High-throughput screening of zwitterion-based coatings towards improved mechanical stability and drug-loading capacity

Biomedical materials are widely used for implantable medical devices due to mechanical properties, corrosion resistance, and biocompatibility, but biofouling from pathogenic contamination remains a major challenge that impacts patient outcomes and device inertness. Highly hydrophilic antifouling hydrogel coatings can mitigate biofouling; HEMA-, PEG-, and zwitterion-based hydrogels are classic antifouling systems. Zwitterionic polymers, inspired by phosphorylcholine headgroups, contain both anionic and cationic groups and reduce water mobility to repel fouling while increasing swelling. However, zwitterionic hydrogel coatings often have inadequate adhesion and mechanical stability. Incorporating HEMA segments can improve toughness and strength. Monomer content, crosslinking degree, and functional group ratios all affect polymerization and final coating properties. Systematically exploring these multidimensional variables via traditional one-by-one experiments is time- and resource-intensive, creating a need for rapid, cost-effective screening. High-throughput methods have enabled exploration of large compositional spaces for drug mixtures, antibacterial compounds, and biomaterial surfaces, facilitating prediction and understanding of composition/structure–function relationships. This study aims to employ high-throughput, miniaturized synthesis and screening to rapidly design SBMA/HEMA antifouling hydrogel coatings with optimized mechanical stability and drug-loading capacity.

Prior efforts to stabilize zwitterionic coatings include: (i) dopamine-bearing copolymers (PMNC) enabling substrate fixation via catechol adhesion (Gong et al.), and (ii) zwitterionic-phosphonic random copolymers immobilized on Ti through strong coordination with phosphonate/phosphonic motifs (Huang et al.). While effective for adhesion, such motifs can compromise intrinsic antifouling performance. High-throughput platforms have been applied to related problems: Levkin’s group used a miniaturized droplet microarray to rapidly screen antibacterial compounds and assess P. aeruginosa drug resistance; Fang et al. used gradient surfaces to optimize peptide-functionalized Ti with improved biocompatibility and antimicrobial activity. These works demonstrate that exploring broad chemical spaces can accelerate optimal materials design and clarify composition/structure–function relationships.

High-throughput combinatorial hydrogel microarrays were fabricated using a non-contact droplet microarray printer (NanoPlotter NP2.1) with picoliter dispensing and real-time imaging. Libraries varied: (1) monomer composition (SBMA:HEMA = 100:0, 75:25, 50:50, 25:75, 0:100), (2) monomer content (10, 20, 30, 40, 50 wt%), (3) crosslinker content (PEGDMA at 0–100 wt% relative to monomers in 5% steps; 21 levels), and (4) crosslinker molecular weight (Mn ≈ 200, 600, 1000). Each microarray contained 315 unique spots; 15 microarrays yielded 1575 total formulations using ~0.6 mL total reagents and ~150 min. Representative dispensing parameters: single drop burst 400 pL; humidity 70%. A typical print: 50 nL SBMA (1 g/mL in PBS, 30 °C), overprinted with 50 nL HEMA (1 g/mL in PBS, 35 °C), then PEGDMA (temperature-controlled up to 75 °C) from 0 to 100 nL per spot (5% step), followed by photoinitiator (2 wt% in PBS) at 200 nL (25 °C). Droplets were UV-cured (365 nm, 60 mW/cm², 3 min). Mixing/homogeneity validation: overprinting induced vortices; Raman spectroscopy of representative spots showed peaks at ~1034 (S=O), 1456 (CH3/CH2), 1732 (C=O), and 2911 cm−1 (C–H), with Raman mapping indicating radially uniform distributions; fluorescence images of fluorescein- (SBMA) and rhodamine B- (HEMA) labeled spots showed even dye distribution. Mechanical stability screening: (i) immersion swelling in PBS 72 h; (ii) flow test simulating arterial flushing using a peristaltic pump at 500 mL min−1 for 72 h; (iii) tape-peeling abrasion using 3M CT-18 (600#) tape pressed with 50 N, peeled at 45°, 10 cycles. After each test, arrays were washed with ethanol (×3), dried with argon, and imaged optically. Drug-loading capacity assay: arrays were immersed in Congo red solution for 24 h, washed with ethanol, dried, and imaged; ImageJ processing included RGB to 8-bit grayscale conversion, inversion, optical density calibration, region delineation, and mean grayscale extraction. Grayscale intensity correlated with dye retention (swellability/drug-loading). Data were visualized via heat maps (stability) and 3D histograms (drug-loading).

