Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications

Abstract

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.

Protease‐Sensitive, VEGF‐Mimetic Peptide, and IKVAV Laminin‐Derived Peptide Sequences within Elastin‐Like Recombinamer Scaffolds Provide Spatiotemporally Synchronized Guidance of Angiogenesis and Neurogenesis

“…The 1:1 IKVAV:ELR ratio, or 60% w/w, was selected based on a previous study by Pavia dos Santos et al., which showed that a 20% w/w IKVAV ELR concentration (in reality 40%, as each ELR backbone contains two IKVAV domains) resulted in longer neurite formation than those containing 10% or 5% IKVAV‐ELR. [ 33 ] An NHS‐Cyanine 5 fluorescent probe (Jena Bioscience GmbH, Germany) was 1:1 conjugated to the RGD‐azide‐ELR and VKV‐cyclooctyne‐ELR to produce the RGD‐azide‐Cy5‐ELR and VKV‐cyclooctyne‐Cy5‐ELR. De novo produced ELRs, namely VKV‐azide‐ELR, VKV‐cyclooctyne‐Cy5‐ELR, RGD‐azide‐IKVAV‐ELR, and RGD‐azide‐Cy5‐ELR, were characterized by proton nuclear magnetic resonance (1H‐NMR), matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) (Figures S6–S21, Supporting Information).…”

Bioresorbable Polymers: Advanced Materials and 4D Printing for Tissue Engineering

“…Amongst the genetically encoded polymeric sequences, ELPs (elastin-like polypeptides), also known as elastin-like recombinamers (ELRs), have been widely used for the development of thermosensitive block copolymers for several biomedical applications, mainly for release systems and tissue engineering [232][233][234][235][236][237][238]. Elastin-like polypeptides can undergo reversible phase transition induced by pH, temperature, or ionic strength, whose transition phase is directly dependent on the ELP's sequence transition temperature (Tt).…”

Amyloids as Building Blocks for Macroscopic Functional Materials: Designs, Applications and Challenges

“…ThT assay confirmed the presence of amyloid fibrils formed from the IKVAV sequence. Both in vitro and in vivo experiments demonstrated that the hydrogel promoted the growth of sensory neuron without noticeable cytotoxicity and inflammatory effects [ 126 ]. Deidda et al fused the amyloid peptide NSGAITIG from adenovirus fiber shaft to the RGD motif [ 127 ].…”