Formulation of Nanospanlastics as a Promising Approach for ‎Improving the Topical Delivery of a Natural Leukotriene Inhibitor (3-‎Acetyl-11-Keto-β-Boswellic Acid): Statistical Optimization, in vitro ‎Characterization, and ex vivo Permeation Study

Figures & data

Table 1 Prescreening Study for Investigating the Effect of Addition of EA on Elasticity of AKBA-Loaded SNVs

Formula	Span 60 (mg)	EA (mg)	Cholesterol (mg)	PS Before Extrusion (nm)	PS After Extrusion (nm)	DI
S1	450	50	–	245.8±4.16	239.2±4.24	7.21 ±0.25
S2	450	50	–	265.2±2.53	259.7±1.79	5.91 ±0.16
S3	450	50	–	288.1±3.26	280.5±2.69	4.38 ±0.24
Niosomes	450	–	225	301.5±2.86	40.33±1.72	0.51 ±0.04

Notes: S1; Spanlastic formulation containing Tween 80, S2; spanlastic formulation containing Tween 40, S3; spanlastic formulation containing Tween 20; the values are expressed as mean ± SD; n = 3.

Abbreviations: EA, edge activator; DI, deformability index; PS, particle size.

Table 2 Prescreening Study for the Formulation of AKBA-Loaded SNVs Using Tween 80 as EA

Formula	Ratio of Span 60 to EA	Rotation Speed (rpm)	Sonication Time (min)	DI
P1	90:10	250	0	4.38 ±0.18
P2	80:20	250	0	5.01 ±0.11
P3	70:30	250	0	6.93 ±0.29
P4	90:10	500	0	5.58 ±0.15
P5	80:20	500	0	6.23 ±0.31
P6	70:30	500	0	7.86 ±0.22
P7	90:10	1000	0	6.99 ±0.24
P8	80:20	1000	0	8.24 ±0.36
P9	70:30	1000	0	9.08 ±0.42
P10	90:10	250	3	7.21 ±0.25
P11	80:20	250	3	8.35 ±0.21
P12	70:30	250	3	10.61 ±0.43
P13	90:10	500	3	8.51 ±0.27
P14	80:20	500	3	9.11 ±0.44
P15	70:30	500	3	11.45 ±0.38
P16	90:10	1000	3	9.11 ±0.39
P17	80:20	1000	3	10.36 ±0.42
P18	70:30	1000	3	11.59 ±0.43
P19	90:10	250	5	9.13 ±0.34
P20	80:20	250	5	10.07 ±0.44
P21	70:30	250	5	12.33 ±0.53
P22	90:10	500	5	10.43 ±0.45
P23	80:20	500	5	11.43 ±0.43
P24	70:30	500	5	12.93 ±0.56
P25	90:10	1000	5	11.92 ±0.44
P26	80:20	1000	5	12.21 ±0.41
P27	70:30	1000	5	14.19 ±0.52

Notes: Spanlatic formulations contained Span60 and EA at 90:10 ratio involves using 450 mg Span 60 and 50 mg EA; 80:20 ratio involves using 400 mg Span 60 and 100 mg EA; 70:30 ratio involves using 350 mg Span 60 and 150 mg EA; The values are expressed as mean ± SD; n = 3.

Abbreviations: EA, edge activator; DI, deformability index.

Table 3 Experimental Runs, Independent Variables and Observed Responses in 2³ Factorial Design for AKBA-Loaded SNVs

Formula	Variables
	Independent			Dependent
	X1	X2	X3	Y1*	Y2*	Y3*
F1	−1	−1	−1	91.23±0.82	63.61±1.68	245.8±1.68
F2	−1	−1	1	74.85±1.57	44.09±2.74	229.7±2.74
F3	−1	1	−1	81.02±2.11	65.78±1.62	233.7±1.62
F4	−1	1	1	62.02±1.24	47.71±1.64	215.5±1.64
F5	1	−1	−1	95.44±1.58	95.18±2.72	275.7±2.72
F6	1	−1	1	80.53±2.22	92.05±1.55	243.3±1.55
F7#	1	1	−1	90.04±0.58	96.87±2.67	255.8±2.67
F8	1	1	1	73.90±2.86	93.49±1.73	238.4±1.73
Independent variables					Low (−1)	High (+1)
X1: Ratio of Span60 to Tween 80					70:30	80:20
X2:Rotation speed (rpm)					500	1000
X3:Sonication time (min)					3	5

Notes: Y1: EE (%), Y2: Q_8h (%), Y3: PS (nm), *the values are expressed as mean ± SD; n = 3, #Optimized Formulation.

Abbreviations: EE, entrapment efficiency; Q_8h, % drug released after 8h; PS, particle size.

Figure 1 ¹H-NMR spectrum of AKBA.

Abbreviations:¹H-NMR, proton nuclear magnetic resonance; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎.

Figure 2 ¹³C-NMR spectrum of AKBA.

Abbreviations:¹³C-NMR, carbon-13 nuclear magnetic resonance; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎.

