Combining Pharmacokinetics and Vibrational Spectroscopy: MCR-ALS Hard-and-Soft Modelling of Drug Uptake In Vitro Using Tailored Kinetic Constraints.

Raman microspectroscopy is a label-free technique which is very suited for the investigation of pharmacokinetics of cellular uptake, mechanisms of interaction, and efficacies of drugs in vitro. However, the complexity of the spectra makes the identification of spectral patterns associated with the drug and subsequent cellular responses difficult. Indeed, multivariate methods that relate spectral features to the inoculation time do not normally take into account the kinetics involved, and important theoretical information which could assist in the elucidation of the relevant spectral signatures is excluded. Here, we propose the integration of kinetic equations in the modelling of drug uptake and subsequent cellular responses using Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and tailored kinetic constraints, based on a system of ordinary differential equations. Advantages of and challenges to the methodology were evaluated using simulated Raman spectral data sets and real Raman spectra acquired from A549 and Calu-1 human lung cells inoculated with doxorubicin, in vitro. The results suggest a dependency of the outcome on the system of equations used, and the importance of the temporal resolution of the data set to enable the use of complex equations. Nevertheless, the use of tailored kinetic constraints during MCR-ALS allowed a more comprehensive modelling of the system, enabling the elucidation of not only the time-dependent concentration profiles and spectral features of the drug binding and cellular responses, but also an accurate computation of the kinetic constants.

Data mining Raman microspectroscopic responses of cells to drugs in vitro using multivariate curve resolution-alternating least squares.

Raman microspectroscopy is gaining popularity for the analysis of time-dependent biological processes such as drug uptake and cellular response. It is a label-free technique which acquires signals from a large variety of components, including cell biomolecules and exogenous compounds such as drugs and nanoparticles, and is commonly employed for in vitro analysis of cells and cell populations with no labelling or staining required. By monitoring the changes to the Raman spectra of the cell as a result of a perturbing agent (e.g. exposure to a drug or toxic agent), one can study the associated changes in cell biochemistry involved in both, the disruption and the subsequent cellular response. The main challenge is that the Raman spectra should be data mined in order to extract the information corresponding to the different actors involved on the process. Here, we study the application of multivariate curve resolution-alternating least squares (MCR-ALS) for extracting kinetic and biochemical information of time-dependent cellular processes. The technique allows the elucidation of the concentration profiles as well as the pure spectra of the components involved. Initially, we used Ordinary Differential Equations (ODE) to simulate drug uptake and 2 responses, which were employed to simulate perturbations to experimental control spectra, creating a dataset containing 36 simulated Raman spectra. Four different scenarios governing the drug exposure-response were evaluated: an undetectable disruption (e.g. radiation), a detectable disruption (e.g. a drug) and disruption with a signal significantly larger than the biological changes induced (e.g. a resonant drug), as well as simultaneous and asynchronous responses. Subsequently, data acquired from the exposure of a pulmonary adenocarcinoma cell line (A549) to Doxorubicin was analysed. The results indicate that MCR-ALS can independently identify and isolate both the spectra of the drug and the cell responses under the different scenarios. The predicted concentrations map out the drug uptake and cellular response curves. The technique shows great potential to investigate non-linear kinetics and modes of action. Advantages and limitations of the technique are discussed, providing guidelines for future analysis strategies.

Two-dimensional correlation analysis of Raman microspectroscopy of subcellular interactions of drugs in vitro.

Two-dimensional (2D) correlation analysis is explored to data mine the time evolution of the characteristic Raman microspectroscopic signatures of the subcellular responses of the nucleoli of human lung cancer cells to the uptake of doxorubicin. A simulated dataset of experimental control spectra, perturbed with systematically time-dependent spectral changes, constituted by a short-term response which represents the initial binding of the drug in the nucleolus, followed by a longer term response of the organelle metabolism, is used to validate the analysis protocol. Applying 2D correlation analysis, the in phase, synchronous correlation coefficients are seen to contain contributions of both response profiles, whereas they can be independently extracted from the out of phase, asynchronous correlation coefficients. The methodology is applied to experimental data of the uptake of doxorubicin in human lung cell lines to differentiate the signatures of chemical binding and subsequent cellular response.

Advancing Raman microspectroscopy for cellular and subcellular analysis: towards in vitro high-content spectralomic analysis.

In the confocal mode, Raman microspectroscopy can profile the biochemical content of biological cells at a subcellular level, and any changes to it by exogenous agents, such as therapeutic drugs or toxicants. As an exploration of the potential of the technique as a high-content, label-free analysis technique, this report reviews work to monitor the spectroscopic signatures associated with the uptake and response pathways of commercial chemotherapeutic agents and polymeric nanoparticles by human lung cells. It is demonstrated that the signatures are reproducible and characteristic of the cellular event, and can be used, for example, to identify the mode of action of the agent as well as the subsequent cell death pathway, and even mechanisms of cellular resistance. Data mining approaches are discussed and a spectralomics approach is proposed.

Doxorubicin kinetics and effects on lung cancer cell lines using in vitro Raman micro-spectroscopy: binding signatures, drug resistance and DNA repair.

