Achieving Controlled Biomolecule-Biomaterial Conjugation

Abstract

The conjugation of biomolecules can impart materials with the bioactivity necessary to modulate specific cell behaviors. While the biological roles of particular polypeptide, oligonucleotide, and glycan structures have been extensively reviewed, along with the influence of attachment on material structure and function, the key role played by the conjugation strategy in determining activity is often overlooked. In this review, we focus on the chemistry of biomolecule conjugation and provide a comprehensive overview of the key strategies for achieving controlled biomaterial functionalization. No universal method exists to provide optimal attachment, and here we will discuss both the relative advantages and disadvantages of each technique. In doing so, we highlight the importance of carefully considering the impact and suitability of a particular technique during biomaterial design.

Figure 1

Key covalent methods by which biomolecule-material conjugation can be achieved. Each shall be discussed in detail during this review.

Figure 2

Major developments in biomaterial functionalization over the past 20 years. Many conjugation techniques have first been reported for the functionalization of proteins with small molecules, before later being translated to biomaterial derivatization.

Figure 3

EDC/NHS couplings are most commonly used to undertake amide couplings via a two-step process, first activating the carboxylic acid at pH 5, and subsequently undertaking conjugation at pH 8. However, the reaction is associated with side reactions which limit conjugation efficiency.

Figure 4

Imine formation is the rate-limiting step during reductive amination. Sodium borohydride is able to reduce the aldehydes which dominate and therefore fails to generate the desired conjugate. In contrast, sodium cyanoborohydride selectively reduces imines, leading to the formation of stable amine-conjugates.

Figure 5

N-terminal specific conjugation can be used to functionalize 2-pyridinecarboxaldheyde (2PCA) hydrogels with native ECM proteins. Increased cell spreading is observed with increased scaffold functionalization with (a) collagen, (b) fibronectin, and (c) laminin. Adapted from ref (95). Copyright 2016 with permission from Elsevier.

Figure 6

The rate of thiol Michael addition is dependent on the electrophilicity of the acceptor. The more electron-deficient an alkene, the more susceptible it is to nucleophilic attack.

Figure 7

Maleimide adducts can undergo retro-Michael addition and thiol-exchange in the presence of exogenous thiols. (a) Decrease in storage modulus of maleimide–thiol cross-linked hydrogels following incubation with different concentrations of glutathione (GSH); (b) increased hydrogel swelling as a result of decreased cross-linking density. Adapted from ref (130) with permission of The Royal Society of Chemistry.

Figure 8

Key advantages and limitations of cycloaddition reactions used for biomolecule-material conjugation. (a) Copper-catalyzed azide–alkyne cycloaddition (CuAAC); (b) strain-promoted azide–alkyne cycloaddition (SPAAC); (c) inverse-electron demand Diels–Alder reaction (IEDDA); and (d) furan-maleimide Diels–Alder reaction.

Figure 9

Furan-functionalized hyaluronic acid hydrogels can be cross-linked with a dimaleimide-functionalized peptide via Diels–Alder cycloaddition. MMP-cleavable peptides enable the migration of seeded breast cancer cells through the gel. Adapted with permission from ref (269). Copyright 2015 John Wiley and Sons.

Figure 10

Addition of amines to aldehydes to form imines is a reversible process, with equilibrium strongly favoring the starting materials in aqueous solution. In contrast, hydrazones and oximes are less susceptible to hydrolysis, providing the possibility for stable, but reversible conjugation.

Figure 11

Reversibility of hydrazone formation enables materials to possess self-healing properties. (a and b) Dye-loaded, hydrazone cross-linked PEG-hydrogels are cut in half; (c–e) incubation for 7 h is able to merge two halves placed together via reformation of hydrazone linkages at the gel interface. Adapted with permission from ref (273). Copyright 2010 American Chemical Society.

Figure 12

HRP/H₂O₂ treatment can lead to the formation of diphenol linkages between tyrosine containing peptides and phenol-capped synthetic polymers. In doing so, hydrogels bearing different levels of pendant RGD peptides can be produced. (a–d) Phase contrast microscopy images of seeded MC3T3-E1 cells at different RGD grafting densities; (e–h) live/dead assay, green, living cells and red, dead cells; (i–l) F-actin (red, phalloidin) and nuclei staining (blue, Hoechst 33258). Adapted from ref (317) with permission of The Royal Society of Chemistry.

Figure 13

Conjugation mechanism of (a) photoacrylate cross-linking; (b) photo thiol–ene reaction; and (c) photo thiol–yne diaddition.

Figure 14

(a) Alloc- and (b) norbornene-handles are most widely used for photo thiol–ene reactions and can be used to create 3D patterns of pendant fluorescently labeled peptides. Panel a is adapted with permission from ref (198). Copyright 2010 American Chemical Society. Panel b is adapted with permission from ref (117). Copyright 2009 John Wiley and Sons.

Figure 15

Two-photon activation can be used to induce photo thiol–ene reactions with precise 3D resolution. In doing so, intricate patterns of multiple fluorescently labeled peptides can be sequentially introduced into an alloc functionalized hydrogel. Scale bar = 100 μm. Adapted by permission from Springer Nature: ref (234). Copyright 2011.

Figure 16

Comparison of (a) lysozyme and (b) TGF-β; bioactivity following UV-irradiation in the presence of either acrylate-cross-linking or thiol–ene photopolymerization reactive species. Irradiation was continued until gelation occurred −180 s for acrylate cross-linking, compared to just 10 s for thiol–ene reaction. Reproduced with permission from ref (420). Copyright 2012 American Chemical Society.

Figure 17

Two-photon induced decaging of thiol-bromocoumarins enables photopatterning of agarose hydrogels. Through sequential deprotection-conjugation steps, multiple peptides/proteins can be patterned with high 3D resolution. (a and b) 3D projections of patterned hydrogels from different angles, labeled with fluorescent barstar (green) and streptavidin (red). Adapted by permission from Springer Nature: ref (463). Copyright 2011.

Figure 18

(a) Illustration of a VEGF-mimicking peptide amphiphile, self-assembling to form nanofibres; (b) quantification of muscle capillary density within a murine hind-leg ischemia model following the administration of peptide amphiphile (VEGF PA), VEGF mimicking peptide, a mutant amphiphile, or control treatment, based on the staining of CD31 positive capillaries; Reproduced with permission from ref (497). Copyright 2011 National Academy of Sciences.

Figure 19

(a) Host–guest interactions between cyclodextrin and naphthyl- or adamantane-functionalized peptides allow alginate functionalization; Confocal microscope images of 3T3 fibroblasts on functionalized substrates: (b) without peptide, (c) 10 μM naphthyl-RGDS, and (d) 10 μM adamantane-RGDS. Scale bar = 10 μm. (e) Quantification of cell spreading on substrates in the presence of various guests. Adapted with permission from ref (506). Copyright 2013 John Wiley and Sons.

Figure 20

Examples of some of the conjugation-handles that can be site-selectively incorporated into proteins via codon reassignment. These include reactive motifs for nucleophilic and photo thiol–ene reactions, cycloadditions, aldehyde functionalization, host–guest chemistry, and photocaging.