Affibody-functionalized gold-silica nanoparticles for Raman molecular imaging of the epidermal growth factor receptor

Abstract

The affibody functionalization of fluorescent surface-enhanced Raman scattering gold-silica nanoparticles as multimodal contrast agents for molecular imaging specific to epidermal growth factor receptor (EGFR) is reported. This nanoparticle bioconjugate reports EGFR-positive A431 tumors with a signal nearly 35-fold higher than EGFR-negative MDA-435S tumors. The low-level EGFR expression in adjacent healthy tissue is 7-fold lower than in the positive tumors. Validation via competitive inhibition reduces the signal by a factor of six, and independent measurement of EGFR via flow cytometry correlates at R(2) = 0.92.

Figure 1

Preparation of NPs: a gold core with Raman-active molecules is coated with a silica shell for stability and SERS intensity. The NPs are first coated with a fluorophore (star) for high throughput cell-based assays (A) and remaining thiols react with a bis-maleimido short (n = 3) PEG (coil) cross-linker (B). After removing excess PEG, affibodies specific to EGFR or Her2 are bound to the NP surface (C). RT denotes room temperature reactions.

Figure 2

Validation of SERS NPs with Cultured Cells: EGFR positive (A431) and negative (MDA-435S) cell lines were labeled with fluorescent SERS NPs coated with Alexa Fluor 647 and EGFR affibody, and analyzed via FC. Controls include an isotype affibody (Her-2) and competitive inhibition (block) of A431.

Figure 3

Raman Intensity Maps: white-light images with Raman intensity maps superimposed indicate differences between various samples types. The dashed line indicates the area within the white-light image subjected to Raman mapping. Here, EGFR positive A431 cells are were incubated with normal saline (A), EGFR affibody unbound to NPs (B), native SERS NPs (C), PEGylated NPs (D), and NPs with an isotype control affibody (E). Both (F) and (G) show A431 cells incubated with NPs targeted to EGFR via an affibody, although (F) was previously incubated with free affibody (25 μg mL⁻¹) as a competitive inhibition control. Sample (H) is stained EGFR-negative MDA-435S cells with the EGFR-targeted NPs. Scale bar = 20 μm.

Figure 4

Independent Verification of EGFR Imaging by SERS NPs: five different cell lines with unique EGFR expression levels were analyzed by both FC (A) and Raman imaging (B), similar to Figure 3. The intensity of the two measurement modalities correlates above R ² = 0.90 suggesting the Raman molecular imaging may be used to quantitatively examine EGFR expression levels.

Figure 5

Validation of SERS NPs with explanted xenograft tumors: A) Representative spectra for A431 tumors labeled ex vivo with three different NP types: targeted (dots; via anti-EGFR affibody), targeted with competitive inhibition (solid), and untargeted (dashes; blank). Specific signals arise from the targeted NPs. B) Collections of spectra create Raman maps from whole tumors and signal quantitated in (C). Error bars represent the standard error and * indicates significance at p < 0.01.