Supporting Information Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy Bieke Van de Broek 1,2, Nick Devoogdt 3,4, Antoine D Hollander 1, Hannah-Laura Gijs 1, Karolien Jans 1, Liesbet Lagae 1,5, Serge Muyldermans 3,4, Guido Maes 2, Gustaaf Borghs 1 1 imec, Bio-Nano Electronics, Functional Nanosystems, Kapeldreef 75, B-3001 Leuven, Belgium 2 Department of Chemistry, Quantum and Physical Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium 3 Department of Cellular and Molecular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium 4 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium 5 Department of Physics, Laboratory of Solid State Physics and Magnetism, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium * Address correspondence to Bieke.Vandebroek@imec.be S1
ICP-OES measurements The gold concentration of the branched gold nanoparticles was determined with ICP-OES. Hereto, branched gold nanoparticles were prepared with different optical densities (OD 0.4 OD 2) and gold standards were prepared from a HAuCl 4 solution with concentrations between 0.1 and 2 ppm. All the samples were dissolved in ten times diluted aqua regia (HCl:HNO 3 /3:1 (v/v)). Hereto, 100 µl of the samples and of the standards were mixed with 4900 µl of the diluted aqua regia. With ICP-OES the gold concentrations of the samples were measured and multiplied by 50 to account for the dilution. The resulting values in ppm were converted to mg/ml and plotted in function of the optical density (Figure S1). Figure S1. Gold concentrations (mg/ml) of branched gold nanoparticles with different optical densities (0.4 2) determined by ICP-OES. This linear relationship between the gold concentration and the optical density is only valid until OD 2. A linear relationship is observed between the gold concentration and the optical density until an optical density of 2. For higher optical density values an error is introduced since the particles can be located along the same optical path, obstructing each other s light absorption. To achieve accurate measurements for higher concentrations, a dilution of the suspension is required. A suspension with an optical density of 4 is actually a suspension with an optical density of 1 after a 4 times dilution. In Table S1 the measured gold concentration (mg/ml) for branched gold nanoparticles with an optical density of S2
1 is shown together with the calculated values for the branched gold nanoparticles with optical densities of 2, 4 and 6. The number of NP/mL is calculated by dividing the gold concentration (mg/ml) by the mass of gold in a single branched nanoparticle. To determine the mass of gold present in a single nanoparticle, the volume of a single branched gold nanoparticle (8.71 x 10-23 m³) was calculated using the average size determined by TEM and multiplied by the density of gold (19.3 x 10 6 g/m³). Dividing the total gold concentration (3.86 x 10-5 g/ml) by the mass of gold present in a single nanoparticle (1.68 x 10-15 g) resulted in a concentration of 2.3 x 10 10 nanoparticles per ml for OD1. The determination of the volume of a single branched Au NP (8.71 x 10-23 m³) is challenging due to the complex morphology and heterogeneity of the nanoparticles. The nanoparticle volume was approximated assuming a spherical form. Using the volume of a sphere based on the maximal diameter of the branched Au NP, which was determined as 60.4 nm, would be an overestimation since it would include a lot of empty (i.e. not filled) spaces. Thus, we preferred to approximate the volume of the branched Au NP by taking that of a sphere with a smaller diameter. On the TEM images we defined this diameter that cuts halfway the edges of the sharp tips of the branched nanoparticle (a diameter of 55 nm). The volume of the tips that is excluded corresponds roughly to the empty volume present between the extrusions of the branched nanoparticle. As such, the volume of a sphere with this diameter is a good approximation for the volume of the branched nanoparticle. Table S1. Calculated gold concentrations (mg/ml) and numbers of NP/mL of branched gold nanoparticles with different optical densities (1, 2, 4, 6). Optical density Gold concentration (mg/ml) Number of NP/mL 1 0.0386 2.3 x 10 10 2 0.0772 4.6 x 10 10 4 0.1544 9.2 x 10 10 6 0.2316 1.4 x 10 11 S3
