Transethosomal gels as carriers for the transdermal delivery of colchicine: statistical optimization, characterization, and ex vivo evaluation

Figures & data

Table 1 Factorial designs for the preparation of transethosomes

	Values
	Low	High
Independent variables
A. Ethanol concentration	20%w/w	35%w/w
B. PL90G concentration	2%w/w	4%w/w
C. Surfactant concentration*	0.2%w/w	0.4%w/w
D. Number of extrusions	10 times	20 times
Dependent variables
The average vesicular size (nm)
Dispersity
Zeta potential (mV)

* Note: Three surfactants Tween 20^®, sodium taurocholate, and Labrafil^® were used separately in each factorial experiment.

Abbreviation: PL90G, Phospholipon 90G^®.

Table 2 Composition of the gel base

Ingredients	Concentration
Carbopol 940^®	2%w/w
Methyl paraben	0.02%w/w
Propyl paraben	0.01%w/w
Propylene glycol	1%w/w
Distilled water	Up to 100%w/w

Table 3 Experimental design and value of each factor and responses for transethosomes with Tween 20^® (TET) design

Code	Factors				Responses
	A	B	C	D	Size (nm)	Đ	ZP (mV)
TET 1	20	2	0.2	10	145.6±3.5	0.164±0.013	−25.97±1.65
TET 2	35	2	0.2	10	110.4±5.9	0.167±0.043	−26.13±0.40
TET 3	20	4	0.2	10	152.0±3.2	0.124±0.040	−19.93±0.60
TET 4	35	4	0.2	10	107.8±5.5	0.143±0.023	−20.93±0.82
TET 5	20	2	0.4	10	129.6±2.3	0.090±0.033	−18.57±2.98
TET 6	35	2	0.4	10	108.5±10.2	0.120±0.022	−26.63±0.74
TET 7	20	4	0.4	10	133.7±12.7	0.112±0.024	−17.50±1.88
TET 8	35	4	0.4	10	89.5±3.2	0.145±0.025	−22.00±0.94
TET 9	20	2	0.2	20	142.4±1.8	0.154±0.007	−25.67±1.60
TET 10	35	2	0.2	20	103.6±3.4	0.119±0.012	−22.30±1.50
TET 11	20	4	0.2	20	146.3±3.7	0.113±0.010	−18.03±1.02
TET 12	35	4	0.2	20	99.6±4.2	0.138±0.041	−19.03±1.58
TET 13	20	2	0.4	20	125.2±2.3	0.085±0.023	−19.97±0.98
TET 14	35	2	0.4	20	109.5±11.9	0.086±0.036	−23.40±1.15
TET 15	20	4	0.4	20	127.7±8.6	0.080±0.011	−17.40±1.34
TET 16	35	4	0.4	20	86.0±3.4	0.150±0.028	−19.10±1.85

Notes: Each value is expressed as mean ± standard deviation (n=3). A, ethanol concentration (%w/w); B, PL90G concentration (%w/w); C, Tween 20^® concentration (%w/w); D, number of extrusions. Formulations with vesicular size ≤150 nm, Đ≤0.3, and ZP ≥−20 mV are shown in bold.

Abbreviations: Đ, dispersity; ZP, zeta potential; PL90G, Phospholipon 90G^®.

Table 4 Experimental design and value of each factor and responses for transethosomes with sodium taurocholate (TENa) design

Code	Factors				Responses
	A	B	C	D	Size (nm)	Đ	ZP (mV)
TENa 1	20	2	0.2	10	166.8±1.0	0.160±0.014	−24.87±2.39
TENa 2	35	2	0.2	10	94.3±3.4	0.131±0.013	−31.30±3.90
TENa 3	20	4	0.2	10	146.4±2.8	0.092±0.041	−26.37±1.72
TENa 4	35	4	0.2	10	90.6±3.1	0.085±0.005	−26.77±1.01
TENa 5	20	2	0.4	10	159.3±7.5	0.129±0.017	−31.17±1.93
TENa 6	35	2	0.4	10	97.4±3.6	0.108±0.020	−37.00±0.93
TENa 7	20	4	0.4	10	127.3±2.7	0.110±0.023	−25.40±0.88
TENa 8	35	4	0.4	10	94.2±2.1	0.154±0.015	−33.47±0.52
TENa 9	20	2	0.2	20	158.0±8.7	0.093±0.034	−21.47±2.11
TENa 10	35	2	0.2	20	85.8±1.9	0.115±0.015	−24.10±0.16
TENa 11	20	4	0.2	20	140.0±3.6	0.110±0.004	−22.63±0.42
TENa 12	35	4	0.2	20	85.4±5.4	0.087±0.007	−25.47±0.98
TENa 13	20	2	0.4	20	152.6±9.2	0.098±0.033	−30.30±0.54
TENa 14	35	2	0.4	20	92.4±3.8	0.111±0.019	−31.17±2.21
TENa 15	20	4	0.4	20	128.8±3.3	0.130±0.041	−26.77±0.29
TENa 16	35	4	0.4	20	88.9±3.9	0.117±0.052	−32.10±1.53

