Additional file 1 of A simple and robust nanosystem for photoacoustic imaging of bladder cancer based on α5β1-targeted gold nanorods
Creators: name: Alfano, Massimo
name: Alchera, Elisa
name: Sacchi, Angelina
name: Gori, Alessandro
name: Quilici, Giacomo
name: Locatelli, Irene
name: Venegoni, Chiara
name: Lucianò, Roberta
name: Gasparri, Anna Maria
name: Colombo, Barbara
name: Taiè, Giulia
name: Jose, Jithin
name: Armanetti, Paolo
name: Menichetti, Luca
name: Musco, Giovanna
name: Salonia, Andrea
name: Corti, Angelo
name: Curnis, Flavio
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Biotechnology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. Biochemical characterization of peptide Iso4 by Ellman’s assay, electrospray ionization mass spectrometry (ESI–MS) analysis, RP-HPLC, nuclear magnetic resonance (NMR) spectroscopy and integrin binding. Table S2. Expression of cell surface integrins on murine MB49-Luc bladder carcinoma cells as determined by FACS analysis. Figure S1. Zoom into the 1H-1D NMR spectrum of peptide Iso4 centered on the Hα resonances of phg and Phg. NMR spectroscopy shows that peptide Iso4 consists of two isomers corresponding to a cyclic head-to-tail peptide with D-phenylglycine (phg) and L-phenylglycine (Phg), the latter accounting for about 30%. The HαD and HαL signals of phenylglycine (each giving rise to a doublet) are indicated. Figure S2. Binding of Iso4-HRP conjugate to microtiter plates coated with or without α5β1, αvβ3 and αvβ5. A Schematic representation of head-to-tail cyclized Iso4 and control peptides (Iso3 and ARA, positive and negative control, respectively). B Binding of peptide-HRP conjugates to microtiter plates coated with α5β1, αvβ3 and αvβ5. Peptide-HRP conjugates were prepared by coupling peptides to maleimide-activated horseradish peroxidase (HRP), as described in Ref. [15]. The binding assay was performed as described previously [15] using the indicated amount of integrins for microtiter plate coating. Mean ± SE, n = 2 wells. The Iso3-HRP and ARA-HRP were used as positive and negative controls, respectively, to assess integrin functionality (Iso3-HRP) and binding specificity (ARA-HRP). The binding curves of Iso4-HRP and ARA-HRP α5β1 are reprinted with the permission of Ref. [15]. Figure S3. Expression of α5-, β1-, β3-, β5-, αv-integrin subunits, and αvβ6 integrin on murine bladder MB49-Luc carcinoma cells. Integrin expression by MB49-Luc cells was analyzed by FACS using the indicated anti-integrin antibodies (5 µg/ml), and appropriate species-specific Alexa Fluor 488-labeled secondary antibodies (5 µg/ml). Binding of an isotype control antibody is also shown (see Table S2 for antibody description). Figure S4. TEM analysis of MB49-Luc cells incubated with GNRs@PEG-Iso4 or GNRs@PEG-Cys. MB49-Luc cells, cultured in a 12-well plate (cell confluency &gt; 90%), were washed twice with 0.9% sodium chloride and then incubated for 5 min with 25 mM Hepes buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 1% w/v BSA. The cells were then incubated for 2 h at 37 °C, 5% CO2, with GNRs@PEG-Iso4 or GNRs@PEG-Cys (1 × 1011 NPs/ml, 500 μl well). The cells were then washed with the same buffer (3 times, 5 min each), fixed, and analyzed by TEM as described in Ref. S1. Figure S5. Characterization of GNRs@PEG-Iso4 stability by α5β1-integrin binding assay. Binding of GNRs@PEG-Iso4 (different lots, called #A and #B, corresponding to freshly prepared NPs or after 4 months of storage at 4 °C, respectively), to microtiter plated coated with or without α5β1. NPs binding was detected as described in Methods. GNRs@PEG-Cys was also included as a negative control. Note that the binding of GNRs@PEG-Iso4 Lot#B is