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. 2006 Jan 31;103(5):1215-20.
doi: 10.1073/pnas.0509739103. Epub 2006 Jan 24.

Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents

Glauco R Souza  1 , Dawn R Christianson, Fernanda I Staquicini, Michael G Ozawa, Evan Y Snyder, Richard L Sidman, J Houston Miller, Wadih Arap, Renata Pasqualini
Affiliations

Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents

Glauco R Souza et al. Proc Natl Acad Sci U S A. .

Abstract

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.

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Figures

Fig. 1.
Fig. 1.
Concept and biological/structural characterization of Au–phage and Au–phage–imid networks. (a) Strategy for Au assembly onto phage nanoparticles. Imid and the yellow spheres [Au nanoparticles (not drawn to scale); the Au particles have a diameter of 44 ± 9 nm, and the pVIII capsid peptide has a thickness of ≈6 nm]. (b) Vials of nanoparticle solutions: Au–phage hydrogel (Left) and suspension of purified Au–phage–imid (Right; suspended from hydrogels precursor). (c and d) Hydrogel formed with RGD-4C-displaying phage. (Scale bar, 20 μm.) (c) C17.2 murine neural stem cells cultured within hydrogel for 24 h. Cell accumulation followed by cell-induced network displacement is shown (arrows point to cells within the network). (d) Control hydrogel (no cells). (e) TEM of purified networks: Au–phage (Upper) and Au–phage–imid (Lower). (Scale bar, 500 nm; inset scale bar, 100 nm.) (f) Bacterial infection with purified Au–phage (Upper) and Au–phage–imid (Lower) networks; TU are shown for purified and functional Au–phage and Au–phage–imid solution and for unbound phage present in the supernatant from centrifuged network solutions.
Fig. 2.
Fig. 2.
Mechanism of assembly for Au–phage networks. (a) Light-absorption spectrum at various phage input (indicated in the legend) in the presence of 0.25 M NaCl (no phage, bottom curve). (b) Light extinction at 710 nm for Au–phage solutions as a function of phage input at various pH levels (10 mM boric acid, pH 5.2; 10 mM sodium borate buffer, pH 6.5; 10 mM sodium borate, pH 9.2; or 10 mM NaOH, pH 14.0). (c) Cartoon illustrating electrostatic interaction of Au (yellow spheres) with phage [elongated structures (not drawn to scale)]. Arrows point to pVIII major capsid protein and pIII minor capsid protein.
Fig. 3.
Fig. 3.
Optical and physical characterization of Au–phage networks. (a) Light-absorption spectrum of purified and suspended Au–phage–imid (dark blue) and Au–phage (red). (b) Temperature as a function of illumination (785-nm laser light) time of Au–phage–imid (blue) and Au–phage (red) solutions; the controls (○) are the solutions of Au (green), Au–imid (orange), and phage (cyan). The concentrations of all solutions carrying Au were normalized according to the area under the absorption region of the spectra (>475 nm). The solution temperature was measured with a digital temperature probe (Teflon-coated cables of type-K beaded sensor coupled to a Fisher traceable double thermometer with computer output from Fisher Scientific) immersed in 300 μl of solution and 5 mm away from the laser focal point. (c) SERS of Au–phage–imid (blue) and Au–phage (red) measured in water.
Fig. 4.
Fig. 4.
Immunofluorescence-based phage binding and internalization assay with cultured melanoma cells. The red color represents fluorescence related to the RGD-4C peptide, and the blue color shows fluorescence of DAPI-stained cell nuclei. Cells were incubated with different phage preparations, all carrying a phage input of 1.0 × 107 TU. (a) Au–RGD-4C-displaying phage. (b) Cells preincubated with the RGD-4C synthetic peptide (1.0 × 10–3 nM for 30 min) followed by addition of Au–RGD-4C. (c) RGD-4C phage (no Au). (d) Au–fd-tet (negative control). (e) Cells preincubated with the RGD-4C synthetic peptide (1.0 × 10–3 nM) followed by addition of Au–fd-tet (negative control). (f) fd-tet phage (negative control).
Fig. 5.
Fig. 5.
Confocal fluorescence. Shown are KS1767 cells incubated with phage preparations (input of 1.0 × 107 TU) and labeled with anti-fd bacteriophage antibody (red, first column), SYTOX green nucleic acid stain (green, second column), and an anti-β1 integrin antibody demarking the cell surface (blue, third column). The fourth column shows merged images: RGD-4C phage, Au–RGD-4C networks, and Au–RGD-4C-imid networks (controls for each of the respective RGD-4C phage preparations are shown in Fig. 8, which is published as supporting information on the PNAS web site). (Scale bar, 10 μm.)
Fig. 6.
Fig. 6.
Dark-field images (real color) of cell-bound Au–phage networks using light from a microscope mercury lamp. Confluent KS1767 cells incubated with phage preparations (input of 1.0 × 107 TU): Au–RGD-4C (gold color) (a) and Au–fd-tet (control insertless phage) networks (b). The blue color shows residual fluorescence from DAPI-stained cell nuclei.
Fig. 7.
Fig. 7.
Targeted cell detection by using SERS. (a) Cell-suspension scheme for SERS detection. Light from a laser diode (785 nm) was delivered through a fiber-optic probe to a suspension of KS1767 cells; the same fiber-optic probe collected and delivered the Raman signal. (b) SERS and spectra of cells incubated with Au–phage: Au–RGD-4C-imid (blue line), Au–fd-tet-imid (red line), and RGD-4C phage (green line; control, no Au) in suspension.
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