MUC1-Targeted Cancer Cell Photothermal Ablation Using Bioinspired Gold Nanorods.

Recent studies have highlighted the overexpression of mucin 1 (MUC1) in various epithelial carcinomas and its role in tumorigenesis. These mucins present a novel targeting opportunity for nanoparticle-mediated photothermal cancer treatments due to their unique antenna-like extracellular extension. In this study, MUC1 antibodies and albumin were immobilized onto the surface of gold nanorods using a “primer” of polydopamine (PD), a molecular mimic of catechol- and amine-rich mussel adhesive proteins. PD forms an adhesive platform for the deposition of albumin and MUC1 antibodies, achieving a surface that is stable, bioinert and biofunctional. Two-photon luminescence confocal and darkfield scattering imaging revealed targeting of MUC1-BSA-PD-NRs to MUC1+ MCF-7 breast cancer and SCC-15 squamous cell carcinoma cells lines. Treated cells were exposed to a laser encompassing the near-infrared AuNR longitudinal surface plasmon and assessed for photothermal ablation. MUC1-BSA-PD-NRs substantially decreased cell viability in photoirradiated MCF-7 cell lines vs. MUC1- MDA-MB-231 breast cancer cells (p < 0.005). Agents exhibited no cytotoxicity in the absence of photothermal treatment. The facile nature of the coating method, combined with targeting and photoablation efficacy, are attractive features of these candidate cancer nanotherapeutics.

mM bicine buffer or ultrapure deionized water) were used. Scans were performed in 10 mm pathlength cuvettes over a 400-1100 nm spectral range in 1 nm increments. AuNR samples were analyzed immediately after synthesis and after up to five cycles of redispersion and centrifugation in water. Longitudinal peak absorbance intensities were normalized to the initial value.

X-Ray Photoelectron Spectroscopy (XPS)
Gold substrates were prepared by sputtering 100 nm of Au onto Si wafer chips modified with a 15 nm Ti adhesion layer (Au/Ti/Si substrates). Samples were incubated in 1 ml of dopamine (0.1 mg/ml) and/or albumin solutions (20 mg/ml) for 30 minute periods with sonication in sealed 24-well plates. Substrates that were subjected to successive coating (BSA-PD) were transferred to a fresh well with each modification. The cationic surfactant is crucial in the seed-mediated synthesis of AuNRs and its positive charge is expected to strongly affect the deposition of the subsequent polydopamine and albumin protein layers. Therefore, to better represent the conditions during gold nanorod surface modification, Au/Ti/Si substrates were first incubated in 0.2 M cetyltetraammonium bromide (CTAB).
An Omicron ESCALAB XPS (Taunusstein, Germany) equipped with a monochromated 300 W Al Kα (1486.8 eV) X-ray source and 1.5 mm circular spot size under ultrahigh vacuum (<10 -8 Torr) was used to acquire spectra at a takeoff angle of 45°. Survey scans were acquired at 0.5 eV resolution over a binding energy range of 0-1100 eV. Detailed scans were acquired at 0.05 eV resolution for C 1s, O 1s, N 1s, and Au 4f emissions. Spectra were calibrated to the C 1s (C-C) peak at 284.6 eV.

Flow Cytometry
MCF7 and MDA-MB-231 cells were grown to confluence and harvested via trypsinization. Cells were rinsed and resuspended at 1x10 6 cells/ml in media. The cell suspension (400 µl) was aliquoted and mixed with 100 µl of modified AuNR solutions prepared by pellet dilution in 2 ml PBS after three centrifugation steps. Propidium iodide was added to each sample to achieve a final concentration of 2 µg/ml. Samples were allowed to incubate at 37°C for 30 minutes prior to acquisition with a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). A minimum of 20,000 events were collected and gated to exclude debris. To confirm MUC1 status, MCF7, MDA-MB-231, and SCC15 cells were harvested, fixed in 4% paraformaldehyde, incubated with PE-conjugated MUC1-N antibodies, washed, and analyzed for shifts in mean fluorescence intensities compared to unlabeled Isotype controlled counterparts. Data was processed with BD FACSDiva (San Jose, CA) and FlowJo (Ashland, OR) software suites.

Gel Electrophoresis
Antibody binding was assessed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Supernatants were separated from BSA-PD-NRs and BSA-NRs treated with 0.4 μg IgG-AlexaFluor 633 via centrifugation. Supernatants were mixed to a final concentration of 2.5% sample and 50% Laemmli sample buffer and heated at 95°C for 5 minutes. Antibody losses in sample supernatants were identified by electrophoresis with a separating gel containing a 4-15% Tris-HCl polyacrylamide gradient. Gels were stained with Sypro Ruby and imaged with a Typhoon 94000 fluorimager (GE Healthcare).