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

Figures & data

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.

Table S1 Draize’s scale of weighted scores for grading the severity of ocular lesions

Assessment	Score
Cornea
A. Opacity – degree of density (most dense area taken for reading)
No ulceration or opacity	0
Scattered or diffuse area; details of iris clearly visible	1
Easily discernible translucent areas; details of iris slightly obscured	2
Opalescent areas; no details of iris visible, size of pupil barely discernible	3
Opaque; iris invisible	4
B. Area of cornea involved
None	0
One-quarter (or less) but not zero	1
Greater than one-quarter, less than one-half	2
Greater than one-half, less than three-quarters	3
Greater than three-quarters, up to whole area	4
	Score = A × B ×5 (range, 0 to 80)
Iris
A. Values
Normal	0
Folds above normal, congestion, swelling and/or circumcorneal injection; iris still reacting to light (sluggish reaction is positive)	1
No reaction to light, hemorrhage and/or gross destruction	2
	Score = A ×5 (range, 0 to 10)
Conjunctivae
A. Redness of palpebral conjunctivae
Normal	0
Vessels definitely injected above normal	1
More diffuse, deeper crimson red; individual vessels not easily discernible	2
Diffuse beefy red	3
B. Chemosis
Normal	0
Any swelling above normal (includes nictitating membrane)	1
Obvious swelling with partial eversion of the lid	2
Swelling with lids about half closed	3
Swelling with lids about half closed to completely closed	4
C. Discharge
Normal	0
Any amount different from normal	1
Discharge with moistening of the lids and hair just adjacent to the lids	2
Discharge with moistening of the lids and considerable area around the eye	3
	Score = (A + B + C) ×2 (range, 0 to 20)
Total score=Sum of all scores obtained for the cornea, iris and conjunctivae

Table S2 Classification of products according to the Draize ocular irritation index (OII)

Average score	Classification
0	Non-irritant
0–15	Slightly irritant
>15–30	Moderately irritant
>30–50	Irritant
>50	Very irritant

Table 1 Characterization of different kinds of CG-GS-PRN-LDH nanocomposites (mean±SD, n=3)

Samples	Particle size (nm)	Zeta potential (mV)	pH	Drug loading (%)
CG-GS-PRN-LDH (1:0.5)-50 mg	155.8±1.4	+45.32±1.96	8.37±0.25	6.05±0.36
CG-GS-PRN-LDH (1:1)-50 mg	158.3±2.6	+43.61±2.37	8.46±0.12	5.97±0.51

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium.

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.

Figure S1 IR spectra of CTS (a), CTS-Fmoc-GS (1:0.5) (b), CTS-Fmoc-GS (1:1) (c), CTS-GS (1:0.5) (d), CTS-GS (1:1) (e), CG-GS (1:0.5) (f) and CG-GS (1:1) (g).

Abbreviations: CTS, chitosan; CG-GS, chitosan-glutathione-glycylsarcosine; IR, infrared.

Figure S2 TEM micrographs of Mg-Al-NO₃-LDH (A) and CG-GS-PRN-LDH (B).

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium; TEM, transmission electron micrography.

Figure S3 Thermogravimetric analyses of PRN, blank LDH, Mg-Al-PRN-LDH nanoparticles, different amounts of CG-GS for CG-GS-PRN-LDH nanocomposites and the physical mixture of CG-GS and PRN-LDH.

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Figure S4 HCEpiC cell viability of CG-GS-PRN-LDH (1:1), CG-GS-LDH (1:1), CG-GS-PRN-LDH (1:0.5) and CG-GS-LDH (1:0.5) nanocomposites at different LDH concentrations. Data represent the mean and SD of triplicate experiments.

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; HCEpiC, human corneal epithelial primary cells; LDH, layered double hydroxides; PRN, pirenoxine sodium.

Figure S5 In vitro cellular uptake of CG-GS-PRN-LDH (1:1) nanocomposites, CG-GS-PRN-LDH (1:0.5) nanocomposites, PRN-LDH nanoparticles, physical mixture of CG-GS and PRN solution (0.00038% [w/v]) and PRN solution at different time points (mean±SD, n=3). *P<0.05 vs PRN-LDH. The bar shown is 25 μm.

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium; UI, uptake index.

Figure S6 The effects of endocytic inhibitors on cellular uptake of CG-GS-FITC-LDH nanocomposites (mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs control group.

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; FITC, fluorescein isothiocyanate Isomer I; LDH, layered double hydroxides.

Figure S7 Histopathology microscopy of cornea (A), iris (B), and conjunctiva (C) after treating with different formulations for 7 days: (1) blank; (2) normal saline; (3) CG-GS-PRN-LDH (1:0.5); (4) CG-GS-PRN-LDH (1:1), n=3. The bar shown is 50 μm. The insert shows the representative images that appears in each figure part. (Hematoxylin-eosin stain; original magnifications ×100; insert ×400).

Abbreviations: CG-GS, chitosan-glutathione-glycylsarcosine; LDH, layered double hydroxides; PRN, pirenoxine sodium.