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Therapeutic Delivery

Skin delivery aspects of benzoyl peroxideloaded solid lipid nanoparticles for acne treatment

Background: Benzoyl peroxide (BPO) has been a mainstay of topical acne treatment for years. However, is frequently accompanied by cutaneous irritation and erythema. To reduce these side effects many novel drug delivery systems have been developed in the past, of which solid lipid nanoparticles (SLN) demonstrate clear dominance. Hence, we developed a facile method to prepare stable SLN of BPO and evaluated their anti-bacterial activity. Results: BPO-SLN optimized using 23 full factorial design provided high occlusion factor, low permeation rate, increased drug deposition, reduced skin irritation and strong anti-bacterial activity in contrast with marketed product. Conclusion: Desired goals were achieved by factorial design approach in shortest possible time with minimum number of experiments. The developed BPO-SLN system provided controlled drug release, thereby reducing the well-known side effects.

Background Acne vulgaris affects almost all adolescents and adults at some point in their life. Worldwide, acne vulgaris has a lifetime prevalence rate higher then 90% in people of all ages, and a point-prevalence of nearly 85% in the population ages 15 to 24 years [1] . Acne vulgaris by definition is one of the commonest chronic inflammatory cutaneous disorders of the pilosebaceous unit (PSU), with a polygenic and multifactorial nature. Four predominant pathogenetic factors that contribute to the development of acne vulgaris lesions are: (1) anomalous sebum production; (2) altered keratinization; (3) Propionibacterium acnes (gram-positive, anaerobic bacterium) follicular colonization; and, (4) liberation of inflammatory mediators into the skin [2] . Among different treatment options available, topical antimicrobial therapy has been a mainstay of acne treatment because it offers localized treatment with minimal risk of systemic adverse effects [3] . Benzoyl peroxide (BPO) is commonly used in topical formulation and was first introduced in 1934 for acne treatment [4] . BPO is available in a variety of over-the-counter and prescription formula-

10.4155/TDE.14.31 © 2014 Future Science Ltd

Varsha B Pokharkar*,1, Charu Mendiratta1, Abhay Y Kyadarkunte1, Siddharth H Bhosale2 & Ganesh A Barhate1 1 Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune – 411038, Maharashtra, India 2 Department of Biochemical Sciences, National Chemical Laboratory, Dr Homi Bhabha Road, Pune – 411008, India *Author for correspondence: Tel.: +91 20 2543 7237 Fax: + 91 20 2543 9383 [email protected]

tions, in concentrations ranging from 2.5 to 10% [5] . However, use of BPO 2.5% exhibited similar benefits to the 5 and 10% with lesser associated notorious side effects [6] . BPO acts through three predominant mechanisms in the control of acne vulgaris: it is bactericidal to P. acnes and also shows comedolytic as well as moderate anti-inflammatory activity [7] . Being lipophilic it concentrates within the sebaceous follicles to produce benzoic acid and reactive oxygen species (ROS). These products are thought to be responsible for bacterial protein oxidation, thereby impeding protein and nucleotide synthesis, metabolic pathways and mitochondrial activity [8,9] . Unlike their counterparts, antibacterial resistance does not appear to occur with BPO [4,10] . BPO monotherapy has shown greater activity than topical tretinoin or isotretinoin against inflammatory lesions [11] . Despite its marked efficacy and lower cost, it causes initial local irritation, itching, burning, stinging and redness of the skin, which is strongly linked to the concentration of drug present in the skin and type of formulation. These problems have led us to investigate suitable topical delivery systems for the encapsulation of BPO.

Therapeutic Delivery (2014) 5(6), 635–652

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ISSN 2041-5990

635

Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate

Key Terms Pilosebaceous unit: Consists of a hair follicle, sebaceous gland and an arrector pili muscle. Comedolytic: Agents or drug products capable of resolving comedones or blocking the emergence of new comedones (imperfections that form when surplus oil, along with dead skin cells become trapped in the pore). Solid lipid nanoparticles: Submicron (50–500 nm) colloidal carriers comprised of physiological lipids solid at room and body temperature, stabilized by aqueous surfactant solutions.

Recently, several attempts have been made by a large number of research groups for effective delivery of BPO employing novel vesicular and particulate drugdelivery systems such as liposomes [12,13] , niosomes [14] , microsponge [15–17] and microspheres [18] . These studies remained, more or less, limited to either controlled release or reduced skin irritation. However, antibacterial activity in terms of minimum inhibitory concentration (MIC) and zone of inhibition against different species associated with acne vulgaris (e.g., P. acnes, Staphylococus aureus [S. aureus; gram positive, aerobic bacterium], Pseudomonas aeruginosa [P. aeruginosa; gram negative, aerobic bacterium], Escherichia coli [E. coli; gram negative, aerobic bacterium], Candida albicans [C. albicans; gram-positive, aerobic fungi], and Aspergillus niger [A. niger; gram-positive, aerobic fungi]) and time-kill studies (in broth as well as on the surface of porcine skin) against P. acne, still remain to be explored. Solid lipid nanoparticles (SLN) have been employed for the encapsulation of different active agents [19–21] . This type of carrier system offers the same multitude of benefits as conventional ones such as liposomes, niosomes and polymeric nanoparticles, with added advantages such as: high biocompatibility due to generally regarded as safe (GRAS) lipidic substance and stabilizers used for the preparation of topical SLN; controlled release of drug; improved physical stability due to their solid state of particle matrix; increased adhesiveness; high occlusion factor F; skin hydration due to submicron size of lipid particles; low-cost, large-scale production owing to its facility of preparation and reproducibility; cosmetic acceptability [22–24] . Finally, SLN systems for topical acne treatment may reduce notorious side effects and, thereby, increase patient compliance. Thus, SLN clearly demonstrate their superiority over conventional nanoparticles. The main goal of the present study was to develop a facile method to encapsulate BPO into SLN and optimize it by employing a 23 full factorial design approach. Furthermore, the influence of some formulation variables on the BPO-SLN particle size and percentage encapsulation efficiency (% EE) were investigated. The

