Formulation And Evaluation Of Nanostructured Lipid Carriers (NLCs) Of 20(S)-Protopanaxadiol (PPD) By Box-Behnken Design

Figures & data

Figure 1 Chemical structure of (A) 20(S)-protopanaxadiol (PPD), an aglycone form of ginsenosides and (B) Compound K.

Table 1 Solubility Of PPD In Various Liquid Lipids And Surfactants

Vehicle	Solubility (mg/mL)*
Liquid lipid	Miglyol 812 N	12.64 ± 1.38
	Capmul MCM	10.60 ± 1.44
	L.A.S	8.06 ± 1.59
	Lauroglycol CC	6.87 ± 0.38
	Labrafac CC	3.80 ± 0.82
Surfactant	Tween 20	5.37 ± 0.89
	Tween 80	3.55 ± 0.64
	Isopropyl myristate	2.53 ± 0.73
	Limonene	1.54 ± 0.26
	PEG 400	0.02 ± 0.01

Note: *Each value is the mean ± SD (n=3).

Abbreviation: L.A.S, PEG-8 Caprylic/Capric Glycerides.

Table 2 Independent And Dependent Variables And Their Levels In Box-Behnken Design

Independent Variables	Levels
	Low	Medium	High
X1 = PPD (%, w/v)	0.05	0.1	0.15
X2 = liquid lipid (%, v/v)	0.5	1	1.5
X3 = Tween 20 (%, v/v)	0	1	2
Transformed values	−1	0	+1
Dependent variables	Criteria:
Y1 = particle size (nm)	Minimize
Y2 = polydispersity index	Close to 0.25
Y3 = entrapment efficiency	Maximize

Abbreviation: PPD, 20(S)-protopanaxadiol.

Table 3 Box-Behnken Design Matrix With Transformed Values And The Measured Response

Run	Independent Variables			Observed Data
	X1	X2	X3	Y1	Y2	Y3
1	−1	0	−1	231.8 ± 2.2	0.179 ± 0.037	70.3 ± 1.5
2	1	−1	0	151.0 ± 2.4	0.290 ± 0.008	39.8 ± 0.9
3	0	0	0	167.2 ± 3.0	0.250 ± 0.007	70.2 ± 2.4
4	0	0	0	164.9 ± 1.3	0.257 ± 0.001	72.6 ± 1.2
5	1	0	1	152.9 ± 1.2	0.290 ± 0.009	75.4 ± 2.7
6	0	0	0	162.4 ± 2.2	0.252 ± 0.022	75.1 ± 0.5
7	0	1	−1	248.5 ± 2.3	0.220 ± 0.030	56.7 ± 1.4
8	0	1	1	179.8 ± 2.9	0.250 ± 0.013	77.3 ± 1.8
9	−1	1	0	190.3 ± 2.6	0.250 ± 0.004	76.5 ± 5.8
0	−1	0	1	147.4 ± 3.1	0.272 ± 0.012	81.2 ± 4.6
11	1	0	−1	247.2 ± 1.6	0.249 ± 0.010	46.9 ± 2.3
12	0	−1	−1	174.2 ± 1.2	0.262 ± 0.010	44.0 ± 1.5
13	−1	−1	0	143.6 ± 1.8	0.280 ± 0.001	74.5 ± 1.9
14	0	−1	1	147.2 ± 0.6	0.298 ± 0.013	68.6 ± 0.9
15	1	1	0	176.8 ± 1.4	0.250 ± 0.013	56.8 ± 0.5

Abbreviations: X₁, percent of 20(S)-protopanaxadiol; X₂, percent of liquid lipid; X₃, percent of Tween 20; Y₁, Particle size; Y₂, polydispersity index; Y₃, encapsulation efficiency.

Table 4 Results Of Regression Analysis

Parameter	Particle Size (Y1)		PDI (Y2)		EE (Y3)
	Coefficient	p-value	Coefficient	p-value	Coefficient	p-value
Intercept	138	0.003	0.261	0.000	68.8	0.000
X1	296	0.739	0.647	0.078	−447	0.001
X2	95.5	0.004	−0.160	0.019	41.4	0.044
X3	−80.7	0.000	0.060	0.005	0.30	0.001
X1X2	−209	0.513	−0.130	0.713	150	0.239
X1X3	−50	0.752	−0.270	0.155	87.8	0.177
X2X2	−14.9	0.650	0.068	0.093	−23.1	0.098
X3X3	25.7	0.014	−0.002	0.814	0.76	0.809
	0.910		0.841		0.904

Abbreviations: PDI, polydispersity index; EE, encapsulation efficiency; X₁, percent of 20(S)-protopanaxadiol; X₂, percent of liquid lipid; X₃, percent of Tween 20; , R-squared.

Figure 2 2D contour plots and 3D response surface plots for the effect of PPD amount, volume of oil, and surfactant amount on (A) particle size, (B) polydispersity index (PDI), and (C) entrapment efficiency (EE).

Table 5 Comparison Between The Predicted Yi Values Obtained By BBD And Their Actual Measurement Values Of The Optimized PPD-Loaded NLC Formulation

Response	Predicted Value (A)	Experimental Value (B)	Relative Percentage (100 x B/A) (%)
Particle size (nm; Y1)	149.2	148.7 ± 1.5	99.6
PDI (Y2)	0.250	0.250 ± 0.014	100.0
EE (%; Y3)	77.3	78.2 ± 3.1	101.2

Note: Data were expressed as mean ± standard deviation (n=3).

Abbreviations: BBD, Box-Behnken design; PPD, 20(S)-protopanaxadiol; NLC, nanostructured lipid carrier; PDI, polydispersity index; EE, encapsulation efficiency.

Figure 3 Optimization plots of Y₁, Y₂ and Y₃ versus X₁, X₂ and X_3.

Figure 4 TEM image of NLC optimized by Box-Behnken design. The scale bars represent 200 nm.

Figure 5 X-ray diffraction patterns of PPD, cetyl palmitate, poloxamer and physical mixture of PPD/cetyl palmitate/poloxamer, freeze-dried NLC without PPD, and freeze-dried NLC with PPD.

Figure 6 In vitro deposition amount of PPD in the Strat-M™ membrane at 3 hr and 6 hr after application of 1.0 mL of aqueous solution, lipid solution or optimized NLC formulation containing 0.5% (w/v) of PPD. Data were expressed as the mean ± standard deviation (n = 4). ***p < 0.0001.

Figure 7 In vitro deposition amount of PPD in the epidermis (A), dermis (B), and total skin (C) of the human cadaver skin at 3 hr and 6 hr after application of 1.0 mL of liquid solution or optimized NLC formulation containing 0.5% (w/v) of PPD. Data were expressed as the mean ± standard deviation (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Table 6 Average Irritation Score Of All Human Subjects (n=32) After Single Dose Application Of The Optimized NLC Formulations With Or Without PPD

	Optimized NLC Formulation
	Without PPD	With PPD
Average human skin irritation score	0.00	0.00
Irritation criteria	No irritancy	No irritancy

Abbreviation: PPD, 20(S)-protopanaxadiol.

Figure 8 Correlation of the in vitro deposition amount of PPD in the Strat-M™ membrane and the human cadaver skin at 3 hr and 6 hr after application of 1.0 mL of liquid solution or optimized NLC formulation containing 0.5% (w/v) of PPD. Data were expressed as the mean ± standard deviation (n = 3). The solid line represents the fitted regression curve.