similar to that of Lot#A, suggesting that no chemical detachment or degradation of the various compounds composing the NPs occurred during the storage for 4 months at 4 °C. Figure S6. Photothermal properties of GNRs@PEG-Iso4. The capability of GNRs@PEG-Iso4 to release heat after illumination (0–15 min) was tested using a custom-made system consisting of: 1) a continuous-wave NIR laser line at 808 nm (LDC220C, TED200C, Thorlabs, laser power, 0.2, 0.3 and 0.4 W), 2) a multimodal optical fiber (1 mm Ø, Thorlabs), 3) a power meter sensor (Thorlabs, cat. s405c); 4) a cuvette holder (Thorlabs), 5) a polystyrene cuvette (Kartell, cat. 01938–00) containing 500 µl of sample, (0–500 µM of Au, final concentration) and 6) a NIR camera (HEIMANN Sensor, cat. HTPA80 × 64dR2L3.9/0.8HiA). A Heating curve of GNRs@PEG-Iso4 (500 µM Au) dispersed in 0.05% HSA (Diluent) and irradiated with an 808 nm laser at the indicated power densities. Right panel inset, the image of the cuvette containing GNRs@PEG-Iso4 after 15 min of laser illumination at 0.4 W is shown. B Thermal stability of GNRs@PEG-Iso4 (125 µM Au) over 2 cycles of a laser on/off experiment at the indicated laser power. The 2nd cycle of a laser illumination was performed after 24 h from the 1st cycle. Figure S7. Expression of α5- and β1-integrin subunit in the MB49-Luc bladder cancer model. Representative immunohistochemistry photomicrographs of the expression of α5- and β1-integrin subunit in different areas of the bladder of a tumor-bearing mice, 15 days after intravesical instillation of MB49-Luc cells. T, MB49-Luc tumor; L, lumen of the urinary bladder; Red dashed rectangle: zoomed areas. Zoomed area 1: healthy bladder, black arrows indicate the urothelial cells. Zoomed area 2: Luminal side of MB49-Luc tumor. Note that this big tumor is minimally covered by layers of α5-negative urothelial cells (blue arrows). Immunostaining was performed as described previously [15]. Adapted and reprinted with the permission of Ref. [15]. Figure S8. GNRs@PEG-Cys does not bind to orthotopic MB49-Luc tumor lesions. A Schematic representation of the experimental procedure. A mouse with orthotopic MB49-Luc tumor lesions underwent PAUS imaging of the bladder before (Step 1) and after (Step 2) intravesical instillation of GNRs@PEG-Cys (26 nmol Au). After bladder washing, additional PAUS imaging of the bladder was then performed before (Step 3) and after (Step 4) instillation of GNRs@PEG-Iso4 (26 nmol Au). B Representative PAUS images (axial 2D) of the bladder after step 1, 2, 3, and 4. Grayscale, co-registered US signal; Green, PA signal; Cyan arrows, specific PA signal (generated by GNRs@PEG-Iso4). Yellow arrows, unspecific PA signal (independent from nanoparticles). Bar, 2 mm. Specific PA signal on tumor lesions was observed only after step 4, suggesting that peptide Iso4 is crucial for tumor recognition by nanoparticles. Figure S9. Effect of a neutralizing anti-α5β1 mAb on the uptake of GNRs@PEG-Iso4 to MB49-Luc bladder tumors. Mice bearing orthotopic MB49-Luc tumors were intravescically administered with a control isotype mAb or a neutralizing anti-α5β1 mAb (clone: RTK2758 and 5H10-27(MFR5), respectively, 20 µg/mouse). After 15 min, the bladders were emptied and subsequently filled with GNRs@PEG-Iso4 (26 nmol Au in 100 µl, ~ 1 × 1011 NPs). After 15 min, the bladders were washed, and PA and US imaged. PA signals associated with whole tumors or adjacent healthy tissues (background) were quantified using VevoLab 5.6.1 software. Box-plots with median, interquartile and 5–95 percentile, in which dots represent well-established tumors. N = 4–5 mice, with one or two tumors per bladder. P, by Mann–Whitney nonparametric test.