A prospective cancer chemo-immunotherapy approach mediated by synergistic CD326 targeted porous silicon nanovectors

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1 Electronic Supplementary Material A prospective cancer chemo-immunotherapy approach mediated by synergistic CD326 targeted porous silicon nanovectors Mohammad-Ali Shahbazi 1 ( ), Neha Shrestha 1, Ermei Mäkilä 1,2, Francisca Araújo 1,3,4, Alexandra Correia 1, Tomás Ramos 5, Bruno Sarmento 3,6, Jarno Salonen 2, Jouni Hirvonen 1, and Hélder A. Santos 1 ( ) 1 Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI Helsinki, Finland 2 Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI Turku, Finland 3 INEB Instituto de Engenharia Biomédica, NewTherapies Group, Universidade do Porto, Rua do Campo Alegre, 823, Porto, Portugal 4 ICBAS Instituto Ciências Biomédicas Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira, Porto, Portugal 5 Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal 6 IINFACTS Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Instituto Superior de Ciências da Saúde-Norte, Department of Pharmaceutical Sciences, CESPU, Rua Central de Gandra, Gandra, Portugal Supporting information to DOI /s Cell Lines and Culture Conditions Dulbecco s modified Eagle s medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% non-essential amino acids, 1% L-glutamine, penicillin (100 IU ml 1 ), and streptomycin (100 mg ml 1 ) (all from HyClone, USA) was used to grow MCF-7 human breast cancer cells. Standard Roswell Park Memorial Institute 1640 media, supplemented with the same aforementioned compounds was used to culture MDA-MB-231 breast carcinoma cells. The cell lines were obtained from American Type Culture Collection and cultured in 75 cm 2 culture flasks (Corning Inc. Life Sciences, USA). Cells were cultured in a standard BB 16 gas incubator (Heraeus Instruments GmbH, Germany) with the conditions of 95% humidity, 5% CO 2, and 37 C. The subculturing was performed when the cells were at 80% of confluency. Trypsin PBS EDTA solution was used for cell detachment prior to passaging, as well as prior to each experiment. The passage numbers used for MCF-7 and MDA-MB-231 were #4 30 and #6 36, respectively. Cell Viability Studies Typically, 100 μl of the cell suspensions with the concentration of cells/ml in the cell media were seeded in 96-well plates and allowed to attach overnight. Thereafter, the cell media was removed and replaced with 100 μl of the NPs at concentrations of 5, 10, 25, 50, 100 and 200 μg ml 1. After 24 and 48 h incubation at 37 C, 100 μl of the reagent assay (CellTiter-Glo Luminescent Cell Viability Assay, Promega, USA) was added Address correspondence to Mohammad-Ali Shahbazi, m.a.shahbazi@helsinki.fi; Hélder A. Santos, helder.santos@helsinki.fi