- Homogeneous mixing of SBMA/HEMA/crosslinker within picoliter droplets was confirmed by real-time imaging, Raman mapping (uniform S=O, CH3/CH2, C=O, C–H distributions), and dual-fluorescence images. - Immersion stability (72 h PBS): Incorporation of HEMA markedly improved stability; SBMA-only coatings showed no intact spots after swelling due to fragility. Among crosslinkers, PEGDMA with Mn ≈ 200 gave superior stability compared to Mn ≈ 600 or 1000. Crosslinker content exhibited an optimum: uncrosslinked spots disappeared; excessive crosslinking caused brittleness; many intact spots occurred at intermediate crosslink densities. Increasing SBMA content generally required higher monomer content to maintain stability. - Flow (500 mL/min, 72 h) and tape-peeling (10 cycles, 50 N, 45°) tests on 208 immersion-stable spots: Formulations with 0–25% HEMA lost integrity under flow/abrasion; stability improved by increasing HEMA fraction and by using lower-MW crosslinker. Notably, 100% HEMA suffered serious damage under flow/peeling, whereas 50:50 and 75:25 HEMA:SBMA compositions retained more intact spots, indicating that an appropriate amount of zwitterionic segments enhances abrasion resistance. - Counts: 208 stable spots (of 1575) after immersion; 70 remained stable after subsequent flow and tape-peeling. - Drug-loading capacity (Congo red): Positively correlated with SBMA fraction and negatively with high crosslinker content within a given composition, showing a rise-then-fall trend versus crosslinker content (low-to-moderate crosslinking favored swellability; too low reduced network integrity). These trends align with known bulk hydrogel behavior. - Final selection: 10 formulations (of 1575) exhibited optimized combinations of mechanical stability and drug-loading capacity across PEGDMA Mn ≈ 200, 600, and 1000 sets.

The high-throughput droplet microarray strategy efficiently mapped composition/structure–function relationships in SBMA/HEMA hydrogel coatings, directly addressing the need to rationally balance antifouling (associated with zwitterionic content and swellability) and mechanical stability (enhanced by HEMA content and appropriate crosslink density). Shorter crosslinkers (PEGDMA Mn ≈ 200) likely promote closer proximity of pendant functional groups, enabling interchain interactions and local friction that dissipate energy under deformation, and potentially activating hydrogen bonding, thereby improving toughness. Crosslink density exhibited a clear optimum: insufficient crosslinking compromised integrity, whereas excessive crosslinking increased brittleness. While SBMA maximized swellability and drug-loading (supporting antifouling and drug-eluting potential), inclusion of HEMA improved resistance to immersion, flow, and abrasion; purely HEMA formulations, however, underperformed in flow/abrasion without the zwitterionic segments. Thus, mixed compositions (notably 50:50 and 75:25 HEMA:SBMA) with lower-MW crosslinker and moderate crosslinking achieved superior overall performance. The approach used minimal reagents and time yet yielded comprehensive heat maps and quantitative dye-retention metrics, enabling rapid, data-driven selection of optimal coatings.

A miniaturized, non-contact droplet microarray platform was established to rapidly synthesize and screen 1575 SBMA/HEMA hydrogel coating formulations using only ~0.6 mL of reagents and ~150 minutes. Systematic variation of monomer composition, monomer content, crosslinker content, and crosslinker molecular weight allowed efficient evaluation of mechanical stability (immersion, flow, tape-peeling) and drug-loading capacity (Congo red uptake). Key trends were identified: HEMA incorporation is essential for mechanical stability; shorter crosslinkers (PEGDMA Mn ≈ 200) yield better stability; crosslink density has an optimal range; higher SBMA fractions enhance drug-loading. From the library, 10 formulations exhibited optimized stability and drug-loading. The demonstrated strategy enables rapid discovery and fine design of antifouling hydrogel coatings and can be extended to broader biomaterial surface optimization.