http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–16 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.855277

ORIGINAL ARTICLE

Development and characterization of niosomal gel for topical delivery of benzoyl peroxide Gagan Goyal, Tarun Garg, Basant Malik, Gaurav Chauhan, Goutam Rath, and Amit K. Goyal

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Department of Pharmaceutics, ISF College of Pharmacy, Moga, Punjab Technical University, Jalandhar, Punjab, India

Abstract

Keywords

Benzoyl peroxide (BPO) is generally considered as first line treatment against acne. Low water solubility and formation of larger clusters and limited skin permeation upon topical application necessitates the application of high amount of drug for desired action which leads to induction of skin irritation. In the present study, we developed BPO-loaded niosomal formulation to improve its permeation through skin. The niosomes were further loaded in the carbopol gel to improve contact time. The results of the skin permeation study, skin retention study revealed that niosomes can effectively improve the drug permeation through skin. Application of niosomal gel significantly reduced the bacterial load after a treatment of four days. This reduction in bacterial load was further resulted in a significant reduction in the inflammation with minimal skin irritation compared with plain drug and the plain niosomal formulation.

Acne, carbopol gel, niosomes, skin permeation

Introduction Benzoyl peroxide (BPO) is the most widely prescribed topical medication for the treatment of acne due to its potent antipropionibacterial activity. BPO is poorly soluble in water gets together to form clusters and crystallize in aqueous environment. These clusters of BPO may be larger than the follicular opening and thus unable to penetrate the follicle. This is the probable reason for the less bioavailability of BPO. That is why BPO is presented at very high concentration of 2.5–10% in the form of cream, lotion or gel despite of its very low minimum inhibitory concentration (MIC) (Bikowski, 2010). Moreover, the current trend is to combine BPO with topical antibiotics, retinoids or other medications to increase the efficacy and decrease the risk for resistance to antibiotics (Tanghetti & Popp, 2009). But retinoids used in combination has a number of adverse effects. Systemic retinoids are contraindicated during pregnancy as they may cause central nervous system (CNS), cranio-facial, cardiovascular and other defects (Garg et al., 2012). The adverse effects with topical retinoid include primary irritant dermatitis, erythema, scaling and burning sensation. BPO monotherapy is frequently used in the form of washes designed to be rinsed off rather than remain on the skin surface where they may be irritating (Zaenglein, 2010). But they have been shown inadequate efficacy in reducing Propionibacterium acnes due to their

History Received 13 August 2013 Revised 10 October 2013 Accepted 10 October 2013

short duration of contact with skin. For the management of truncal acne, there is extremely limited existing data regarding the efficacy of BPO. Still, BPO washes are frequently recommended while showering due to easiness of application to large areas (Zaenglein & Thiboutot, 2006). However, there is some concern that washes may not provide sufficient contact time, particularly on the back where longer contact times are typically required. Niosomes are the non-ionic surfactant-based vesicular system which may enhance anti-acne efficacy by improving bioavailability and intra-follicular penetration of the BPO active. Encapsulation of the drug in the niosomes can improve the drug permeation. However, it cannot extend the contact time with the skin. Therefore, in the present study, we prepared BPO-loaded niosomes which were further incorporated in the carbopol 934 gel to alter the contact time to gain maximum benefits of the treatment.

Materials and methods Materials BPO and span 60 (Sorbitan monostearae) were kindly obtained from Central Drug House, New Delhi. Phosphatidyl choline, cholesterol, chloroform and all other products were analytical grade and were purchased from Sigma Aldrich (St. Louis, MO). Vesicular preparation

Address for correspondence: Amit K. Goyal, Department of Pharmaceutics, ISF College of Pharmacy, Moga 142001, Punjab, India. Email: [email protected]

Non-ionic surfactant vesicular system, i.e. niosomes, was prepared by the thin film hydration method as reported by Ruckmani & Sankar (2010) with slight modifications. Drug, surfactant (span 60) and cholesterol were dissolved

2

G. Goyal et al.

Drug Deliv, Early Online: 1–16

in chloroform in a round bottom flask. The chloroform was evaporated at 40  C under reduced pressure using rotary evaporator (R215, Superfit India) at 60 rpm. The dried thin film was hydrated at temperature 55  C with aqueous phase. The prepared dispersion after hydration was subjected to bath sonication that leads to formation of small unilamellar vesicles. The formulation variables were optimized on the basis of size and entrapment efficiency.

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Optimization of BPO-loaded niosomes by design of experiment Box–Behnken statistical screening design was used to optimize the formulation parameters. This design was specifically selected since it requires fewer runs than a central composite design in cases of three or four variables (Li et al., 2008). The dependent and independent variables selected are shown in Table 1 along with their low, medium and high levels for the preparation of final optimized formulation. The independent variables like surfactant:cholesterol ratio, hydration volume and drug amount were analyzed by the design for two responses (size and entrapment efficiency of niosome). Design matrix comprising of 15 experimental runs with range of three variables, i.e. surfactant:cholesterol (X1), hydration volume (X2) and drug content (X3) and the respective observed responses were size (Y1) and entrapment efficiency (Y2). The responses accomplished after the preparation of these formulations were analyzed by the design of experiment (DOE) (Design ExpertÕ Version 8.0.7.1, Stat-Ease Inc., Minneapolis, MN). The best fitting model was selected. The run which revealed the minimal size range of the niosomes and maximum range of entrapment efficiency was found to be optimized. Finally optimized was selected for further characterization. Vesicle characterization Surface morphology The niosome vesicles were characterized for their shape and surface morphology at Panjab University Chandigarh using transmission electron microscope (TEM) Hitachi (H-7500, Shiga, Japan). A drop of niosomal dispersion was smeared to a carbon film-covered copper grid and was stained with a 1% phospho-tungstic acid. Then, samples were examined and photographed with TEM at an accelerating voltage of 100 kV.

