Conjugation of antibodies to gold nanorods through Fc portion: synthesis and molecular specific imaging

Abstract

Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions, as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential, and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral, optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection.

Figure 1

Schematic representation of gold nanorods bioconjugation procedures from left to right: electrostatic adsorption of antibodies to CTAB layer on gold nanorods; coating of CTAB nanorods by charged polymers followed by electrostatic adsorption of antibodies; replacement of the CTAB layer using bifunctional ligands followed by covalent attachment of antibodies; the directional conjugation method proposed here that consist of replacement of the CTAB layer with mPEG-thiol molecules followed by directional attachment of antibodies.

Figure 2

Schematic illustration of the directional conjugation synthesis proposed here.

Figure 3. Schematic of the experimental set-up used to obtain combined ultrasound and photoacoustic images

Figure 4

a) Transmittance electron microscopy images of as prepared CTAB coated gold nanorods (left) and the nanorods after modification with mPEG-thiol molecules (right). b) Extinction spectra of as prepared gold nanorods (blue) and the nanorods after modification with either 2 kDa mPEG-thiol (red); clone c225 (purple) and RG16 (green) antibodies. c) Comparison of extinction spectra of as prepared gold nanorods (blue) and nanorods after ligand exchange using small molecular weight, 300 Da MW, mPEG-thiol molecules (orange). Aggregation of nanorods is evident from profound spectral changes (orange spectrum).

Figure 5

Cytotoxicity of as prepared CTAB coated and 2 kDa mPEG-thiol coated nanorods in different cell lines. The y-axis shows normalized cell viability. The viability of control samples is normalized to 100. Values statistically different (p < 0.05) from controls are labeled by *;values that do not show statistically significant difference from controls (p > 0.05) are labeled by #.

Figure 6

Optical characterization of molecular specificity of antibody conjugated gold nanorods in cell cultures. Columns from left to right: dark-field transmittance images; transmittance hyperspectral image with color coded peak wavelength in the 500 - 800 nm spectral region; transmittance hyperspectral image with color coded integrated absorbance in the 500 - 800 nm region; absorbance spectra integrated over the regions highlighted by red boxes in the previous column.

Figure 7

Combined ultrasound (US) and photoacoustic (PA) images of tissue mimicking cell phantoms. The phantom on the left consists of A431 cells labeled with anti-EGFR gold nanorods and the phantom on the right has unlabeled A431 cells. The plot at the bottom shows PA signal intensity integrated over the phantom area as a function of excitation wavelength.