Development and characterization of niosomal gel for topical delivery of benzoyl peroxide

Figures & data

Table 1. Variables in Box–Behnken design.

Factor	Levels used
Independent variables	Low (−1)	Medium (0)	High (+1)
X1 Surfactant (span 60): Chol.	6:4	7:3	8:2
X2 Hydration vol. (ml)	4	6	8
X3 Drug content (mg)	2	4	6
Dependent variables	Constraints
Y1 Size (nm)	Minimum
Y2 Entrapment efficiency (%)	Maximum

Table 2. Parameters used for spreadability determination of gel.

S. No.	Parameter	Respective value
1	Test type	Compression
2	Target	7.5 mm
3	Hold time	5 s
4	Trigger load	3 gm
5	Test speed	0.5 mm/s
6	Return speed	0.5 mm/s

Table 3. Different animal groups for in vivo study.

Group no.	Group name	No. of animals
1	Control group	5
2	Plain drug solution	5
3	Blank NG	5
4	Drug-loaded NG	5

Table 4. Observed responses for BPO-loaded niosomes (Box–Behnken design).

		Independent variables			Response
Std	Run	(A) Surf: Chol.	(B) Hydration volume (ml)	(C) Drug content (mg)	Response Y1 size (nm)	Response Y2 entrapment efficiency (%)
14	1	0	0	0	534	49.4
13	2	0	0	0	538	48.1
4	3	1	1	0	481.3	47.38
3	4	−1	1	0	697	47.4
8	5	1	0	1	634.5	53.2
15	6	0	0	0	549.8	48.9
1	7	−1	−1	0	579.2	44.6
10	8	0	1	−1	431.8	57.6
5	9	−1	0	−1	606.5	52.8
7	10	−1	0	1	466.4	53.8
9	11	0	−1	−1	615.3	46.7
12	12	0	1	1	622.5	52.3
2	13	1	−1	0	697	45.9
11	14	0	−1	1	563.8	50.2
6	15	1	0	−1	437	54.72

Table 5. Summary of results of regression analysis for responses Y1, for fitting to quadratic model.

Quadratic model	R^2	Adjusted R^2	Predicted R^2	SD	% CV
Response (Y1) size	0.9778	0.9378	0.6630	21.10	3.74

Regression equations of the fitted models

Table 6. Summary of results of regression analysis for responses Y2, for fitting to quadratic model.

Quadratic model	R^2	Adjusted R^2	Predicted R^2	SD	%CV
Response (Y2) entrapment efficiency	0.9605	0.8894	0.4390	4.839	2.42

Regression equations of the fitted model

Figure 1. Contour plots showing the interaction effect. (Blue region is small size region.).

Figure 2. Response surface plot showing effect of interaction between surfactant:cholesterol ratio (A) and hydration volume (B) with low level of drug content (C) on response (size).

Figure 3. Response surface plot showing effect of surfactant:cholesterol (A) and drug content (C) interaction with high level of hydration volume (B) on response (size).

Figure 4. Response surface plot showing effect of hydration volume (B) and drug content (C) interaction with high level surfactant:cholesterol (A) of on response (size).

Figure 5. Contour plots showing the interaction effect for entrapment efficiency (orange yellow region shows more entrapment efficiency).

Figure 6. Response surface plot showing effect of interaction between surfactant:cholesterol ratio (A) and hydration volume (B) with low level of drug content (C) on response (entrapment efficiency).

Figure 7. Response surface plot showing effect of interaction between surfactant:cholesterol ratio (A) and drug content (C) with high level of hydration volume (B) on response (entrapment efficiency).

Figure 8. Response surface plot showing effect of interaction between hydration volume (B) and drug content (C) with high level of surfactant:cholesterol ratio (A) on response (entrapment efficiency).

Table 7. Composition of checkpoint formulations, expected and observed value for response variable of niosomal size.

	Composition
Formulation code	Surf: Chol.	Hydration volume (ml)	Drug content (mg)	Expected value	Observed value ± S.D
S1	8:2	7	2	336.64	341.9 ± 17.5
S2	8:2	7.5	2	309.06	315.2 ± 15.46
S3	8:2	8	2	290.96	308.9 ± 23.12

Figure 9. (a) Noisome image under motic digital microscope and (b) TEM image.

Figure 10. Size and size distribution of optimized niosomes.

Figure 11. Zeta potential of noisome.

Figure 12. In vitro drug release profile of noisome.

Figure 13. Viscosity profile of niosome gel.

Figure 14. In vitro drug release of NG.

Figure 15. In vitro permeation of niosomal and plain gel.

Table 8. Skin permeation and retention study of NG.

Formulation	% Drug permeated	% Drug remaining on skin surface	% Drug retained in the skin
NG	29.54 ± 1.513	16.74 ± 0.832	53.72 ± 1.461
Plain gel (equivalent drug)	20.73 ± 0.94	41.66 ± 1.289	37.61 ± 1.125

Table 9. MIC of BPO.

	MIC (µ g/ml)
Compound	S. aureus	P. acnes
BPO	15.6 µ g/ml	62.5 µ g/ml

Table 10. Zone of inhibition against S. aureus.

		Zone of inhibition against S. aureus (cm)
Dilutions(µg/ml)	Equivalent drug (µg)	Niosome	NG	Plain BPO
5	1	–	–	–
10	2	–	–	–
20	4	–	–	–
40	8	–	–	–
100	20	1.1	1.0	0.8
200	40	1.4	1.2	1.1

Table 11. Zone of inhibition of formulations against P. acnes equivalent drug (µg).

		Zone of inhibition against P. acnes (cm)
Dilutions (µg/ml)	Equivalent drug (µg)	Niosome	NG	Plain BPO
100	20	–	–	–
200	40	–	–	–
300	60	1.1	0.9	0.8
400	80	1.4	1.3	1.2
500	100	1.6	1.5	1.3
750	150	2.1	1.9	1.6

Figure 16. Percentage inhibition of inflammation by test formulations.

Table 12. % inhibition of inflammation after epicutaneous application of formulation.

				Daily epicutaneous application of test formulation up to four days
				% increase in Inflammation remaining
Group	Normal Ear thickness (mm)	Thickness after 24 h of injection (mm)	% increase in Inflammation	Day 1 (%)	Day 2 (%)	Day 3 (%)	Day 4 (%)	% inhibition of inflammation
Control	0.32	0.60	87.5	96.8	92.8	86.7	84.3	3.65
Plain drug solution	0.28	0.56	100	70.7	63.4	56.7	49.1	50.9
Blank NG	0.26	0.47	80.7	92.3	80.7	76.3	75.1	6.93
Drug-loaded NG	0.32	0.57	78.1	59.4	49.7	42.3	35.8	54.16

Figure 17. (a) Mice ear after P. acnes injection (b) mice ear after four days treatment with NG.

Figure 18. Log CFU count of P. acnes after epicutaneous application of test formulations.

Table 13. Log CFU count of P. acnes after epicutaneous application of test formulation.

	CFU count after one day		CFU count after two days		CFU count after four days
Group	CFU/ml	Log CFU/ml	CFU/ml	Log CFU/ml	CFU/ml	Log CFU/ml
Control group	1.6 × 10^9	9.20	1.3 × 10^9	9.11	1.2 × 10^9	9.0
Plain drug solution	2.1 × 10^7	7.32	0.9 × 10^7	6.95	1.4 × 10^6	6.14
Blank NG	1.3 × 10^9	9.11	1.1 × 10^9	9.04	0.9 × 10^9	8.95
Drug -loaded NG	3.3 × 10^7	7.52	1.0 × 10^7	7.0	2.9 × 10^5	5.46