Synthesis and Characterization of an Array of Elastin-like Polypeptide–Polyelectrolyte Conjugates with Varying Chemistries and Amine Content for Biomedical Applications

Abstract

Many structural variants of elastin-like polypeptides (ELPs), the genetically engineered equivalents of part of human elastin, currently are being investigated for drug delivery and tissue engineering. Here, we report preparation of six different aminated ELP conjugates via two strategies. In the first, a direct linking strategy was used to couple hydrophobic ELP with either polyethyleneimine, polylysine, or polyarginine. In the second, conjugates were made by attaching ELP onto the reactive polymer, poly(2-vinyl-4,4-dimethyl azlactone), and then exhaustively reacting residual azlactone groups with either ethylenediamine, 1,4-butanediamine, or arginine. Molecular size and chemistry of the resulting six aminated-ELP conjugates were confirmed through gel electrophoresis, FTIR spectroscopy, and mass spectrometry. Dynamic light scattering analysis showed that the conjugates prepared using the "direct reaction scheme" formed small aggregates as well as retained their inverse volume-phase transition temperature behavior. The conjugates prepared using the "reactive polymer linker scheme" also retained this transition temperature behavior. o-Phthalaldehyde assay was used to measure the relative primary amine content of the ELP conjugates. Overall, we prepared an array of aminated-ELPs with independently varying amine content and chemistry (i.e., the same amine content for different materials and different amine contents for the same material). Synthesis of such amphiphilic ELP structures that otherwise cannot be prepared through genetic engineering has the potential to further extend the versatility of the ELPs for many biomedical applications.

Effect of amine content and chemistry on long‐term, three‐dimensional hepatocyte spheroid culture atop aminated elastin‐like polypeptide coatings

“…In the so‐called “Direct Reaction Scheme,” ELP (MW = 17,000 Da) was directly conjugated to different polycations, including PEI (Sigma, St Louis, MO, MW = 1200 Da), polylysine (Sigma, MW = 1000–5000 Da), or polyarginine (Sigma, MW = 5000–10,000 Da) to create an Aminated‐ELP conjugate. In the “Reactive Polymer Linker Scheme,” ELP was conjugated to the reactive polymer, poly(2‐vinyl‐4,4‐dimethylazlactone) (PVDMA), which was synthesized by reversible addition‐fragmentation chain transfer polymerization and characterized as described previously (MW = 5420 Da, approximately 30 azlactone moieties, dispersity = 1.13) . Analysis by comparison to protein ladder assay shows that nucleophilic addition of ELP to PVDMA tethers 1 or 2 ELPs to the PVDMA, which allows small‐molecule (protected) electrolytes—including N‐Boc ethylenediamine, N‐Boc‐1,4‐butanediamine, or l ‐arginine (all Sigma)—to be reactively coupled through nucleophilic addition, which after deprotection yields the ELP–PVDMA conjugates.…”

“…In the “Reactive Polymer Linker Scheme,” ELP was conjugated to the reactive polymer, poly(2‐vinyl‐4,4‐dimethylazlactone) (PVDMA), which was synthesized by reversible addition‐fragmentation chain transfer polymerization and characterized as described previously (MW = 5420 Da, approximately 30 azlactone moieties, dispersity = 1.13) . Analysis by comparison to protein ladder assay shows that nucleophilic addition of ELP to PVDMA tethers 1 or 2 ELPs to the PVDMA, which allows small‐molecule (protected) electrolytes—including N‐Boc ethylenediamine, N‐Boc‐1,4‐butanediamine, or l ‐arginine (all Sigma)—to be reactively coupled through nucleophilic addition, which after deprotection yields the ELP–PVDMA conjugates. It is worth noting that the nucleophilic addition of primary amines to the azlactone rings of PVDMA proceeds without by‐product formation .…”

“…Analysis by comparison to protein ladder assay shows that nucleophilic addition of ELP to PVDMA tethers 1 or 2 ELPs to the PVDMA, which allows small‐molecule (protected) electrolytes—including N‐Boc ethylenediamine, N‐Boc‐1,4‐butanediamine, or l ‐arginine (all Sigma)—to be reactively coupled through nucleophilic addition, which after deprotection yields the ELP–PVDMA conjugates. It is worth noting that the nucleophilic addition of primary amines to the azlactone rings of PVDMA proceeds without by‐product formation . OPA fluorescence assay (Thermo Scientific, Pittsburgh, PA) was used to measure primary amine content of Aminated‐ELPs.…”

“…In the “Direct Reaction Scheme,” ELP was directly conjugated to PEs to yield ELP‐PEI, ELP‐polylysine, and ELP‐polyarginine conjugates. In the second strategy, we refer to as “Reactive Polymer Linker Scheme,” ELP was conjugated to the reactive polymer, poly(2‐vinyl‐4,4‐dimethyl azlactone (PVDMA), which was subsequently reacted with various small electrolyte molecules to yield ELP‐PVDMA‐ethylenediamine, ELP‐PVDMA‐butanediamine, and ELP‐PVDMA‐arginine conjugates . The small electrolyte molecules were purposefully chosen to create equivalent molecular structures between the Direct Reaction Scheme and the Reactive Polymer Linker Scheme.…”

Responsive hybrid (poly)peptide–polymer conjugates

“…106 Another recent study explored the conjugation of ELPp to pol(lysine ) , poly(arginine), and poly(ethyleneimine), as well as to poly(2-vinyl-4,4-dimethyl azlactone) to form nanoparticles with potential in drug delivery. 107 While SPPS, ROP and recombinant methods are successful, the development and application of hydrophobic peptides and their inclusion into peptide-polymer conjugates remains a synthetic challenge. 42 …”