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Therapeutic Delivery (2014) 5(6)

study also encompasses evaluation of the BPO-SLN and marketed product (Benzac AC 2.5%, Galderma®) for skin delivery aspects such as in vitro drug release, occlusion test, ex vivo skin permeation, drug deposition, retention and skin irritation antibacterial activity in terms of MIC, as well as the zone of inhibition against different species associated with acne vulgaris and time-kill studies (in broth as well as on the surface of porcine skin) against P. acnes. We hypothesized that preparation of a ‘drug-enriched core’ type of SLN would facilitate controlled BPO release and skin targeting of the BPO-SLN system, and thus reduce notorious side effects and increase patient compliance. Materials & methods Materials

BPO was purchased from Sigma Aldrich, India. Precirol ATO 5 was received as a gift sample from Gattefosse, France. Tween 80 and Chloroform was purchased from Merck Limited, Mumbai, India. Carbopol 934 NF was a gift sample from Lubrizol Inc. India. Nutrient broth, nutrient agar, Sabouraud dextrose broth, Sabouraud dextrose agar and Brain’s heart infusion broth were purchased from Himedia Laboratories, India. P. acnes (MTCC 1951) was procured from IMTECH, Chandigarh, India. S. aureus (NCIM 2079), P. aeruginosa (NCIM 2493), E. coli (NCIM 2345), C. albicans (NCIM 523) and A. niger (NCIM 592) were procured from NCL, Pune, India. Selection of components

The solubility of BPO was determined in several solid lipids and organic solvents. For lipid solubility of BPO, 1 g of lipid was taken in a vial, the drug was added in steps of 0.010 g, and the vial was heated in temperature-controlled water bath set at 75°C. The amount of BPO dissolved in solid lipid was estimated. Solvent solubility of BPO was determined by adding an excess amount of BPO to the given volume of the solvent, vortexed for 2 min and equilibrated at 32°C ± 1°C (skin temperature) for 24 h. The contents were subsequently centrifuged (Allegra™ 64R Centrifuge, Beckman Coulter, CA, USA) at 10,000 rpm for 15 min. The clear supernatant was filtered using 0.45 μm membrane filter and diluted suitably with chloroform (2 ml) and analyzed spectrophotometrically (Jasco V-530, Japan) at 235 nm. Preparation of BPO-SLNs

BPO-SLNs were prepared by using the solvent evaporation method. Briefly, Precirol ATO 5 and BPO were dissolved in chloroform. Resulting solution was rapidly injected through an injection needle into aqueous surfactant solution of Tween 80 (maintained at 25°C)

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Skin delivery aspects of benzoyl peroxide-loaded solid lipid nanoparticles for acne treatment 

under ultrasonication (Sonics vibra-cell, Ultrasonic processor VCX 750 watt; Sonics and materials, Newtown, CT, USA) for 3 min. Following ultrasonication, solvent was evaporated using rotary evaporator (Superfit Continental Pvt. Ltd, India) for 1 h at 40°C. At last, the obtained BPO-SLN dispersion was shifted to airtight amber-colored glass vials and instantly sealed with rubber closures, which were covered by an aluminum cap. BPO-SLN was optimized using a 23 factorial design approach. Preparation of BPO-SLN gel

On the basis of a factorial design approach, optimal BPO-SLN dispersion (batch F3) was selected and converted into gel and mentioned hereafter as BPOSLN gel. 0.25% gel was prepared using Carbopol® 934. Briefly, the required amount of Carbopol® 934 was added into water and kept overnight for complete humectation of polymer chains. Batch F3 was added to hydrated carbopol solution to give a final concentration of 0.25% (w/w). Gelling was induced by neutralizing the dispersion to pH 6.8–7.0 using 18% (w/v) NaOH solution. Effect of variables

To study the effect of variables on BPO-SLNs characteristics and performance, different batches were prepared using a 23 full factorial design. The amount of lipid:drug ratio, surfactant and sonication amplitude were chosen as three independent factors at two levels (+1 and -1). The particle size and EE were selected as dependent variables. Independent factors and observed responses (dependent variables) for each run were depicted in Table 1. Characterization of BPO-SLN

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(TEM; JEM-2010, JEOL, Tokyo, Japan) at an acceleration voltage of 200 kV. The samples were placed on copper grids with films. The grids were dried at room temperature and then observed by TEM. Encapsulation efficiency

Encapsulation efficiency (EE; %) was determined by minicolumn centrifugation as described in our previous work [25] . Sephadex® G25M solution (10%, w/v) was prepared in DDW and was kept aside for 24 h for swelling. To prepare minicolumns, a Whatman filter pad was inserted in 1 ml syringe and swelled Sephadex was added slowly. Care was taken to avoid air entrapment in the column. Excess water was removed by spinning the column at 2000 rpm for 3 min using centrifuge machine. BPO-SLNs (100 μl) were added slowly on prepared column and centrifuged as earlier. The procedure was repeated on the same column by adding 100 μl of DDW. Free drug remained bound to the gel, while SLNs travelled the gel and were collected from the first and second stage of centrifugation. Obtained eluted nanoparticles were ruptured using a sufficient volume of chloroform and percentage encapsulation was calculated from total amount of BPO present in 100 μl of SLN by UV spectrophotometer set at 235 nm using the following equation: EE = e

Qe 100 Qt o # Equation 1

where, Qe is the amount of encapsulated BPO and Qt is the amount of BPO present in 100 μl of SLN. The method was validated by applying free drug solution instead of SLN.

