Effective Ocular Delivery of Eplerenone Using Nanoengineered Lipid Carriers in Rabbit Model

Figures & data

Table 1 Experimental Runs, Independent Variables, and Measured Responses of the 3¹×4¹ Full Factorial Experimental Design of Epl-loaded MNLCs

F (Sorted by Standard Order)	Type of Surface Modifier	Concentration of Surface Modifier (% w:v)	Y1: PS (nm)	Y2: PDI	Y3: ZP (mV)	Y4: EE% (% w:w)	Y5: Viscosity (cP) at Shear Rate (20 [1/s])
MNLC-1	Hydrogenated collagen	0.3	152.75±15.20	0.333±0.01	−28.1±0.42	62.945±1.10	39.24±0.1
MNLC-2	COSL	0.3	282.25±32.31	0.382±0.01	+15.8±0.85	69.948±0.77	0±0
MNLC-3	Hyaluronic acid	0.3	167±42.57	0.416±0.16	−44.25±1.63	81.285±1.63	186.39±0.32
MNLC-4	Hydrogenated collagen	0.5	155.6±4.10	0.362±0.07	−24.1±0.57	63.865±1.89	39.24±0.56
MNLC-5	COSL	0.5	236.15±26.38	0.428±0.06	+20.65±0.49	70.583±9.08	78.48±0.98
MNLC-6	Hyaluronic acid	0.5	262.75±84.07	0.449±0.03	−48.1±1.13	79.025±1.05	333.54±0.15
MNLC-7	Hydrogenated collagen	0.7	127.773±9.15	0.381±0.13	−27.95±0.21	67.281±8.39	49.05±0.17
MNLC-8	COSL	0.7	222.95±11.10	0.433±0.03	+29.3±0.28	70.26±0.08	29.43±2.11
MNLC-9	Hyaluronic acid	0.7	261.75±66.40	0.434±0.04	−53.2±1.13	88.445±2.80	824.04±1.65
MNLC-10	Hydrogenated collagen	0.9	161.2±5.23	0.393±0.04	−30.944±0.93	76.905±7.96	39.24±1.24
MNLC-11	COSL	0.9	397.1±158.11	0.428±0.16	+46.35±1.77	74.357±6.73	9.81±0.98
MNLC-12	Hyaluronic acid	0.9	277.45±36.56	0.475±0.01	−54.4±0.49	90.775±3.57	1373.4±0.47

Notes: Data presented as means ± SD (n=3). All systems contained 1.25 mg Epl/1 mL.

Abbreviations: PS, particle size; PDI, polydispersity index; ZP, ζ-potential; EE, entrapment efficiency.

Figure 1 (I) Procedural steps for preparing the hen’s egg on chorioallantoic membrane test (A–D). The egg was candled to check embryo viability (A) the air space marked (B), the external shell removed (C), and the inner membrane removed (D); (II) the observed responses to different systems application (E–H). Negative control (0.9% NaCl) (E); positive control (10% NaOH) (F); Epl aqueous suspension (G); Epl- loaded COSL-MNLCs (H); Epl loaded HA-MNLCs (I).

Table 2 Independent Variables, their Levels, and Summary Statistics of the 3¹×4¹ Full Factorial Design Used for Optimization of Epl-loaded MNLCs

Factors (independent variables)
Type of surface modifier	Chitosan oligosaccharide lactate	Hyaluronic	Hydrogenated collagen
Concentration of surface modifier (% w:v)	0.3	0.5	0.7	0.9
Responses (dependent variables)	R^2	Adjusted R^2	Predicted R^2	Constraints	P	Adequate precision
Y1: PS (nm)	0.9985	0.9915	0.8459	Minimize	0.0069	41.924
Y2: PDI	0.9185	0.8879	0.8243	Less than 0.5	0.0001	16.330
Y3: ZP (mV)	0.9703	0.9456	0.8164	Maximize	0.0002	19.364
Y4: EE%	0.9161	0.8846	0.8061	Maximize	0.0001	15.254
Y5: Viscosity (cP)	0.9756	0.9553	0.8493	Maximize	0.0001	19.891

Abbreviations: PS, particle size; PDI, polydispersity index; ZP, ζ-potential; EE, entrapment efficiency.

Figure 2 Line plots of significant effects of type of surface modifier and its concentration on (A) PS, (B) PDI, (C) ZP, (D) EE%, and (E) viscosity.

Table 3 Predicted Values of Responses for Optimized Systems (Epl-loaded HA-MNLCs and Epl-loaded COSL-MNLCs) Compared to Actual Values, along with Prediction Intervals

	Validity Parameters	Y1: PS (nm)	Y2: PDI	Y3: ZP (mV)	Y4:EE (%)	Y5: Viscosity (cP)
Epl-loaded HA-MNLCs	Predicted value	265.54	0.464	−54.47	88.94	1190.78
	95% prediction interval	240.72–290.36	0.44–0.48	48.76–60.18	84.75–93.12	1,025.4–1,356.16
	Actual values	255±36.55	0.40±0.01	−54.2±0.494	90.77±3.57	1,200±9.81
Epl-loaded COSL-MNLCs	Predicted value	318.92	0.437828	40	75.2155	38.0875
	95% prediction interval	294.37–343.48	0.419–0.45	34.46–45.53	71.09–79.33	36.78–40.03
	Actual values	285±5.03	0.42±0.15	30±0.28	70.11±0.08	40.24±7.12

Figure 3 Cumulative release profile of Epl from loaded unmodified NLC system, Epl loaded HA-MNLCs and Epl loaded COSL-MNLCs.

Figure 4 Mucoadhesion results: (A) PS measurements; (B) ZP measurements.

Figure 5 Transmission electron microscopy: (A) Epl loaded HA-MNLCs; (B) Epl loaded COSL-MNLCs.

Figure 6 Photographs showing normal rabbit eyes after instillation of 0.1 mL physiological saline in a control eye, Epl loaded HA-MNLCs, and Epl loadedCOSL-MNLCs (instillation repeated every hour for 6 hours).

Figure 7 Histopathological stained sections (hematoxylin and eosin) of normal untreated rabbit eyes (a₁–c₁)and rabbit eyes treated with Epl loaded HA-MNLCs (a₂–c₂) and Epl loaded COSL-MNLCs (a₃–c₃), where “a” represents the outer layer of the cornea, “b” the irisand ciliary process, and “c” the retina, choroid, and sclera at magnification 40×.

Figure 8 (I) Confocal laser-scanning microscopy of rabbit eyes at the corneal and scleral surfaces for RhB-labeled Epl-loaded unmodified NLCs, RhB-labeled Epl loaded HA-MNLCs, and RhB-labeled Epl loaded COSL-MNLCs; (II) confocal laser-scanning microscopy of rabbit corneas showing the depth of RhB; (III) mean fluorescence intensity of RhB-labeled Epl-loaded unmodified NLCs, RhB labeled Epl loaded HA-MNLCs, and RhB-labeled Epl loaded COSL-MNLCs at the time points indicated on the bar graph.