A sandcastle worm-inspired strategy to functionalize wet hydrogels

The study addresses the challenge of endowing hydrogels with diverse biofunctions without pre-installing specific reactive handles on the polymer network. Conventional approaches functionalize polymers before or during gelation by introducing matched reactive groups (e.g., azide–alkyne), limiting flexibility across different hydrogel chemistries and requiring custom syntheses for both substrates and ligands. Inspired by the sandcastle worm Pc-1 adhesive protein, which features a 1:1 molar ratio of DOPA and lysine, the authors designed a minimal adhesive tripeptide, DbaYKY (dibutylamine–DOPA–lysine–DOPA), hypothesizing it could universally tether functional molecules to wet hydrogels through catechol- and amine-mediated interactions. The goal is a simple, one-step, aqueous modification applicable to various hydrogels to impart antimicrobial activity, cell adhesion, and wound-healing functions with tunable degree and stable attachment.

The authors review two prevailing hydrogel functionalization routes: pre-gel polymer modification and in situ functionalization during gelation, both typically requiring orthogonal reactive sites (e.g., azide/alkyne), thus constraining generality. They note extensive work on mussel-inspired catechol chemistry for surface modification, where DOPA or DOPA–lysine motifs enable adhesion and can confer antifouling, antimicrobial, or cell-adhesive properties. However, direct, universal modification of wet hydrogels lacking specific reactive groups has remained unclear. Lessons from marine adhesives (mussels and sandcastle worms) indicate catechol–amine synergy enhances wet adhesion, and that adding amine groups (e.g., lysine) strengthens interactions. Pc-1 from sandcastle worms with 1:1 DOPA:lysine inspired a short sequence design; prior studies also show increased adhesion with longer Lys–DOPA sequences and that peptide order is less critical than composition in some adhesive polymers.

Design and synthesis: An amine-terminated DbaYKY tripeptide (DbaYKY–NH2) was synthesized on gram scale (5.3 g, 43% yield over 4 steps from Fmoc-DOPA(acetonide)-OH) and via a solid-phase route. DbaYKY–NH2 was converted in one step into initiators for four polymerizations: ATRP (initiator 1), RAFT (initiator 2), α-amino acid N-carboxyanhydride (NCA) polymerization (initiator 3), and anionic ring-opening polymerization of β-lactams (AROP; initiator 4). These produced functional polymers (Pol-1 to Pol-8) bearing terminal DbaYKY.

Hydrogel substrates: Five representative hydrogels were prepared: PEG (20 wt% PEGDA UV-crosslinked), PHEMA (HEMA with MBAA UV-crosslinked), PSBMA (zwitterionic SBMA with MBAA UV-crosslinked), PVA (10 wt% freeze–thaw physically crosslinked), and alginate (5 wt% ionically crosslinked with CaCl2). Discs of 4.5–8 mm diameter, 1 mm thick.

One-step modification protocol: Hydrogels (typically 6 mm) were immersed in 60 µL polymer solution (4.0 mg/mL; 0.093–0.27 mM) for 24 h at room temperature: water-soluble polymers in 100 mM Tris pH 8.5; insoluble polymers in Tris/MeOH (4:6). Afterward, thorough washing with water (three immediate rinses plus five exchanges over 24 h). For bulk functionalization, hydrogels were lyophilized prior to modification to enlarge pores.

Adhesion quantification by AFM-SMFS: DbaYKY (and controls YY, YKY, KYK, KKYY) were tethered to AFM cantilevers via PEG linkers. Single-molecule force spectroscopy measured rupture forces against PEG hydrogels in degassed 100 mM Tris pH 8.5, with 2 s contact at 1000 nm/s approach/retract. Non-specific and multiple-contact events were excluded.

Characterization of modification: XPS and ATR-FTIR on freeze-dried hydrogel surfaces verified successful tethering. Kaiser test qualitatively detected amine-containing polymers within PHEMA hydrogels. Stability was assessed by fluorescence retention using DbaYKY–OEG8–rhodamine after PBS shaking at room temperature and proteinase K incubation at 37 °C up to 7 days.

