Functional intercalated nanocomposites with chitosan-glutathione-glycylsarcosine and layered double hydroxides for topical ocular drug delivery

Abstract

Background: To enhance ocular bioavailability, the traditional strategies have focused on prolonging precorneal retention and improving corneal permeability by nano-carriers with positive charge, thiolated polymer, absorption enhancer and so on. Glycylsarcosine (GS) as an active target ligand of the peptide tranpsporter-1 (PepT-1), could specific interact with the PepT-1 on the cornea and guide the nanoparticles to the treating site.

Purpose: The objective of the study was to explore the active targeting intercalated nanocomposites based on chitosan-glutathione-glycylsarcosine (CG-GS) and layered double hydroxides (LDH) as novel carriers for the treatment of mid-posterior diseases.

Materials and methods: CG-GS-LDH intercalated nanocomposites were prepared by the coprecipitation hydrothermal method. In vivo precorneal retention study, ex vivo fluorescence images, in vivo experiment for distribution and irritation were studied in rabbits. The cytotoxicity and cellular uptake were studied in human corneal epithelial primary cells (HCEpiC).

Results: CG-GS-LDH nanocomposites were prepared successfully and characterized by FTIR and XRD. Experiments with rabbits showed longer precorneal retention and higher distribution of fluorescence probe/model drug. In vitro cytological study, CG-GS-LDH nanocomposites exhibited enhanced cellular uptake compared to pure drug solution. Furthermore, the investigation of cellular uptake mechanisms demonstrated that both the active transport by PepT-1 and clathrin-mediated endocytosis were involved in the internalization of CG-GS-LDH intercalated nanocomposites. An ocular irritation study and a cytotoxicity test indicated that these nanocomposites produced no significant irritant effects.

Conclusions: The active targeting intercalated nanocomposites could have great potential for topical ocular drug delivery due to the capacity for prolonging the retention on the ocular surface, enhancing the drug permeability through the cornea, and efficiently delivering the drug to the targeted site.

Keywords: active targeting; glycylsarcosine; intercalated nanocomposites; layered double hydroxides; peptide transporter-1.

Figure 1

¹H NMR spectra of CG-GS (1:1) (a), CG-GS (1:0.5) (b), GSH (c), CTS-GS (1:1) (d), CTS-GS (1:0.5) (e), CTS-Fmoc-GS (1:1) (f), CTS-Fmoc-GS (1:0.5) (g), CTS (h), Fmoc-GS (i) and GS (j). Abbreviations: CTS, chitosan; CG-GS, chitosan-glutathione-glycylsarcosine; Fmoc, ; GSH, l-glutathione reduced form; ¹H NMR, proton nuclear magnetic resonance.

Figure 2

IR patterns of Mg-Al-NO₃-LDH (a), Mg-Al-PRN-LDH (b), CG-GS-PRN-LDH (1:0.5) (c), CG-GS (1:0.5) (d), CG-GS-PRN-LDH (1:1) (e), CG-GS (1:1) (f) and PRN (g). Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; IR, infrared; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Figure 3

XRD pattern of blank LDH (a), Mg-Al-PRN-LDH (b), CG-GS-PRN-LDH (1:0.5)-50 (c), CG-GS-PRN-LDH (1:1)-37.5 (d), CG-GS-PRN-LDH (1:1)-50 (e), CG-GS-PRN-LDH (1:1)-75 (f) and physical mixture of Mg-Al-PRN-LDH and CG-GS (1:0.5)-50 (g). Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium; XRD, X-ray diffractometer.

Figure 4

Release profiles of PRN, PRN-LDH, different DS of GS (A) and the amount of CG-GS (B) for CG-GS-PRN-LDH nanocomposites in artificial tears (mean±SD, n=3). Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; DS, degree of substitution; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Figure 5

Fluorescence microscopy images of HCEpiC incubated with CG-GS-FITC-LDH (1:1) nanocomposites (a), CG-GS-FITC-LDH (1:0.5) nanocomposites (b), physical mixture of CG-GS and FITC solution (0.00038% [w/v]) (c), FITC-LDH nanoparticles (d) and FITC solution (e) at different times. Scale bar =25 μm. Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; FITC, fluorescein isothiocyanate Isomer I; HCEpiC, human corneal epithelial primary cells; LDH, layered double hydroxides.

Figure 6

The effects of different concentrations of GS on cellular uptake of CG-GS-FITC-LDH (1:0.5) nanocomposites (A) and CG-GS-FITC-LDH (1:1) nanocomposites (B) (mean±SD, n=3); **P<0.01 versus control group. Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; FITC, fluorescein isothiocyanate Isomer I; LDH, layered double hydroxides.

Figure 7

The concentration–time curves of PRN of different nanocomposite eye drops in rabbit tears (mean±SD, n=6). Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Figure 8

Ex vivo fluorescence imaging of rabbit ocular tissues from rabbit treated with FITC: blank (a), FITC-LDH (b), CG-GS-FITC-LDH (1:0.5)-50 (c), CG-GS-FITC-LDH (1:1)-50 (d) and physical mixture of CG-GS and FITC solution (e). Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; FITC, fluorescein isothiocyanate Isomer I; LDH, layered double hydroxides.

Figure 9

Concentrations of PRN in ocular tissues of aqueous humor (A), cornea (B), iris-ciliary body (C), sclera (D), and crystalline lens (E) at different time points (0.5, 2, 4, 6 and 8 h) after topical administration. (mean±SD, n=3). *P<0.05 versus commercial product group, **P<0.05 versus PRN-LDH group. Abbreviations: Con, concentration; CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Scheme 1

Schematic reaction for the synthesis of chitosan-glutathione-glycylsarcosine (CG-GS). Abbreviations: CTS, chitosan; CG-GS, chitosan-glutathione-glycylsarcosine; EDC, carbodiimide hydrochloride; Fmoc-OSu, Fmoc N-hydroxysuccinimide ester; GSH, l-glutathione reduced form; NHS, N-hydroxysuccinimide.