Notes: Each value is expressed as mean ± standard deviation (n=3). A, ethanol concentration (%w/w); B, PL90G concentration (%w/w); C, sodium taurocholate concentration (%w/w); D, number of extrusions. Formulations with vesicular size ≤150 nm, Đ≤0.3, and ZP ≥−20 mV are shown in bold.

Abbreviations: Đ, dispersity; ZP, zeta potential; PL90G, Phospholipon 90G^®.

Table 5 Experimental design and value of each factor and responses for transethosomes with Labrafil^® (TEL) design

Code	Factors				Responses
	A	B	C	D	Size (nm)	Đ	ZP (mV)
TEL 1	20	2	0.2	10	168.7±3.9	0.113±0.008	−25.60±3.48
TEL 2	35	2	0.2	10	111.5±1.5	0.197±0.006	−22.57±1.65
TEL 3	20	4	0.2	10	159.1±1.5	0.183±0.011	−17.37±1.20
TEL 4	35	4	0.2	10	114.3±2.6	0.262±0.021	−25.17±8.09
TEL 5	20	2	0.4	10	168.2±2.9	0.178±0.019	−20.90±1.16
TEL 6	35	2	0.4	10	169.9±4.2	0.230±0.004	−20.30±4.17
TEL 7	20	4	0.4	10	159.2±6.8	0.152±0.018	−24.40±7.30
TEL 8	35	4	0.4	10	101.1±2.0	0.194±0.009	−20.07±0.57
TEL 9	20	2	0.2	20	167.1±1.1	0.132±0.024	−23.80±1.02
TEL 10	35	2	0.2	20	94.5±2.9	0.168±0.027	−18.53±0.92
TEL 11	20	4	0.2	20	159.1±0.6	0.179±0.013	−18.03±1.07
TEL 12	35	4	0.2	20	109.3±4.8	0.220±0.030	−18.30±1.73
TEL 13	20	2	0.4	20	162.3±5.5	0.180±0.015	−22.90±2.82
TEL 14	35	2	0.4	20	133.1±2.7	0.150±0.008	−10.58±3.44
TEL 15	20	4	0.4	20	149.2±1.7	0.130±0.021	−16.13±4.57
TEL 16	35	4	0.4	20	104.0±3.6	0.154±0.033	−12.93±1.92

Notes: Each value is expressed as mean ± standard deviation (n=3). A, ethanol concentration (%w/w); B, PL90G concentration (%w/w); C, Labrafil^® concentration (%w/w); D, number of extrusions. Formulations with vesicular size ≤150 nm, Đ≤0.3, and ZP ≥−20 mV are shown in bold.

Abbreviations: Đ, dispersity; ZP, zeta potential; PL90G, Phospholipon 90G^®.

Table 6 Results of regression analysis for the responses of the size, Đ, and ZP for all the designs

Design	Responses	R^2	Adjusted R^2	Predicted R^2	Adequate precision	Significant factors
TET	Size (nm)	0.9051	0.8856	0.8562	21.096	A, C, D, AB, AC, BC
	Đ	0.4695	0.3766	0.236	7.007	A, C, AB, BC
	ZP (mV)	0.8068	0.7611	0.6918	11.683	A, B, C, D, AC, AD, BC, ABC
TENa	Size (nm)	0.9721	0.9672	0.9598	35.389	A, B, C, D, AB, AC
	Đ	0.4258	0.2504	−0.0207	5.79	BC, ABD
	ZP (mV)	0.861	0.8079	0.723	14.922	A, B, C, D, BC, BD, ABC
TEL	Size (nm)	0.9847	0.9776	0.9656	30.928	A, B, C, D, AB, AC, AD, BC, BD, CD, ABC, ABD, BCD, ABCD
	Đ	0.7858	0.7204	0.6192	12.784	A, B, D, AC, AD, BC
	ZP (mV)	0.5751	0.376	0.0441	5.942	A, C, D, AB

Note: A, B, C, and D refer to the independent variables selected for every factorial design experiment as illustrated in Table 1.

