Development and Characterization of PLGA Nanoparticle-Laden Hydrogels for Sustained Ocular Delivery of Norfloxacin in the Treatment of Pseudomonas Keratitis: An Experimental Study

Figures & data

Table 1 The Independent Variables and the Characterization Data of the Factorial Design-Derived NFX-Loaded PLGA NPs

NP Formulae	Independent Variables		Characterization Data									Desirability Indices
	Polymer Type	Drug:Polymer Ratio	PS (nm)	PDI	ZP (mV)	EE (%)	Q30min (%)	Q12h (%)	P30min (%)	P12h (%)	HL (%)
NP1	Ester-terminated PLGA	1:0.5	356.13±2.13	0.14±0.02	−22.52±0.23	37.80±1.13	12.12±0.92	63.21±2.10	20.21±1.5	59.41±2.11	82.60±0.27	0.23
NP2		1:1	392.02±1.33	0.17±0.07	−30.43±1.50	79.24±0.91	10.96±1.42	58.23±1.52	17.39±1.13	53.31±1.92	82.55±0.17	0.60
NP3		1:1.5	438.21±2.49	0.19±0.05	−25.61±0.41	68.52±1.31	8.17±2.27	52.01±2.71	14.13±1.62	45.19±2.33	82.66±0.33	0.43
NP4		1:2	485.05±1.83	0.24±0.05	−26.83±0.67	53.68±0.72	6.57±1.61	43.15±1.91	10.06±1.62	40.12±2.22	83.12±0.14	0.31
NP5	Acid-terminated PLGA	1:0.5	176.07±2.62	0.17±0.06	−26.51±1.60	59.63±1.43	19.31±0.78	78.23±1.73	25.02±0.73	70.12±1.81	82.08±0.33	0.18
NP6		1:1	190.51±1.23	0.18±0.09	−33.62±0.18	91.72±1.23	16.65±1.22	71.20±2.24	21.05±0.43	65.01±1.73	81.93±0.14	0.57
NP7		1:1.5	245.13±3.28	0.19±0.09	−27.67±0.41	78.54±0.81	13.26±2.32	65.32±1.82	18.17±1.42	56.54±1.71	82.48±0.34	0.53
NP8		1:2	256.12±2.94	0.20±0.08	−28.62±0.86	60.47±1.12	9.09±1.43	55.32±2.53	13.31±1.61	52.12±2.11	82.96±0.13	0.51

Notes: Data are represented as mean ± SD, n = 3.

Abbreviations: NFX, norfloxacin; PLGA, Poly(lactic-co-glycolic acid); NPs, nanoparticles; PS, particle size; PDI, polydispersity index; ZP, zeta potential; EE%, drug entrapment efficiency percentage; Q_30min, drug percentage released after 30 minute; Q_12h, drug percentage released after 12 hours; P_30min, drug percentage permeated after 30 minutes; P_12h, drug percentage permeated after 12 hours.

Table 2 The Composition and Characterization Data of the Investigated NFX-Loaded PLGA NP-Laden Hydrogels

Hydrogels	Composition		Characterization Data
	NPs	HPMC Concentration (%, w/w)	pH	Travelled Distance (cm)	ŋmin (cp)	ŋmax  (cp)	Hysteresis Loop Area (cm²)	Farrow’s Constant (N)	Flow Behavior	Zone of Inhibition (mm)
G1	NP2	1.5	7.03±0.13	7.16±0.30	652±10	1120±44	21.03±1.14	4.84	Pseudoplastic	27.0±0.5
G2	NP6		6.95±0.11	6.92±0.20	634±13	1005±23	21.38±1.08	4.68		37.1±1.3
G3	NP2	3	7.50±0.09	4.43±0.40	1553±47	4530±120	31.35±2.33	5.25		23.6±0.9
G4	NP6		7.17±0.08	4.03±0.25	1338±32	4387±114	27.12±1.58	5.03		33.7±1.4

Notes: Data are represented as mean ± S.D., n = 3.

Abbreviations: NFX, norfloxacin; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; HPMC, Hydroxypropyl methylcellulose; K4M; ŋ, viscosity.