Raman micro-spectroscopy is a non-invasive analytical tool, whose potential in cellular analysis and monitoring drug mechanisms of action has already been demonstrated, and which can potentially be used in pre-clinical and clinical applications for the prediction of chemotherapeutic efficacy. To further investigate such potential clinical application, it is important to demonstrate its capability to differentiate drug mechanisms of action and cellular resistances. Using the example of Doxorubicin (DOX), in this study, it was used to probe the cellular uptake, signatures of chemical binding and subsequent cellular responses, of the chemotherapeutic drug in two lung cancer cell lines, A549 and Calu-1. Multivariate statistical analysis was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Biomarkers related to DNA damage and repair, and mechanisms leading to apoptosis were also measured and correlated to Raman spectral profiles. Results confirm the potential of Raman spectroscopic profiling to elucidate both drug kinetics and pharmacodynamics and differentiate cellular drug resistance associated with different subcellular accumulation rates and subsequent cellular response to DNA damage, pointing towards a better understanding of drug resistance for personalised targeted treatment.

In vitro label-free screening of chemotherapeutic drugs using Raman microspectroscopy: Towards a new paradigm of spectralomics.

This overview groups some of the recent studies highlighting the potential application of Raman microspectroscopy as an analytical technique in preclinical development to predict drug mechanism of action and in clinical application as a companion diagnostic and in personalised therapy due to its capacity to predict cellular resistance and therefore to optimise chemotherapeutic treatment efficacy. Notably, the anthracyclines, doxorubicin and actinomycin D, elicit similar spectroscopic signatures of subcellular interaction characteristic of the mode of action of intercalation. Although cisplatin and vincristine show markedly different signatures, at low exposure doses, their signatures at higher doses show marked similarities to those elicited by the intercalating anthracyclines, confirming that anticancer agents can have different modes of action with different spectroscopic signatures, depending on the dose. The study demonstrates that Raman microspectroscopy can elucidate subcellular transport and accumulation pathways of chemotherapeutic agents, characterise and fingerprint their mode of action, and potentially identify cell-resistant strains. The consistency of the spectroscopic signatures for drugs of similar modes of action, in different cell lines, suggests that this fingerprint can be considered a "spectralome" of the drug-cell interaction suggesting a new paradigm of representing spectroscopic responses.

An in vitro study of the interaction of the chemotherapeutic drug Actinomycin D with lung cancer cell lines using Raman micro-spectroscopy.

The applications of Raman microspectroscopy have been extended in recent years into the field of clinical medicine, and specifically in cancer research, as a non-invasive diagnostic method in vivo and ex vivo, and the field of pharmaceutical development as a label-free predictive technique for new drug mechanisms of action in vitro. To further illustrate its potential for such applications, it is important to establish its capability to fingerprint drug mechanisms of action and different cellular reactions. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 48 and 72 hours exposure of lung cancer cell lines, A549 and Calu-1, after exposure to Actinomycin D (ACT) and Raman micro-spectroscopy was used to track its mechanism of action at subcellular level and subsequent cellular responses. Multivariate data analysis was used to elucidate the spectroscopic signatures associated with ACT chemical binding and cellular resistances. Results show that the ACT uptake and mechanism of action are similar in the 2 cell lines, while A549 cells exhibits spectral signatures of resistance to apoptosis related to its higher chemoresistance to the anticancer drug ACT. The observations are discussed in comparison to previous studies of the similar anthracyclic chemotherapeutic agent Doxorubicin. A, Preprocessed Raman spectrum of ACT stock solution dissolved in sterile water and mean spectrum with SD of (B) nucleolus, (C) nucleus and (D) cytoplasm of A549 cell lines after 48 hours exposure to the corresponding IC50 .

Differentiating responses of lung cancer cell lines to Doxorubicin exposure: in vitro Raman micro spectroscopy, oxidative stress and bcl-2 protein expression.

The potential of Raman micro spectroscopy as an in vitro, non-invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu-1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl-2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu-1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action. PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu-1 with corresponding loadings of PC1 and PC2.

Monitoring doxorubicin cellular uptake and trafficking using in vitro Raman microspectroscopy: short and long time exposure effects on lung cancer cell lines.

Raman microspectroscopy is a non-invasive, in vitro analytical tool which is being increasingly explored for its potential in clinical applications and monitoring the uptake, mechanism of action and cellular interaction at a molecular level of chemotherapeutic drugs, ultimately as a potential label-free preclinical screening and companion diagnostic tool. In this study, doxorubicin (DOX), a "gold standard" chemotherapeutic drug, is employed as a model in the in vitro lung cancer cell line A549 in order to demonstrate the potential of Raman microspectroscopy to screen and identify spectroscopic markers of its trafficking and mechanism of action. Confocal laser scanning microscopy (CLSM) was used in parallel to illustrate the uptake and subcellular localisation, and cytotoxicity assays were employed to establish the toxicity profiles for early and late exposure times of A549 to DOX. Multivariate statistical analysis, consisting of principal components analysis (PCA), partial least squares regression (PLSR) and independent component analysis (ICA), was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Raman spectroscopic profiling illustrates both drug kinetics and its pharmacodynamics in the cell and associated biochemical changes, demonstrating that DOX is mainly localised in the nuclear area, saturating the nucleolus first, within ~6 h of exposure, before the surrounding nuclear areas after ~12 h, and only accumulates in the cytoplasm after 48 h. PLSR over varying time intervals enables identification of DOX-DNA binding at early stages of exposure (0-12 h), while regression over longer time periods (24-72 h) reveals spectroscopic signatures associated with the metabolic cellular response. Graphical Abstract Subcellular uptake of doxorubicin, and changes in biomolecular signatures in the nucleolus, as monitored by Raman spectroscopy.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking.

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488nm and 785nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles.

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473 ± 41 nm), polydispersity index (0.3 ± 0.2), zeta potential (34 ± 4 mV), drug content (76 ± 7 µM) and encapsulation efficiency (95 ± 1 %). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX-loaded nanoparticles within the cells and the subsequent dose- and time-dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells.

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling.

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non-continuous matrix, providing a better understanding of the limited barrier function.