Particle size & zeta potential determination

Crystallographic investigations

The mean particle size and size distributions of BPO-SLNs were determined by dynamic laser light scattering (DLS) using a 90 PLUS particle size analyzer (Brookhaven Instruments Corporation, NY, USA) at a fixed scattering angle 90° and at a temperature of 25 ± 1°C. Each value reported is the mean of three runs; the duration of each run was 2 min. Values reported are mean diameter ±SD for three replicate samples. Electrophoretic mobility and zeta potential were determined by 90 PLUS zeta size analyzer. For the zeta potential determination samples were diluted with double distilled water (DDW) and placed in the electrophoretic cell.

x-ray diffraction (XRD) patterns were captured using a Philips PW 1729 x-ray diffractometer (Philips, The Netherlands). Samples (BPO, Precirol ATO 5 and BPO encapsulated in SLN [i.e., Qe]) were irradiated with monochromatized Cu Kα radiation and were measured at angles 2 Theta from 5 to 30º. The voltage and current used were 30 kV and 30 mA, respectively. The range and the chart speed were 5 × 103 cps and 10 mm/2-Theta, respectively. The differential scanning calorimeter (DSC) measurements were performed using a METLER DSC 821e module controlled by STARe software (METLER Toledo GmbH, Switzerland). Samples (BPO, Precirol ATO 5 [4–12 mg each] and BPO encapsulated in SLN [i.e., Qe, 40 μl]) were placed into a calorimetric pan and hermetically sealed, before heating under nitrogen flow (20 ml/min) at a scanning rate of 10°C/min, over the temperature range

Transmission electron microscopy

The morphology of optimal BPO-SLN dispersion was examined by transmission electron microscopy

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate

Table 1. The independent factors and the observed responses (dependent variables) for each run of factorial design. Batch Lipid:drug Sonication code ratio X1  amplitude (%) X 2 

Tween 80 Particle size (% w/v) X 3  (nm) ±SD

ζ (mV) ±SD

PI

EE (%) ± SD

F1

15:1 (-1)

70 (-1)

4 (-1)

401 ± 5.67

-6.19 ± 1.35

0.198

58.04 ± 2.31

F2

20:1 (1)

70 (-1)

4 (-1)

783 ± 9.23

-6.23 ± 1.92

0.215

48.00 ± 3.92

F3

15:1 (-1)

90 (1)

4 (-1)

283 ± 7.39

-7.66 ± 0.94

0.174

64.61 ± 2.38

F4

20:1 (1)

90 (1)

4 (-1)

576 ± 6.90

-6.05 ± 2.01

0.196

58.66 ± 4.02

F5

15:1 (-1)

70 (-1)

6 (1)

246 ± 4.20

-6.53 ± 1.29

0.242

30.33 ± 1.78

F6

20:1 (1)

70 (-1)

6 (1)

547 ± 6.21

-5.89 ± 2.68

0.219

26.81 ± 4.39

F7

15:1 (-1)

90 (1)

6 (1)

267 ± 5.03

-5.34 ± 2.17

0.278

26.65 ± 3.18

F8

20:1 (1)

90 (1)

6 (1)

254 ± 4.85

-5.21 ± 1.94

0.209

28.34 ± 5.26

The values represent mean ±SD; n = 3. ζ: Zeta potential; EE: Encapsulation efficiency; PI: Polydispersity index; SD: Standard deviation.

of 0 to 200°C. An empty aluminum pan was used as reference. In vitro drug-release studies

Release studies (n = 3) were carried out for optimal BPO-SLN dispersion, BPO-SLN gel, marketed product and plain BPO solution using jacketed Franz cells. Prior to study, cellophane dialysis membrane (MW cutoff 12,000 Da, Hi-media Labs, India) was equilibrated in release medium (ethanol/water, 80:20, v/v) for 12 h and installed in between the donor and receptor chamber. Subsequently, aliquots containing a 1 mg equivalent dose of BPO and BPO encapsulated in SLN were gently placed in the donor chamber. The receptor chamber was filled with 25 ml of ethanol/water, 80:20, v/v and stirred with a magnetic bead. The temperature of the assay was maintained at 32 ± 1°C in order to mimic human skin conditions. At predetermined time intervals over 24 h (0.5, 1, 2, 4, 8, 16 and 24 h), 2 ml samples were withdrawn from the receptor chamber (and immediately replaced with equal volume of fresh medium). The % BPO release was analyzed by UV spectrophotometer at 235 nm with suitable dilutions. In vitro occlusion test

The in vitro occlusion test for optimized BPO-SLN, BPO-SLN gel and marketed product was performed as described by de Vringer [26] . Briefly, glass beakers (100 ml) were filled with 50 g of water, covered with filter paper (cellulose filters, 9 cm, circle, Whatman grade 6, cutoff size: 3 μm, USA) and sealed. 200 mg of sample was evenly distributed with a spatula on the filter surface (18.8 cm2). A visible film formation on top of Key Term Dermatomed skin: Stratum corneum and epidermis with partial layer of dermis, thickness about 500 μm.

