Novel cationic lipid nanoparticles as an ophthalmic delivery system for multicomponent drugs: development, characterization, in vitro permeation, in vivo pharmacokinetic, and molecular dynamics studies

Figures & data

Table 1 Independent variables and levels of experiment design

Independent variables	Levels
	−2	−1	0	1	2
X1: amount of PUE (mg)	2	3.5	4	6.5	8
X2: amount of SCU (mg)	3	5.25	6	9.75	12
X3: lecithin/cholesterol (%)	1	3	5	7	9
X4: Tween^®80 (mL)	0.1	0.575	2	1.525	2

Abbreviations: PUE, puerarin; SCU, scutellarin.

Table 2 Analysis of variance of the regression coefficients of the quadratic equations

Parameters	Regression coefficients
	EE1 (%)	DL1 (%)	EE2 (%)	DL2 (%)
A0	40.02	1.04	53.91	1.72
A1	1.35	0.17	−0.23	−0.24
A2	0.076	−6.667E–0.03	−2.55	0.34
A3	−1.95	−0.062	1.46	−0.028
A4	9.10	0.22	11.61	0.42
A5	2.07	0.041	2.60	−0.016
A6	0.92	8.750E–0.03	1.34	0.12
A7	1.26	0.033	0.84	−0.060
A8	−2.53	−0.092	−3.46	−0.22
A9	1.97	0.12	2.44	0.28
A10	−0.87	−0.11	−1.68	−0.072
A11	1.53	0.043	1.52	0.10
A12	1.19	0.021	0.47	9.167E–0.03
A13	−2.15	−0.095	1.65	−0.13
A14	−2.34	−0.061	−2.30	−0.053
R	85.98	88.38	94.09	92.22
p-value	0.0204	0.0072	0.0001	0.0006

Note: A₀–A₁₄: coefficient of independent variables.

Abbreviations: EE₁, EE₂, encapsulation efficiencies of puerarin and scutellarin; DL₁, DL₂, drug loading of puerarin and scutellarin.

Figure 1 Three-dimensional response surface plots for EE and DL.

Notes: (A) Response surface plot showing the effect of the amount of PUE (X₁) and Tween^®80 (X₄) on the EE and DL of PUE (EE₁, DL₁). (B) Response surface plot showing the effect of the amount of SCU (X₂), Tween^®80 (X₄), and SCU (X₂), lecithin/cholesterol (X₃) on the EE of SCU (EE₂).

Abbreviations: PUE, puerarin; SCU, scutellarin; EE, entrapment efficiency; DL, drug loading.

Figure 2 (A) Particle size distribution, (B) zeta potential distribution, and (C) transmission electron micrograph.

Figure 3 In vitro release profiles of PUE and SCU from PUE-SCU CLN formulations (n=3).

Abbreviations: PUE, puerarin; SCU, scutellarin; CLN, cationic lipid nanoparticle.

Figure 4 Fluorescence images of rabbit eyes after administration (M1: corneal surface; M2: inner canthus and nasolacrimal duct region).

Notes: (A) RhB solution; (B) RhB-CLNs.

Abbreviations: CLNs, cationic lipid nanoparticles; RhB, rhodamine B.

Table 3 Pharmacokinetic parameters of PUE in aqueous humor after topical administration in the conscious rabbits $(\overline{X}\pm \text{SD},\mathrm{n}=3)$

Preparation	Tmax (h)	Cmax (μg·mL^−1)	AUC (μg·mL^−1·h)	T1/2 (h)	MRT (h)	VRT (h)
Solution	0.33	0.49±0.12	1.01±0.23	0.53±0.16	1.13±0.35	0.81±1.43
Formulation 1	0.67	0.59±0.30*	2.95±0.25**	0.12±0.21	1.06±0.31	1.68±1.28
Formulation 2	0.67	0.64±0.09*	2.22±0.36**	0.13±0.22	1.48±0.33	1.68±0.65
Formulation 3	0.67	0.83±0.28*	2.35±0.35**	1.92±0.06**	1.23±0.24	1.56±1.21

Notes: Solution was PUE and SCU mixed solution; formulation 1 was lipid nanoparticles; formulation 2 was lipid nanoparticles with QACMC as a cationic material; and formulation 3 was lipid nanoparticles with QACMC and Gelucire^®44/14 as a cationic material and penetration enhancer, respectively.

* p<0.05 versus PUE solution;

** p<0.01 versus PUE solution.

Abbreviations: T_max, time to peak concentration; C_max, maximum concentration of PUE; AUC, area under the curve; T_1/2, half-life time; MRT, average residence time; VRT, retention time variance; PUE, puerarin; SCU, scutellarin; SD, standard deviation; QACMC, octadecyl quaternized carboxymethyl chitosan.

Table 4 Pharmacokinetic parameters of SCU in aqueous humor after topical administration in the conscious rabbits $(\overline{X}\pm \text{SD},\mathrm{n}=3)$

Preparation	Tmax (h)	Cmax (μg·mL^−1)	AUC (μg·mL^−1·h)	T1/2 (h)	MRT (h)	VRT (h)
Solution	0.33	0.50±0.37	0.72±0.21	0.43±0.22	0.65±0.28	0.66±1.15
Formulation 1	0.67	0.57±0.05*	1.42±0.18**	1.63±0.23**	0.91±0.35	1.43±0.98
Formulation 2	0.33	0.52±0.14*	1.09±0.23*	0.79±0.33*	1.50±0.18	1.45±1.02
Formulation 3	0.67	0.56±0.28*	1.67±0.15**	1.82±0.33**	1.08±0.24	1.39±0.53

Notes: Solution was PUE and SCU mixed solution; formulation 1 was lipid nanoparticles; formulation 2 was lipid nanoparticles with QACMC as a cationic material; and formulation 3 was lipid nanoparticles with QACMC and Gelucire^®44/14 as a cationic material and penetration enhancer, respectively.

* p<0.05 versus SCU solution;

** p<0.01 versus SCU solution.

Abbreviations: T_max, time to peak concentration; C_max, maximum concentration of SCU; AUC, area under the curve; T_1/2, half-life time; MRT, average residence time; VRT, retention time variance; PUE, puerarin; SCU, scutellarin; SD, standard deviation; QACMC, octadecyl quaternized carboxymethyl chitosan.

Figure 5 PUE and SCU concentration–time profiles following a 200-μL topical administration at a dose of 2.0 mg·mL⁻¹ in the aqueous humor (n=3).

Notes: (A) PUE; (B) SCU.

Abbreviations: PUE, puerarin; SCU, scutellarin.

Figure 6 (A, B) Free energy profiles of PUE and SCU passing across POPC bilayer. (C, D) The final structure of PUE at the lowest and highest energies. (E, F) The final structure of SCU at the highest and lowest energies.

Abbreviations: PUE, puerarin; SCU, scutellarin.

Figure 7 The MSD of PUE and SCU at 100 ns.

Abbreviations: PUE, puerarin; SCU, scutellarin; MSD, mean square deviation.