pH-Responsive Liposome-Hydrogel Composite Accelerates Nasal Mucosa Wound Healing

Abstract

Objectives: Nasal mucosa wound healing faces challenges such as acidic microenvironments and bacterial proliferation. Persistent mucosal defects predispose to complications such as nasal septal perforation. Conventional drug delivery systems suffer from nonspecific release and short-term efficacy. This study aimed to develop a pH-responsive liposome-hydrogel composite (HYD-Lip/DXMS@HG) to integrate pH-triggered dexamethasone (DXMS) delivery, antifouling properties, and mechanical support for refractory injuries. Methods: The composite combined acylhydrazone-modified liposomes with a hydrogel synthesized from hydroxyethylacrylamide (HEAA) and diethylacrylamide (DEAA). In vitro assays evaluated DXMS release kinetics, RPMI 2650 cell migration/proliferation, and antibacterial properties. In vivo rabbit nasal mucosal injury models assessed healing efficacy via histology analyses. RNA sequencing was performed to identify key signaling pathways. Results: HYD-Lip/DXMS@HG exhibited sustained DXMS release in acidic conditions, accelerating cell migration/proliferation in vitro. In rabbits, the composite reduced TNF-α expression and CD45+ leukocyte infiltration, while enhancing collagen alignment and epithelial thickness. RNA sequencing identified upregulated ECM receptor interaction, Hippo, TGF-β, and PI3K-Akt pathways, linked to collagen remodeling, anti-apoptosis, and angiogenesis. Conclusions: This multifunctional platform synergizes pH-triggered drug delivery, mechanical support, and antibacterial activity, offering a promising therapeutic strategy for refractory nasal mucosal injuries and postoperative recovery.

Keywords: acylhydrazone bond; antibacterial; dexamethasone; hydrogel; liposome; nasal mucosa; pH-responsive.

Figure 1

Schematic diagram of the preparation of HYD-Lip/DXMS@HG and application in rabbits. Abbreviations: LAP: lithium phenyl-2,4,6-trimethylbenzoylphosphinate; MPEG2K: Methoxy polyethylene glycol 2000; HYD: Hydrazone; DSPE: Distearoylphosphatidylethanolamine.

Figure 2

Characteristics of liposomes. (a) Transmission electron micrograph of acylhydrazone bond modified DXMS liposome. Scale bar: 100 nm; (b) The hydrodynamic size of DXMS liposome; (c) The UV spectrum of DXMS standard.

Figure 3

Characteristics of hydrogels. (a) Scanning electron micrograph of @HG and HYD-Lip/DXMS@HG. Scale bar: 100 nm; (b) FTIR spectrum of HEAA, DEAA, Bis, LAP, HYD-Lip/DXMS, HYD-Lip/DXMS@HG, @HG.

Figure 4

Gelation kinetics of @HG and HYD-Lip/DXMS@HG with varied cross-linking time. (a) Storage moduli of @HG with varied cross-linking time (e.g., 30 s, 1 min, 5 min) where G′ and G″ represent storage and loss moduli, respectively; (b) Storage moduli of HYD-Lip/DXMS@HG with varied cross-linking time. Data are expressed as mean ± SD (n = 3). One-way variance analysis followed by Tukey’s post hoc test was used to indicate the significance, and *** p < 0.001, **** p < 0.0001, not significant (ns).

Figure 5

Stress–strain curves of @HG and HYD-Lip/DXMS@HG. (a) Compressive stress–strain curves of @HG and HYD-Lip/DXMS@HG with varied cross-linking times; (b) Cyclic compression behavior (3 cycles) of @HG and HYD-Lip/DXMS@HG under varied cross-linking times.

Figure 6

Adhesion test of @HG and HYD-Lip/DXMS@HG. (a,b) Rheological behavior of @HG adhered to mucosa and cartilage; (c,d) Rheological behavior of HYD-Lip/DXMS@HG adhered to mucosa and cartilage.

Figure 7

Cryo-TEM images of liposomes released from hydrogel. (a) Intact liposomes released at pH 7.5; (b) Ruptured liposomes released at pH 6.0.

Figure 8

(a) DXMS release of HYD-Lip/DXMS@HG in pH 1.5, pH 5.5, pH 6.0, pH 6.5, pH 7.5; (b) Swelling ratio of @HG and HYD-Lip/DXMS@HG.

Figure 9

In vitro biocompatibility evaluation and antibacterial testing of hydrogels. (a) Images and quantitative analysis of live/dead fluorescence staining. Scale bar: 50 μM; (b) Effect of HYD-Lip/DXMS@HG on RPMI 2650 evaluated by the wound healing experiments. The red dot lines are used to mark the scratch area for facilitating the observation of cell migration at 0 h, 24 h, and 48 h under different conditions (CTRL, @HG, HYD-Lip/DXMS@HG). Scale bar: 100 μM; (c) Statistical analysis of proliferation rate of RPMI 2650 after different treatments; (d) Statistical analysis of RPMI 2650 mobility after different treatments; (e,f) Antibacterial properties of materials. *** p < 0.001, **** p < 0.0001.

Figure 10

Results of rabbit nasal mucosa after repair. (a) Schematic diagram of animal experiments; (b) Repaired mucosae obtained from different treatment groups on day 7. The yellow circles highlight the size of the nasal mucosal wounds. Scale bar: 1 cm; (c) Hematoxylin and eosin (H&E) staining, Masson staining and TNF-α immunohistochemistry staining at the wound site on day 14 after different treatments. Scale bar: 200 μM; (d) On day 14, the ratio of mucosal thickness on the injured side to the full thickness of the nasal septum; (e) Relative content of collagen in nasal mucosa by Masson staining on day 14; (f) Quantitative data of relative average optical density (AOD) of TNF-α. *** p < 0.001, **** p < 0.0001, not significant (ns).

Figure 11

Results of immunofluorescence staining. (a–e) CD31, α-SMA, CD45, PCNA, and Tubulin immunofluorescence staining pictures taken on day 14 after different treatments. Scale bar: 100 μM; (f–j) Quantitative data of relative fluorescence intensity of CD31, α-SMA, CD45, PCNA and Tubulin. * p < 0.05, **** p < 0.0001.

Figure 12

Analysis of RNA sequencing results of nasal injury and repair sites in rabbits at early stage (7 days). (a) Principal component analysis (PCA) of RNA sequencing results at 7 days for the control group and other experimental groups; (b) Volcano plot depicting differentially expressed genes between the HYD-Lip/DXMS@HG group and the control group at 7 days; (c,d) Top 20 upregulated signaling pathways in the KEGG enrichment analysis for the HYD-Lip/DXMS@HG group and the control group at 7 days; (e) Gene expression levels related to ECM remolding, Immune homeostasis and Epitheliosis at 7 days; (f) Heatmap of gene expression related to ECM remolding, Immune homeostasis and Epitheliosis in the HYD-Lip/DXMS@HG group and the control group at 7 days. Data are shown as mean ± SEM (n = 3).