Mumbai, India) based on photon correlation spectroscopy at temperature 25  C. Zeta potential of the vesicles directs the stability of the niosomes and was measured by Zetasizer (Beckman Coulter India Pvt Ltd., Mumbai, India), working on the principle of electrophoretic light scattering. Entrapment efficiency of niosome vesicles The free drug was removed by passing the niosomal formulation through a Sephadex G-50 column. The vesicles were centrifuged at 5000 rpm for 15 min and absorbance of supernatant was checked at 236.5 nm using UV–Vis spectrophotometer (Ganta et al., 2010). In vitro drug release study of niosomes loaded with BPO In vitro release of BPO from niosomes was implemented using the dialysis bag (molecular weight 12 kDa, Sigma Aldrich) method as reported by Balasubramaniam et al. (2002) with slight modifications. The study was carried out using methanolic phosphate buffer (pH 5.6) as release medium. Two milliliters of the niosomal dispersion was filled in dialysis bag and was suspended in a conical flask containing 50 ml of release medium. The temperature was maintained at about 37  C in the shaking incubator with 100 rpm. Quantitative analysis was carried out immediately after withdrawal of samples at predetermined time intervals, using UV–Vis spectrophotometer under validated conditions. Percentage cumulative drug released was plotted against time. Preparation and characterization of niosomal gel Preparation of carbopol gel The very low viscosity often exhibited by niosome is not suitable for topical use. The viscosity can be increased by adding thickening agents, which also change the appearance of the system, usually influencing drug release. As a vehicle for incorporation of niosomes for skin delivery, the niosome formulation was loaded in 1% carbopol 934. Hundred milligrams of the carbopol 934 was dispersed in 10 ml of distilled water and kept in the dark for 24 h to allow the complete swelling of carbopol. Drug-loaded niosomes were slowly added in carbopol gel under constant stirring. The mixture was neutralized by adding a drop of triethanolamine to adjust the pH of the gel. Mixing was continued until a transparent gel appeared.

Vesicle size and zeta potential The mean vesicle size and its distribution were estimated by using particle size analyzer (Beckman Coulter India Pvt Ltd., Table 1. Variables in Box–Behnken design. Factor Independent variables X1 Surfactant (span 60): Chol. X2 Hydration vol. (ml) X3 Drug content (mg) Dependent variables Y1 Size (nm) Y2 Entrapment efficiency (%)

Levels used

Characterization of niosomal gel The prepared gel formulation was inspected visually for their color, homogeneity and consistency. The pH value of the prepared gel was determined by using pH meter (Digital SV-4, India). Gel rheology

Low (1)

Medium (0)

High (þ1)

6:4 4 2

7:3 6 4 Constraints Minimum Maximum

8:2 8 6

Viscosity measurements were conceded out using Brookfield R/S plus viscometer (Lorch, Germany). A specific amount of the formulation was used and the speed of the spindle (R3-C75) was adjusted to 100 rpm. A single run was performed at a temperature of 25  2  C with shear rate ranging from 10 to 100 s1.

Niosomal gel for topical delivery of benzoyl peroxide

DOI: 10.3109/10717544.2013.855277

Drug content uniformity The content uniformity was determined by analyzing drug concentration in gel taken from three different points, i.e. top, middle and bottom from the container. One gram of the gel sample was dissolved in methanol and BPO was extracted by sonication for 15 min. After sonication the solution was filtered through Whatman filter paper and suitably diluted with ethanol. BPO concentration was analyzed using UV–Vis spectrophotometer at 236.5 nm against blank as ethanol.

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Spreadability of the gel Spreadability of the gel was analyzed using CT-3 texture analyzer (Brookfield, Germany). Standard female and male cone attachments were used. For the analysis, female cone was filled with the optimized gel formulations and male cone was inserted into it under a set of protocol (Table 2). In vitro drug release study of niosomal gel An in vitro drug release study was performed using modified Franz diffusion cell of capacity 60 ml. Dialysis membrane (molecular weight 12 kDa, Sigma Aldrich) was placed between receptor and donor compartments. Niosomal gel (NG) equivalent to 1 g was placed in the donor compartment and because of the very low solubility of BPO in water; methanolic phosphate buffer (pH 5.6) was used as receptor compartment. The diffusion cells were maintained at 37  2  C with magnetic stirring at 100 rpm throughout the experiment. One milliliter of aliquots were withdrawn at different time intervals up to 24 h from receiver compartment and replaced with the same amount of fresh methanolic phosphate buffer solution (PBS) to maintain the sink conditions. The samples were analyzed using UV spectrophotometry at wavelength 236.5 nm. In vitro skin permeation study of NG In vitro skin permeation studies were performed using Franz diffusion cell with an effective diffusional area of 1.2 cm2 and 60 ml of receiver chamber capacity using goat skin. The hair on the skin was carefully trimmed. The skin was mounted between the donor and receiver compartment of the Franz diffusion cell while keeping the stratum corneum toward donor compartment. The receiver compartment was filled with methanolic phosphate buffer (pH 5.6). The Franz diffusion cell was placed on magnetic stirrer rotating at a speed of 100 rpm with temperature maintained at 37  1  C. One gram of NG formulation was placed into donor compartment and sealed with paraffin film to provide occlusive conditions. One milliliter of aliquots were withdrawn at regular intervals up to 24 h and were analyzed for drug Table 2. Parameters determination of gel. S. No. 1 2 3 4 5 6