Table 3 The Factorial Design Analysis Data of the Responses of NFX-Loaded PLGA NPs

Responses	Sum of Squares	Mean Sum of Squares	F value	R²	Adjusted R²	Predicted R²	Adequate Precision
Ps	94,188.875	23,547.2188	110.564	0.993	0.984	0.952	26.4711
ZP	74.923	18.731	34.184	0.979	0.949	0.847	17.502
EE%	1955.875	488.969	23.484	0.969	0.928	0.779	13.729
Q30min	124.677	31.169	16.447	0.956	0.898	0.689	11.952
Q12h	732.5	183.125	4.381	0.853	0.658	0.039	5.527
P30min	161.058	40.265	189.826	0.996	0.991	0.972	40.929
P12h	586.893	146.723	4.777	0.864	0.683	0.035	5.779

Abbreviations: NFX, norfloxacin; PLGA, Poly(lactic-co-glycolic acid); NP, nanoparticle; PS, particle size; ZP, zeta potential, EE%, drug entrapment efficiency percentage; Q_30min, drug percentage released after 30 minute; Q_12h, drug percentage released after 12 hours; P_30min, drug percentage permeated after 30 minutes; P_12h, drug percentage permeated after 12 hours.

Figure 1 (A) In vitro release profiles of NFX from NFX-loaded PLGA NPs and NFX suspension in simulated tear fluid (pH 7.4), mean ± S.D., n = 3. (B) Ex vivo goat corneal permeation profiles of NFX from NFX-loaded PLGA NPs and NFX suspension in simulated tear fluid (pH 7.4), mean ± S.D., n = 3.

Abbreviations: NFX, norfloxacin; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles.

Figure 2 Transmission electron micrographs of NP2 (A) and NP6 (B).

Abbreviation: NP, nanoparticles.

Figure 3 (A) FT-IR spectra of NFX (1), ester terminated PLGA (2), NFX-ester terminated PLGA physical mixture (3) and lyophilized NFX-loaded NP2 (4). (B) FT-IR spectra of NFX (1), acid terminated PLGA (2), NFX-acid terminated PLGA physical mixture (3) and lyophilized NFX-loaded NP6 (4).

Abbreviations: FT-IR, Fourier transform infrared; NFX, norfloxacin; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles.

Figure 4 Thixotropic behavior of NFX-loaded PLGA NP-laden HPMC hydrogels (G1 (A), G2 (B), G3 (C) and G4 (D).

Abbreviations: NFX, norfloxacin; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; HPMC, hydroxypropyl methylcellulose K4M.

Figure 5 (A) In vitro release profiles of NFX from NFX-loaded PLGA NP-laden HPMC hydrogels in simulated tear fluid (pH 7.4), mean ± S.D., n = 3. (B) Ex vivo goat corneal permeation profiles of NFX from NFX-loaded PLGA NP-laden HPMC hydrogels in simulated tear fluid (pH 7.4), mean ± S.D., n = 3.

Abbreviations: NFX, norfloxacin; PLGA, poly(lactic-co-glycolic acid), NPs, nanoparticles; HPMC, Hydroxypropyl methylcellulose K4M.

Table 4 Comparison of the Clinical and Histopathological Findings of the Excised Corneal Tissue Sections in the Investigated Groups

Points of Comparison	Control Values	Groups
		Infected	Drug Suspension	G3	G4
Corneal opacity	–	Grade 3	Grade 1	Grade 1	Grade 1
Ulcer size	–	9 mm	2 mm	2 mm	3 mm
Conjunctival hyperemia	–	Severe	Severe	Severe	Severe
Stromal edema	–	Severe	Moderate	Moderate	Moderate
Covering epithelial damage	–	+++	+	+	+
Collagen fibers disorganization	–	+++	++	++	++
Neovascularization	–	++	++	++	++
Inflammatory cells infiltrate	–	+++	+++	++	+++

Note: Symbols: (-) nil, (+) mild, (++) moderate and (+++) severe.

Figure 6 Conjunctival hyperemia and corneal edema in rabbits with Pseudomonas-induced keratitis pre- (A) and 10 days post- (B) non-treatment in the infected group (a) or treatment in drug suspension- (b), G3- (c), G4- (d) groups.

Figure 7 Histopathological changes observed, under two magnifications, in the sections of the excised corneal tissues in different groups [control (A), infected (B), drug suspension (C), G3 (D), G4 (E)]. The black arrow points to the epithelial layer, the green arrow shows the proliferating fibroblasts, and the red arrow refers to the polymorphonuclear inflammatory cells infiltrate. The stars denote the stromal collagen lamellar morphology (A), neovascularization (B), congested/dilated blood vessels (C–E).