Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy

Abstract

Quantitative drug imaging in live cells is a major challenge in drug discovery and development. Many drug screening techniques are performed in solution, and therefore do not consider the impact of the complex cellular environment in their result. As such, important features of drug-cell interactions may be overlooked. In this study, Raman microscopy is used as a powerful technique for semi-quantitative imaging of Strathclyde-minor groove binders (S-MGBs) in mammalian cells under biocompatible imaging conditions. Raman imaging determined the influence of the tail group of two novel minor groove binders (S-MGB-528 and S-MGB-529) in mammalian cell models. These novel S-MGBs contained alkyne moieties which enabled analysis in the cell-silent region of the Raman spectrum. The intracellular uptake concentration, distribution and mechanism were evaluated as a function of the pK _a of the tail group, morpholine and amidine, for S-MGB-528 and S-MGB-529, respectively. Although S-MGB-529 had a higher binding affinity to the minor groove of DNA in solution-phase measurements, the Raman imaging data indicated that S-MGB-528 showed a greater degree of intracellular accumulation. Furthermore, using high resolution stimulated Raman scattering (SRS) microscopy, the initial localisation of S-MGB-528 was shown to be in the nucleus before accumulation in the lysosome, which was demonstrated using a multimodal imaging approach. This study highlights the potential of Raman spectroscopy for semi-quantitative drug imaging studies and highlights the importance of imaging techniques to investigate drug-cell interactions, to better inform the drug design process.

Fig. 1. Analysis of S-MGBs using Raman spectroscopy. (A) General design and chemical structures of truncated S-MGBs, S-MGB-528 (morpholine tail group) and S-MGB-529 (amidine tail group). (B) The Raman scattering of the alkyne-tagged S-MGBs in a solid state. (C) Thermal melt analysis of S-MGB-528 or S-MGB-529 bound to gDNA. Exemplar melt curve from one experimental repeat, which has been fitted with a Boltzmann distribution and inset table including average thermal melts from at least 2 experimental repeats. The errors of the average thermal melts are within ±0.5 °C.

Fig. 2. Ratiometric Raman analysis of S-MGBs in mammalian cells. Representative Raman maps of live (A and C) and fixed (B and D) PNT2 and HeLa cells after the treatment with either S-MGB-528 or S-MGB-529 (10 μM, 24 h, 37 °C, 5% CO₂). The chemical contrast is shown by the ratio 2850 cm⁻¹/(2850 + 2930) cm⁻¹ indicative of CH₂ and CH₃ respectively. The drug localisation is shown through the normalised alkyne Raman intensity (i.e. 2105–2930 cm⁻¹). Quantification of the normalised Raman scattering intensity of the alkyne tag (~2105 cm⁻¹) relative to the symmetric C–H stretching (2930 cm⁻¹) in live and fixed cells, (E) PNT2 and (F) HeLa cells, respectively. Raman data were collected using 532 nm, 10 mW laser power, 0.5 s integration time, 60× immersive objective lens, 1 μm step size in x and y, 1 accumulation. Data representative of the mean ± SD. A minimum of three replicate images were acquired for each condition.

Fig. 3. Investigating the temperature dependence of the S-MGBs uptake. (A) Representative Raman maps of live HeLa cells after the treatment with S-MGB-528 and S-MGB-529 (10/20 μM, 4 h, 4 °C/26 °C/37 °C, in presence of HEPES or bicarbonate buffer). The chemical contrast is shown by the ratio 2850 cm⁻¹/(2850 + 2930) cm⁻¹ indicative of CH₂ and CH₃ respectively. The drug localisation is shown through the normalised alkyne Raman intensity (i.e. 2104/2930 cm⁻¹). (B) Normalised alkyne intensity of S-MGB-528 and S-MGB-529 in HeLa and PNT2 cells. The Raman data were collected using 532 nm, 0.5 s integration time, 10 mW laser power, 60× objective immersive lens, step size 1 μm in x and y, 1 accumulation. A minimum of three replicate images were acquired for each condition. Data represents mean ± SD (****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns p > 0.05, Student's t test).

Fig. 4. Investigating the kinetics of the uptake of the S-MGBs into live HeLa cells. Alkyne scattering of S-MGB-528 and S-MGB-529 in live HeLa cells for the whole cells (A and B) and cytoplasm and nuclear regions (C and D). HeLa cells were incubated with increasing concentration of either S-MGB-528 or S-MGB-529 (240 min). Following drug treatment and imaging, the alkyne Raman intensities for each concentration, and the whole cell, nucleus and cytoplasm, were measured and normalised to the symmetric stretching of the C–H at 2930 cm⁻¹, then, the normalised Raman intensities were averaged and with the relative standard deviations divided by the time (240 min) to generate the kinetics of drug uptake associated with S-MGB-528 and S-MGB-529 in HeLa cells at the cellular and subcellular level. Lastly, the kinetics associated with the uptake of S-MGB-528 and S-MGB-529 and the relative standard deviation were plotted against the drug concentration fitting the Michaelis–Menten model whilst the kinetics of drug accumulation at the cytoplasmic and nuclear level were plotted against the drug concentration. Raman data were collected using 532 nm, 0.5 s integration time, 10 mW laser power, 60× immersive objective lens, step size 1 μm in x and y, 1 accumulation. A minimum of three replicate images were acquired for each condition. Data represents mean ± SD.

Fig. 5. Investigating S-MGBs uptake using stimulated Raman scattering. (A) Time-dependent uptake of S-MGB-528. Representative SRS images of live HeLa cells after the treatment with S-MGB-528 (10 μM) for either 30 min or 6 h. The samples were imaged using SRS microscopy at the following: 2930 cm⁻¹ (proteins), 2850 cm⁻¹ (lipids), 2106 cm⁻¹ (alkyne) and 2000 cm⁻¹ (off-resonance). An image achieved by merging the channels of proteins and alkyne is also shown, highlighting the time-dependent subcellular localisation of the S-MGB-528. Lookup table: 0–3000 a.u. (Lipids and proteins), 0–1500 a.u. (on–off resonance) (B) The progressive lysosomal accumulation of S-MGB-528 in live HeLa cells using SRS microscopy. Representative SRS images of live HeLa cells after the treatment with S-MGB-528 (10 μM) for either 0.5, 2 and 4 h. The samples were imaged using SRS microscopy at the following: 2930 cm⁻¹ (proteins), 2850 cm⁻¹ (lipids) 2106 cm⁻¹ (alkyne) and 2000 cm⁻¹ (off-resonance). The time-lapse study highlights the progressive time-dependent accumulation of S-MGB 528 within the cytoplasm. Lookup table: 0–3000 a.u. (proteins and lipids), 0–1500 a.u. (on–off resonance). Scale bar size: 10 μm.