Nanorods synthesis, SiO2 coating, and characterization.
Gold nanorods (AuNRs) in CTAB (Cetyltrimethylammonium Bromide) were synthesized using salicylic acid as an additive as previously described [36,37]. For coating AuNRs with silica, CTAB on the surface was exchanged with mPEG-SH. Excess CTAB was washed by repeated centrifugation, AuNRs were resuspended in Milli-Q water and a solution of mPEG-SH (50 molecules/nm2) was added dropwise. After 1 h of stirring AuNRs were washed and finally redispersed in ethanol. Silica coating was performed by mixing AuNRs ([Au] = 0.5 mM) with the appropriate amount of H2O (10.55 M), NH3 (0.2M), and tetraethyl orthosilicate (TEOS) solution (1 mM) and allowing reaction for 6 h at RT . The fluorophore Rhodamine B isothiocyanate (RBITC) was conjugated to APMS and added in subsequent shell re-growth steps allowing its conjugation to SiO2 . Fluorescently coated AuNRs were washed by repeated cycles of centrifugation/redispersion in ethanol. All reagents were obtained from Sigma-Aldrich, except ethanol (96%) that was purchased from Scharlab. Ethanol-dispersed nanorods were adsorbed onto a Lacey copper grid (400 mesh, EM Resolutions) and characterized using a JEM 1011 (JEOL) transmission electron microscope (Figure S1).
Gene synthesis, protein expression, and purification.
ShTxB:6xHis recombinant gene construct was synthesized by General Biosystems, Inc. (Morrisville, USA) and was cloned in pET-15b plasmid systems (Novagen, Merck KGaA, Spain). One Shot™ BL21(DE3) E. coli (NZYTech, Portugal) cells were transformed with the expression vector. Bacterial cultures were grown in Luria-Bertani (LB) broth supplemented with 100 μg/mL ampicillin and 35 μg/mL chloramphenicol until A600 ca. 0.6. The expression of the protein was induced by adding 0.1 mM isopropyl b-D thiogalactopyranoside (IPTG). Cells were collected after 4 hours by centrifugation and were resuspended in 50 mM NaPi, 300 mM NaCl, pH 8.0 with 1 mg/mL lysozyme, and protease inhibitors (Pierce, Thermo Fischer, Spain).
Bacterial cell lysates were obtained by probe sonication (5 ´ 15 s pulses at 130 W, 65% amplitude, with 15 s intervals, at 4 °C), and insoluble material was removed by centrifugation. Bacterial soluble protein lysate was loaded onto pre-equilibrated Ni-TED columns (Protino® Ni-TED, Macherey-Nagel GmbH & Co., Düren, Germany). The recombinant His-tagged protein was eluted in buffer supplemented with 250 mM imidazole. Finally, PD-10 desalting Columns (GE Healthcare, Chicago, USA) were used to remove the imidazole and to exchange buffer for PBS.
Protein bioconjugation on particles, SDS-PAGE protein analysis.
Nanorods were functionalized with 0.5 mg/mL of the protein solution (saturating amounts) in PBS during 3 ´ 2 s mild sonication at room temperature. For SDS-PAGE, the protein functionalized on the particles was stripped using Laemmli sample buffer 1´ (BioRad) at 90 ºC for 2 min. SDS-PAGE electrophoresis was performed using Mini-Protean® precast gels (BioRad). Protein analysis was performed on Coomassie blue-stained gels that were scanned using the BioRad GelDoc EZ system.
Cell culture and compacted pellet (micro tumor) conditions.
Detroit 562 cells, human pharynx epithelial carcinoma cells derived from a metastatic pleural effusion, and MCF-7, human breast cancer cells, were obtained from ATCC (Ref. CCL-138 and HTB-22 respectively). Detroit 562 cells were grown in Modified Eagle´s Medium and MCF-7 cells in Dulbecco´s Modified Eagle´s Medium (Sigma) containing 10% bovine serum. Both cell lines were kept in standard conditions. MCF7 cells were used as controls (GB3-ve) after characterization with the anti-GB3 antibody (Figure S4). A total of 500.000 cells were washed, trypsinized, and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and pellets were re-suspended in 50 μL of culture media to create a cell pellet that was used as a microtumor.
Staining, fluorescent, and electron microscopy imaging.
Cells were exposed to 5 µg/mL of bioconjugated AuNRs@SiO2:RBTIC@ShTxB particles for 3 hours. Cells were fixed with 4% paraformaldehyde, were stained with Hoechst 33258 (Sigma-Aldrich®), Acridine Orange (Sigma-Aldrich®) for live cells, or ethidium bromide (Sigma) for dead cells. Alexa 647-anti-Human CD77 (BD Pharmingen™, UK) was used for immunostaining the GB3 receptor. Confocal images were taken with a Nikon A1R microscope. All fluorescent images are pseudo-colored. Cell samples processed for electron microscopy were fixed with 3% glutaraldehyde in 0.12 M PBS for 24 hours and were post-fixed in 2% buffered osmium tetroxide, dehydrated in a graded acetone series, and embedded in Araldite. Ultrathin sections of ca. 70 nm thick, were obtained on an LKB ultramicrotome, stained with lead citrate and uranyl acetate. TEM was performed using a JEOL JEM 1011 operated at 100 kV.