Quantification of grafting: Two approaches were used. (1) Fluorescamine assay for Pol-7 on dissolvable hydrogels (PVA in hot water; alginate in 5% sodium citrate). Concentrations were calculated from calibration curves and normalized to dry or wet hydrogel weights and to initial polymer dose to estimate efficiency. (2) Rhodamine assay for DbaYKY–OEG8–Rh across all hydrogels; fluorescence induced under acidic conditions (0.1 N HCl) was quantified with calibration to compute bound mass per hydrogel, functionalization degree (relative to wet/dry weight), and efficiency. Concentration dependence (0.0625–1 mg/mL) probed tunability.

Mechanistic probe: DbaYKY–Ac was incubated in Tris pH 8.5 up to 24 h and analyzed by HPLC, UV–Vis, and MALDI-TOF to detect catechol transformations (e.g., Michael additions) occurring under modification conditions.

Functional evaluations:

• Antibacterial activity: PEG and PVA hydrogels modified with Pol-1 or Pol-3 were challenged with E. coli (ATCC 25922) and S. aureus (ATCC 6538) suspensions (2 × 10^6 CFU/mL, 40 µL) for 2.5 h at 37 °C. Surviving CFU quantified; killing efficacy calculated versus unmodified controls. Durability assessed after pre-incubation with bacteria for 1, 3, and 7 days followed by re-challenge.
• Cell adhesion assays: NIH 3T3 fibroblasts were seeded on bare and Pol-7–modified hydrogels (all five types). LIVE/DEAD staining used for viability and coverage; for PEG, long-term adhesion and spreading were assessed at 1, 3, and 7 days with immunostaining for actin (FITC–phalloidin), vinculin (Alexa Fluor 555), and nuclei (DAPI). Controls included polymers lacking DbaYKY (e.g., Pol-11) to rule out entanglement.
• Interior functionalization: Lyophilized PHEMA hydrogels modified with Pol-7 were sectioned; cell adhesion on interior surfaces was assessed. Confocal imaging using DbaYKY–OEG8–rhodamine visualized penetration with/without lyophilization.
• In vivo wound healing: Full-thickness 8 mm circular dorsal wounds were created in SD rats (n=3; 6 wounds/rat; total n=18; groups: control, bare PVA, Pol-7–modified PVA; n=6 per group). Dressings applied with Tegaderm. Wound areas imaged on days 0, 3, 6, 9, 12; closure rates computed. Day 12 tissues harvested for H&E histology. Statistics: t-test and one-way ANOVA with Tukey post-test.

• DbaYKY adhesion strength: Single-molecule rupture force to PEG hydrogels had a median of ~223 pN (range ~40–760 pN). Control peptides showed lower adhesion: YY ~82 pN; YKY ~164 pN; KYK ~172 pN; KKYY ~203 pN. Two amines (Dba + Lys) and two catechols (two DOPAs) synergistically enhance adhesion; sequence order less critical than composition.
• Successful one-step modification: XPS and ATR-FTIR confirmed tethering of DbaYKY-terminated polymers (Pol-1, Pol-3, Pol-7) to PEG (and PVA) hydrogels, evidenced by increased N1s and characteristic Br3d/F1s/Cl2p peaks and new O–H, N–H, and amide C=O stretches. Polymers lacking DbaYKY (Pol-9, Pol-10, Pol-11) did not appreciably tether.
• Quantitative grafting levels and tunability: • Pol-7 on dissolvable hydrogels (fluorescamine): PVA ~0.0515% (dry wt), ~0.003% (wet wt), efficiency ~0.360%; ALG ~0.203% (dry wt), ~0.017% (wet wt), efficiency ~1.97%. • DbaYKY–OEG8–Rh on five hydrogels (0.5 mg/mL; rhodamine assay): dry-weight basis—PEG ~0.051%, PHEMA ~0.104%, PSBMA ~0.046%, PVA ~0.020%, ALG ~0.083%; wet-weight basis—PEG ~0.004%, PHEMA ~0.058%, PSBMA ~0.022%, PVA ~0.001%, ALG ~0.007%. Functionalization efficiency: PEG ~3.92%, PHEMA ~54.6%, PSBMA ~20.6%, PVA ~1.10%, ALG ~6.44%. Increasing modifier concentration increased grafting amount, demonstrating tunable functionalization. • Stability: Minimal loss of fluorescent signal after 1–7 days in PBS or in proteinase K at 37 °C, indicating stable attachment.
• Mechanistic observations: Under pH 8.5, DbaYKY formed new species consistent with catechol–amine Michael additions (UV–Vis peak ~360 nm; MALDI evidence of intra/intermolecular adducts), consistent with cross-linking that can increase modification density; no Schiff base intermediate observed by MALDI.
• Antibacterial efficacy: Pol-1 or Pol-3 modified PEG and PVA hydrogels achieved >99% killing of E. coli and S. aureus after 2.5 h exposure. After prior incubation with bacteria for 1, 3, or 7 days, re-challenged hydrogels retained high activity (>95% killing; most >99%), demonstrating durability.
• Cell adhesion: All bare hydrogels (PEG, PHEMA, PSBMA, PVA, ALG) were antifouling; Pol-7 modification induced robust NIH 3T3 adhesion and spreading across all types. On PEG, cells remained adherent and proliferated with healthy morphology through 7 days with actin stress fibers and vinculin focal adhesions. Pol-7 enabled adhesion whereas Pol-11 (no DbaYKY) did not, confirming DbaYKY-dependent tethering. Lyophilization enabled bulk functionalization, evidenced by cell adhesion on cut interior surfaces and rhodamine penetration into cross-sections only for lyophilized samples.
• Wound healing in vivo: Pol-7–modified PVA dressings significantly accelerated wound closure versus bare PVA and no-treatment controls, with near-complete closure by day 12 and restored epidermis on H&E. Statistical significance reported (#P<0.05, *P<0.01).

