Ginger Extract–Loaded Transethosomes for Effective Transdermal Permeation and Anti-Inflammation in Rat Model

Figures & data

Table 1 Composition of ginger extract (GE) transethosome (TRE) nanovesicles per 10 mL colloidal dispersion, and composition of GE transethosomal and nontransethosomal hydrogel formulations

Transethosome formulations	GE (mg)	Phospholipid (mg)	Cholesterol (mg)	Span 80 (mg)	Tween 80 (mg)	Sodium deoxycholate (mg)	Ethanol (mL)	Water (mL)
TRE1	30	300	20	30	0	0	3	7
TRE2	30	300	20	0	30	0	3	7
TRE3	30	300	20	0	0	30	3	7
Hydrogel formulations	Content
F1	GE-NaAlg (4% w:v)
F2	GE-CS (2% w:v)
F3	GE-HPMC (3% w:v)
F4	GE-TRE3-NaAlg (4% w:v)
F5	GE-TRE3-CS (2% w:v)
F6	GE-TRE3-HPMC (3% w:v)

Note: GE equivalent to 0.3% w:v in hydrogel formulations.

Abbreviations: CS, chitosan; HPMC, hydroxypropyl methylcellulose; GE-TRE3, GE-loaded TREs prepared using sodium deoxycholate; GE-TRE3-NaAlg (4%), NaAlg hydrogel containing GE-loaded TREs prepared using sodium deoxycholate; CS-TRE3, CS (2%) hydrogel containing GE-loaded TREs prepared using sodium deoxycholate; HPMC-TRE3, HPMC hydrogel (3%) GE-loaded TREs prepared using sodium deoxycholate.

Table 2 Characterization of ginger extract (GE)-loaded transethosome (TRE) nanovesicles. Data presented as means ± SD (n=3)

	Particle size (nm)	PDI	ζ-Potential (mV)	EE%	DL%
TRE1	254.8±30.84	0.45±0.02	−7.8±0.125	79.26±0.60	7.67±0.04
TRE2	286.23±10.54	0.327±0.012	−21.1±0.265	84.81±0.73	8.20±0.04
TRE3	188.3±7.66*	0.276±0.021	−38.6±0.08*	91.0±0.24*	8.8±0.03

Note: *P<0.05 compared to TRE1 and TRE2.

Abbreviations: TRE1, GE-loaded TREs prepared using Span 80; TREs 2, GE-loaded TREs prepared using Tween 80; TRE3, GE-loaded TREs prepared using sodium deoxycholate; EE, encapsulation efficiency; DL, drug loading; PDI, polydispersity index.

Figure 1 (A) Transmission electron microscopy and (B) size distribution of GE-loaded transethosomes prepared using sodium deoxycholate. Magnification 72,000×, scale bar 100 nm.

Figure 2 FT-IR spectra of (A) pure ginger extract, (B) blank transethosomes, and (C) ginger-extract transethosomes prepared using sodium deoxycholate (TRE3).

Table 3 Physical appearance, mean vesicle size, polydispersity index (PDI) and entrapment efficiency (EE) of GE-loaded TREs after 1-month storage at 4°C and 25°C. Data presented as means ± SD (n=3)

	Zero time		30 Days
Temperature	4°C	25°C	4°C	25°C
Visual appearance	No sedimentation	No sedimentation	No sedimentation	No sedimentation
EE%	91.0±0.24	91.0±0.24	89.16±0.42^NS	89.10±0.9^NS
PDI	0.276±0.04	0.276±0.04	0.321±0.03	0.34±0.021
Size (nm)	188.3±7.66	188.3±7.66	210.21±10.63	215.41±13.18

Abbreviation: NS, not significant.

Table 4 Characterization of ginger extract transethosomal and nontransethosomal hydrogel formulations. Results presented as a means ± SD (n=3)

	pH	Viscosity (mPa)	Clarity	Homogeneity	Drug content	Spreadability (g.cm/s)	Skin irritation
F1	7±0.05	3666.66±208.0	Clear	Good	99.1±1.2	7.63±0.23	Null
F2	6.9±0.03	2500±189.0	Clear	Good	99.0±0.98	8.51±0.18	Null
F3	7.2±0.06	1800±190.0	Clear	Good	99.1±1.12	9.95±0.40	Null
F4	7.1±0.03	6100±232.0*	Slightly opaque	Good	98.0±0.95	9.83±0.67	Null
F5	7.1±0.05	4700±153.17*	Slightly opaque	Good	97.6±0.93	12.6±0.16	Null
F6	7.0±0.07	4000±325.38*	Slightly opaque	Good	99.4±1.21	13.71±0.17	Null^#

Notes: *P<0.05 (transetheosome hydrogel vs nontransethosome hydrogel); ^#skin irritation evaluated visually after application of the tested formulations in comparison with irritated skin in positive-control rats that received formalin 0.8% v:v as reference irritant for 3 days.

Figure 3 Rheological profiles of GE transethosomal and nontransethosomal hydrogel formulations at 25°C (n=3).