used

for

spreadability

Parameter

Respective value

Test type Target Hold time Trigger load Test speed Return speed

Compression 7.5 mm 5s 3 gm 0.5 mm/s 0.5 mm/s

3

content by UV–Vis spectrophotometer. Percentage cumulative amount of drug permeated versus time was plotted and compared with the plain gel of BPO. Skin retention studies Skin retention study was done for the estimation of drug disposition in the skin. After permeation studies, skin was removed carefully from diffusion cell. Skin was cleaned with cotton soaked in phosphate buffer (pH 5.6) in order to remove the excess of the formulation. Cleaned skin piece was cut into small pieces and added in 10 ml of methanol. Above solution was sonicated in bath sonicator for 15 min with methanol in order to extract BPO. Then, the resulting solution was centrifuged and passed through 0.25 mm filter paper. Drug content in filtrate was determined spectrophotometrically. Anti-microbial assay Anti-microbial activity of BPO was tested against two microbial strains Staphylococcus aureus (MTCC 96) and P. acnes (MTCC 1951). The microbial cultures were maintained in their appropriate agar slants at 4  C throughout the study and used as stock cultures. Microbiological screening Anti-microbial activity of NG was evaluated by MIC and the agar well diffusion method and compared with the plain BPO solution. Both these studies were done in accordance with standard National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Minimum inhibitory concentration MIC value of BPO was evaluated for both the bacterial strains using the micro-dilution method. This experiment was done in 24-well micro-titer plates. Stock solution of 1000 mg/ml was prepared for BPO in 5% dimethyl sulphoxide (DMSO) in phosphate buffer solution (pH 5.6) (Candan et al., 2003). The stock solution of BPO was diluted with 5% DMSO in PBS in an arithmetic progression to obtain concentration of BPO in range from 1.95 to 250 mg/ml with constant volume of 800 ml of culture media in each well. After the samples were diluted, a standard amount of bacterial suspension (100 ml) in culture medium with turbidity equivalent to a 0.5 McFarland standard (1.5  108 colony forming unit (CFU)/mL) was inoculated onto micro-titer plates so that the inoculum density in the wells was equal to 106 CFU/ml. Immediately after inoculation, the microtiter plates were covered properly to prevent the culture medium from drying out. After 24 h incubation at 37  C, the UV absorbance of the samples was taken at 620 nm. The absorbance measured was directly related to the concentration of bacterial suspension. MIC was recorded as the lowest concentration of the agent inhibiting the growth of micro-organisms. Determination of in vitro anti-bacterial activity Anti-bacterial potential of developed formulations was established by an agar well diffusion bioassay against two bacterial strains: S. aureus and P. acnes. Sterilized tryptone soya agar medium was used for the growth of S. aureus. For the growth of P. acnes bacteria, Reinforced clostridial agar media was

4

G. Goyal et al.

Drug Deliv, Early Online: 1–16

prepared. Bacterial suspensions of S. aureus and P. acnes were prepared in their respective broth and incubated at temperature 37  C for 24 and 72 h, respectively. Preparation of dilutions of formulations for S. aureus and P. acnes Dilutions of niosome and NGs were prepared in the range of 5–200 mg/ml against S. aureus. For P. acnes dilutions prepared were ranges from 100 to 750 mg/ml. The dilutions of niosomes were prepared in methanolic PBS. Dilutions of NG were prepared in 5% DMSO in PBS (pH 5.6). Dilutions of plain BPO in 5% DMSO in PBS were also prepared.

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Application of dilutions of formulations After the spreading of the 100 ml of bacterial strain onto the prepared agar plates, two wells were made on each petri plate with the help of sterile tissue borer or cork borer. Then, 200 ml of above prepared dilutions were applied into the wells with the help of sterile syringe. Control experiments comprising inoculums without drug were set up. Then, the plates of S. aureus were incubated at 37  C for 24 h whereas the culture plates of P. acnes were incubated at 37  C for 72 h. Subsequently, anti-bacterial activity was determined by measuring the diameter of zone of inhibition (in centimeters) and compared with a positive control of BPO. In vivo anti-microbial activity study Swiss albino mice of either sex were used for the in vivo study. Protocol for studies was approved by Committee for the purpose of control and supervision of experiments on animals (CPCSEA)/Institutional Animal Ethical Committee (IAEC). The experiments were conducted as per CPCSEA (Committee for Prevention, Control and supervision of Experimental Animal) guidelines.

polypropylene cages, with free access to a standard laboratory diet and water ad libitum. Gel formulation with drug equivalent to 1 mg was applied to the left ear of the mice, while treating right ear as a control. The development of erythema was monitored for six days using the method of Van-Abbe et al. (1975). In vivo anti-acne activity Induction of infection To induce inflammation in vivo, 25 ml (1  109 CFU) of living P. acnes were intra-dermally injected into the ear of albino mice of all the groups. Ear swelling was observed in P. acnes injected ear after 24 h of the injection (Nakatsuji et al., 2009). Evaluation parameters The test formulations (drug equivalent to 1 mg) were applied epicutaneously on the mice ear after the 24 h of the P. acnes infection. The efficacy of the formulations was evaluated based upon various parameters, including inflammation, histological analysis and bacterial multiplication rate (CFU count/ml). Thickness of albino mice ear was estimated using digital vernier callipers before and after injecting P. acnes. The percentage reduction in inflammation at different time intervals of epicutaneous application of formulation up to four days was calculated.

Ear thickness.

After four day treatment with test formulation, the mice ear was cross-sectioned and stained with haematoxylin and eosin (Sigma) for observation of histological changes. The results were compared with the ear of untreated mice.

