Multifunctional organic–inorganic hybrid nanoparticles and nanosheets based on chitosan derivative and layered double hydroxide: cellular uptake mechanism and application for topical ocular drug delivery

Figures & data

Figure 1 Diagrams of three types of organic–inorganic hybrid nanocomposites based on polymer, LDHs and drug.

Notes: (A) Organic-coated inorganic hybrid LDH nanoparticles. (B) Organic-intercalated inorganic hybrid LDH nanoparticles. (C) Organic–inorganic hybrid LDH nanosheets.

Abbreviation: LDH, layered double hydroxide.

Table 1 Characterization of different types of CG-VV-LDH nanosheets

m(CG-VV): m(LDH)	Size (nm)	PI	Zeta potential (mV)
0:1	47.5±12.1	0.210±0.021	+35.4±0.9
1:1	81.7±2.4	0.230±0.011	+39.3±4.0
2:1	84.6±1.9	0.197±0.009	+50.4±4.4
4:1	102.8±2.7	0.252±0.013	+51.4±4.3
8:1	162.2±5.9	0.705±0.031	+45.1±2.8

Note: Each value represents mean ± SD (n=3).

Abbreviations: CG-VV, chitosan–glutathione–valine–valine; LDH, layered double hydroxide; PI, polydispersity index; SD, standard deviation.

Figure 2 Cumulative release–time profiles of PRN and CG-VV-PRN-LDH nanosheets.

Note: Each value represents mean ± SD (n=3).

Abbreviations: PRN, pirenoxine sodium; CG-VV, chitosan–glutathione–valine–valine; LDH, layered double hydroxide; SD, standard deviation.

Table 2 Stability of CG-VV-PRN-LDH nanosheet eye drops

Time (days)	Appearance	Particle size (nm)	Zeta potential (mV)	pH	Content (%)
0	No precipitation	108.4±2.1	+46.1±0.3	7.38±0.11	99.4±0.2
5	No precipitation	109.7±3.7	+43.8±0.8	7.45±0.14	99.1±0.8
10	No precipitation	106.1±1.9	+46.0±0.6	7.38±0.07	98.1±0.4

Notes: Each value represents mean ± SD (n=3). pH, hydrogen ion concentration.

Abbreviations: CG-VV, chitosan–glutathione–valine–valine; PRN, pirenoxine sodium; LDH, layered double hydroxide; SD, standard deviation.

Table 3 Pharmacokinetic parameters of commercial PRN eye drops and CG-VV-PRN-LDH nanosheet eye drops

Sample	AUC0–6 h (μg/mL min)	MRT (min)	CL (mL/min)
Commercial PRN eye drops	34.05±3.13	18.69±3.68	0.074±0.007
CG-VV-PRN-LDH nanosheet eye drops	172.88±24.67**	34.62±1.93**	0.015±0.002**

Notes: Each value represents mean ± SD (n=6).

** P<0.01 vs commercial PRN eye drops group.

Abbreviations: PRN, pirenoxine sodium; CG-VV, chitosan–glutathione–valine–valine; LDH, layered double hydroxide; AUC_{0–6 h}, area under the concentration vs time curve from 0 to 6 h; MRT, mean retention time; CL, clearance; SD, standard deviation.

Figure 3 The concentration–time curve of PRN in tear samples of commercial PRN eye drops and CG-VV-PRN-LDH nanosheet eye drops.

Note: Each value represents mean ± SD (n=6).

Abbreviations: PRN, pirenoxine sodium; CG-VV, chitosan–glutathione–valine– valine; LDH, layered double hydroxide; SD, standard deviation.

Figure 4 In vitro corneal permeation profiles of commercial PRN eye drops and CG-VV-PRN-LDH nanosheet eye drops in GBR solution.

Note: Each value represents mean ± SD (n=6).

Abbreviations: PRN, pirenoxine sodium; CG-VV, chitosan–glutathione–valine– valine; LDH, layered double hydroxide; GBR, glutathione-sodium bicarbonate Ringer; SD, standard deviation.

Figure 5 In vitro cytotoxicity of LDH nanoparticles, LDH nanosheets, CG-VV-LDH nanoparticles and CG-VV-LDH nanosheets against (A) HCEpiC, (B) ARPE-19 cells; (C) in vitro cytotoxicity of PRN-LDH nanoparticles, CG-VV-PRN-LDH nanoparticles and CG-VV-PRN-LDH nanosheets against HCEpiC after 12 h incubation, respectively; cell viability was determined by MTT assay.

Note: Each value represents mean ± SD (n=4).

Abbreviations: LDH, layered double hydroxide; CG-VV, chitosan–glutathione–valine–valine; HCEpiC, human corneal epithelial primary cells; ARPE-19, retinal pigment epithelial; PRN, pirenoxine sodium; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; SD, standard deviation.

Figure 6 Fluorescence microscopy images of (A) HCEpiC and (B) ARPE-19 cells incubated with the following: a, FITC-LDH nanoparticles; b, CG-VV-FITC-LDH nanoparticles; c, CG-VV-FITC-LDH nanosheets; d and e, CG-VV-FITC solution (0.008% and 0.00038% [w/v]); f, free FITC at 37°C for 1, 2 and 4 h, respectively.

Note: Scale bar =25 μm.

Abbreviations: HCEpiC, human corneal epithelial primary cells; ARPE-19, retinal pigment epithelial; FITC, fluorescein isothiocyanate; LDH, layered double hydroxide; CG-VV, chitosan–glutathione–valine–valine.

Figure 7 Flow cytometry measurement of intracellular uptake of (A) FITC-LDH nanoparticles, (B) CG-VV-FITC-LDH nanoparticles, (C) CG-VV-FITC-LDH nanosheets treated with specific endocytic inhibitors on HCEpiC; flow cytometry measurement of intracellular uptake of (D) FITC-LDH nanoparticles, (E) CG-VV-FITC-LDH nanoparticles, (F) CG-VV-FITC-LDH nanosheets treated with specific endocytic inhibitors on ARPE-19 cells.

Notes: HCEpiC or ARPE-19 cells without treatment with specific endocytic inhibitors were taken as the control (*P<0.05, **P<0.01, ***P<0.001 vs control group, n=3).

Abbreviations: FITC, fluorescein isothiocyanate; LDH, layered double hydroxide; CG-VV, chitosan–glutathione–valine–valine; HCEpiC, human corneal epithelial primary cells; ARPE-19, retinal pigment epithelial.