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Therapeutic Delivery (2014) 5(6)

the filter paper was observed during the experiment. Later, the samples were immediately stored at 32 ± 1°C and 60 ± 5% relative humidity (RH) for 48 h in order to simulate the temperature of the skin surface. The weight of the water present in the beakers was weighed at 6, 12, 24 and 48 h. The glass beakers covered with filter paper, but without applied sample, served as a reference. Each experiment was performed in triplicate (n = 3). The occlusion factor (F) was calculated using the following equation: F = ` A - B j # 100 A

Equation 2

where, A = water loss without sample (reference) and B = water loss with sample. An F-value of 0 means no occlusive effect compared with the reference, whereas an F-value of 100 means maximum occlusive factor. Stability studies

According to International Conference on Hormonisation (ICH) guidelines, samples of optimal BPO-SLN dispersion were stored in airtight amber-colored glass vials at 25 ± 2°C/60 ± 5% RH and at 40 ± 2°C/75% ± 5% RH in a stability chamber for a period of 3 months. Particle size, encapsulation efficiency, zeta potential and polydispersity index of the samples were measured at regular intervals (initial, 7 days, 15 days, 30 days and 90 days) after their preparation. Ex vivo skin permeation & deposition of BPO

Permeation studies (n = 3) were carried out using porcine ear skin (obtained from local abattoir) as a model of human skin. Briefly, dermatomed skin was installed in between the donor and receptor chamber of vertical, jacketed Franz cells (receptor volume of 25 ml, ethanol/water 80:20, v/v) with an effective perme-

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Skin delivery aspects of benzoyl peroxide-loaded solid lipid nanoparticles for acne treatment 

ation area 3.37 cm2, constantly stirred with a magnetic bead and thermostatted at 32 ± 1°C. Later, the test formulations (approximately 800 μl of optimal BPOSLN dispersion, 800 mg of BPO-SLN gel [i.e., BPO encapsulated in SLN ‘Qe’] and 40 mg of marketed product) equivalent to 1 mg of BPO were gently placed in the donor chamber. At predetermined time intervals over 12 h (0.5, 1, 2, 4, 6 and 12 h), samples (2 ml) were withdrawn from the receptor chamber (immediately replaced with an equal volume of fresh medium) and % BPO permeated in receptor chamber was analyzed by UV spectrophotometer at 235 nm with suitable dilutions. At the end of the experiment the Franz diffusion cell was dismantled and % BPO remaining on the skin was determined. The skin surface was washed three times with diffusion medium. These washings were filtered and analyzed by UV spectrophotometer at 235 nm with suitable dilutions.% BPO deposited onto skin was estimated after washing skin with phosphatebuffered saline (PBS) pH 7.4. In the next step skin was cut into small pieces and 5 ml of ethanol was added to the tube, and drug present in the skin was extracted using tissue homogenizer (T25 Ultra Turrax, IKA, Staufen, Germany) for 2 min at 5000 rpm, followed by bath sonication for 15 min. Following this, skin homogenate was centrifuged, and the supernatants were analyzed by UV spectrophotometer at 235 nm with suitable dilutions. All the animal investigations were performed as per the protocol approved by the Institutional Animal Ethical Committee (IAEC) and Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA) No.100-1999 Poona College of Pharmacy, Pune. Draize skin irritation test

The irritation potential of BPO-SLN gel in contrast with marketed product was evaluated by carrying out the Draize patch test on albino rabbits [27] . For this study, animals of 2.5–3.0 kg were divided into four groups (n = 3) as follows: • Group 1: Control; • Group 2: Marketed product; • Group 3: BPO-free SLN (placebo); • Group 4: BPO-SLN gel. Briefly, backs of the rabbits were shaved 24 h before the application of formulations. Accurately measured quantities of formulations (equivalent to 5 mg of drug), were applied to the skin under 2.54 cm gauze patches that were secured with adhesive tapes. To prevent any volatile constituents form evaporating, the trunks of

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the rabbits were wrapped in rubberized cloth and the animals were placed in restrainers and patches left in contact with the skin for 24 h. The skin was observed for any visible change such as erythema at 0 (initial), 24, 48 and 72 h after the application of formulation. The mean erythema scores (MES) were recorded ranging from 0 to 4 (0 = no erythema; 1 = slight erythema [barely perceptible-light pink]; 2 = moderate erythema [dark pink]; 3 = moderate to severe erythema [light red]; and, 4 = severe erythema [extreme redness]) depending on the degree of erythema [27] . Antibacterial activities of developed BPO-SLN system Against anaerobic bacteria

P. acnes, gram-positive bacterium is one of the main causative agents of acne vulgaris. Lyophilized culture of P. acnes was revived in aseptic condition. Briefly, one loop full of P. acnes culture was aseptically transferred to reinforced brain’s heart infusion (BHI) broth (5 ml), allowed to reach around OD600 = 1 (log phase of the growth) and termed as mother culture. Anaerobic condition was created in the in-house designed apparatus using 100% nitrogen gas. A minimum inhibitory concentration (MIC) of optimal BPO-SLN dispersion and BPO-SLN gel were compared with marketed product. These samples were suspended in DMSO (0.05%, v/v), incubated with P. acnes (107 CFU/ml) and transferred to BHI broth. Twofold serial dilutions of the same were performed in BHI broth to obtain optimum concentrations for marketed product, optimal BPO-SLN dispersion and BPO-SLN gel. Control tube received 0.05% (v/v) of DMSO only; clindamycin phosphate and BPO-free SLN (placebo) served as positive and negative control, respectively. The tubes were incubated under anaerobic atmosphere at 37°C for 48 h followed by determination of OD600 to estimate the bacterial growth [28] . Lowest concentration of formulation exhibiting growth inhibition of P. acnes was termed as MIC. Against aerobic bacteria

