Preparation of an antibacterial and anti-inflammatory sulfonated lignin/poly (vinyl alcohol) nanofiber membrane and its application in subconjunctival inflammation intervention

Objective  To prepare a structurally stable sulfonated lignin (SL)/poly(vinyl alcohol) (PVA) composite nanofiber membrane as a bio-based ocular carrier material with intrinsic antioxidant and antibacterial activities, and verify its role in the intervention of subconjunctival inflammation.

Methods  SL suitable for electrospinning was prepared from natural lignin by sulfonation to introduce -SO ₃H groups, thereby improving the hydrophilicity and solubility. SL and PVA were blended at a mass ratio of 25∶75 and processed into SL/PVA composite nanofiber membrane by co-electrospinning. Morphology, chemical composition, and mechanical properties of the membrane were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and tensile testing. Antioxidant capacity of the membrane was assessed using DPPH radical- and hydroxyl radical (OH)-scavenging assays. Cytocompatibility was evaluated by cell counting kit-8 and Calcein-AM/propidium iodide (PI) live/dead staining. Antibacterial activity of the membrane against Staphylococcus aureus ( S. aureus) was examined using live/dead bacterial staining. Intracellular reactive oxygen species (ROS) in CCL-20.2 cells were measured using the DCFH-DA probe. The expression of interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-1β in RAW264.7 macrophages was determined by immunofluorescence staining. A rat subconjunctival injury model was established to evaluate conjunctival congestion and edema scores at different time points after the membrane implantation. Hematoxylin-eosin (HE) staining on post-modelling day 7 was used to assess inflammatory cell infiltration and tissue damage in subconjunctival tissues. All animal experiments complied with the Regulations on the Management of Laboratory Animals and relevant ethical regulations, and had been approved by the Experimental Animal Ethics Committee of Henan Eye Hospital (No. HNEECA-2024-18).

Results  The SL/PVA nanofiber membrane exhibited a uniform and continuous nanofibrous architecture with favorable mechanical performance and structural stability. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed successful incorporation of -SO ₃H groups and interactions between SL and PVA. SL/PVA membrane extracts showed concentration-dependent scavenging of DPPH radical and OH over 0.1, 0.2, 0.4 and 0.8 mg/ml, with OH scavenging reaching a plateau beyond 0.4 mg/ml. After co-culture with the membrane for 24, 36, and 48 hours, cell viability remained greater than 90%. The SL/PVA group showed significantly lower intracellular ROS levels than the hyperosmotic model and PVA groups (both P<0.05). Live/dead bacterial staining results showed that the bacterial survival rates in the control group, PVA group and SL/PVA group were (93.82±2.15)%, (83.23±2.42)% and (8.63±0.84)%, respectively, with a significant overall difference ( F=1 727.47, P<0.001). The bacterial survival rate in SL/PVA group was significantly lower than that in the control group and PVA group (both P<0.001). Immunofluorescence analysis showed significantly reduced mean fluorescence intensities of TNF-α, IL-8, and IL-1β in the SL/PVA group compared with the lipopolysaccharide group and PVA group (all P<0.001). In vivo, rat conjunctival congestion and edema scores in the SL/PVA group were significantly lower than those in the control group on postoperative days 7 and 14 (all P<0.01), and inflammatory cell counts in subconjunctival tissues were significantly reduced in comparison with the control group and PVA group on postoperative day 7 (both P<0.01).

Conclusions  A structurally stable and multifunctional SL/PVA composite nanofiber membrane with excellent antioxidant activity, antibacterial performance, and cytocompatibility has been successfully developed. The membrane can downregulate pro-inflammatory cytokine expression and alleviate subconjunctival inflammatory responses in vivo, indicating its potential as a bioactive carrier material for ophthalmic applications. The membrane can significantly down-regulate the expression of inflammatory factors and relieve subconjunctival inflammatory reactions.