106 107 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Soft CondensedMatter Group Beamline B22 Viewing drug action in a cell by infrared nanoprobe Related publication: Chan K. L. A., Lekkas I., FrogleyM. D., Cinque G., Altharawi A., Bello G. &Dailey L. A. Synchrotron Photothermal Infrared Nanospectroscopy of Drug-Induced Phospholipidosis inMacrophages. Anal. Chem. 92 , 8097–8107 (2020). DOI: 10.1021/acs.analchem.9b05759 Publication keywords: RE-AFM-IR; Synchrotron IR; Photothermal nanospectroscopy; NanoFTIR; PTIR D etecting metabolic changes inside a biological cell as a result of drug treatment is fundamental in pharmaceutical development. Currently, most techniques require the addition of dye (staining) to reveal some chemical changes, which may alter the natural process and potentially produce misleading results. Infrared (IR) microspectroscopy can probe chemical changes in biological matter without the use of dye or chemical label, but the images produced optically so far are too coarse to see clearly inside a single cell. Macrophages are a type of white blood cell that defend against infection. They producemany tiny fatty droplets when exposed to drugs, but the exact reason for this response was not clear. Knowing the chemical composition of these droplets will help scientists better understand the process and could help develop better medicines. A team of researchers investigated a new Synchrotron IR spectroscopic imaging method, developed on Diamond Light Source’s Multimode InfraRed Imaging AndMicrospectroscopy (MIRIAM) beamline (B22), to probe themolecular changes insidemacrophages. The results showed that IR nanospectroscopy can measure a drug’s effect inside a single mammalian cell by clearly identifying the chemical changes within the tiny fatty droplets before and after the application of the drug. Researchers will be able to use this powerful newmolecular imaging tool to understand the responses of macrophages exposed to different drugs. This will help to identify new drug candidates that are more suitable for further development versus those that are for example ineffective or toxic, improving the chance of success indeliveringbettermedicines in the near future. In the development of inhaled medicines, poor understanding of alveolar macrophage cellular responses to new drug compounds have been identified as a key barrier 1 . Phospholipidosis or adaptive responses has been associated with the appearance of ‘foamy macrophages’ but the exact mechanism remains poorly understood. Xenobiotics (e.g. amiodarone, poorly soluble drugs, polymeric nanoparticles) could induce the 'foamy macrophage' phenotype whereby cells express with a granular or vacuolated cytoplasmic appearance (Fig. 1). As a result, within the context of pre-clinical safety studies, the appearance of foamy macrophage is usually sufficient to halt compound development contributing to an already high rate of attrition in drug development. An electron micrograph of the foamy macrophage is shown in Fig. 1 highlighting the well-defined vacuoles of 0.5-2 μm in diameter. A chemically- resolved analytical technique with a nanoscale spatial resolution would shed new light in the understanding of macrophages' responses to drug treatment. Resonance-Enhanced Infrared Atomic Force Microscopy (RE-AFM-IR) developed with Synchrotron Radiation at Diamond beamline B22 is a near- field IR probe capable of measuring infrared spectra (thereby the chemical information)froma100nmspot 3 offeringanidealtooltoprobethecomposition inside the vacuoles, potentially solving a major obstacle in inhaled drug discovery. The specific advantage of using a synchrotron source over tunable IR lasers 4,5 is that the light source covers a broader IR range, the spectral profile is more uniform and the whole spectrum can be collected simultaneously. These advantages are important for exploratory analysis of biological matters, where the samples are complex and often molecular changes can only be detected by the analysis of multiple spectral bands. In this work, we have applied the novel method for the first time at B22 on biological matter to highlight its potential in the study of drug induced phospholipidosis (DIPL). Randomly selected untreated (UT) and amiodarone-treated (AM) cells were characterised via SR RE-AFM-IR. The integrated RE-AFM-IR intensity map (i.e. molecular response to all IR wavelengths indiscriminately) has revealed a sub-micrometre structure within the cytoplasm of the cell (Fig. 2) that is not the same as the AFM (topographical height) map. The ratio of the RE-AFM-IR map against the AFMmap, which removes the contribution due to topography, has