The findings validate a universal, one-step aqueous strategy to functionalize diverse wet hydrogels without pre-installed reactive handles by exploiting the synergistic adhesion of catechol and amine groups in a minimal DbaYKY tripeptide. Strong single-molecule adhesion and robust XPS/ATR-FTIR signatures confirm effective tethering. Functionalization levels are tunable via modifier concentration and are stable under physiological-like conditions and protease exposure, enabling persistent bioactivity (antibacterial action and cell adhesion) for at least a week. The approach generalizes across chemically crosslinked (PEG, PHEMA, PSBMA) and physically crosslinked (PVA, alginate) hydrogels, and can be extended to bulk functionalization through lyophilization to increase pore size. Mechanistically, multiple interactions—hydrogen bonding, cation–π interactions, and electrostatics—likely contribute, while mild oxidative/crosslinking chemistry at pH 8.5 can increase graft density. The method addresses the flexibility limitations of conventional click/bio-orthogonal strategies by decoupling substrate chemistry from ligand design, broadening applications in antimicrobial surfaces, tissue-engineering scaffolds, and wound dressings.

A sandcastle worm-inspired DbaYKY tripeptide serves as a versatile adhesive handle for direct, one-step functionalization of wet hydrogels. By converting DbaYKY into initiators for multiple polymerizations, diverse functional polymers with terminal DbaYKY were synthesized and tethered onto or into various hydrogels, imparting strong antibacterial activity, durable cell adhesion, and enhanced wound healing in vivo. Functionalization degree is tunable and stable for at least 7 days under PBS and protease conditions. This strategy simplifies and generalizes hydrogel biofunctionalization without substrate-specific reactive groups, offering a practical toolkit for biomedical applications. Future work could optimize conditions (pH, concentration, time) for monolayer versus multilayer deposition, expand the library of functional cargos (e.g., growth factors, enzymes), evaluate long-term in vivo performance and biocompatibility, and further develop bulk functionalization approaches for 3D tissue engineering and cell encapsulation.

• Quantitative grafting by fluorescamine was feasible only for hydrogels that can be dissolved (PVA and alginate); absolute grafting levels on insoluble hydrogels relied on a fluorescent surrogate.
• Penetration into hydrogel interiors is limited by pore size; bulk functionalization required a lyophilization pretreatment and may not be applicable to all hydrogel systems without affecting mechanical properties.
• Mechanistic analysis indicates catechol-derived crosslinking at pH 8.5, which may influence the exact chemistry of the graft and potential multilayer formation; monolayer control may require tighter condition optimization.
• In vivo evaluation was limited to a short (12-day) wound-healing model in rats; long-term stability, immunogenicity, and performance in other models and under dynamic physiological conditions remain to be assessed.
• Only selected functional outputs (antibacterial, cell adhesion) and polymer classes were demonstrated; broader functional cargo scope and scaling/manufacturability were not addressed in detail.