Figure 2. Nanoscopic presentation of VCAM-1 facilitates attachment and diminishes spreading of human melanoma cells.

(a) Representative scanning electron microscopy (SEM) image of gold nanopatterns (Au-NPs) on glass (d=98 ±11nm). (b) Schematic of biofunctionalization: Via its C-terminal 6His tag, VCAM-1 is immobilized in a site-directed manner to NTA-groups bound on gold nanoparticles (Au-NPs). (c) The subsequent steps of biofunctionalization on gold were monitored by Quartz Crystal Microbalance with Dissipation (QCMD) measurements. The frequency/dissipation changes show successful conjugation of the VCAM-1-molecule. (d) Immunofluorescence image of the border-region between nanostructured and non-nanostructured areas using a fluorescent antibody directed against VCAM-1, which creates a sharply demarcated line (margin of functionalization; left photomicrograph). The clear contrast indicates immobilization of VCAM-1 on the Au-NPs and shows a low nonspecific background. The right hand phase contrast image again depicts the margin of functionalization and demonstrates that A375-melanoma cells adhere almost exclusively to the side functionalized with VCAM-1 nanopatterns but not to the pegylation side. (e) Defined gold nanoparticle matrices were bio-functionalized with VCAM-1 yielding ligand presentations at the indicated densities. The spaces between the ligand sites were passivated by (PLL-g-PEG) without RGD. Human melanoma cells adhered to these matrices for 45 min, and the matrices were then washed in a standardized fashion. (f) Melanoma cells which had firmly adhered to the matrices depicted in (e) were quantitated microscopically. The values shown represent mean numbers from 10 standardized microscopic fields (SD). *indicates p<0.05, and *** indicates p<0.001. The experiment was repeated with similar results. (g) Matrices with nanoscopically defined VCAM-1 (left), RGD motifs in-between the passivating pegylation (middle) or both (right, bifunctional matrix) were generated as detailed in Material and Methods. Representative phase-contrast photomicrographs of A375-melanoma cells on nanoscopic-VCAM-1 (65 nm) after a washing-step with PBS for 10 minutes showing attachment but no spreading on nanoscopic VCAM-1 (left, upper panel), full spreading when RGD motifs are interspersed within the pegylation (middle panel) and reduced spreading when nanosopic VCAM-1 and RGD are combined in a bifunctional matrix (right), respectively. (h) Flow cytometry histograms depicting the melanoma cell expression of integrins relevant for binding to RGD (α₅β₁, α_Vβ₃) or VCAM-1 (α₄β₁).