Histopathology study.

CFU count/ml. P. acnes load was analyzed in this study after

Experimental protocol Animals will be divided into six groups. Each group consists of five numbers of animals with average weight of 25–40 g. Different animal groups used for the in vivo study are depicted in Table 3. Skin irritation test Skin irritation test was performed to confirm concentration of materials suitable for topical drug delivery. Van-Abbe et al. mentioned that a value between 0 and 9 indicates that the applied formulation is generally not irritant to human skin. Swiss albino mice weighing 20–25 g were used as animal model for this study. The animals were kept under standard laboratory conditions, with temperature of 25  1  C and relative humidity of 55  5%. The animals were housed in Table 3. Different animal groups for in vivo study. Group no. 1 2 3 4

Group name

No. of animals

Control group Plain drug solution Blank NG Drug-loaded NG

5 5 5 5

a specific regimen of formulations application. After 24 h of P. acnes infection, one mice from each group was sacrificed and analyzed for bacterial load. This protocol was repeated up to four days. To determine P. acnes number in the ear, the ear was cut-off after one day of epicutaneous application of formulation and was cleaned to remove the excess of the formulation from the ear surface. It was then homogenized in 10 ml of sterile PBS with a hand tissue grinder. CFU of P. acnes in the ear were enumerated by plating serial dilutions (1:102– 1:106) of the homogenate on a Brucella agar plate. To count colonies, the plate was anaerobically incubated for 72 h at 37  C. Colonies grown on the Brucella agar plate were counted to calculate the number of CFU per milliliter. The percentage reduction in CFU count/ml at different time intervals up to four days was calculated and compared with the basal percentage reduction in CFU of untreated mice.

Results and discussion Optimization and characterization of niosomes Optimization of niosomes by DOE All the niosome formulations were prepared according to the experimental design and the effect of various formulation

Niosomal gel for topical delivery of benzoyl peroxide

DOI: 10.3109/10717544.2013.855277

5

size. On the other hand, the parameter C, i.e. drug content possesses a direct proportionality with niosomal size. Coefficients with higher-order terms or more than onefactor term in the regression equation represent quadratic relationships or interaction terms, respectively. It also shows that the relationship between responses and factors is not always linear. Used at different levels in the experiment or when more than one factors are changed simultaneously, a factor can produce different degree of response. The interaction effect of A&C, B&C has unfavorable (Increasing size) for response Y1 while that of A&B was favorable (decreasing size).

variables: surfactant:cholesterol ratio, hydration volume and drug content on niosome size and percentage entrapment efficiency was observed (Table 4). The combination of different independent variables resulted in different niosome size and entrapment efficiency. The niosome size and entrapment efficiency (%) were found to be varying between 431.8–697 nm and 44.6–57.6%, respectively. Analysis of responses All the responses observed for 15 formulations prepared were simultaneously fitted to first order, second order and quadratic models using Design ExpertÕ .

Response Y2 (entrapment efficiency)

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Response Y1 (niosome size)

Variation in entrapment efficiency is a function related to the rate at which drug is retaining into the vesicles during their in situ formation. Thus, a transformation is required while analyzing this response. Inverse transformation was applied and the best-fitted model was observed to be the quadratic and the comparative values of R2, SD and %CV are given in Table 6 along with the regression equation generated for this response. Only statistically significant (p50.05) coefficients are included in the equation. On the basis of regression equations, allusion can be drawn from the magnitude and

Analyzing this response required no transformation, the bestfitted model was observed to be the quadratic and the comparative values of R2, SD and %CV are given in Table 5 along with the regression equation generated for this response. On the basis of regression equations, allusion can be drawn from the magnitude and mathematical sign of the each coefficient. The regression equation of the niosomal size clearly explains that the impact of surfactant:cholesterol ratio (A) and hydration volume (B) is inversely related to niosomal

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

Run

(A) Surf: Chol.

14 13 4 3 8 15 1 10 5 7 9 12 2 11 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0 0 1 1 1 0 1 0 1 1 0 0 1 0 1

Response

(B) Hydration volume (ml)

(C) Drug content (mg)

Response Y1 size (nm)

Response Y2 entrapment efficiency (%)

0 0 1 1 0 0 1 1 0 0 1 1 1 1 0

0 0 0 0 1 0 0 1 1 1 1 1 0 1 1

534 538 481.3 697 634.5 549.8 579.2 431.8 606.5 466.4 615.3 622.5 697 563.8 437

49.4 48.1 47.38 47.4 53.2 48.9 44.6 57.6 52.8 53.8 46.7 52.3 45.9 50.2 54.72

Table 5. Summary of results of regression analysis for responses Y1, for fitting to quadratic model. Quadratic model Response (Y1) size

R2

Adjusted R2

Predicted R2

SD

% CV

0.9778

0.9378

0.6630

21.10

3.74

Regression equations of the fitted models Niosome size ðY1Þ ¼ 540:60  12:41A  27:84B þ 24:57C 83:37AB þ 84:40AC þ 60:55BC þ 25:39A2 þ 47:64B2  29:89C2 Table 6. Summary of results of regression analysis for responses Y2, for fitting to quadratic model. Quadratic model Response (Y2) entrapment efficiency

R2

Adjusted R2

Predicted R2

SD

%CV

0.9605

0.8894

0.4390

4.839

2.42

Regression equations of the fitted model 1=entrapment EfficiencyðY2Þ ¼ 0:020  1:351A  8:571B þ 5:456Cþ 1:610AB þ 2:185AC þ 8:131BC þ 1:522A2 þ 9:563B2  1:997C2