Abbreviations: Đ, dispersity; ZP, zeta potential; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate; TEL, transethosomes with Labrafil^®.

Table 7 Coded equations of the size, Đ, and ZP for all the designs

Design	Equations for vesicular size	Equations for Đ	Equations for ZP
TET	Size = +119.84 − 17.97A − 2.01B − 6.12C − 2.28D − 4.12AB + 2.64AC − 2.49BC − 2.00ABC	Đ = +0.12 + 9.283E−003A + 1.294E−003B − 0.016C − 8.627E−003D + 9.323E−003AB + 7.701E−003AC + 0.012BC	ZP = −21.41 − 1.03A + 2.17B + 0.84C + 0.80D + 6.250E−003AB − 1.18AC + 0.69AD − 0.60BC + 0.66ABC
TENa	Size = +119.26 − 28.15A − 6.56B − 1.64C − 2.77D + 5.21AB + 3.74AC − 1.24BC	Đ = +0.11 − 8.569E−004A − 3.665E−003B + 5.121E−003C − 6.806E−003D + 1.065E− 003AB + 8.361E−004AD + 0.012BC + 7.122E− 003BD + 1.142E−003CD − 9.853E−003ABD − 5.864E−003BCD	ZP = −28.15 − 2.02A + 0.78B − 2.77C + 1.40D − 0.054AB − 0.49AC + 0.57AD + 0.71BC − 0.77BD − 0.56CD − 0.78ABC − 0.53ABD + 0.40ACD
TEL	Size = +139.40 − 22.20A −7.50B + 3.96C − 4.60D − 2.53AB + 5.85AC − 2.40AD − 7.49BC + 3.07BD − 1.62CD − 6.93ABC + 3.40ABD + 0.16ACD + 1.36BCD + 2.10ABCD	Đ = +0.18 + 0.021A + 7.936E−003B − 5.342E−003C − 0.012D + 2.633E−003AB − 9.533E−003AC − 0.012AD − 0.021BC − 1.199E−003BD − 5.365E−003CD + 4.768E−003ABD	ZP = −19.85 + 1.29A + 0.80B + 1.32C + 2.20D − 1.36AB + 1.26AC + 1.27AD − 0.66BC + 0.50BD + 0.69CD + 0.69ABC − 0.47ABD + 0.051ACD + 0.46BCD − 1.13ABCD

Note: A, B, C, and D refer to the independent variables selected for every factorial design experiment as illustrated in Table 1.

Abbreviations: Đ, dispersity; ZP, zeta potential; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate; TEL, transethosomes with Labrafil^®.

Figure 1 Response surface plots showing influences of the independent factors on the response parameters in TET design.

Abbreviations: PL90G, Phospholipon 90G^®; T20, Tween 20^®; TET, transethosomes with Tween 20^®.

Figure 2 Response surface plots showing influences of the independent factors on the response parameters in TENa design.

Abbreviations: PL90G, Phospholipon 90G^®; NaT, sodium taurocholate; TENa, transethosomes with sodium taurocholate.

Figure 3 Response surface plots showing influences of the independent factors on the response parameters in TEL design.

Abbreviations: PL90G, Phospholipon 90G^®; Lab, Labrafil^®; TEL, transethosomes with Labrafil^®.

Table 8 The effect (increase/decrease) of individual factors on the vesicular size, Đ, and ZP in each design

Design	Size		Đ		ZP
	Increase	Decrease	Increase	Decrease	Increase	Decrease
TET	–	A*, B, C*, D*	A*, B	C*, D	A*	B*, C*, D*
TENa	–	A*, B*, C*, D*	C	A, B, D	A*, C*	B*, D*
TEL	C*	A*, B*, D*	A*, B*	C, D*	–	A*, B, C*, D*

Notes: A, B, C, and D refer to the independent variables selected for every factorial design experiment as illustrated in Table 1.

* Statistically significant (p<0.05).

Abbreviations: Đ, dispersity; ZP, zeta potential; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate; TEL, transethosomes with Labrafil^®.