Absorbance (a.u.) Nanobody coupling to branched gold nanoparticles The optimal concentration to couple the nanobodies to the branched gold nanoparticles was determined by adding different nanobody concentrations. The (in)stability of the branched gold nanoparticles upon addition of different nanobody concentrations was evaluated by UV-Vis absorption spectroscopy. For the anti-her2 nanobodies, aggregation of the nanoparticles was observed upon addition of 10 and 100 µg/ml (disappearance of the plasmon absorption band) (Figure S2). The nanoparticles remain stable upon addition of 0.1 and 1 µg/ml of anti-her2 nanobodies. The optimal anti-her2 nanobody concentration is located between 1 and 10 µg/ml. The UV-Vis spectra from the branched gold nanoparticles after coupling of anti-her2 nanobodies with concentrations between 1 and 10 µg/ml indicate that 5 µg/ml is the maximum nanobody concentration that can be coupled without inducing aggregation (Figure S2). For the anti-psa nanobodies, the branched gold nanoparticles remain stable upon addition of 0.1, 1 and 10 µg/ml (Figure S3). 1,2 1 0,8 0,6 Branched NP Branched NP + maleimide 0.1 µg/ml anti-her2 1 µg/ml anti-her2 10 µg/ml anti-her2 100 µg/ml anti-her2 0,4 0,2 0 300 400 500 600 700 800 900 1000 1100 Wavelength (nm) Figure S2. UV-Vis absorption spectra of branched gold nanoparticles, maleimide functionalized branched gold nanoparticles, and maleimide functionalized branched gold nanoparticles coupled with different concentrations of the anti-her2 nanobody (0.1, 1, 10 and 100 µg/ml). S4
Absorbance (a.u.) Absorbance (a.u.) 1,4 1,2 1 0,8 0,6 Branched NP Branched NP + maleimide 0.625 µg/ml anti-her2 1.25 µg/ml anti-her2 2.5 µg/ml anti-her2 5 µg/ml anti-her2 10 µg/ml anti-her2 0,4 0,2 0 300 400 500 600 700 800 900 1000 1100 Wavelength (nm) Figure S3. UV-Vis absorption spectra of branched gold nanoparticles, maleimide functionalized branched gold nanoparticles, and maleimide functionalized branched gold nanoparticles coupled with different concentrations of the anti-her2 nanobody (0.625, 1.25, 2.5, 5 and 10 µg/ml). 1,4 1,2 1 0,8 Branched NP Branched NP + maleimide 0.1 µg/ml anti-psa 1 µg/ml anti-psa 10 µg/ml anti-psa 0,6 0,4 0,2 0 300 400 500 600 700 800 900 1000 1100 Wavelength (nm) Figure S4. UV-Vis absorption spectra of branched gold nanoparticles, maleimide functionalized branched gold nanoparticles, and maleimide functionalized branched gold nanoparticles coupled with different concentrations of the anti-psa nanobody (0.1, 1 and 10 µg/ml). S5
HER2 positive and HER2 negative expression of SKOV3 and CHO cells HER2 positive or negative expression was confirmed by incubating both cell lines with an anti-her2 monoclonal antibody labeled with a fluorescent dye, phycoerythrin (PE), which emits at 570 nm. In Figure S5 the FACS results are presented by the percentage of measured cells plotted versus the fluorescent signal intensity. The anti-her2-labeled SKOV3 cells show an increased fluorescence signal compared to the isotype control signal confirming their HER2-expression (Figure S5). This isotype control signal is emitted by the cells after incubation with a nonspecific antibody from the same isotype. The anti-her2-labeled CHO cells do not exhibit a significant increase in fluorescent signal, confirming their HER2-negative expression (Figure S5). Figure S5. FACS results of (a) HER2+ SKOV3 cells and (b) HER2- CHO cells after labeling with PE anti-her2 monoclonal antibody compared to the isotype control. The percentage of measured cells is plotted versus the fluorescent signal intensity. S6