Cell interactions with hierarchically structured nano-patterned adhesive surfaces

Abstract

The activation of well-defined numbers of integrin molecules in predefined areas by adhesion of tissue cells to biofunctionalized micro-nanopatterned surfaces was used to determine the minimum number of activated integrins necessary to stimulate focal adhesion formation. This was realized by combining micellar and conventional e-beam lithography, which enabled deposition of 6 nm large gold nanoparticles on predefined geometries. Patterns with a lateral spacing of 58 nm and a number of gold nanoparticles, ranging from 6 to 3000 per adhesive patch, were used. For α(v) β(3)-integrin activation, gold nanoparticles were coated with c(-RGDfK-)-thiol peptides, and the remaining glass surface was passivated to prevent non-specific protein adsorption and cell adhesion. Results show that focal adhesion formation is dictated by the underlying hierarchical nanopattern. Adhesive patches with side lengths of 3000 nm and separated by 3000 nm, or with side lengths of 1000 nm and separated by 1000 nm, containing approximately 3007 ± 193 or 335 ± 65 adhesive gold nanoparticles, respectively, induced the formation of actin-associated, paxillin-rich focal adhesions, comparable in size and shape to classical focal adhesions. In contrast, adhesive patches with side lengths of 500, 250 or 100 nm, and separated from adjacent adhesive patches by their respective side lengths, containing 83 ± 11, 30 ± 4, or 6 ± 1 adhesive gold nanoparticles, respectively, showed a significant increase in paxillin domain length, caused by bridging the pattern gap through an actin bundle in order to mechanically, synergistically strengthen each single adhesion site. Neither paxillin accumulation nor adhesion formation was induced if less than 6 c(-RGDfK-)-thiol functionalised gold nanoparticles per adhesion site were presented to cells.

Fig. 1

A typical substrate design of hierarchically patterned substrates: (A) a schematic drawing of the substrate design. (B) A SE micrograph of the pattern fields: each field has a side length of approximately 100 μm. (C) A phase-contrast micrograph of REF52 cells plated for 3 h on hierarchical patterns shown in (B). (D) A close-up of the pattern field containing 3000 nm large squares, separated by 3000 nm. (E)–(H) Gold nanoparticles arranged on 1000 nm, 500 nm, 250 nm and 100 nm squares. The number in the top right corner corresponds to the numbers given in (B) and (C). (I) A schematic for the biofunctionalization of interfaces for single integrin activation.

Fig. 2

(A) A live cell fluorescence microscopy image of a REF52-YFP-paxillin cell plated for 2 h on a 50 μm square divided into 500 × 500 nm squares, separated by 500 nm. (B) A close-up of (A), illustrating the contact site formation on such squares. (C) The paxillin fluorescence intensity distribution on cellular adhesion sites.

Fig. 3

Close-ups of FA and cytoskeleton formation of REF52-YFP-paxillin cells plated for 3 h on hierarchically structured nanopatterns. Side lengths of squares: 3000 nm (row A), 1000 nm (row B), 500 nm (row C), 250 nm (row D), 100 nm (row E) and an extended nanopattern (row F). The cells were either fixed and fluorescently stained on glass substrates (columns 3) or fixed and critical point dried on silicon wafers (columns 4 and 5) on the respective patterns. The red lines and the yellow squares in column 3 highlight the positions of each adhesive patch. FA size is restricted by the underlying pattern geometry if patch sizes are 3 μm or 1 μm, as indicated by the red arrow in A1. On patch sizes ≤500 nm, adjacent paxillin domains are bridged by an overlying actin fiber, see the red arrow in C1. The borders between neighboring paxillin sites are blurred due to the spatial resolution of optical microscopy.

Fig. 4

(A) A phase-contrast micrograph of a fixed REF52 cell plated for 24 h on extended patterned interfaces including 6 nm-sized gold nanoparticles with lateral spacings of ~58 nm. (B)–(D) Scanning electron micrographs show parts of cells on such biofunctionalized extended nanopatterns. The inset in (C) shows a close-up view of ultra-small cellular protrusions with diameters of 10–20 nm, and lengths of 30–50 nm, interacting with the activated gold nanoparticles. (D) SEM image, recorded with a tilt angle of 40°, depicting ultra-small cellular protrusions interacting with the c(-RGDfk-) adhesion sites. (E)–(G) Scanning electron micrographs of filopodial structures on biofunctionalized hierarchical nanopatterns (500 nm squares separated by 1000 nm, tilt angle 45°). The white arrow indicates an early filopodial structure, including its bending (indicated by the yellow arrow), red arrows indicate mature contact structures, and blue arrows show ultra-small cellular protrusions in contact with the adhesive gold nanoparticles.

Fig. 5

The length of actin-connected paxillin domains on different pattern fields: (A) 3 × 3 μm²), (B) 1 × 1 μm², (C) 0.5 × 0.5 μm ², (D) 0.25 × 0.25 μm², and (E) 0.1 × 0.1 μm², compared to (F) an extended nanopatterned substrate, with interparticle spacings of 58 nm.