Table 9 Characteristics of transethosomes loaded with 0.2%w/w of colchicine

No	Code	Size (nm)	Đ	ZP (mV)	EE%
1	TET 1-0.2	150.8±4.5	0.108±0.003	−22.16±1.35	65.07±0.52
2	TET 2-0.2	92.7±2.9	0.113±0.008	−19.33±2.22	78.54±3.41
3	TET 4-0.2	95.6±1.4	0.106±0.015	−10.11±3.32	65.58±3.35
4	TET 6-0.2	87.5±1.6	0.085±0.009	−18.93±1.37	75.72±2.25
5	TET 8-0.2	93.2±0.4	0.108±0.009	−14.33±2.33	72.06±0.67
6	TET 9-0.2	148.9±3.3	0.130±0.003	−14.63±0.61	72.40±3.75
7	TET 10-0.2	82.7±1.5	0.118±0.003	−18.6±0.49	70.78±5.15
8	TET 14-0.2	79.8±1.9	0.083±0.004	−14.26±2.10	83.57±0.84
9	TENa 2-0.2	95.4±0.4	0.163±0.032	−30.43±0.41	66.26±1.85
10	TENa 3-0.2	145.7±4.9	0.174±0.029	−25.23±1.38	64.30±1.18
11	TENa 4-0.2	96.2±0.8	0.145±0.010	−27.46±3.51	75.21±0.41
12	TENa 6-0.2	99.2±0.8	0.154±0.023	−31.3±3.31	66.52±1.57
13	TENa 7-0.2	141.4±1	0.223±0.012	−26.86±1.30	68.22±0.52
14	TENa 8-0.2	94.2±1.6	0.139±0.014	−31.66±2.49	68.31±0.75
15	TENa 10-0.2	89.9±2.5	0.196±0.017	−27.13±0.20	74.53±3.47
16	TENa 11-0.2	142.6±5.3	0.165±0.027	−22.6±0.96	69.24±1.88
17	TENa 12-0.2	89.9±2.1	0.134±0.012	−20.43±0.47	77.34±0.24
18	TENa 14-0.2	94.8±2	0.149±0.006	−30.16±2.50	71.55±0.63
19	TENa 15-0.2	133.1±2.6	0.153±0.002	−23.56±2.18	66.94±1.73
20	TENa 16-0.2	84.2±0.6	0.133±0.010	−27.46±1.51	67.20±0.52
21	TEL 2-0.2	108.5±1.4	0.240±0.003	−11.73±1.19	72.74±6.08
22	TEL 4-0.2	118.9±2.8	0.183±0.006	−25.13±2.74	60.04±0.52
23	TEL 8-0.2	172.1±20	0.692±0.042	−26.63±1.91	69.07±0.83

Notes: Each value is expressed as mean ± standard deviation (n=3). All formulations shown in bold had EE% values >75% and were selected for further studies.

Abbreviations: Đ, dispersity; ZP, zeta potential; EE%, entrapment efficiency; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate; TEL, transethosomes with Labrafil^®.

Table 10 Characteristics of transethosomes loaded with 0.3%w/w of colchicine

No	Code	Size (nm)	Đ	ZP (mV)	EE%
1	TET 2-0.3	91.0±3.6	0.111±0.015	−19.86±0.77	78.84±0.65
2	TET 6-0.3	89.1±4.3	0.131±0.022	−23.00±1.66	73.44±0.57
3	TET 14-0.3	87.6±3.0	0.197±0.040	−15.16±0.57	76.74±0.77
4	TENa 4-0.3	90.4±2.2	0.090±0.004	−24.76±2.29	76.45±0.85
5	TENa 12-0.3	87.6±1.4	0.109±0.009	−20.86±1.09	79.75±1.04

Notes: Each value is expressed as mean ± standard deviation (n=3). All formulations shown in bold had EE% values >75% and were selected for further studies.

Abbreviations: Đ, dispersity; ZP, zeta potential; EE%, entrapment efficiency; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate.

Table 11 Characteristics of transethosomes loaded with 0.5%w/w of colchicine

No	Code	Size (nm)	Đ	ZP (mV)	EE%
1	TET 2-0.5	104.0±0.2	0.138±0.002	−17.00±1.34	69.37±1.46
2	TET 6-0.5	114.1±3.3	0.148±0.02	−18.00±2.08	65.21±0.68
3	TET 14-0.5	110.2±2.8	0.125±0.011	−17.73±1.73	65.34±0.56
4	TENa 4-0.5	142.1±0.6	0.132±0.003	−13.83±1.38	61.83±0.77
5	TENa 12-0.5	138.2±1.3	0.106±0.005	−10.35±1.90	63.98±1.12

Note: Each value is expressed as mean ± standard deviation (n=3).