Figure 3 EI/MS fragmentation pattern of AKBA.

Abbreviations: EI/MS, electron ionization/mass spectrometer; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎.

Figure 4 The chemical structure of AKBA and other major bioactive triterpenoids isolated from Boswellia carterii oleo-gum resin.

Abbreviation: AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎.

Figure 5 Linearity plots of AKBA-loaded SNVs shown as observed versus predicted values of (A) EE%, (B) Q_8h, and (C) PS.

Abbreviations: EE, entrapment efficiency; Q_8h, % drug released after 8h; PS, particle size.

Figure 6 The effect of different independent variables (A) ratio of Span 60 to Tween 80, (B) stirring speed and (C) sonication time on EE% of AKBA-loaded SNVs.

Abbreviations: EE, entrapment efficiency; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎; SNVs, spanlastic nanovesicles.

Figure 7 The in vitro release profile of AKBA-loaded SNVs, (n = 3).

Abbreviations: AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎; SNVs, spanlastic nanovesicles.

Figure 8 The effect of different independent variables (A) ratio of Span 60 to Tween 80, (B) stirring speed and (C) sonication time on Q_8h of AKBA-loaded SNVs.

Abbreviations: Q_8h, % drug released after 8h; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid.

Table 4 The Calculated Correlation Coefficients for the in vitro Release of AKBA-Loaded SNVs Employing Different Kinetic Orders

Formula	Zero Order	First Order	Higuchi Model	Hixson Crowell	Korsmeyer- Pappas
F1	0.9872	−0.9944	0.9971	0.9926	0.9957
F2	0.9648	−0.9726	0.9894	0.9700	0.9812
F3	0.9477	−0.9617	0.97468	0.95718	0.96658
F4	0.9255	−0.9311	0.9626	0.9293	0.9364
F5	0.9765	−0.9812	0.9952	0.9946	0.9926
F6	0.9732	−0.9781	0.9897	0.9878	0.9851
F7	0.9842	−0.9793	0.9988	0.9969	0.9953
F8	0.9880	−0.9682	0.9951	0.9866	0.9791

Figure 9 The effect of different independent variables (A) ratio of Span 60 to Tween 80, (B) stirring speed and (C) ‎sonication time on PS of AKBA-loaded SNVs.

Abbreviations: PS, particle size; AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid.

Figure 10 Scanning electron micrograph (A), transmission electron micrograph (B), particle size distribution curve (C) and zeta potential (D) of the optimized AKBA-loaded SNVs.

Figure 11 FTIR spectrum of (A) AKBA, (B) Tween 80, (C) Span 60, (D) Plain spanlastic and (E) the optimized AKBA-loaded SNVs.

Abbreviations: AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎; SNVs, spanlastic nanovesicles.

Figure 12 DSC thermogram of (A) AKBA, (B) Tween 80, (C) Span 60, (D) Plain spanlastic and (E) the optimized AKBA-loaded SNVs.

Abbreviations: AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎; SNVs, spanlastic nanovesicles.

Table 5 Effect of Storage on the Properties of the Optimized AKBA-Loaded SNVs (F7)

Parameter	Fresh F7	Stored F7
*Drug content (%)	98.54±0.65	95.94±0.37
*EE (%)	90.04±0.58	86.85±0.44
*Q8h (%)	96.87±0.82	94.65±1.47

Notes: *Each value represents mean ± SD (n = 3). F7: the optimized AKBA-loaded SNVs.

Abbreviations: EE, entrapment efficiency; Q_8h, % drug released after 8h; PS, particle size.

Table 6 Ex vivo Permeation Parameters of the Optimized AKBA-Loaded SNVs and AKBA Dispersion

Formula	*t lag (h)	*Jss (µg cm^−2 hr^−1)	*KP (cm hr^−1)	ER
AKBA dispersion	0.63±0.05	7.84±0.45	0.0078±0.15	–
F7	0.16±0.02	26.21±1.53	0.0262±0.12	3.34

Notes: *Each value represents mean ± SD (n = 3). F7: the optimized AKBA-loaded SNVs.

Abbreviations: t _lag, lag time; J_ss, steady state flux; K_P, permeability coefficient; ER, enhancement ratio.

Figure 13 Ex vivo permeation profile through hairless rat skin of AKBA dispersion and the optimized AKBA-loaded SNVs, (n = 3).

Abbreviations: AKBA, ‎3-‎acetyl-11-keto-β-boswellic acid‎; SNVs, spanlastic nanovesicles.

Table 7 The Calculated Correlation Coefficients for the ex vivo Permeation of the Optimized AKBA-Loaded SNVs and AKBA Dispersion Employing Different Kinetic Orders

Formula	Zero Order	First Order	Higuchi Model	Hixson Crowell	Korsmeyer- Pappas
AKBA dispersion	0.9918	−0.9898	0.9696	0.9905	0.9580
F7	0.9946	−0.9884	0.9822	0.9920	0.9626

Note: F7: the optimized AKBA-loaded SNVs.