Figure 4 (A) Cumulative in vitro release profiles of GE from NaAlg hydrogel (F1), CS hydrogel (F2), HPMC hydrogel (F3), and GE aqueous solution in phosphate buffer (pH 6.8 for 24 h at 37°C). Data presented as means ± SD (n=3). (B) Cumulative in vitro release profiles of GE from NaAlg TRE3 hydrogel (F4), CS-TRE3 hydrogel (F5), HPMC-TRE3 hydrogel (F6), and GE-TRE3 suspension in phosphate buffer (pH 6.8 for 24 h at 37°C). Data presented as means ± SD (n=3).

Figure 5 Cumulative in vitro release profiles of GE from TRES3 dispersion, HPMC hydrogel (F3), and HPMC-TRE3 hydrogel (F6) in phosphate buffer (pH 7.4 for 24 h at 37°C). Data presented as means ± SD (n=3).

Figure 6 Ex vivo permeation profiles (A) and skin-deposition percentages (B) of ginger extract HPMC hydrogel (F3) and HPMC-TRE3 hydrogel (F6) in comparison with drug solution in phosphate buffer (pH 7.4 for 24 h at 37°C). Data presented as means ± SD (n=3).

Table 5 Physicochemical evaluation of ginger extract transethosome hydrogel (HPMC-TRE3 gel; F6) during the stability study (means ± SD, n=3)

	Zero time		30 Days
Storage temperature	4°C	25°C	4°C	25°C
Surface pH	7.0±0.07	7.0±0.07	7.1±0.02	7.13±0.06
Viscosity (mPa.S) (pH 7.4, 37°C)	4000±325.38	4000±325.38	3800±225.18^NS	3650±135.10^NS
Visual appearance	Homogeneous	Homogeneous	Homogeneous	Homogeneous
Drug content	99.4±1.21	99.4±1.21	98.4±0.51^NS	98.1±1.72^NS
Spreadability (g.cm/s)	13.71±0.17	13.71±0.17	12.51±0.23	11.90±0.21

Notes: NS, not significant compared to measurements at zero time (P>0.05).

Figure 7 Percentage swelling (A) and inhibition of induced paw edema (B) in rats after treatment with free ginger-extract HPMC hydrogel (F3), transethosomal HPMC hydrogel (F6), and commercial gel (ketoprofen, 1%) compared with unmedicated group (carrageenan-induced paw edema). *Significantly different from free ginger-extract HPMC hydrogel (GE-HPMC) at P<0.05. Data presented as means ± SEM of five experimental rats per group.

Figure 8 (A) Effect of GE-HPMC-TRE3 hydrogel and ketoprofen gel (1%) on serum TNFα level in paw tissue. Means ± standard errors of the mean (SEM). *P<0.05, carrageenan vs control group; ^#P<0.05, carrageenan + GE-HPM-TREs and carrageenan ketoprofen group vs carrageenan group. (B) Effect of GE-HPMC-TRE3 hydrogel and ketoprofen gel (1%) on serum PGE₂ level in paw tissue. Data presented as means ± SEM. *P<0.05, carrageenan vs control group; ^#P<0.05, carrageenan + GE-HPM-TREs and carrageenan + ketoprofen group vs carrageenan group.

Figure 9 (A) ROS production in paw tissue in carrageenan-induced edema group was increased in compared with the control rats. GE-HPMC-TRE hydrogel and ketoprofen gel produced a significant decrease in ROS production compared to the control. (B) Paw-tissue homogenates of carrageenan-treated group showed a significant increase in MDA, and treatment with GE-HPMC-TRE hydrogel and ketoprofen gel inhibited this effect.

Notes: Values represent mea ns ± SEM. P<0.05, carrageenan group vs control group; ^#P<0.05, carrageenan + GE-HPMC-TREs and carrageenan + ketoprofen groups vs carrageenan group.

Figure 10 I and II: Histological examination (H&E, 100×) of rat-paw skin. Histological examination of paw skin in rat model of carrageenan-induced paw edema using H&E staining. (I) Thin-skin photomicrographs; (II) thick-skin photomicrographs:. IA and IIA: Control sections with normal histological structure in both thin and thick skin. IB and IIB: Sections obtained from positive control (carrageenan-induced edema without treatment) with detachment of epidermal layer (arrow), severe vacuolation, and edematous reaction in thin and thick skin (stars) and severe dermal inflammatory reaction in thin skin (double-headed arrow), as well as severe inflammatory reaction in dermal and muscular layers in thick skin (double-head arrow). IC and IIC: Sections obtained from animals treated with GE-HPMC-TRE3 hydrogel showed moderate dermal edematous and inflammatory reactions (stars). ID and IID: Sections obtained from animals pretreated with ketoprofen gel showed mild dermal edematous and inflammatory reaction (star). III and IV: Results of statistical analysis of inflammation scores of experimental groups for thin and thick skin.

Notes: (I) Thin skin and (II) thick skin. Values represent mea ns ± SEM. *P<0.05, carrageenan vs control group; ^#P<0.05, carrageenan + GE-HPMC-TRES and carrageenan + ketoprofen gel vs carrageenan group.