Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin‐like polypeptide

Abstract

Abbreviations: A99, laminin α1 chain RGD-containing peptide (AGTFALRGDNPQG); A99-ELP-C, chemically conjugated CGG-A99 containing peptide to I30; A99-ELP-R, recombinant A99 fused to I30; ECM, extracellular matrix; ELP, elastin-like polypeptide; FOV, field of view; HDF, human dermal fibroblast; I30, elastin-like polypeptide with a sequence of (VPGIG) 30 ; LCST, lower critical solution temperature; MW, molecular weight; OD, optical density; RT, room temperature; T t , transition temperature. AbstractThe extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering. K E Y W O R D S biomaterial, elastin-like polypeptide, laminin peptide, synthetic biology, tissue engineering 6730 | TRUONG eT al.

Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

“…As a type of important biofunctional protein in the ECM, laminins can determine the properties of basement membranes and serve a crucial role in several cellular behaviors (20). Across the five types of laminin α chains, laminin α1 has been demonstrated to have a wide expression in human tissues, including skin, retina, neural stem cells and testes, and may have an important role in the biological behaviors of cells (40)(41)(42)(43). Furthermore, increasing studies have indicated that laminin α1 is upregulated in fibrotic diseases, such as pulmonary fibrosis, liver fibrosis and keloid fibrosis (44)(45)(46).…”

Cell-binding peptides on the material surface guide stem cell fate of adhesion, proliferation and differentiation

“…Although ECM proteins such as recombinant vitronectin, laminin-511, or laminin-521 are frequently immobilized on TCP dishes for hPSC culture and differentiation by the coating method, some researchers have also immobilized nonmodified peptides. 72,92,98,108,129,[173][174][175][176][177][178][179] There are also some reports that peptides were conjugated with synthetic polymers or proteins by chemical or genetic engineering methods; the resulting polymers are used as coating materials. Several studies in which stem cells are cultured on peptide-coated surfaces are summarized in Table S9 (ESI †).…”

“…A similar design of the engineered peptides was developed by several researchers. 175,176 For example, Truong et al developed the engineered peptide (A99-ELP-R), which contains laminin α1 chain-derived peptide (A99, AGTFALRGDNPQG) and elastin-like peptide ((VPGIG) 30 ) with a GMG joint segment (Fig. 7).…”

“…7). 175 Lee et al engineered globular domains 1–3 of laminin α5 with fusion to ELP, 176 an engineered peptide that should be called a protein because of its high molecular weight (70 kDa). The TCP dishes coated with 1 μg mL −1 of the engineered peptide (protein) could support hMSC adhesion with favorable stem cell characteristics.…”

Phase separation in synthetic biology

“…ELPs are de novo designed stimuliresponsive peptide polymers composed of repeat domains [157][158][159]. Proteins fused with ELPs have the property of thermoresponsiveness that is the lower critical solution temperature (LCST) phase behavior [156,160,161]. This approach is a good way to implement phase separation of target proteins.…”