The study of the response of cancer cells to chemotherapy drugs is of high importance due to the specificity of some drugs to certain types of cancer and the resistance of some specific cancer types to chemotherapy drugs. Our aim was to develop and apply the label-free and non-destructive Fourier transform infrared (FTIR) method to determine the sensitivity of three different cancer cell-lines to a common anti-cancer drug doxorubicin at different concentrations and to demonstrate that information about the mechanism of resistance to the chemotherapy drug can be extracted from spectral data. HeLa, PC3, and Caco-2 cells were seeded and grown on an attenuated total reflection (ATR) crystal, doxorubicin was applied at the clinically significant concentration of 0.1-20 μM, and spectra of the cells were collected hourly over 20 h. Analysis of the amide bands was correlated with cell viability, which had been cross validated with MTT assays, allowing to determine that the three cell lines had significantly different resistance to doxorubicin. The difference spectra and principal component analysis (PCA) highlighted the subtle chemical changes in the living cells under treatment. Spectral regions assigned to nucleic acids (mainly 1085 cm(-1)) and carbohydrates (mainly 1024 cm(-1)) showed changes that could be related to the mode of action of the drug and the mechanism of resistance of the cell lines to doxorubicin. This is a cost-effective method that does not require bioassay reagents but allows label-free, non-destructive and in situ analysis of chemical changes in live cells, using standard FTIR equipment adapted to ATR measurements.

A number of amino acids (AA) has been investigated as promising hydrotropes to improve the solubility of biopharmaceutics classification system (BCS) class II drugs carbamazepine (CBZ) and indomethacin (IND) via specific complexations in aqueous solution. The aim of this work is to understand the molecular basis of these hydrotropic interactions by investigating the two model drugs combined with 12 amino acids including phenylalanine, tryptophan, isoleucine, proline, valine, glycine, serine, threonine, arginine, lysine, histidine and aspartic acid in water at 25 °C, 30 °C and 45 °C. The amino acids were chosen based on their different side chains (neutral aromatic, aliphatic, polar charged or uncharged) to investigate their hydrotropic performance. A linear solubility curve was observed between indomethacin and mono-neutral hydrophobic amino acids (phenylalanine, tryptophan, isoleucine, proline and valine) well beyond 1:1 molar ratio indicating the interaction is predominantly non-ionic between the drug and the hydrotropes. Interestingly, the aqueous solubility of carbamazepine (a neutral compound) was enhanced by neutral, charged basic or acidic amino acids, confirming the presence of hydrophobic interactions that involve H-bonds, H/π and π/π stacking and the results were confirmed by UV-Vis spectroscopy. A combination of multiple neutral amino acids showed additive hydrotropic effect in indomethacin solubility with up to 7-folds increases. This study demonstrates for the first time the potential of amino acids as hydrotropes to improve aqueous solubility of poorly water-soluble drugs, which is important for pharmaceutical development.

Amino acids have shown promising abilities to form complexes with poorly water-soluble drugs and improve their physicochemical properties for a better dissolution profile through molecular interactions. Salt formation via ionization between acidic drugs and basic amino acids is known as the major contributor to solubility enhancement. However, the mechanism of solubility enhancement due to non-ionic interactions, which is less pH-dependent, remains unclear. The aim of this study is to evaluate non-ionic interactions between a model acidic drug, indomethacin (IND), and basic amino acids, arginine, lysine and histidine, in water. At low concentrations of amino acids, IND–arginine and IND–lysine complexes have shown a linear relationship (A_L-type phase solubility diagram) between IND solubility and amino acid concentration, producing ∼1 : 1 stoichiometry of drug-amino acid complexes as expected due to the strong electrostatic interactions. However, IND–histidine complexes have shown a nonlinear relationship with lower improvement in IND solubility due to the weaker electrostatic interactions when compared to arginine and lysine. Interestingly, the results have also shown that at high arginine concentrations, the linearity was lost between IND solubility and amino acid concentration with a negative diversion from linearity, following the type-A_N phase solubility. This is indicative that the electrostatic interaction is being interrupted by non-electrostatic interactions, as seen with histidine. The IND–lysine complex, on the other hand, showed a complex curvature phase solubility diagram (type B_S) as lysine self-assembles and polymerizes at higher concentrations. The freeze-dried drug–amino acid solids were further characterized using thermal analysis and infrared spectroscopy, with results showing the involvement of weak non-ionic interactions. This study shows that the solubility improvement of an insoluble drug in the presence of basic amino acids was due to both non-ionic and ionic interactions.

Fourier transform infrared spectroscopy (FTIR) has been shown to be a promising tool for identifying the mode of action of drugs. However, most previous studies have focused on the analysis of fixed or dried cells. The measurement of living cells has the advantage of obtaining time series data, and the in situ approach eliminates the need for fixing or drying the cells. In this study, the potential of live-cell FTIR method for the identification of the mode of action of drugs was demonstrated. Four different drugs were tested, with two of the drugs having the same mode of action (tamoxifen and toremifene) and the other two having different modes of action (imatinib and doxorubicin). Live cells were treated in the four drugs at and below the IC₅₀ level (i.e. the concentration of drug required to inhibit the growth of cells by 50%), and the changes to their spectra after the addition of drugs were monitored over a 24-hour period. Principal component analysis (PCA) of the spectral data shows that drugs with different modes of action are well-separated, while the drugs with the same mode of action are grouped together. The results also show that at IC₅₀, the separation appears to be the clearest at 2 hours for imatinib and tamoxifen/toremifene and 6 hours for doxorubicin. However, at 50% of the IC₅₀ drug concentration, the separation appears to be the best at longer incubation time, i.e. 24 hours, for all four drugs. In conclusion, live-cell FTIR has shown to be able to distinguish and group spectral signatures of cells treated with drugs of known modes of action after a relatively short time of exposure. Further collection of live-cell data would enable an algorithm to be developed for the prediction of the modes of action of novel drugs, which can help in the preclinical drug screening process.

FTIR imaging is a label-free, non-destructive method valuably exploited in the study of the biological process in living cells. However, the long wavelength/low spatial resolution and the strong absorbance of water are still key constrains in the application of IR microscopy ex vivo. In this work, a new retrofit approach based on the use of ZnS hemispheres is introduced to significantly improve the spatial resolution on live cell FTIR imaging. By means of two high refractive index domes sandwiching the sample, a lateral resolution close to 2.2 μm at 6 μm wavelength has been achieved, i.e. below the theoretical diffraction limit in air and more than twice the improvement (to ~λ/2.7) from our previous attempt using CaF₂ lenses. The ZnS domes also allowed an extended spectral range to 950 cm⁻¹, in contrast to the cut-off at 1050 cm⁻¹ using CaF₂. In combination with synchrotron radiation source, microFTIR provides an improved signal-to-noise ratio through the circa 12 μm thin layer of medium, thus allowing detailed distribution of lipids, protein and nucleic acid in the surround of the nucleus of single living cells. Endoplasmic reticula were clearly shown based on the lipid ν(CH) and ν(C=O) bands, while the DNA was imaged based on the ν(PO₂⁻) band highlighting the nucleus region. This work has also included a demonstration of drug (doxorubicin) in cell measurement to highlight the potential of this approach. Graphical abstract