Max Semmling1, Oliver Kreft2, Almdena Muñoz Javier3, Gleb B. Sukhorukov2,4, Josef Käs1*, Wolfgang J. Parak3* 1 Institut für Experimentelle Physik I and Paul Flechsig Institut, Universität Leipzig, Leipzig, Germany 2 Max-Plank-Institut für Kolloid- und Grenzflächenforschung, Golm, Germany 3 Fachbereich Physik, Phillips Universität Marburg, Marburg, Germany 4 QMUL London, United Kingdom * wolfgang.parak@physik.uni-marburg.de Flow cytometry based assay for the analysis of the uptake of polyelectrolyte capsules

SUPPORTING INFORMATION (I) Cell culture and capsule uptake by cells (II) FACS measurements of capsules (III) FACS measurement of cells incubated with capsules

(I) Cell culture and capsule uptake by cells Culture media The following culture medium was used: MCF-7 culture medium MEM 500 ml, Product No. M5650, Sigma-Aldrich Chemie GmbH, Munich, Germany L-glutamine 0.146 g bovine insulin (stock solution in HCl 2 mg/ml) 2.5 ml sodium pyruvate (stock solution in H2O 100 mg/ml) 0.55 ml FCS 50 ml MCF-10a culture medium DMEM/Ham’s F-12 with L-Glutamine, Product No. E15-813, PAA Laboratories GmbH 10 mM HEPES buffer 10 µg/ml insulin 20 ng/ml hEGF 100 ng/ml cholera toxin 0.5 µg/ml hydrocortisone 5% horse serum SV-T2 and BALB/3t3 culture medium DMEM with L-Glutamine, without Phenol Red, Product No. E15-877, PAA Laboratories GmbH 10% calf serum HL-60 culture medium RMPI 1640 with L-Glutamine, without Phenol Red, Product No. E15-848, PAA Laboratories GmbH 10% fetal bovine serum The adherent cell were planted in 12-well-multidishes (Product No. 665180, greiner bio-one GmbH) with 2 ml of prewarmed culture medium the day before the measurement started. The density was adjusted to the morphology: epithelial-like cells: 2.5•104 cells/cm² fibroblasts-like cells: 104 cells/cm² The HL-60 cells were added to 24-well-multidishes (Product No. 662160, greiner bio-one GmbH) with 0.5 ml of prewarmed culture medium the day before the measurement started, with a density of 5*10^5 cells/ml. For every measuring point a separate dish was prepared to avoid interruptions during the incubation.

Cells with incorporated capsules In the following several photos of cells and capsules are shown in order to visualize the size of the capsules compared to the size of cells. The capsules have (PAH/PSS)4 geometry and had be synthesized around 3,2µm diameter CaCO3 template cores. In the images on the left hand side only cells, on the right hand side cells with capsules are shown.

In the following 2 images one MCF-7 cell and one SV-T2 cell is shown with (PAH/PSS)4 capsules around 3,2µm diameter CaCO3 template cores. The images visualize the size of the capsules versus the size of a cell.

(II) FACS measurements of capsules

Figure SI-1: 2-dimensional density plot of the events recorded with FACS of two samples with dispersed SNARF-loaded capsules. In the first sample the capsules were dispersed in buffer with pH = 5, and in the second sample with pH = 8. The intensities of the red (Ired) and green (Igreen) fluorescence signal of each event in which a capsule is detected are visualized as 2-dimensional density plot. The number of events is color-coded from blue (low frequency) to red (high frequency). In the bottom an overlay of both plots is shown. In the overlay two populations of events can be distinguished, events with higher red fluorescence and events with higher green fluorescence which can be associated with capsules dispersed in medium with pH = 8 and pH = 5, respectively.

(III) FACS measurement of cells incubated with capsules

Figure SI-2: 2-dimensional density plot of the events recorded with FACS of one sample of cells that had been incubated with capsules. The experimental data correspond to the data shown in Figure 1 in the main manuscript. The density plot of the forward scattering (IFSC) and the sideward scattering (ISSC) signal are shown. Each recorded event is shown as point in the diagram. The density of events is color coded from blue (low frequency) to red (high frequency). The diagram can be divided into 2 populations. Events with high forward scattering signal (IFSC > 110) and high sideward scattering signal (ISSC > 5•100) correspond to events involving cells. Other events with lower forward or sideward scattering signal correspond to freely dispersed capsules and debris. Integration over all events in the first quadrant yields to the number of events Ncell, integration over all events in the other quadrants yields the number of events Ndebris+caps. The shown plot of the forward scattering and sideward scattering does not resolve the number of populations Ncell w caps, Ncell w/o caps, Ncaps, and Ndebris, Ncell w caps(out), and Ncell w caps(in) that are resolved in Figure 1 in the main manuscript.

Figure SI-3: FACS data obtained with MCF-10 cells. The plots correspond to different incubation times t. a) 5min, b) 15min, c) 30min, d) 45min, e) 1h, f) 1.5h, g) 2h, h) 3h. The fluorescence plots are not gated and all event are included. In a) the population of freely suspended capsules and cells with adherent capsules is marked red. In d) the population of cells with internalized capsules has emerged and is marked green. In h) finally a population is marked yellow that can not be separated. The yellow population might correspond to cells that have adherent and internalized capsules as well i.e. as colocalisation. The aggregation of these events marked yellow is not present for the overlay of FACS measurements of freely suspended capsules (Figure SI-1) where only distinct red or green events are shown.

Figure SI-4: FACS data obtained with a) MCF-10 b) MCF-7, c) BALB-3T3, d) SV-T2, and e) HL-60 cells. Each data point corresponds to the result of one single FACS measurement of which the number of cells with internalized capsules Ncell w caps(in), the number of cells Ncell = Ncell w caps + Ncell w/o caps, and the total number of events Ntot has been derived as shown in Figure 1. Data were recorded for different incubation times t and a constant number of capsules added per cell at the beginning of the incubation (t=0). The data are based on the same data set as shown in Figure 3 in the main manuscript.