Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Abstract

Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.

Collagen tethering of synthetic human antimicrobial peptides cathelicidin LL37 and its effects on antimicrobial activity and cytotoxicity

“…Peptide sequence and structure has been shown to influence AMP flexibility [17,19], secondary structure [20,22], orientation [17,20,22,24,25], and charge density [18,58]. Previous work on CBD-mediated collagen tethering has involved either high molecular weight, uncharged or globular molecules such as growth factors [34,35,60] and streptavidin [37], or peptides too small to adopt significant structure on their own, such as fibronectin-derived RGD peptides [61,62]. In these cases, CBD acted like an anchor without influencing peptide activities.…”

“…CBDs may increase the stability and retention of AMPs on collagen, by limiting peptide exposure to proteases [12,15,[17][18][19]66] and providing high affinity [50], specific binding onto collagen [50,61,67]. This increased stability is highly sought-after currently in both soluble and tethered AMP technologies [6,12,15,66].…”

“…Its lower cytotoxicity could be related to decreased overall charge of LL37 caused by addition of fCBD (net -1), or structural alterations caused by aggregation of fCBD moieties [61,65]. Despite the observed reduction in antimicrobial activity, the minimum therapeutic index of fCBD is still 2x (> 20 µM cytotoxicity / 10 µM MIC).…”

Dip‐pen nanolithography on SiOx and tissue‐derived substrates: comparison with multiple biological inks

“…It is important to note that preparation protocol did not alter the activity of the collagen before and after the DPN process. This has been verified through in vitro cell culture assay (Sistiabudi et al 2009). …”

Current Treatment Limitations in Age-Related Macular Degeneration and Future Approaches Based on Cell Therapy and Tissue Engineering

“…Although initially surface modification was performed in synthetic scaffolds, in the last years, same strategies have started to be translated into natural origin scaffolds. An example of that is provided by Sistiabudi and colleagues where they modify the inner collagenous layer of Bruch's membrane with a collagen binding motif, where bioactive molecules are anchored [167]. …”