Development and Characterization of Dual-Loaded Niosomal Ion-Sensitive In Situ Gel for Ocular Delivery

Abstract

The study investigates the development and characterization of dual-loaded niosomes incorporated into ion-sensitive in situ gel as a potential drug delivery platform for ophthalmic application. Cannabidiol (CBD) and epigallocatechin-3-gallate (EGCG) simultaneously loaded niosomes were prepared via the thin film hydration (TFH) method followed by pulsatile sonication and were subjected to comprehensive physicochemical evaluation. The optimal composition was included in a gellan gum-based in situ gel, and the antimicrobial activity, in vitro toxicity in a suitable corneal epithelial model (HaCaT cell line), and antioxidant potential of the hybrid system were further assessed. Dual-loaded niosomes based on Span 60, Tween 60, and cholesterol (3.5:3.5:3 mol/mol) were characterized by appropriate size (250 nm), high entrapment efficiency values for both compounds (85% for CBD and 50% for EGCG) and sustained release profiles. The developed hybrid in situ gel exhibited suitable rheological characteristics to enhance the residence time on the ocular surface. The conducted microbiological studies reveal superior inhibition of methicillin-resistant Staphylococcus aureus (MRSA) adhesion by means of the niosomal in situ gel compared to the blank gel and untreated control. Regarding the antioxidant potential, the dual loading of CBD and EGCG in niosomes enhances their protective properties, and the inclusion of niosomes in gel form preserves these effects. The obtained outcomes indicate the developed niosomal in situ gel as a promising drug delivery platform in ophthalmology.

Keywords: HaCaT cells; cannabidiol; epigallocatechin-3-gallate; gellan gum; ocular delivery; rheological studies; sol gel transition.

Figure 1

Size distributions of empty and drug-loaded niosomes.

Figure 2

Cryo-TEM images of (a) empty niosomes (N6); (b) CBD-loaded niosomes (N5).

Figure 3

Viscosity as a function of the shear rate of G6, G6N, and G6N:CBD:EGCG formulations at 25 °C.

Figure 4

Variation in elastic (G′) and loss (G″) moduli as a function of shear stress (τ) of G6, G6N, and G6N:CBD:EGCG formulations. All measurements were carried out at 35 °C.

Figure 5

In vitro release profile of CBD and EGCG from optimal niosomal formulation (N3) and its hybrid in situ gelling system (G6N:CBD:EGCG). N3:CBD, G6N:CBD denote cannabidiol release from niosomal suspension and hybrid niosomal gel, respectively, whereas N3:EGCG and G6N:EGCG represent the release profiles of epigallocatechin-3-gallate from niosomes and its hybrid niosomal gel formulation. Each value is presented as mean ± SD (n = 3).

Figure 6

Quantitative evaluation of MRSA biofilm formation after exposure to blank (G6N) or hybrid niosomal drug-loaded gel G6N:CBD:EGCG (1/0.5 mg/mL). Legend: Co—untreated control; Dilution 1:8 = 0.125/0.06125 mg/mL; Dilution 1:16 = 0.0625/0.03125 mg/mL; Dilution 1:32 = 0.03125/0.0156 mg/mL. Each value is presented as mean ± SD (n = 4).

Figure 7

Cytotoxicity on HaCaT cells of: (A) empty niosomes (N6); (B) free cannabidiol (CBD); (C) free epigallocatechin (EGCG); (D) combination of free cannabidiol and free epigallocatechin (CBD + EGCG); (E) dual-loaded CBD and EGCG vesicles (N:CBD:GCG, formulation) niosomes and (F) niosomal in situ gel based on double-loaded niosomes (G6N:CBD:EGCG), measured by MTT assay. All groups were compared statistically vs. untreated controls by one-way ANOVA with Dunnet’s post hoc test. The results are expressed as means ± SD of triplicate assays (n = 8). *** p < 0.001 vs. control (CTRL, untreated control cells).

Figure 8

Protective effects of (A) empty niosomes; (B) free CBD; (C) free EGCG; (D) combination of free CBD and free EGCG (CBD + EGCG); (E) dual-loaded CBD and EGCG (N:CBD:EGCG) niosomes and (F) niosomal in situ gel based on double-loaded niosomes (G6N:CBD:EGCG) in a H₂O₂-induced damage model in human keratinocyte HaCaT cell line. The results are expressed as means ± SD of triplicate assays (n = 8). ANOVA with Dunnett’s post-test. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. H₂O₂ CTRL (untreated control cells); H₂O₂ cells treated with H₂O₂ (200 µM).