Sorafenib-loaded nanostructured lipid carriers for topical ocular therapy of corneal neovascularization: development, in-vitro and in vivo study

Figures & data

Table 1. Independent variables and the correspondent values in coded and physical form.

Factors	Levels
(Independent variables)	−1.682	−1	0	1	1.682
X1(Km, S/CoS)	3	3.8	5	6.2	7
X2(Mixed lipid, g)	0.2	0.36	0.6	0.84	1
X3(Drug/the mixed lipid)	0.008	0.013	0.02	0.027	0.032
Responses (dependent variables)	Desirability constraints
Y1: Drug content (mg/ml)	Maximize
Y2: Particle size (nm)	Minimize

S: surfactants; CoS: cosurfactants.

Table 2. Solubility of SRB in solid lipids, liquid oils, surfactants, and cosurfactants (mean ± SD, n = 3).

Materials	Name	Solubility (mg/g)
Solid lipids	Monolaurin	12< S < 13
	Monostearin	7 < S < 8
	Glyceryl tripalmitate	2< S < 3
	Palmitin	2 < S < 3
	Glyceryl stearate	4< S < 5
Liquid lipids	Capryol-90	9.58 ± 0.14
	Lauroglycol 90	4.51 ± 0.32
	Lauroglycol FCC	2.43 ± 0.46
	MCT	1.24 ± 0.02
	Monoolein	0.89 ± 0.24
	Castor oil	1.48 ± 0.31
	Ricinoleic acid	2.36 ± 0.23
	Oleic acid	2.61 ± 0.29
	Ethyl oleate	0.24 ± 0.03
	Olive oil	0.13 ± 0.01
	Corn oil	0.13 ± 0.03
	Propylene glycol dicaprylate	1.20 ± 0.27
	Triacetin	2.01 ± 0.05
	Isopropyi myristate	1.13 ± 0.09
Surfactants	CRH 40	71.81 ± 1.05
	HS 15	74.36 ± 1.53
	EL 35	56.72 ± 6.37
	Tween 80	50.63 ± 2.42
	Triton X 100	27.99 ± 1.64
Co-surfactants	PEG 400	76.72 ± 1.21
	Transcutol^®P	119.83 ± 1.53

Figure 1. Left: DSC melting curves of the different ratios of monolaurin and Capryol-90 ratios. Right: Detailed DSC thermograms of MC mixtures.

Table 3. Emulsification efficiency of CRH 40 and HS 15 (mean ± SD, n = 3).

Surfactants	Transmittance%
CRH 40	82.97 ± 1.98
HS 15	73.97 ± 3.98

Figure 2. Pseudo ternary phase diagrams prepared with mixed lipid (solid lipid to liquid oil is 2:1), CRH 40 (surfactant) and Transcutol^®P (cosurfactant).

Figure 3. Actual versus predicted value.

Table 4. Experimental design for SRB-NLC.