shown the nano-scale heterogeneity within the cytoplasm region. The AM cells has shown a more heterogeneous chemical distribution as a result of the formation of increase in number and size of vacuoles than the UT cells, which is in good agreement with the micrograph shown in Fig. 1. Spectra extracted from the RE-AFM-IR map, which shows specific spectral discriminations, are shown in Fig. 3A,B revealing the local chemical compositions from the 100 nm spot. The RE-AFM-IR spectra were compared to the optical IR microscopy, confirming that spectral bands that correspond to various functional groups are all clearly captured by the novel method. It is important to highlight that the far-field spectra were captured in 5 μm spot in contrast to 100 μm spot by the RE-AFM-IRmeasurement. The improved spatial resolution enables the comparison of chemical differences between different sub-cellular regions (e.g. the nuclear and perinuclear regions of UT and AM cells). Results show that the AM- perinuclear scans have consistently higher carbonyl absorbance from the phospholipid than those of the AM-nucleus region and UT cells. Furthermore, the phosphate peaks have shown on average an increase for the AM cells but with a higher variance across the scanned area because of the more heterogeneous cytosol of the AM cells, where many tiny lipid droplets were produced as shown in Fig. 2.Taken together, the RE-AFM-IR measurement has shown a general increase in absorption for all phospholipid bands suggests that the cytosol (perinuclear) region of the AM-treated cells have elevated phospholipid that are heterogeneously distributed, which is not observed in the UT cells. The data was further analysed using principal component analysis, showing a statistically significant difference between the UT/AM cells and the nucleus/perinucleus area in the phospholipid spectra regions, confirming the results observed made from the changes in band intensities. In summary, synchrotron-based RE-AFM-IR was shown to be capable of directly measuring the entire mid-infrared absorption spectra of mammalian cells and their internal molecular composition at the 100 nm scale. To our knowledge, it is the first successful application worldwide of synchrotron RE- AFM-IR to interrogate biological soft matter at subcellular level, in this case on a cellular model of DIPL. References: 1. Forbes B. et al. Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses. Adv. Drug Deliv. Rev. 71 , 15–33 (2014). DOI: 10.1016/j.addr.2014.02.001 2. Chan K. L. A. et al. Synchrotron Photothermal Infrared Nanospectroscopy of Drug Induced Phospholipidosis in Macrophages. Anal. Chem. 92 , 8097–8107 (2020). DOI: 10.1021/acs. analchem.9b05759 3. Donaldson P. M. et al. Broadband near-field infrared spectromicroscopy using photothermal probes and synchrotron radiation. Opt. Express 24 , 1852 (2016). DOI: 10.1364/ oe.24.001852 4. Dazzi A. et al. AFM-IR: Technology and applications in nanoscale infrared spectroscopy and chemical imaging. Chem. Rev. 117 , 5146–5173 (2017). DOI: 10.1021/ acs.chemrev.6b00448 5. Giliberti V. et al. Protein clustering in chemically stressed HeLa cells studied by infrared nanospectroscopy. Nanoscale 8 , 17560–17567 (2016). DOI: 10.1039/c6nr05783g Funding acknowledgement: KLAC acknowledges EPSRC for support (EP/L013045/1). Ali Atharawi thanks Prince Sattam Bin Abdulaziz University (Saudi Arabia) for his PhD sponsorship. Diamond Light Source is acknowledged for beamtime at MIRIAM beamline B22 – commissioning/collaborative call 2019 (SM21061). A special thanks to Dr Ioannis Lekkas and Dr Mark Frogley for crucial experimental support and data analysis help. Corresponding authors: Dr Ka Lung Andrew Chan, King’s College London,
[email protected] ; Dr Gianfelice Cinque, Diamond Light Source,
[email protected] Figure 1: Microscopic characterisation of untreated and amiodarone-treated cells. The spatial distribution of phospholipid inclusion bodies is shown by: (A) light microscopy with hematoxylin and eosin staining, (B) TEM. Figure 3: Averaged synchrotron RE-AFM-IR spectra (solid lines) from 15-20 scan points in three UT (A) or AM (B) cells compared to synchrotron-FTIR spectra from a single cell (dotted lines). The aliphatic lipid band (3020-2800 cm -1 ), the lipid carbonyl n (C=O) band (1774-1709 cm -1 ), the phosphate bands at (1310-1147 cm -1 ) and (1142-1020 cm -1 ) have been used to show an increased phospholipid content (a biomarker of DIPL). Figure2:AFMmicrographsdepictingtopology featuresarecomparedwithoverallandnormalised integratedRE-AFM-IRamplitudemaps forUTandAMcells. AFM topography Integrated RE-AFM-IR intensity RE-AFM-IR intensity/AFM topography