Cell death experiments were performed using 106 Detroit and MCF-7 cells. Following manufacturer instructions, these cells were stained with Annexin V-FITC kit (Immunostep) for 15 min. and then passed through the cytometer (CytoFLEX, Beckman Coulter). Quantitative GB3 expression was validated and measured in a total of 10.000 Detroit and MCF-7 cells immunostained with Alexa 647-anti-Human CD77 (BD Pharmingen™, UK) for 30 minutes and then fixed in 4% paraformaldehyde (Figure S4). Qualitative interaction of AuNRs@SiO2@ShTxB in cells was performed using 10.000 Detroit 562 and MCF-7 cells. Cells were exposed to the nanoparticles for 3h, washed, and passed through the cytometer. Data were analyzed using the CytExpert software.
Murine model and tissue processing.
In vivo experiments were designed and performed to minimize the use of animals. Ethical permissions for this study were requested, approved, and obtained from the Bioethics Committee University of Cantabria, General Directorate of Livestock, Government of Cantabria (Project Ref PI-11-21). C57BL/6 mice (12 weeks old) were housed with a 12 h light/dark cycle with free provision of food and water at the Experimentation Service of the University of Cantabria. The animals were maintained, handled, and sacrificed following the directive 2010/63/UE. The preclinical model of oral carcinogenesis was produced using the carcinogen 4-Nitro-quinoline-1-oxide (4-NQO, Sigma Aldrich) as previously described [33–35]. In brief, C57BL/6 mice were drinking water with 100 μg/mL of 4-NQO for 16 weeks (water was changed once a week). After 16 weeks of carcinogen treatment, the mice with visible lesions in the oral cavity were treated with 5 µg/mL of AuNRs@SiO2@ShTxB in the drinking water (approximately 1 mg/Kg mice total) for 24 hours before sacrifice. For the laser irradiation, mice were humanely sacrificed and the tissues were dissected and immediately irradiated as indicated in the text. The irradiation procedure was performed only once. The tissues were fixed in 10% formalin, embedded in paraffin, sectioned (4 μm thick), deparaffinized, stained with hematoxylin-eosin, or preserved in optimal cutting temperature compound (OCT) at -80 oC to be cryosectioned. Approximately 6 μm cryostat sections were fixed in paraformaldehyde 4%, immunostained with Alexa 647-anti-Human CD77 (BD Pharmingen™, UK), and with Hoechst.
Human biopsies and cell culture of head and neck squamous carcinomas.
Ethical permissions for this study were requested, approved, and obtained from the Ethical Committee for Clinical Research of Cantabria Council, Spain (Ref. 2017.259). In all cases, human tissue material discarded after surgery was obtained with written consent presented by clinicians to the patient and was treated anonymously. Biopsy sample cell cultures were grown from biopsies of malignancies localized at the floor of the mouth (MASCC, moderately aggressive, intermediate grade), and retromolar trigone (ASCC, high grade, aggressive). The grade of aggressiveness was characterized by the clinician. Control cells were isolated from a healthy human biopsy (cheek inner mucosa). Biopsies were cut into pieces and washed twice with PBS‐EDTA, 100 U/mL penicillin‐streptomycin (Pen/Strep), and 0.5 μg/mL Amphotericin B. Pieces were then washed twice with 1x PBS, 100 U/mL Pen/Strep, and 0.5 μg/mL Amphotericin B and incubated with agitation at 37 °C for 4 rounds of 15 min with 0.075% trypsin, 2.5 mg/mL collagenase P, 1´ PBS, 100U/mL Pen/Strep and 0.5 μg/mL Amphotericin B. Isolated keratinocytes were co-cultured with a feeder layer of (3T3)-J2 mouse fibroblast feeder layer (previously inactivated with mitomycin C).
HNSCC and healthy primary keratinocytes were cultured in modified Rheinwald FAD medium [39,40]: 3:1 v/v Dulbecco’s Modified Eagle Medium – Ham’s F12, 5% fetal bovine serum, 0.5 μg/mL hydrocortisone, 5 ng/mL epidermal growth factor, 9 ng/mL cholera toxin, 1.8´10-4 M adenine, 5 µg/mL insulin, 2 mM L‐glutamine, 0.75 mM sodium pyruvate, and 100 U/mL Pen/Strep. Mouse fibroblast 3T3-J2 cell line used as feeder layer was cultured in Dulbecco medium with 10% donor calf serum and 100 U/mL Pen/Strep.
Laser microtumor irradiation.
Detroit 562 and control MCF-7 cells were exposed to 5 µg/mL and 10 µg/mL of AuNRs@SiO2@ShTxB for 3h. Then cells were washed, pelleted into microtumors, and irradiated with an unfocused NIR laser (808 nm LD, 1 W) for 10 min. After irradiation cells were placed back in culture plates in the incubator for another 24 hours. After this period, cells were stained with a dead cell apoptosis kit with AnnexinV-FITC and PI, for flow cytometric analysis and confocal microscopy at 0 and 24h.
2D/3D culture irradiation.
Cultures of neoplastic or normal cells from oral biopsies cells were treated with 5 µg/mL AuNRs@SiO2@ShTxB for 3 h. After removal of the particles in the media, cultures were irradiated with the 808 nm LD for 10 min directly on the plate. In vivo tumor irradiation: Once verrucous cancerous lesions were visible in the oral cavity, the mice were supplied with water containing 10 μg/mL of the AuNRs@SiO2:RBTIC@ShTxB particles for 12h. The mice were euthanized, and the tongues were dissected and photographed. Then, tongues were irradiated with an 808 laser for 5 min. Tissue sections after irradiation were processed and analyzed as previously described.
Student’s t-test analysis was used for statistical analysis. In Figure 3, significance that was established for a (*) p < 0.025 or a (**) p < 0.005. Quantitative results are expressed as mean values with their corresponding standard error bars. These statistical analyses were done using SPSS, version 19.0.