MUC1-Targeted Cancer Cell Photothermal Ablation Using Bioinspired Gold Nanorods

<div><p>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.</p></div

Schematic illustration of MUC1 antibody-conjugated gold NRs (anti-MUC1-BSA-PD-NRs) with a polydopamine adlayer and BSA coating.

<p>MUC1 antibodies serve as novel targeting constructs in the application of plasmonic photothermal therapy (A). Probing MUC1 targeting at underglycosylated N- and C-terminal domains in various epithelial carcinomas will expand our cancer-targeting repertoire with the potential for synergistic therapeutic effects (B, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.ref012" target="_blank">12</a>]).</p

Bovine serum albumin (BSA) coating on polydopamine-primed gold NRs.

<p>(A) Electron microscopy of BSA-PD-NRs in secondary electron mode. Scale bar = 600 nm; Inset = 50 nm; (B) Circular dichroism of modified vs. unmodified gold NRs. BSA-modified NRs were modified with 10 or 20 mg/mL BSA, as indicated. The concentration of the BSA control was 0.25 mg/mL. Protein denaturation into β-sheet formation is indicated on PD-treated NRs modified with a sub-optimal concentration of BSA. Otherwise, BSA secondary structure is preserved, as quantified by the respective α-helical propensity of each modification.</p

Photothermal therapy of breast and oral cancer cells.

<p>Photoablation of cells with NKT Photonics SuperK Versa near-infrared (NIR) light source (480–850 nm). Cells were imaged with calcein AM (live, green) and propidium iodide (dead, red) to visualize photoablation efficiency following treatment with increasing doses of AuNRs. Due to the delamination of dead cells, no red staining is present in the treated regions of interest. In the MCF-7 cell line, this dead staining was occasionally observed prior to delamination (Fig I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.s001" target="_blank">S1 File</a>). Scale bar = 100 μm.</p

Darkfield imaging.

<p>AuNR uptake by cells detected by darkfield scattering imaging of MUC1-overexpressing SCC15 oral or MCF-7 breast cancer cells following treatment with MUC1-modified NRs. Scale bar = 50 μm.</p

AuNR zeta-potential measurements.

<p>Decreasing zeta-potential measurements verify successive surface modifications.</p><p>*IgG—AlexaFluor 633 goat anti-mouse immunoglobulin.</p><p>AuNR zeta-potential measurements.</p

Anti-MUC1 conjugation efficiency.

<p>(A) LSPR peak red shifting is observed with sequential BSA and anti-MUC1 modifications. (B) ELISA reveals total incorporation of MUC1-N and—C antibodies was achieved (* p < 0.05) only in PD-primed NRs additionally stabilized via a BSA layer. Near 100% loading in MUC1-C-BSA-NR samples has no practical significance as antibody addition induced complete NR aggregation (#). To quantify the differential MUC1 expression profiles in our cell lines, cells were treated with PE-conjugated MUC1 antibodies and analyzed via flow cytometry for their mean fluorescence intensities (MFI) against cells labeled with isotype controls. Both MCF-7 (MUC1⁺⁺) and SCC15 (MUC1⁺) cells demonstrated an enhanced MFI in agreement with literature reports [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.ref067" target="_blank">67</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.ref068" target="_blank">68</a>]. As expected, MUC1-deficient MDA-MB-231 (MUC1^-) cells did not show an appreciable increase in anti-MUC1 PE labeling (Fig F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128756#pone.0128756.s001" target="_blank">S1 File</a>).</p

Two-photon luminescence imaging.

<p>Two-photon luminescence (TPL) confocal imaging of MCF-7 and MDA-MB-231 breast cancer cells 24 h post treatment with NRs. Brightfield (BF, grayscale) and AuNR TPL images (red) reveal specific targeting in MUC1⁺ MCF-7 cells. Scale bar = 50 μm.</p

Quantification of breast and oral cancer photothermal therapy.

<p>Analysis of normalized viable cell areas reveals MUC1-dependent therapy vs. non-targeted BSA-PD-NRs in MCF-7 and SCC15 cell lines (* p < 0.05, ** p < 0.01, *** p < 0.001). Two-way ANOVA reveals a statistically significant impact of dose (p < 0.0001) and treatment (p < 0.0001). The interaction of dose and treatment was found to be statistically significant only for MCF-7 cells (p < 0.001).</p