6

G. Goyal et al.

Drug Deliv, Early Online: 1–16

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

mathematical sign of the each coefficient. The regression equation of the entrapment efficiency clearly explains that the impact of surfactant:cholesterol ratio (A) and hydration volume (B) is directly related to entrapment efficiency. On the other hand, the parameter C, i.e. drug content possesses inverse proportionality with entrapment efficiency. Coefficients with higher-order terms or more than onefactor term in the regression equation represent quadratic relationships or interaction terms, respectively. It also shows that the relationship between responses and factors is not always linear. Used at different levels in the experiment or when more than one factor are changed simultaneously, a factor can produce different degree of response. The interaction effect of A&C, B&C as well as A&B has unfavorable (decreasing entrapment efficiency) for response Y2. Contour plots and response surface analysis Two-dimensional contour plots and three-dimensional response surface plots are very useful to study the interaction effects of the factors on the responses. These types of plots are useful in study of the effects of two factors on the response at one time, while the third factor was kept at a constant level. Contour model graph for niosome size The interaction between the two independent variables at constant value of third independent variables is shown

in Figure 1. (a) surfactant:cholesterol (A) and hydration volume (B) at three constants (low, medium and high) levels of drug content (C). (b) surfactant:cholesterol (A) and drug content (C) at three constants (low, medium and high) levels of hydration volume (B). (c) hydration volume (B) and of drug content (C) at three constants (low, medium and high) levels of surfactant:cholesterol (A). Effect of drug content variation on size of niosomes Contour plot analysis showed that in the case of interaction between the surfactant:cholesterol ratio (A) and hydration volume (B), the effect of increased drug content on the size response was quite arbitrary. At lower levels of drug content, the prevalence of acceptable blue region was quite high. But this suitable zone was observed only at higher levels of both the interacted parameters, i.e. surfactant:cholesterol ratio (A) and hydration volume (B). With the gradient inclination of drug content, the acceptable blue region was diminishing, which represents that the size was significantly increased. The probable explanation for this stance of observation is accounted to a basic phenomenon of vesicle formation. Lipophilic drug resided in the dry surfactant–cholesterol film while initial phase of preparation. During hydration, this film folds to attain a spherical structure pertaining to its high free surface energy. During this conformational alterations, the film flexibility and texture play a decisive role, which

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

DOI: 10.3109/10717544.2013.855277

ultimately depends upon the film composition. The observations concluded that with high amount of drug concentration in surfactant–cholesterol film, the flexibility was compromised, which finally effects the formation of vesicles with finer size (as the curvature of small size globule is sharper than that of large, so more flexibility is required to attain smaller size vesicle).

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Effect of hydration volume on niosome size Contour plot analysis showed that in the case of interaction between the surfactant:cholesterol ratio (A) and drug content (C), the effect of increased hydration volume on the size response was quite linear. With increase in the hydration volume, the acceptable blue region was augmented resulting in low size range. This observation accounts to a better dispersibility of niosome ingredients which ultimately results in smaller vesicular size. Effect of surfactant:cholesterol ratio Contour plot analysis showed that in the case of interaction between the hydration volume (B) and drug content (C), the effect of increased surfactant:cholesterol ratio (A) on the size response was reasonably in line. With increase in the surfactant:cholesterol ratio, the acceptable blue region was increased resulting in low size range of niosomes. This observation can be explained by the fact that the vesicle formation process is preeminent at an optimum ratio of the two ingredients. At this ratio intercalation of cholesterol molecules in surfactant chain is ideal to attain a vesicle structure with minimum size. In this case, 8:2 was observed to be the best ratio providing the smallest size range to continue with. Response surface analysis for niosomal size Contour plot analysis further explained that in all probable case of interactions, low level of drug content (C), and high levels of surfactant:cholesterol ratio (A) as well as hydration volume (B) was providing favorable range of minimum niosomal size. Surface response curve in Figures 2–4 further justified the above made conclusion.

Niosomal gel for topical delivery of benzoyl peroxide

7

Contour model graph for entrapment efficiency The interaction between the two independent variables at constant value of third independent variables is shown in Figure 5. (a) Surfactant:cholesterol (A) and hydration volume (B) at three constants (low, medium and high) levels of drug content (C). (b) Surfactant:cholesterol (A) and drug content (C) at three constants (low, medium and high) levels of hydration volume (B). (c.) Hydration volume (B) and of drug content (C) at three constants (low, medium and high) levels of surfactant:cholesterol (A). Effect of drug content variation on entrapment efficiency Contour plot analysis showed that in the case of interaction between the surfactant:cholesterol ratio (A) and hydration volume (B), the effect of increased drug content on the entrapment efficiency response was reasonably partial. At lower levels of drug content, the prevalence of acceptable region (orange) was relatively high in context to the cases with increased drug content. But this suitable zone was predominant at higher levels of both the interacted parameters, i.e. surfactant:cholesterol ratio (A) and hydration volume (B). With the gradient inclination of drug content, the acceptable orange region was diminishing, which represents that the entrapment efficiency was significantly decreased. The explanation is again related to the compromised film flexibility at higher drug concentrations. With low flexibility the likelihoods of small vesicle formation is quite less as well as the chances of the membrane irregularities and even vesicle rupture are quite high. Effect of hydration volume on entrapment efficiency Contour plot analysis showed that in the case of interaction between the surfactant:cholesterol ratio (A) and drug content (C), the effect of increased hydration volume on the entrapment efficiency response was quite linear. With increase in the hydration volume, the acceptable orange region was augmented resulting in high entrapment efficiency. This observation directly relates to the above explanation of size reduction with the increasing hydration volume.