Antibacterial activities of optimal BPO-SLN dispersion, BPO-SLN gel and marketed preparation was carried on skin infective aerobic bacteria such as S. aureus, P. aeruginosa and E. coli, and fungi such as C. albicans and A. niger using agar gel diffusion method. The respective bacterial cultures were grown in nutrient broth at 37˚C for 24 h. Fungal cultures were seeded in sabouraud dextrose broth followed by incubation at 25˚C, for 48 h. Optical density of the culture was adjusted to 0.1 with sterile nutrient broth. From this 0.1 ml of the culture was seeded in 25 ml molten nutrient agar, mixed and poured into sterile petri plate, and

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639

Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate allowed to solidify. The wells were bored with 8 mm borer in seeded agar followed by addition of different concentrations of the formulation in each well. The control plate received respective seed cultures only; clindamycin phosphate and placebo were served as the positive and negative control, respectively. Plates were allowed to normalize to room temperature and further incubated at 37˚C for 24 h. The diameter of the zones of inhibition were measured and recorded in millimeters (mm) using a vernier caliper [28] . Time-kill assay To investigate further, in vitro time-kill studies (n = 3) of optimal BPO-SLN dispersion, BPO-SLN gel and marketed product were performed in broth, as well as on the surface of porcine skin. Evaluation of bactericidal reduction in broth was performed in a specially designed test tube using ten-times the MIC. Samples were prepared at two-times the final test concentration in DDW, and a culture suspension in 2 × broth was added in a 1:1 ratio to achieve the final BPO concentration and inoculum (P. acnes log phase prepared at 107 CFU/ml in BHI at 37°C in an anaerobic conditions). At predetermined time intervals over 6 h (0, 0.5, 1, 2, 4 and 6 h), samples were sequentially diluted and 100 μl of each sample was plated onto BHI. Colony counts were assessed after 3 to 5 days under anaerobic conditions. The solution with no BPO-SLN was used as control. Time-kill studies were also performed after incubation of P. acnes on the surface of porcine ear skin (obtained from local abattoir). Porcine skin was used because the structure, permeability and metabolic properties are similar to humans. Briefly, the skin was gently cleaned and the subcutaneous fat was removed manually such that the PSU were left intact and then excised. Glass rings (height of 10 mm and diameter of 20 mm) were glued to the skin surface and installed in 6-well culture plates (surface area 9.6 cm2/well, Becton Dickinson and Company, NJ, USA) and hydrated with 0.5 ml of sterile water and stored at 2–8° C for 24 h prior to use. After 24 h, water was removed and 30 μl of P. acnes culture was gently placed on the porcine skin surface within the limits of the glass ring to a concentration of 6 to 7 log of CFU/cm2. Inoculated skin samples were incubated under anaerobic conditions for 1 h in order to allow absorption of bacteria to the skin surface. After 1 h of equilibration, 50 μl of BPO was applied to the skin samples (17.7 μl/cm2 of skin); this dose of BPO provided an application similar to the topical applications of BPO used in humans. Treated skin samples were incubated under anaerobic conditions and at predetermined time intervals over 6 h (0, 0.5, 1, 2, 4 and 6 h) samples were taken out from the incubator. All skin samples were rinsed with 1 ml of cold

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Therapeutic Delivery (2014) 5(6)

saline (twice) in order to accumulate any viable bacteria still living on the skin surface. The merged rinses were combined and sequentially diluted, and 100 μl of all dilutions, including the initial merged rinse, was plated on BHI and incubated anaerobically at 35–37°C. Statistical data analysis In the present study, all the experiments were carried out in triplicate and the data reported as mean ± standard deviation (SD). The significance of differences was evaluated using ANOVA (Dunnett’s multiple comparison test) at the probability level of 0.05. Results & discussion Selection of components

In order to establish the solid lipids and solvents that can dissolve the maximum amount of BPO, a solubility study of BPO in various solid lipid and solvent was performed. Among the solid lipids and solvents screened, the maximum solubility of BPO was found in Precirol ATO 5 and chloroform (Table 2) . With the aim to prepare ‘drug-enrich core’ type SLN, Precirol ATO 5 was finally selected for preparation of BPOSLNs. In addition, the higher solubility of BPO in Precirol ATO 5 would allow facile encapsulation of drug cargo with high efficiency. Chloroform was selected as it gets fast evaporated (in contrast with other solvents tested) from lipid nanoparticles and shortens the time for the fabrication of BPO-SLNs. Experimental design & statistical data

Initial experiments indicated that the independent variables were the key factors affecting particle size and % EE of the BPO-SLNs. Therefore, a 23 full factorial design approach was employed in order to investigate the impact of these three key independent variables on particle size and % EE of the prepared SLN. For each factor, the experimental scale was selected based on the results of initial experiments and the practicability of preparing the SLN at the utmost value. The value scale of the variables was lipid:drug ratio (15:1 and 20:1), Tween 80 concentrations (4 and 6%) and sonication amplitude (70 and 90%). Evaporation time (1 h) and temperature (40°C) were kept constant for the study. A total of nine batches were carried out. Responses of different batches obtained using factorial design are displayed in Table 1. The main effect terms (X1, X 2 and X 3) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X1X 2, X1X 3, and X 2 X 3) show how the response changes when two factors are simultaneously changed. The effects of the variables are interpreted considering the magnitude of coefficient and the mathematical sign it carries.

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Skin delivery aspects of benzoyl peroxide-loaded solid lipid nanoparticles for acne treatment 

Y = b0 + b1 X1 + b2 X 2 + b3 X 3 + b12 X1 X 2 + b13 X1 X3 + b23 X 2 X3

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Table 2. Solubility data of benzoyl peroxide in different solid lipids and solvents. Solid lipid

Solubility (mg/gm)

Solvent

Solubility (mg/ml)

Obtained data were subjected to multiple regression analysis followed by Contour plots generated using the PC software MODDE 9.1 demo version (Umetrics, AB, Umeå, Sweden). The data were fitted in (Equation 3) . Results of multiple regression analysis for all parameters studied are summarized in Table 3. Analysis of data was carried out using ANOVA to check the adequacy of the fitted model.