Abbreviations: Đ, dispersity; ZP, zeta potential; EE%, entrapment efficiency; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate.

Table 12 Characterization of various colchicine-loaded transethosomal gels

Parameter	TET 2-0.3	TET 14-0.3	TENa 4-0.3	TENa 12-0.3
Size (nm)	111.4±1.6	112.7±1.1	110.5±1.1	108.4±1.5
Đ	0.157±0.01	0.159±0.012	0.162±0.014	0.153±0.01
ZP (mV)	−40.56±5.32	−41.25±3.24	−43.54±2.56	−43±1.64
pH	6.74±0.05	6.75±0.05	6.81±0.04	6.72±0.03
Viscosity (Pa·s)	66.25±2.75	60.22±0.51	55.68±0.76	53.56±1.43
Yield (Pa)	131.91±1.94	128.55±0.98	122.71±0.99	119.85±1.98

Notes: Each value is expressed as mean ± standard deviation (n=3). All formulations shown in bold were selected for further studies.

Abbreviations: Đ, dispersity; ZP, zeta potential; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate.

Figure 4 Representative rheograms of the prepared transethosomal gels.

Notes: (A) TET 2-0.3 gel; (B) TET 14-0.3 gel; (C) TENa 4-0.3 gel; (D) TENa 12-0.3 gel.

Figure 5 Ex vivo skin permeation of colchicine from various gels.

Note: The data are represented as mean ± standard deviation (n=3).
Abbreviations: NE, non–ethosomal; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate.

Table 13 The flux at steady state (J_ss) and the permeability coefficient (Kp) of colchicine from various gels through rat skin

Code	Jss (µg/cm^2/h)	Kp (cm/h)
TET 2-0.3 gel	7.233±1.1	0.0031±0.0005
TENa 4-0.3 gel	1.084±0.04	0.0005±1E–05
NE gel	0.063±0.01	0.00003±2E–06

Note: Each value is expressed as mean ± standard deviation (n=3).

Abbreviations: NE, non–ethosomal; TET, transethosomes with Tween 20^®; TENa, transethosomes with sodium taurocholate.

Figure 6 TEM images of TET 2-0.3 transethosomes in its suspension and transethosomal gel forms.

Abbreviations: TEM, transmission electron microscopy; TET, transethosomes with Tween 20^®.

Table 14 Stability study results of the TET 2-0.3 gel stored at 4°C±2°C

Parameters	0 months	4°C±2°C
		1st month	2nd month	3rd month
Size (nm)	111.4±1.6	119.4±1.9	130.5±1.8	141.7±1.8
Đ	0.157±0.01	0.145±0.013	0.152±0.015	0.156±0.012
ZP (mV)	−40.56±5.32	−39.2±2.23	−38.3±4.64	−47.3±1.2
pH	6.74±0.05	6.77±0.14	6.8±0.04	6.88±0.09
Drug content	99.94±0.32	99.98±0.36	99.06±1.16	99.16±0.47
Viscosity (Pa·s)	66.25±2.75	66.84±2.36	66.69±2.48	66.81±1.9
Yield (Pa)	131.91±1.94	132.17±1.38	131.6±1.26	132.03±1.25

Note: Each value is expressed as mean ± standard deviation (n=3).

Abbreviations: Đ, dispersity; ZP, zeta potential; TET, transethosomes with Tween 20^®.

Table 15 Stability study results of the TET 2-0.3 gel stored at 25°C±2°C/60%±5% RH

Parameters	0 months	25°C±2°C/60%±5% RH
		1st month	2nd month	3rd month
Size (nm)	111.4±1.6	140.7±1.4	150.1±1.3	162.1±2.1
Đ	0.157±0.01	0.173±0.013	0.155±0.021	0.176±0.021
ZP (mV)	−40.56±5.32	−36±2.62	−45±2.57	−44.4±1.35
pH	6.74±0.05	6.76±0.11	6.85±0.05	7.07±0.14
Drug content	99.94±0.32	99.34±0.4	97.62±0.74	95.96±0.61
Viscosity (Pa·s)	66.25±2.75	66.86±2.12	66.51±1.92	66.01±2.6
Yield (Pa)	131.91±1.94	131.94±1.11	131.54±1.31	132.01±2.22

Note: Each value is expressed as mean ± standard deviation (n=3).

Abbreviations: Đ, dispersity; ZP, zeta potential; RH, relative humidity; TET, transethosomes with Tween 20^®.