Adhesion of retinal cells to gold surfaces by biomimetic molecules

Abstract

Background: Neural cell-electrode coupling is crucial for effective neural and retinal prostheses. Enhancing this coupling can be achieved through surface modification and geometrical design to increase neuron-electrode proximity. In the current research, we focused on designing and studying various biomolecules as a method to elicit neural cell-electrode adhesion via cell-specific integrin mechanisms.

Methods: We designed extracellular matrix biomimetic molecules with different head sequences (RGD or YIGSR), structures (linear or cyclic), and spacer lengths (short or long). These molecules, anchored by a thiol (SH) group, were deposited onto gold surfaces at various concentrations. We assessed the modifications using contact angle measurements, fluorescence imaging, and X-ray Photoelectron Spectroscopy (XPS). We then analyzed the adhesion of retinal cells and HEK293 cells to the modified surfaces by measuring cell density, surface area, and focal adhesion spots, and examined changes in adhesion-related gene and integrin expression.

Results: Results showed that YIGSR biomolecules significantly enhanced retinal cell adhesion, regardless of spacer length. For HEK293 cells, RGD biomolecules were more effective, especially with cyclic RGD and long spacers. Both cell types showed increased expression of specific adhesion integrins and proteins like vinculin and PTK2; these results were in agreement with the adhesion studies, confirming the cell-specific interactions with modified surfaces.

Conclusion: This study highlights the importance of tailored biomolecules for improving neural cell adhesion to electrodes. By customizing biomolecules to foster specific and effective interactions with adhesion integrins, our study provides valuable insights for enhancing the integration and functionality of retinal prostheses and other neural implants.

Keywords: RGD; YIGSR; biomimetics; cell-adhesion; neural electrode interface; regenerative medicine; retinal prostheses; tissue engineering.

FIGURE 1

Molecular structure of the biomolecules studied in this study. (A) Short linear RGD; Cys-Gly-Gly-Arg-Gly-Asp-D-Phe-Lys (CGGRGDfK). (B) Short cyclic RGD; Cys-Gly-Gly-cyclo(Arg-Gly-Asp-D-Phe-Ly), (CGG-c(RGDfK)). (C) Long-cyclic RGD; Cys-PolyProline(10)-cyclo(Arg-Gly-Asp-D-Phe-Ly), (C-PolyPro-c(RGDfK)). (D) Short YIGSR; Cys-Gly-Gly-Tyr-Ile-Gly-Ser-Arg (CGGYIGSR). (E) Long YIGSR; Cys-PolyProline(10)-Tyr-Ile-Gly-Ser-Arg (C-PolyPro-YIGSR). Red–Cys–an anchor to the gold surface, Black–short and long spacers (GG and PolyProline(10), respectively) and blue–the ligand head group (RGDfK and YIGSR).

FIGURE 2

Assembly of the biomolecules to gold surfaces. STED Fluorescent images of (A) untreated and (B) fluorescent RGD-NBD-coated glass-gold patterned surfaces revealed RGD assembly mainly in the gold areas. Scale bar 300 µm.

FIGURE 3

Contact angle measurements presenting gold surface modifications. (A–C) Contact angle images of untreated (A), short linear RGD coated (B), and short linear YIGSR (C) coated gold surfaces. (D) The water contact angle of gold surfaces coated with the various molecules (N = 3). *p < 0.05 compared with bare gold. (E) The effect of the biomolecule concentration on the contact angle (N = 3), p < 0.05.

FIGURE 4

Effect of various biomolecule coatings on the HEK293 cells. (A) Short linear RGD, (B) short cyclo-RGD, (C) long cyclo-RGD, (D) short YIGSR, (E) long YIGSR, and (F) DDW. Cells were seeded on the coated gold surface with a biomolecule concentration of 1/3 mg/mL and incubated for 24 h, followed by fixation and Hoechst staining. Scale bar 200 µm. (N = 18).

FIGURE 5

Effect of various biomolecule coatings on the retinal cells. (A) Short linear RGD, (B) short cyclo-RGD, (C) long cyclo-RGD, (D) short YIGSR, (E) long YIGSR, and (F) DDW only. Cells were seeded on the coated gold surface with a biomolecule concentration of 1/3 mg/mL and incubated for 24 h, followed by fixation and Hoechst staining. Scale bar 200 µm.

FIGURE 6

The effect of biomolecules on cell density. (A) HEK 293 cells and (B) rat retinal cells. Cells were seeded on gold surfaces coated with various biomolecules at several concentrations. Cell density was evaluated using a Leica LMD7 microscope and a binarization ImageJ algorithm. For each biomolecule, the cell density was normalized to a cell density of DDW. *Red star denotes significance compared to control, p<<0.05. **Black star denotes significance compared to a long cyclo-RGD [Poly-Pro-c (RGD)]-coated surface, p<<0.05.

FIGURE 7

The effect of the coating molecules on the cell surface area. (A) HEK293 cells and (B) rat retinal cells. The cells were incubated for 72 h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/mL) followed by fixation and staining. The average surface area from each biomolecule-coated surface was normalized to that of DDW. *p < 0.05, ***p < 0.001, compared to untreated (bare) gold.

FIGURE 8

Confocal images of HEK293 cells presenting the effect of coating biomolecules on focal adhesion spots. Cells were incubated for 72 h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/mL) followed by fixation and staining. (A) Short linear RGD, (B) short cyclo-RGD, (C) long cyclo-RGD, (D) short YIGSR, (E) long YIGSR, and (F) DDW only. Biomolecule concentration of 1/12 mg/mL. Yellow arrows denote spots of focal adhesions. Hoechst (Blue), genetically encoded GFP (green), Vinculin (FA, red), f-actin (magenta). Scale bar 20 µm.

FIGURE 9

Confocal images of rat-dissociated retinal cells presenting the effect of coating biomolecules on the focal adhesion spots. Cells were incubated for 72 h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/mL), followed by fixation and staining. (A) Short linear RGD, (B) short cyclo-RGD, (C) long cyclo-RGD, (D) short YIGSR, (E) long YIGSR, and (F) DDW only. Biomolecule concentration of 1/12 mg/mL. Yellow arrows denote spots of focal adhesions. Hoechst (blue), ViaFluor488 (green), Vinculin (FA, red), f-actin (magenta). Scale bar 25 µm.

FIGURE 10

The effect of coating molecules on the focal adhesion spots. (A) HEK293 cells and (B) retinal cells. The cells were incubated for 72 h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/mL) followed by fixation and staining. The number of bright Vinculin spots was counted manually. *p < 0.03, **p < 0.001.

FIGURE 11

qPCR analysis of the Effect of surface coating on the Relative normalized gene expression levels of adhesion integrins and focal adhesion proteins. (A) HEK293 cells. (B) Rat-dissociated retinal cells. Cells were incubated for 72 h after being seeded on various biomolecule-coated gold surfaces (1/6 mg/mL) before RNA extraction. Expression levels were normalized to the expression level of the GAPDH gene, used as a reference gene. *p < 0.05 compared to DDW.