Stearic acid

10 ± 4.0

Benzyl alcohol

86 ± 5.0

Precirol ATO 5

300 ± 5.0

Toluene

1.9 ± 2.0

Dynasan 116

30 ± 3.2

Ethyl acetate

1.9 ± 1.5

Dynasan 114

23 ± 1.0

Benzyl benzoate

75 ± 3.0

 

 

Propylene glycol

42 ± 2.9

 

 

Ethanol

80 ± 3.2

 

 

Chloroform

150 ± 6.3

Effect on particle size

Values represent mean ±standard deviation; n = 3.

Equation 3

Particle size (D90) evaluated immediately after preparation of the BPO-SLN was observed to be in the range of 246 ± 4.20 to 783 ± 9.23 nm (Table 1) . To understand the effect of lipid:drug ratio, sonication amplitude and Tween 80 concentrations on particle size, coefficients observed were fitted in (Equation 3) to generate (Equation 4) . Y = 419.625 + 120.375 X1 - 74.625 X 2 - 91.125 X3 - 50.375 X1 X 2 - 48.375 X1 X3 + 6.625 X 2 X3 Equation 4

When the coefficients of the three independent variables in (Equation 4) were compared (Table 3), the value for X1 (β1 = 120.375) was found to be maximum and hence it was considered to be a major contributing factor affecting particle size of the BPO-SLN. The contour plots were plotted to evaluate the effect of different variables on particle size and are illustrated in Figure 1. From the contour plots it was observed that as lipid:drug ratio increased, particle size increased (Figure 1A–D) ; this may be attributed to a greater amount of lipid getting deposited as a coat around the drug particles [29] , also when sonication amplitude (X 2) and Tween 80 (X 3) increases the particle size gets decreased (Figure 1E–F) due to the presence of the Tween 80 (tensioactive material) – such material reduces the interfacial tension and facilitates the further droplet division during sonication (thereby forming smaller droplets) [30] . Effect on encapsulation efficiency

% EE is expressed as a fraction of drug incorporated into the nanoparticle relative to the total amount of drug used. Determination of % EE is an important parameter in case of SLN as it may affect the drug release and skin deposition. The % EE ranged from 26.65 ± 3.18 to 64.61 ± 2.38 (Table 1). The high % EE might prove beneficial to reduce the skin irritation as it avoids the direct contact between drug and skin surface [31] .

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To study the effect of % EE, (Equation 5) was generated after fitting the observed coefficient in (Equation 3) . Y = 42.68 - 2.2275 X1 + 1.885 X2 - 14.6475 X3 + 1.1625 X1 X 2 + 1.77 X1 X3 - 2.4225 X 2 X3 Equation 5

When the coefficients of the three independent variables in equation 5 were compared, the value for X 3 (β3 = - 14.647) was found to be maximum and, hence, it was considered to be a major contributing factor affecting encapsulation efficiency of the BPO-SLN. The contour plots were plotted to evaluate the effect of different variables on % EE and are illustrated in Figure 2. From the contour plots it was observed that as lipid:drug ratio and Tween 80 concentration increased, % EE decreased (Figure 2A–D) since the solubility of drug increases in presence of Tween 80 [32] . Optimal BPO-SLN dispersion (Batch F3) was selected as the optimized batch as it showed smaller particle size and high encapsulation efficiency (Table 1) . Table 3. Summary of regression analysis results for measured responses. Coefficients

Particle size

Encapsulation efficiency

β0

419.625

42.68

β1

120.375

-2.2275

β2

-74.625

1.885

β3

-91.125

-14.647

β12

-50.375

1.162

β13

-48.375

1.77

β23

6.625

-2.422

2

r

0.9777

1

p

0.0426

0.002

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate

Particle size (nm)

Particle size (nm)

B

Particle size (nm)

C

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

0.2

0.2 0 -0.2

Lipid:Drug ratio

0.8

Lipid:Drug ratio

0 -0.2

0 -0.2

-0.4

-0.4

-0.6

-0.6

-0.6

-0.8

-0.8

-0.8

-1

-1 Sonication amplitude Particle size (nm)

Particle size (nm)

E

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 Tween 80 Particle size (nm)

F

0.8

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.4

Sonication amplitude

Lipid:Drug ratio

-1 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 Sonication amplitude

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8

D

-0.4

0.2 0 -0.2 -0.4

Sonication amplitude

Lipid:Drug ratio

A

0.2 0 -0.2

-0.4

0.2 0 -0.2 -0.4

-0.6

-0.6

-0.6

-0.8

-0.8

-0.8

-1

-1

-1 -1 -0.8 -0.6 -0.4-0.2 0 0.2 0.4 0.6 0.8 Tween 80

-1 -0.8-0.6-0.4 -0.2 0 0.2 0.4 0.6 0.8 Tween 80

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 Tween 80

Figure 1. Contour plots of optimal benzoyl peroxide-solid lipid nanoparticle dispersion for particle size (nm) with respective to lipid:drug ratio and sonication amplitude. (A) low (-1) and (B) high (1) level of factor X 3 (Tween 80 concentration); lipid:drug ratio and Tween 80 at (C) low (-1) and (D) high (1) level of factor X 2 (sonication amplitude %). Sonication amplitude and Tween 80 at (E) low (-1) and (F) high (1) level of factor X1 (lipid:drug ratio). Optimal benzoyl peroxide-solid lipid nanoparticle dispersion: Batch F3.