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).

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

8

G. Goyal et al.

Drug Deliv, Early Online: 1–16

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).

Better dispersibility of niosome ingredients leading to smaller vesicular size is directly responsible for high entrapment of drug in these vesicles.

small size vesicles and second is the increased percentage of span 60 moiety, i.e. a hydrophobic surfactant. Response surface analysis of entrapment efficiency

Effect of surfactant:cholesterol ratio Contour plot analysis showed that in the case of interaction between the hydration volume (B) and drug content (C), the effect of increased surfactant:cholesterol ratio (A) on the entrapment efficiency response was reasonably in line. With increase in the surfactant:cholesterol ratio, the acceptable orange region was increased resulting in low size range of niosomes. This observation can be explained by two different facts: first being the preferential formation of

Response surface analysis further explained that in all probable case of interactions, low level of drug content (C), and high levels of surfactant:cholesterol ratio (A) as well as hydration volume (B) was providing favorable range of maximum entrapment efficiency. Surface response curve in Figures 6–8 further justified the above made conclusion. On the basis of contour plots and response surface analysis final conclusion recommended those high levels of surfactant:cholesterol ratio (A) as well as hydration volume (B) and

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

DOI: 10.3109/10717544.2013.855277

Niosomal gel for topical delivery of benzoyl peroxide

9

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).

low level of drug content (C) in order to achieve favorable range of minimum niosome size and maximum entrapment efficiency. Further selection is based on the validation check points generated by the software.

Validation of design To validate the DOE, the niosomal size of three checkpoint formulations S1, S2 and S3 (Table 7) was compared with their

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

10

G. Goyal et al.

Drug Deliv, Early Online: 1–16

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 S1 S2 S3

Surf: Chol.

Hydration volume (ml)

Drug content (mg)

Expected value

Observed value  S.D

8:2 8:2 8:2

7 7.5 8

2 2 2

336.64 309.06 290.96

341.9  17.5 315.2  15.46 308.9  23.12

expected values. There was an insignificant difference in the observed and the expected values which clearly indicated the validity of the Box–Behnken design. Formulation S3 resulted in significantly smaller niosomal size compared with the formulations S2 and S1. Therefore, formulation S3 was selected for further study. The optimum formulation of BPO-loaded niosomes was selected based on the criteria of attaining the minimum niosome size and maximum entrapment efficiency. The formulation composition with surfactant:cholesterol (8:2), hydration volume (8 ml) and drug content 2 mg was found to fulfill requisites of an optimum formulation.

Niosomal gel for topical delivery of benzoyl peroxide

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

DOI: 10.3109/10717544.2013.855277

11

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

Figure 10. Size and size distribution of optimized niosomes.

The optimized formulation had the minimum niosomal size and maximum entrapment efficiency.

and niosomal size distribution of optimized formulation is shown in Figure 10.

Characterization of optimized niosomes

Zeta potential

Surface morphology of niosomes

Zeta potential of the optimized niosome was found to be 45.62 mV and is shown in Figure 11. This depicts the high stability of the optimized vesicular formulation.

Optimized BPO niosomes were observed under a Motic digital microscope (DMWB B1, USA) and TEM for surface morphology. Images of BPO niosomes are shown in Figure 9(a) and (b). TEM images revealed that uniform size niosomal vesicles were formed. Niosome size and distribution The size of the optimized niosome was found to be 308.9 nm with polydispersity index (PDI) of 0.332. Size

Entrapment efficiency of niosome Drug entrapment of the optimized niosomes loaded with BPO was determined at wavelength 236.5 nm with the help of UV spectroscopy. Entrapment efficiency was found to be 58%, which describes an appreciable drug entrapment in niosomes vesicles.

G. Goyal et al.

Drug Deliv, Early Online: 1–16

Figure 11. Zeta potential of noisome.

80 % Cumulative drug released

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

12

Viscosity and pH of gel

70

Rheology is an important parameter as it affects the spreadability and adherence of transdermal formulations to the skin surface. The average viscosity of the NG was observed to be 2.166 Pas (Figure 13). The pH value of the prepared gel was determined by using a Digital SV-4 pH meter (Arvind Industries, India). The pH of the NG was observed to be 6.2  0.162 which is compatible with the skin pH.

60 50 40 30 20 10 0 0

5

10

15

20

25

30

Time (hours)

Figure 12. In vitro drug release profile of noisome.

In vitro drug release study Drug release of the niosomes loaded with BPO was carried out in methanolic phosphate buffer (pH 5.6) and was found to be 72.86  2.31% after 24 h. An appreciable release was observed with approximate first-order kinetics for up to 8 h and then gradually tills toward a pseudo-first-order phase. The in vitro BPO release profile was depicted in Figure 12. Characterization of NG Physical examination A small quantity of gel was pressed between the thumb and index finger, and the consistency and homogeneity of the gel were observed. It was found that there were no coarse particles in the optimized gel formulation. The color of NG was observed to be translucent white.

Drug content uniformity The content uniformity was determined by analyzing drug concentration in gel taken from three different points, i.e. top, middle and bottom from the container. The content uniformity of the niosomes gel was observed to be 91.3%. Spreadability of gel The spreadability of formulation is inversely influenced by the viscosity. Results justified this statement as the spreadibility of the NG was observed to be 18.2  1.217 (g cm/s). In vitro drug release of NG The ability of gel formulation to deliver BPO was examined by determining its drug release rate. Methanolic phosphate buffer (pH 5.6) was used in receiver compartment and sink conditions were maintained. Figure 14 shows the cumulative percentage release of BPO from NG at different sampling intervals. After 24 h, in vitro drug release from NG was found to be 47.12  1.91%, respectively.