Formulation	Independent variables			Dependent variables				Another variable
				Y1 (mg/mL)		Y2 (nm)		DL%
	X1	X2(g)	X3	Actual	Predicted	Actual	Predicted	Actual
1	5	0.6	0.02	0.60 ± 0.01	0.61	118.50 ± 0.72	118.75	1.95 ± 0.02
2	6.2	0.84	0.027	0.60 ± 0.03	0.60	142.20 ± 0.52	138.17	1.42 ± 0.02
3	3.8	0.36	0.027	0.35 ± 0.02	0.34	113.60 ± 0.31	111.08	1.92 ± 0.02
4	6.2	0.36	0.027	0.49 ± 0.01	0.49	46.30 ± 0.14	42.08	2.55 ± 0.08
5	5	0.6	0.032	0.72 ± 0.04	0.72	138.00 ± 0.70	142.54	2.31 ± 0.04
6	6.2	0.36	0.013	0.23 ± 0.01	0.23	14.26 ± 0.12	10.84	1.28 ± 0.01
7	3.8	0.84	0.027	0.99 ± 0.16	0.99	148.50 ± 0.59	144.76	2.23 ± 0.03
8	5	1	0.02	0.10 ± 0.01	0.10	200.00 ± 0.67	204.57	0.15 ± 0.03
9	5	0.6	0.02	0.61 ± 0.03	0.61	118.60 ± 0.83	118.75	1.98 ± 0.02
10	5	0.6	0.02	0.61 ± 0.01	0.61	119.80 ± 1.03	118.75	1.94 ± 0.04
11	3.8	0.36	0.01	0.18 ± 0.01	0.10	96.92 ± 1.15	86.37	1.06 ± 0.01
12	5	0.2	0.02	0.19 ± 0.02	0.19	13.23 ± 0.03	17.74	1.82 ± 0.02
13	5	0.6	0.02	0.61 ± 0.02	0.61	118.30 ± 0.91	118.75	1.97 ± 0.03
14	6.2	0.84	0.01	0.40 ± 0.02	0.32	107.60 ± 1.50	91.92	0.94 ± 0.01
15	7	0.6	0.02	0.42 ± 0.02	0.42	67.33 ± 0.89	71.96	1.37 ± 0.03
16	3.8	0.84	0.013	0.43 ± 0.01	0.44	101.80 ± 1.73	99.01	1.02 ± 0.01
17	5	0.6	0.02	0.61 ± 0.01	0.61	119.20 ± 1.19	118.75	1.90 ± 0.05
18	5	0.6	0.008	0.24 ± 0.01	0.24	59.99 ± 0.21	64.53	0.79 ± 0.03
19	3	0.6	0.02	0.51 ± 0.01	0.51	63.08 ± 0.42	67.56	1.66 ± 0.03
20	5	0.6	0.02	0.60 ± 0.04	0.61	119.01 ± 0.60	118.75	1.95 ± 0.02

X₁: Km, X₂: Mixed lipid, X₃: (Drug/mixed lipid), Y₁: Drug content, Y₂: Particle size.

Figure 4. The TEM images of blank NLC (left) and SRB-NLC (right).

Figure 5. The particle size distribution of optimized SRB-NLC.

Figure 6. DSC diagrams of the SRB, SRB-NLC and Blank NLC samples.

Table 5. Characterization of optimized SRB-NLC (n = 3, Mean ± SD).

PS (nm)	PDI	ZP (mV)	pH	Osmolarity (mOsm/kg)	EE (%)
111.87 ± 0.93	0.15 ± 0.01	−0.35 ± 0.08	5.67 ± 0.14	296.89 ± 2.54	99.20 ± 0.86

Table 6. Short-term stability study of SRB-NLC (n = 3, mean ± SD).

Storage situation	PS (nm)	PDI	pH	EE (%)	Drug content (%)
4 °C
0M	112.67 ± 0.34	0.15 ± 0.11	5.50 ± 0.12	98.23 ± 0.46	99.32 ± 0.78
1M	141.42 ± 1.23	0.33 ± 0.12	5.46 ± 0.23	78.96 ± 0.23	81.47 ± 1.27
2M	198.00 ± 2.32	1.56 ± 0.16	5.42 ± 0.13	56.39 ± 0.35	60.52 ± 3.16
3M	265.36 ± 2.98	3.47 ± 0.13	5.40 ± 0.32	41.09 ± 0.64	51.02 ± 2.33
25 °C
0M	115.09 ± 0.23	0.14 ± 0.01	5.48 ± 0.03	99.01 ± 0.46	99.92 ± 0.69
1M	114.23 ± 0.59	0.16 ± 0.01	5.47 ± 0.03	98.98 ± 0.79	99.89 ± 0.33
2M	116.87 ± 1.34	0.12 ± 0.01	5.47 ± 0.03	98.23 ± 0.46	99.13 ± 0.71
3M	119.47 ± 0.49	0.17 ± 0.01	5.49 ± 0.02	97.97 ± 0.75	98.79 ± 0.89

Figure 7. Comparative drug in vitro release profile of optimized SRB-NLC and SRB-Susp. Data represented as mean ± SD, n = 3. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. SRB-Susp).

