Enzyme-Instructed Self-Assembling Peptide Hydrogels for Sustained Subconjunctival Delivery of Dexamethasone: A Stereochemical Comparison of L- and D-Enantiomeric Peptides for Postoperative Ocular Anti-Inflammatory Therapy.

Sustained postoperative anti-inflammatory therapy following intraocular surgery remains an unmet clinical need, as conventional topical corticosteroid eye drops suffer from poor bioavailability, patient non-compliance, and an inability to maintain therapeutic drug levels over the multi-week inflammatory resolution period. Here we report the development and characterization of alkaline phosphatase (ALP)-responsive enzyme-instructed self-assembling (EISA) peptide hydrogels based on the ultrashort amphiphilic sequence NapFFKY(p)-OH, covalently conjugated with dexamethasone (DEX) via an ester-cleavable succinyl linker, in both L- and D-enantiomeric forms, as injectable platforms for subconjunctival drug delivery. Both conjugates, L-NapFFK(DEX)Y(p)-OH and D-NapFFK(DEX)Y(p)-OH, were synthesized with chemical purity (87.3% and 89.8%, respectively) and theoretical DEX loading of 29.58% w/w. ALP-triggered gelation was confirmed for both enantiomeric gels at 2% w/v, with rheological characterization of the platform establishing a storage modulus plateau of approximately 294 Pa, a G'/G″ ratio of ~10:1, and gel failure at approximately 578% shear strain. Transmission electron microscopy revealed stereochemistry-dependent differences in nanofiber network architecture, with the D-enantiomer forming a comparatively looser, more porous fibrillar matrix than its L-counterpart. In vitro DEX release over 28 days demonstrated that covalent conjugation eliminates the burst release associated with physical drug entrapment (41.1% for the L physical mixture versus 14.2% and 8.3% for L- and D-conjugated systems, respectively, at 2% w/v), while D-amino acid stereochemistry provides an additional level of sustained release restraint. Proteinase K stability studies confirmed the exceptional proteolytic resistance of the D-enantiomer, retaining 92.6 ± 2.2% of intact peptide at 6 h compared with 25.3 ± 0.8% for the L-enantiomer. Ex vivo scleral permeation studies demonstrated a trend towards greater cumulative DEX permeation (10.6 vs. 6.7 µg) and tissue retention (5.6 vs. 3.2 µg) for the D vs. L-formulation over 24 h. Both formulations exhibited injection forces within acceptable limits for a 30-gauge needle for ophthalmic administration, with the D-enantiomer requiring lower force than its L-counterpart. Cell viability of Dex-conjugated peptide sequence demonstrated non toxic in ARPE-19 human retinal pigment epithelial cells across all conditions tested, confirming biocompatibility. These findings establish the backbone stereochemistry independently controls network mechanics, proteolytic stability, and drug release rate in ALP-responsive EISA hydrogels. Cumulative release over 28 days remains below that required to cover a 4-6-week postoperative course, and this work is therefore presented as a mechanistic proof-of-concept defining design principles for the platform rather than a clinically optimized formulation. Drug loading, peptide sequence, and linker chemistry are identified as the levers through which release rate may be matched to intended clinical interval.