Niosomal gel for topical delivery of benzoyl peroxide

DOI: 10.3109/10717544.2013.855277

Figure 13. Viscosity profile of niosome gel.

13

Viscosity Profile of Niosomal Gel

6

Viscosity (Pa.S)

5 4 3 2 1 0 0

20

40

60

80

100

120

% Cumulative Drug Release in Methanolic Phosphate Buffer

Table 8. Skin permeation and retention study of NG.

% Cumulative drug released

60

% Drug permeated

% Drug remaining on skin surface

% Drug retained in the skin

29.54  1.513 20.73  0.94

16.74  0.832 41.66  1.289

53.72  1.461 37.61  1.125

50

Formulation 40

NG Plain gel (equivalent drug)

30 20 10

Table 9. MIC of BPO.

0 0

5

10

15

20

25

MIC (m g/ml)

Time (hours)

Compound Figure 14. In vitro drug release of NG. 40 % Cumulative drug permeated

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Shear rate 1/S

BPO

Niosomal Gel Plain Gel

35

S. aureus

P. acnes

15.6 m g/ml

62.5 m g/ml

well-known effect. Lower size range of niosomes also accounted to this penetration enhancement.

30 25

Skin retention studies

20 15 10 5 0 0

5

10

15

20

25

The percentage drug retained in the skin for NG was found to be more as compared with plain gel (with equivalent amount of drug) as shown in Table 8. The probable reason for the more skin retention of NG may be that it provides large surface area for drug transfer to skin due to the small size of the niosomes in comparison with plain gel.

Time (Hours)

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

In vitro skin permeation of NG In vitro skin permeation studies were performed to determine the permeation rate of BPO from NG using goat skin. Methanolic phosphate buffer (pH 5.6) was used as receiver compartment and sink conditions were maintained. The in vitro skin permeation of NG is shown in Figure 15. Results demonstrated that in vitro skin permeation was found to be more in case of NG as compared with plain gel loaded with equivalent amount of drug, after 24 h. This probably was the effect of surfactant present in NG. Penetration enhancement by surfactants is a

In vitro anti-microbial activity Anti-microbial activity of BPO was tested against two microbial strains S. aureus (MTCC 96) and P. acnes (MTCC 1951). Minimum inhibitory concentration The MIC of BPO was determined and it was depicted in Table 9. Zone of inhibition Zone of Inhibition of BPO against P. acnes and S. aureus was determined using the agar well diffusion method. Tables 10 and 11 depict the zone of inhibition of test formulations

14

G. Goyal et al.

Drug Deliv, Early Online: 1–16

against S. aureus and P. acnes. Results of zone of inhibition against S. aureus and P. acnes revealed that niosome and noisome-based gel has better anti-bacterial activity profile as compared with plain BPO solution. The anti-microbial effect

in case of niosome formulations is justified by the small size of niosome results in more penetration of BPO inside the bacteria in comparison to plain BPO solution. In vivo study

Table 10. Zone of inhibition against S. aureus.

Skin irritation test

Zone of inhibition against S. aureus (cm) Dilutions (mg/ml)

Niosome

NG

Plain BPO

1 2 4 8 20 40

– – – – 1.1 1.4

– – – – 1.0 1.2

– – – – 0.8 1.1

Table 11. Zone of inhibition of formulations against P. acnes equivalent drug (mg). Zone of inhibition against P. acnes (cm) Dilutions (mg/ml) 100 200 300 400 500 750

Equivalent drug (mg)

Niosome

NG

Plain BPO

20 40 60 80 100 150

– – 1.1 1.4 1.6 2.1

– – 0.9 1.3 1.5 1.9

– – 0.8 1.2 1.3 1.6

Induction of infection To induce inflammation in vivo, 25 ml (1  109 CFU) of living P. acnes were intra-dermally injected into the ear of albino mice of all the groups. Ear swelling was observed in P. acnes injected ear after 24 h of the injection. Evaluation parameters The NG (drug equivalent to 1mg) was applied epicutaneously on the mice ear after 24 h of the P. acnes infection. The efficacy of the formulations was evaluated based upon various parameters, including inflammation,

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 Control Plain drug solution Blank NG Drug-loaded NG

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

0.32 0.28 0.26 0.32

0.60 0.56 0.47 0.57

87.5 100 80.7 78.1

96.8 70.7 92.3 59.4

92.8 63.4 80.7 49.7

86.7 56.7 76.3 42.3

84.3 49.1 75.1 35.8

3.65 50.9 6.93 54.16

Figure 16. Percentage inhibition inflammation by test formulations.

60

of % inhibition of inflammation

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

5 10 20 40 100 200

Equivalent drug (mg)

The skin irritation test was performed to confirm the safety of the topical formulation. Van-Abbe et al. mentioned that a value between 0 and 9 indicates that the applied formulation is generally not an irritant to skin. The mean skin irritation score for NG and plain BPO solution was observed to be 1.13  0.21 and 4.57  0.62, respectively. From this, it was concluded that the optimized NG formulation cause very less irritation in comparison with plain BPO solution and was safe to be used for transdermal drug delivery.

50 40 30 20 10 0 Control group

Plain drug solution

Blank niosomal gel

Formulations

drug loaded niosomal gel

Niosomal gel for topical delivery of benzoyl peroxide

DOI: 10.3109/10717544.2013.855277

histological analysis and bacterial multiplication rate (CFU count/ml). Mice ear thickness measurement After 24 h of the P. acnes injection on the mice ear, the increase in ear thickness was measured using digital vernier calliper. Then, after 24 h, the test formulations (drug equivalent to 1 mg) were epicutaneously applied on the mice ear of their respective groups up to four days. No formulation was applied to the control. The percentage inhibition of inflammation was calculated for all the animal groups by using the formula given as follows: The percentage inhibition of inflammation of mice ear after four days is shown in Table 12 and Figure 16.