2 to each well as described elsewhere [1]. The luminescence of the wells was measured using a Varioskan Flash (Thermo Fisher Scientific Inc., USA). Negative (HBSS buffer solution) and positive (1% Triton X-100) control wells were also used and treated similarly as described above. The viability of the negative control was taken as 100%. The results are shown as the average of at least three independent measurements. IL-2 Secretion measurements Briefly, 100 μl of the both types of cancer cells were seeded in 96-well plates at a density of cells/ml. After 24 h, the supernatants were removed and replaced with 75 μl of the medium. Afterwards, 50 μl of the UnTHCPSi or Un-Ab, at concentrations of 25 and 100 μg ml 1, were added to the wells. Samples containing free Ab, equal to the amount of Ab-conjugated to the both tested concentrations of Un-Ab, were also tested to clarify if its potency was affected after conjugation with the NPs. Next, FcγRIIIa positive effector immune T cell subsets were used as effector immune cells and added to the well plates before leaving in incubator for 24 h at 37 C. Then, 100 μl of the supernatant of each well was added to the ELISA Ab pre-coated IL-2 kits. Next, 50 μl of the ELISA diluent was added to each well. The samples were then incubated at RT for 2 h, followed by washing for 5 times. Afterwards, 100 μl of Detector Ab was introduced to each well and incubated for 1 h at RT. The washing step was again repeated seven times, and then, 100 μl of TMB substrate was added to each well and the samples incubated for 30 min at RT in the dark. Finally, 50 μl of the stop solution was used and the amount of IL-2 present in each well was determined spectrophotometrically by measuring the absorbance with a Varioskan Flash at 450 nm. The amount of IL-2 secreted by the effector immune cells was then calculated from the standard curve established by a serial dilution of known concentrations of human IL-2. All samples were prepared at least in triplicates. SEM Imaging of immune-cancer cells association To identify if the ADCC activity results from the interaction between the immune and cancer cells mediated by the Un-Ab NPs, SEM imaging experiments were carried out. For this study, cancer cells were treated with 50 μl of the Un-Ab at the concentration of 100 μg ml 1. Then, of FcγRIIIa positive effector immune T cell subsets were added to each sample before incubating for 24 h at 37 C. Next, the cells were fixed in 2.5% glutaraldehyde for 1.5 h and post-fixed in 1% osmium tetroxide in 0.1 m NaCac buffer (ph 7.4) for 1 h. The cells were then dehydrated in increasing concentrations of 50, 70, 96, and 100% of ethanol for 5, 10, 20 and 15 min, respectively. Finally, the cell suspensions were dropped onto plastic coverslips, dried, and sputter coated with platinum before being observed under SEM (Zeiss DSM 962).

3 Figure S1 Protocol used for the preparation of UnTHCPSi nanoparticles. Figure S2 XRD diffractogram obtained from UnTHCPSi NPs. Si diffraction planes are identified. The strong reflections at ca. 43 and 50 are due to the Cu sample holder. Figure S3 Step sequence for drug loading, surface Ab-grafting of the UnTHCPSi NPs, and the potential of the developed nanocarrier for cancer cell targeting. The THCPSi NPs were immersed into undecylenic acid for 16 h at 120 C (Fig. S1) to produce UnTHCPSi NPs. Carboxyl groups act as a functional linker to allow Ab immobilization on the surface of the particles. Nano Research

4 Figure S4 In vitro release profiles of SFN-UnTHCPSi NPs and pure SFN in PBS medium (ph 7.4) with and without 10% FBS at 37 C. Data represent mean ± s.d. (n 3). Figure S5 TEM images of MCF-7 cells treated with 50 µg ml 1 of bare and Ab-conjugated UnTHCPSi NPs for 2 h. Figure S6 Confocal fluorescence microscopy analysis of the MCF-7 cells incubated with HBSS buffer as control, 50 µg ml 1 of UnTHCPSi and Un-Ab at 37 C for 3 h. CellMask was used for staining of the cell membranes and is shown in red color. The merged panel shows the NPs outside of the cell membrane in green color and those interacting with the cells in yellow color.

5 Figure S7 Confocal fluorescence microscopy analysis of the MDA-MB-231 cells incubated with HBSS buffer as control, 50 µg ml 1 of UnTHCPSi and Un-Ab at 37 C for 3 h. CellMask was used for staining of the cell membranes and is shown in red color. The merged panel shows the NPs outside of the cell membrane in green color and those interacted with the cells in yellow color. Figure S8 Mediated cellular association of immune cells and MCF-7 cells by the recognition of Un-Ab on the surface of cancer cells with immune cells. References [S1] Santos, H. A.; Riikonen, J.; Salonen, J.; Mäkilä, E.; Heikkilä, T.; Laaksonen, T.; Peltonen, L.; Lehto, V. P.; Hirvonen, J. In vitro cytotoxicity of porous silicon microparticles: Effect of the particle concentration, surface chemistry and size. Acta Biomater. 2010, 6, Nano Research