Zeta potential

The values of zeta potential of BPO-SLN stabilized by Tween 80 ranged from -5.21 ± 1.94 to -7.66 ± 0.94 mV (Table 1) . A possible explanation for the lower

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Therapeutic Delivery (2014) 5(6)

zeta potential could be due to the partial hydrolysis of Tween 80 [33] ; and Tween 80 being a non-ionic surfactant provides steric stability for maintaining the stability of BPO-SLN [34] .

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0.8

0.6

0.6

0.4

0.4

0.2

0.2

-0.2

-0.6

-0.8

-0.8

-1

-1

.0 -1

-0

-0

-0

-1

.6

-0.6

.4 -0 .2 0. 0 0. 2 0. 4 0. 6 0. 8

-0.4

.8

-0.4

Tween 80

Tween 80 Encapsulation efficiency (%)

Encapsulation efficiency (%)

D

0.8

0.6

0.6

0.4

0.4 Sonication amplitude

0.8

0.2 0.0 -0.2

0.2 0.0 -0.2 -0.4

-0.6

-0.6

-0.8

-0.8

-1

-1 Tween 80

-1 .0 -0 .8 -0 .6 -0 .4 -0 .2 0. 0 0. 2 0. 4 0. 6 0. 8

-0.4

-1 .0 -0 .8 -0 .6 -0 .4 -0 .2 0. 0 0. 2 0. 4 0. 6 0. 8

Sonication amplitude

C

.8

-0.2

0.0

-0

0.0

0.8

-0

Lipid:Drug ratio

0.8

.0

Lipid:Drug ratio

Encapsulation efficiency (%)

B

.6 -0 .4 -0 .2 0. 0 0. 2 0. 4 0. 6 0. 8

Encapsulation efficiency (%)

A

Research Article

Tween 80

Figure 2. Contour plots of optimal benzoyl peroxide-solid lipid nanoparticle dispersion for encapsulation efficiency (%) with respect to lipid:drug ratio and Tween 80. (A) low (-1) and (B) high (1) level of factor X 2 (sonication amplitude %). Sonication amplitude and Tween 80 at (C) low (-1) and (D) high (1) level of factor X1 (lipid:drug ratio). Optimal benzoyl peroxide-solid lipid nanoparticle dispersion: Batch F3.

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate more, the closer observation of the TEM image illustrates the presence of a thin layer encircling the particle, which suggests the formation of ‘drug-enriched core’ type of SLN. This finding is in agreement with the previous studies, which showed that this type of SLN is generally formed when the melting point of the drug is more than the melting point of the lipid [35] . Melting point of BPO is around 103°C and melting point of Precirol ATO 5 is around 56°C. ‘Drugenriched core’ type of SLN will have its impact on skin drug-delivery aspects such as occlusion factor F, skin permeation, drug deposition and skin irritation, as discussed later on. Crystallographic investigations 100 nm Figure 3. Transmission electron microscopy of optimal benzoyl peroxidesolid lipid nanoparticle dispersion. Optimal benzoyl peroxide-solid lipid nanoparticle dispersion: Batch F3.

TEM

TEM was performed to investigate the morphology and internal structure of optimal BPO-SLN dispersion. Figure 3 provides a close-up view of the internal structure of optimal BPO-SLN dispersion. It was observed that particles were uniform spherical in shape and no drug crystals were found. The absence of drug crystal (free drug) correlated well with the results of EE and certifies the suitability of the method. FurtherA

The XRD patterns of BPO, Precirol ATO 5 and optimal BPO-SLN are shown in Figure 4A. The XRD pattern of BPO presented several sharp and distinct diffraction peaks indicating the crystalline nature of the drug. Peaks for crystallinty were observed in pure BPO at 2θ values of 11.20°, 11.6°, 15°, 20.7°, 21.3°, 22.2° and 27.4°. The solid lipid Precirol ATO 5 showed peaks at 18.25°, 19.12°, 24.27° and 25.97°. On the contrary, the XRD pattern of optimal BPO-SLN showed a weak peak intensity of drug when compared with the BPO and Precirol ATO 5 suggesting that the degree of the crystallinity is lower in the nanoparticles. Such partial loss of crystallinity may be observed due to amorphization of the lipid, which restricts drug expulsion from BPO-SLN [36] . These results also suggest that the crysB BPO-SLNs

T (%)

Intensity

BPO-SLNs Precirol ATO 5

Precirol ATO 5

BPO

BPO 5

10

15

20

2 - Theta (˚C)

25

30

30

40

50

60

90 100 110 120 70 80 Temperature (˚C)

Figure 4. Crystallographic investigations. (A) x-ray diffractograms and (B) differential scanning calorimetry scans of BPO, Precirol ATO 5 and optimal BPO-SLN. BPO: Benzoyl peroxide; SLN: Solid lipid nanoparticle.

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In vitro drug-release studies

depicts the in vitro release profile of optimal BPO-SLN dispersion, BPO-SLN gel, marketed product and plain BPO solution. As expected, plain BPO solution exhibited an initial burst release of 67.29 ± 1.49% in the first 3 h of study. On the contrary, optimal BPO-SLN dispersion (35.48 ± 2.26%) and BPO-SLN gel (28.21 ± 1.43) exhibited a controlled release pattern in contrast with marketed product (44.53 ± 1.29%) over a period of 24 h. This controlled release pattern was mainly attributed to the formation of a BPO-enriched core encircled by a lipid layer (Figure 3) , and due to the hydrophobic long chain fatty acids of the triglycerides (Precirol ATO 5, palmitic [C16] and stearic acid [C18] > 90%) that retained the BPO [39] . This finding is in agreement with previous studies, which state that drug loaded in an SLN system provides a controlled release pattern [40–42] .