15

in the number of infiltrated inflammatory cells as shown in Figure 17(a) and were decreased after the four days of the epicutaneous application of BPO-loaded NG as shown in Figure 17(b). CFU count/ml After the epicutaneous application of our test formulations to their respective groups, P. acnes count was calculated in the mice ear after 1, 2 and 4 days and compared with the P. acnes count of plain drug solution. Bacterial count of P. acnes after 1, 2 and 4 days is shown in Table 13 and Figure 18. From the bacterial count studies of P. acnes, it was observed that the log CFU/ml was reduced from values 9.2 to 5.46 by drug15 Concentration of P.acne log cfu/ml

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

% inhibition ¼ % inflammation ðcontrolÞ  % inflammation ðformulationÞ= % inflammation ðcontrolÞ Inhibition of inflammation was found to be quite more in the groups in which drug-loaded NG was applied. The drug-loaded NG inhibits the inflammation up to 54.16% in four days. Plain BPO solution inhibits the inflammation up to 50.9% in four days. Based on anti-inflammatory studies, it can be concluded that BPO-loaded NG show more inhibition of inflammation than the plain BPO solution. Histopathology studies

Control Plain drug solution Blank niosomal gel Drug loaded niosomal gel 10

5

0

Mice ear after P. acnes injection and after four days treatment with drug-loaded NG formulation is shown in Figure 17(a) and (b), respectively. Histological observation revealed that injection with P. acnes induced a considerable increase

1

2 Days

4

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

Figure 17. (a) Mice ear after P. acnes injection (b) mice ear after four days treatment with NG. Table 13. Log CFU count of P. acnes after epicutaneous application of test formulation. CFU count after one day Group Control group Plain drug solution Blank NG Drug -loaded NG

CFU/ml 9

1.6  10 2.1  107 1.3  109 3.3  107

Log CFU/ml 9.20 7.32 9.11 7.52

CFU count after two days CFU/ml 9

1.3  10 0.9  107 1.1  109 1.0  107

Log CFU/ml 9.11 6.95 9.04 7.0

CFU count after four days CFU/ml 9

1.2  10 1.4  106 0.9  109 2.9  105

Log CFU/ml 9.0 6.14 8.95 5.46

16

G. Goyal et al.

loaded NG. After two days of treatment with formulation, there was insignificant difference between the log CFU/ml of the NG and plain solution. But after four days treatment, the NG was found to reduce more CFU count of P. acnes as compared with plain BPO solution due to the sustain release of drug from niosomes. Therefore, the drug-loaded NG was more effective in reducing the CFU count/ml after four days as compared with plain BPO solution. Therefore, from the in vivo study, it can be concluded that BPO-loaded NG shows more anti-inflammatory effect and causes more reduction in CFU count of P. acnes in mice ear than the plain BPO.

Drug Delivery Downloaded from informahealthcare.com by University of Connecticut on 10/10/14 For personal use only.

Conclusion From this study, we can conclude that encapsulation of BPO in carrier system, i.e. niosome gel is advantageous because it enhances the transdermal permeation of the drug, control the release of the drug and prevent the degradation of BPO by protecting it from the direct exposure to environment. Skin irritation side effects of the drug were also reduced. In vivo study concluded that BPO-loaded NG shows more anti-inflammatory effect and causes more reduction in CFU count of P. acnes in mice ear than the plain BPO.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Drug Deliv, Early Online: 1–16

References Balasubramaniam A, Kumar VA, Pillai KS. (2002). Formulation and in vivo evaluation of niosome-encapsulated daunorubicin hydrochloride. Drug Dev Ind Pharm 28:1181–93. Bikowski J. (2010). A review of the safety and efficacy of benzoyl peroxide (5.3%) emollient foam in the management of truncal acne vulgaris. J Clin Aesthet Dermatol 3:26–9. Candan F, Unlu M, Tepe B, et al. (2003). Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae). J Ethnopharmacol 87:215–20. Ganta S, Deshpande D, Korde A, et al. (2010). A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers. Mol Membr Biol 27:260–73. Garg T, Singh O, Arora S, et al. (2012). Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst 29:1–63. Li PC, Zhang BC, Chen ZG. (2008). [Optimization for extraction technology of polysaccharide in root of Adenophora potaninii from Taibai mountain in China by orthogonal experimental design]. Zhongguo Zhong Yao Za Zhi 33:1266–8, 1312. Nakatsuji T, Kao MC, Fang JY, et al. (2009). Antimicrobial property of lauric acid against Propionibacterium acnes: its therapeutic potential for inflammatory acne vulgaris. J Invest Dermatol 129: 2480–8. Ruckmani K, Sankar V. (2010). Formulation and optimization of Zidovudine niosomes. AAPS PharmSciTech 11:1119–27. Tanghetti EA, Popp KF. (2009). A current review of topical benzoyl peroxide: new perspectives on formulation and utilization. Dermatol Clin 27:17–24. Van-Abbe NJ, Nicholas P, Boon E. Exaggerated exposure in topical irritancy and sensitization testing. J Soc Cosmet Chem 1975;26:173–87. Zaenglein AL. (2010). Making the case for early treatment of acne. Clin Pediatr (Phila) 49:54–9. Zaenglein AL, Thiboutot DM. (2006). Expert committee recommendations for acne management. Pediatrics 118:1188–99.