tumour model using handheld surface enhanced spatially offset Resonance Raman Spectroscopy

In order to improve patient survival and reduce the amount of unnecessary and traumatic biopsies, non-invasive detection of cancerous tumours is of imperative and urgent need. Multicellular tumour spheroids (MTS) can be used as an ex vivo cancer tumour model, to model in vivo nanoparticle (NP) uptake by the enhanced permeability and retention (EPR) effect. Surface enhanced spatially offset Raman spectroscopy (SESORS) combines both surface enhanced Raman spectroscopy (SERS) and spatially offset Raman spectroscopy (SORS) to yield enhanced Raman signals at much greater sub-surface levels. By utilizing a reporter that has an electronic transition in resonance with the laser frequency, surface enhanced resonance Raman scattering (SERRS) yields even greater enhancement in Raman signal. Using a handheld SORS spectrometer with back scattering optics, we demonstrate the detection of live breast cancer 3D multicellular tumour spheroids (MTS) containing SERRS active NPs through 15 mm of porcine tissue. False color 2D heat intensity maps were used to determine tumour model location. In addition, we demonstrate the tracking of SERRS-active NPs through porcine tissue to depths of up to 25 mm. This unprecedented performance is due to the use of red-shifted chalcogenpyrylium-based Raman reporters to demonstrate the novel technique of surface enhanced spatially offset resonance Raman spectroscopy (SESORRS) for the first time. Our results demonstrate a significant step forward in the ability to detect vibrational fingerprints from a tumour model at depth through tissue. Such an approach offers significant promise for the translation of NPs into clinical applications for non-invasive disease diagnostics based on this new chemical principle of measurement

Through barrier detection of ethanol using handheld Raman spectroscopy — Conventional Raman versus spatially offset Raman spectroscopy (SORS)

Spatially offset Raman spectroscopy (SORS) provides chemical analysis at depth even when obscuring barriers such as plastic or tissue are present. As the collection probe is moved further away from the point of laser excitation, scattered photons from deeper layers begin to dominate the acquired spectra, thus giving rise to through barrier detection. Here, we demonstrate the potential of conventional Raman (CR) and SORS for through barrier detection using handheld spectrometers. We report the collection of Raman signals from an ethanol solution through plastic at thicknesses of up to 21 mm using SORS in combination with multivariate analysis. SORS is compared to CR, where we also demonstrate impressive through barrier detection of ethanol at depths up to 9 mm. We also highlight the advantage of applying multivariate analysis for through barrier detection using CR or SORS, particularly when peaks with similar spectral features are present in both the barrier and analyte spectra. In addition, to the best of our knowledge, this is the first report of the assessment of the maximum level of through barrier detection using handheld CR and SORS instruments with a backscattering geometry

A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy

Intracellular pH (pHi) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pHi are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pKa(H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm-1) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pHi in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pHi, complementary to fluorescence microscopy

Targeted SERS nanosensors measure physicochemical gradients and free energy changes in live 3D tumor spheroids.

Use of multicellular tumor spheroids (MTS) to investigate therapies has gained impetus because they have potential to mimic factors including zonation, hypoxia and drug-resistance. However, analysis remains difficult and often destroys 3D integrity. Here we report an optical technique using targeted nanosensors that allows in situ 3D mapping of redox potential gradients whilst retaining MTS morphology and function. The magnitude of the redox potential gradient can be quantified as a free energy difference (ΔG) and used as a measurement of MTS viability. We found that by delivering different doses of radiotherapy to MTS we could correlate loss of ΔG with increasing therapeutic dose. In addition, we found that resistance to drug therapy was indicated by an increase in ΔG. This robust and reproducible technique allows interrogation of an in vitro tumor-model's bioenergetic response to therapy, indicating its potential as a tool for therapy development.Leverhulme Trust (Grant ID: RPG-2012-680), Jamie King Cancer Research FundThis is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C6NR06031

Multiplex imaging of live breast cancer tumour models through tissue using handheld surface enhanced spatially offset resonance Raman spectroscopy (SESORRS)

Through utilizing the depth penetration capabilities of SESORS, multiplexed imaging and classification of three singleplex nanotags and a triplex of nanotags within breast cancer tumour models is reported for the first time through depths of 10 mm using a handheld SORS instrument