Diels–Alder “Clickable” Biodegradable Nanofibers: Benign Tailoring of Scaffolds for Biomolecular Immobilization and Cell Growth

Abstract

Biodegradable polymeric nanofibers have emerged as promising candidates for several biomedical applications such as tissue engineering and regenerative medicine. Many of these applications require modification of these nanofibers with small ligands or biomolecules such as peptides and other growth factors, which necessitates functionalization of these materials in mild and benign fashion. This study reports the design, synthesis, and functionalization of such nanofibers and evaluates their application as a cell culture scaffold. Polylactide based copolymers containing furan groups and triethylene glycol (TEG) units as side chains were synthesized using organocatalyzed ring opening polymerization. The furan moiety, an electron rich diene, provides "clickable" handles required for modification of nanofibers since they undergo facile cycloaddition reactions with maleimide-containing small molecules and ligands. The TEG units provide these fibers with hydrophilicity, enhanced biodegradability, and antibiofouling characteristics to minimize nonspecific adsorption. A series of copolymers with varying amounts of TEG units in their side chains were evaluated for fiber formation and antibiofouling characteristics to reveal that an incorporation of 7.5 mol % TEG-based monomer was optimal for nanofibers containing 20 mol % furan units. Facile functionalization of these nanofibers in a selective manner was demonstrated through attachment of a dienophile containing fluorophore, namely, fluorescein maleimide. To show efficient ligand-mediated bioconjugation, nanofibers were functionalized with a maleimide appended biotin, which enabled efficient attachment of the protein, Streptavidin. Importantly, the crucial role played by the TEG-based side chains was evident due to lack of any nonspecific attachment of protein to these nanofibers in the absence of biotin ligand. Furthermore, these nanofibers were conjugated with a cell adhesive cyclic peptide, cRGDfK-maleimide, at room temperature without the need of any additional catalyst. Importantly, comparison of the cell attachment onto nanofibers with and without the peptide demonstrated that fibers appended with the peptides promoted cells to spread nicely and protrude actin filaments for enhanced attachment to the support, whereas the cells on nonfunctionalized nanofibers showed a rounded up morphology with limited cellular spreading.

Achieving Controlled Biomolecule–Biomaterial Conjugation

“…Furan-maleimide additions have predominantly found use as a method to cross-link synthetic or natural polymers for the construction of core biomaterial scaffolds ( Figure 8 d). 172 , 262 − 265 Examples of such reactions being used for biomolecule conjugation are rare, 266 , 267 in part due to the rapid and well-established reaction of maleimides with thiols, as described above in section 4.2.1 . Diels–Alder conjugations are therefore not suitable for functionalization in the presence of thiolated biomolecules or materials.…”

Smart ECM-Based Electrospun Biomaterials for Skeletal Muscle Regeneration

“…In a recent work, polylactide (PLA)-based copolymers containing furan groups and triethylene glycol (TEG) were synthesized and electrospun to yield nanofibers [ 82 ]. After electrospinning, these nanofibers were conjugated with cyclic peptide, cRGDfK- maleimide via Diels-Alder reaction ( Figure 2 e).…”

“…After electrospinning, these nanofibers were conjugated with cyclic peptide, cRGDfK- maleimide via Diels-Alder reaction ( Figure 2 e). The presence of RGD peptide promoted cell adhesion and proliferation of L929 mouse fibroblasts suggesting that the proposed scaffolds were biocompatible and also provided a highly cytocompatible environment [ 82 ].…”

Orthogonally “Clickable” Biodegradable Nanofibers: Tailoring Biomaterials for Specific Protein Immobilization

“…In our recent study, a furan-bearing PLA was synthesized via organo-catalyzed ROP and electrospun into Diels–Alder clickable nanofibers. 23 These nanofibers were modified with RGD peptide and found to promote cell attachment and proliferation. Although the Diels–Alder reaction with a furan-based system offers several advantages, it is limited in its ability to conjugate molecules bearing maleimide groups.…”