Figure 8. Results of rabbit eye irritation after single instillation of SRB-NLC (0.05%) and saline. The ocular conditions of each group were observed at different time points.

Figure 9. Results of CCK8 evaluation (n = 6). Cell viabilities of human corneal epithelial cells under (a) 0.25 h, (b) 1 h, (c) 2 h, and (d) 4 h cultivation with a series of Blank NLC and SRB-NLC solution (5 μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. blank NLC).

Figure 10. Concentration-time profiles of SRB in rabbit corneas (a), conjunctivas (b), and tear (c) following the single topical instillation of SRB-NLC and SRB-Susp (mean ± SD, n = 6). (*P < 0.05, **P < 0.01, ***P < 0.001 vs. SRB-Susp).

Figure 11. SRB promoted corneal epithelial recovery after treatment. (a) Representative images of the mice corneas with fluorescein staining. (b) The area of epithelial defect. (L: 0.0125% SRB-NLC, M: 0.025% SRB-NLC, H: 0.05% SRB-NLC) (*P < 0.05, **P < 0.01, ***P < 0.001 vs saline).

Figure 12. Inhibitory effect of SRB-NLC on corneal neovascularization (CNV). (a) Representative images of CNV on days 1, 3, and 7 after treatment. (b) Representative images of corneal flat-mounts are displayed under each group. (c) The area of CNV in the five groups at different checkpoints. The area of CNV in the high-dosage group reduced significantly compared with the control group. (L: 0.0125% SRB-NLC, M: 0.025% SRB-NLC, H:0.05% SRB-NLC) (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline).

Figure 13. Histopathological examination of mice cornea. (a) Hematoxylin and eosin (HE) staining of corneal sections in the normal group (400×); (b) HE staining of corneal sections in the saline group (400 ×); (c) HE staining of corneal sections in the 0.0125% (L group) (400 ×); (d) HE staining of corneal sections in the 0.025% SRB-NLC group (M group) (400×); (e) HE staining of corneal sections in the 0.05% SRB-NLC group (H group) (400×); (f) HE staining of corneal sections in the glucocorticoid group (DEX group) (400×). The arrow indicates corneal neovascularization.

Figure 14. Protein expression in cornea on the day 3 and day 7 after treatment: the levels of VEGF-A (a) and PDGF-AB (b) were determined by Elisa in the cornea tissue respectively. (L: 0.0125% SRB-NLC, M: 0.025% SRB-NLC, H:0.05% SRB-NLC) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. saline).

Figure 15. Three-dimensional surface plot for showing effect of the interaction of (a) Km and total lipid on drug content, (b) Km and total lipid on size.

Table 7. Correlation coefficients (R²) and constant values for the different mathematical models applied to the release of SRB.

Formulae	Mathematical models
	Zero order		First-order		Higuchi		Korsmeyer-Peppas
	K (/$h$)	R^2	K (/$h$)	R^2	K (/$h^{1/2}$)	R^2	K (/$h$)	N	R^2
SRB-NLC	0.53	0.96	0.02	0.98	4.8	0.98	0.72	0.44	0.99
SRB-Susp	0.13	0.94	1.98	0.93	0.35	0.87	0.04	0.97	0.93

Table 8. Ocular pharmacokinetic parameters of SRB-NLC and SRB-Susp.

Pharmacokinetic parameters	Cornea		Conjunctiva		Tears
	SRB-NLC	SRB-Susp	SRB-NLC	SRB-Susp	SRB-NLC	SRB-Susp
C max (μg/g)	1.41 ± 0.21	0.36 ± 0.08	1.66 ± 0.47	0.71 ± 0.08	85.89 ± 11.13	45.10 ± 6.79
T max (h)	0.08	0.08	0.08	0.08	0.08	0.08
t1/2(h)	6.46	4.39	6.5	5.17	1.14	0.71
AUC(0-12h）(μg/g·h^−1)	7.13	1.05	4.14	3.34	52.48	27.54