Image-based tracking of anticancer drug-loaded. environments using a fast benchtop mid infrared

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1 Supporting Information Image-based tracking of anticancer drug-loaded nano-engineered polyelectrolyte capsules in cellular environments using a fast benchtop mid infrared (MIR) microscope Rabah Mouras 1, Mohamed R. Noor 2, Laura Pastorino 3, Enrico Bagnoli 2,3,##, Aladin Mani 1, Edel Durack 2, Alexei Antipov 4, Francesca D'Autilia 5, Paolo Bianchini 5, Alberto Diaspro 5, Tewfik Soulimane 2, Christophe Silien 1, Carmelina Ruggiero 3, Syed A. M. Tofail 1,* 1 Department of Physics, Bernal Institute, University of Limerick, Castletroy, Limerick V94 T9PX, Ireland 2 Department of Chemical Sciences, Bernal Institute, University of Limerick, Castletroy, Limerick V94 T9PX, Ireland 3 Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genova, Via Opera Pia, 13, Genoa Genova, ITALY 4 PlasmaChem GmbH, Schwarzschildstr. 10, Berlin, Germany 5 Nanophysics, Istituto Italiano di Tecnologia, Via Morego, 30, Genova, Italy # CÚRAM Centre for Research in Medical Devices, National University of Ireland, Galway S1

2 1. Microscope calibration and sub-wavelength resolution In order to determine the imaging parameters of our MIR microscope, we first calibrated the setup by taking images of a calibration sample consisting of a sliver thin film with micro-sized holes deposited on a microscope slide. We tuned our laser to 3.45 µm (2900 cm-1) corresponding to peak absorption of NPCs. An example of obtained images is presented in figure S1. We can see clearly that the holes are very well resolved and separated by 3 µm corresponding to 0.8λ indicating the sub-wavelength resolution imaging capability of our microscope. Figure S1. (a) Microscope calibration using MIR scattering of holes in silver slide taken at 2900 cm -1 (λ = 3.45 µm) wavelength. Image size 30 µm x 30 µm. (b) Corresponding line profile is shown. We can see that the holes are separated by ~ 3 µm (HWM) which is 0.8λ confirming the subwavelength resolution of our microscope. 2. Fast tuneability and wavelength dependent chemical imaging To demonstrate the tuneability and chemical imaging capabilities of our MIR microscope we imaged the NPCs at different wavelengths. We acquired two successive images at µm (3200 cm -1 ) and at 3.45 µm (2900 cm -1 ). These data demonstrate the multi-wavelength imaging capability of our MIR microscope. S2

3 Figure S2. MIR images of empty NPCs dispersed on CaF 2 substrate taken at 3200 cm -1 (a) and at 2900 cm -1 (b). Line profile (c) shows the size of NPCs empty capsule ~ 4 µm. We can see clearly that the images taken at 2900 cm -1 is more contrasted due to the high absorption at this band which is in good agreement with FTIR data. Image size 100 µm x 100 µm 3. Cytotoxicity analysis and MTT assays Prior to performing a complete cell culture experiment, we tested the potential cytotoxic effects of the non-biodegradable polymers PSS and PAH. The solution with polyelectrolytes was added to 50μl of DMEM and cells. The volume was then brought to 100μl with DMEM. So the most concentrated sample was made by 50 μl of cells and media and 50μl of polyelectrolytes for a total concentration of 1 mg/ml of PSS or PAH. At 1 mg/ml the toxicity of the polyelectrolytes is expected to be greater than the one of the NPCs as they are free in the solution. Indeed the poly-ions are freer to move in the media and to harm cells when they are by themselves, not bonded with the counter poly-ions. The results obtained in incubating MCF-7 for 24, 48, 72 hours with PSS and PAH at 1 mg/ml and 0.5 mg/ml are shown in figure S3-a. It is clear that only PAH displayed a high cytotoxicity towards the MCF7 cells, with viability always less than 40%, which is in line with the literature reports 1. The PSS shows a good biocompatibility especially at a lower dose of 0.5 mg/ml at which the toxicity effects are completely mitigated after 72 hours. S3

4 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 2-3 was then repeated using sterilized functionalized and empty NPCs. The NPCs were built with an initial concentration of 5 mg/ml that is the concentration of the carbonate templates. Different concentration of NPCs (50, 25, 12.5 μl) were added to 50 μl of MCF-7 cells and media, then the required volume of to reach the final volume of 100 μl was added to each concentration. Figure S3. Cell viability results for MCF-7 cells incubated with PSS and PAH polyelectrolytes for 3 days (a) and with empty and DTX loaded NPCs after 12 hours (b). In this study the release of the drug from the capsule was not triggered by any stimuli, but during the incubation period some drug molecules escape from the core due to the change in the permeability of the polymeric shell. Obtained results show that DTX-loaded NPCs have a greater toxicity compared to the empty ones, as in line with our expectations. This is clear in Figure S3- b where the MTT results are reported for empty and functionalized capsules after 12 hours of incubation. Cell viability increases linearly with the decrease of concentration for both kinds of particles, but in general DTX-loaded microcapsules are significantly more harmful for MCF-7 than the empty ones. S4

5 References (1) Kharlampieva, E.; Kozlovskaya, V., CHAPTER 6 Cytocompatibility and Toxicity of Functional Coatings Engineered at Cell Surfaces. In Cell Surface Engineering: Fabrication of Functional Nanoshells, The Royal Society of Chemistry: 2014; pp (2) Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 1983, 65 (1-2), (3) Gerlier, D.; Thomasset, N., Use of MTT colorimetric assay to measure cell activation. Journal of immunological methods 1986, 94 (1), S5