100 90 80 % BPO release

talline BPO is dissolved and embedded in Precirol ATO 5; hence it is suitable as lipid matrix for the production of SLN. The DSC thermograms of BPO and Precirol ATO 5, as well as optimal BPO-SLN, are shown in Figure 4B. The thermal curve of BPO showed an exothermic peak at 110°C due to its decomposition [37] . The thermal curve for Precirol ATO 5 exhibits a melting endotherm at 52°C. However, in case of optimal BPO-SLN, the characteristic peak at 51°C showed melting of Precirol ATO 5 but no melting peak for BPO was detected, indicating that BPO was dissolved in the Precirol ATO 5, which makes them an ideal excipient in the production of SLN. We observed characteristic peak at 115°C, which may be due to the flash point of Tween 80 [38] . The exothermic decomposition peak of BPO disappeared in the thermogram of Optimal BPO-SLN. It can be concluded that encapsulation of BPO in the SLN system prevented the decomposition of BPO.

Research Article

70 60 50 40 30

Optimal BPO-SLN dispersion BPO-SLN gel Marketed product Pure BPO solution

20 10 0 0

2

4

6

8 10 12 14 16 18 20 22 24 Time (h)

Figure 5. In vitrorelease profile of optimal benzoyl peroxide-solid lipid nanoparticle dispersion, benzoyl peroxide-solid lipid nanoparticle gel, marketed product and plain benzoyl peroxide solution over a 24 h period. The values represent mean ±standard deviation; n = 3. Optimal BPO-SLN dispersion: Batch F3. BPO-SLN gel: Batch F3 dispersion converted to 0.25% gel. Marketed product: Benzac AC 2.5%, Galderma®. BPO: Benzoyl peroxide; SLN: Solid lipid nanoparticle.

Figure 5

In vitro occlusion test

The extent of the occlusive effect of SLN depends on various factors such as particle size, applied sample volume and lipid concentration [43] . In vitro occlusion tests revealed a higher occlusion factor F (p < 0.05) for optimal BPO-SLN dispersion and BPO-SLN gel in contrast with marketed product at different time intervals (Table 4) . BPO-SLN has a melting point of approximately 51°C, which means it is still solid at both room and skin temperature, whereas marketed products melt at these temperatures. The increased occlusion factor F is mainly attributed to the high lipid concentration (1 part of BPO and 15 parts of Precirol ATO 5) and adhesive feature of optimal BPO-SLN dispersion and BPO-SLN gel, which reduces evaporation of water through the filter paper. Therefore, an occlusive effect has been claimed [26] .

Table 4. Occlusion factor F (%) of optimal benzoyl peroxide-solid lipid nanoparticle dispersion, benzoyl peroxide-solid lipid nanoparticle gel and marketed product at different time intervals. Formulations 

Occlusion factor F (%) 6h

12 h

24 h

48 h

Marketed product

27.25 ± 6.15

25.64 ± 3.48

21.92 ± 5.12

19.38 ± 2.49

Optimal BPO-SLN dispersion

40.30 ± 3.57

41.28 ± 4.17

37.81 ± 4.81

35. 91 ± 2.98

BPO-SLN gel

45.73 ± 2.98

43.97 ± 1.37

39.02 ± 3.18

36.65 ± 1.86

The values represent mean ±SD; n = 3. Optimal BPO-SLN dispersion: Batch F3; BPO-SLN gel: Batch F3 dispersion converted to 0.25% gel; Marketed product: Benzac AC 2.5%, Galderma®. BPO: Benzoyl peroxide; SD: Standard deviation; SLN: Solid lipid nanoparticle.

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate

Table 5. Particle size, zeta potential, PI and encapsulation efficiency of optimal benzoyl peroxidesolid lipid nanoparticle dispersion over a period of 3 months stored at varying temperature. ζ (mV) ± SD

PI

EE (%) ± SD

25 ± 2°C/60 ± 5% RH  

 

 

 

Initial

283.46 ± 7.39

− 7.66 ± 0.94

0.174

64.61 ± 2.38

7 days

280. 82 ± 4.28

− 7.47 ± 0.25

0.168

65.72 ± 3.58

15 days

281. 92 ± 3.19

− 8.01 ± 0.38

0.169

63.98 ± 1.96

30 days

280.94 ± 2.68

− 8.31 ± 0.97

0.173

67.20 ± 2.04

90 days

281 ± 3.06

− 8.40 ± 0.26

0.175

63.08 ± 1.83

40 ± 2°C/75 ± 5% RH

 

 

 

 

Initial

283.37 ± 7.39

− 7.66 ± 0.94

0.174

64.61 ± 2.38

7 days

281.02 ± 2.64

− 7.38 ± 0.87

0.172

63.84 ± 6.29

15 days

286.61 ± 5.27

− 7.87 ± 0.09

0.170

62.73 ± 7.03

30 days

287.03 ± 3.92

− 8.38 ± 0.25

0.174

62.94 ± 3.97

90 days

288.20 ± 4.76

− 8.41 ± 0.49

0.189

61.05 ± 2.89

Time (days)

Particle size (nm) ± SD

The values represent mean ±SD; n = 3. Optimal benzoyl peroxide-solid lipid nanoparticle dispersion: Batch F3. ζ: Zeta potential; EE: Encapsulation efficiency; PI: Polydispersity index; RH: Relative humidity; SD: Standard deviation.

The higher occlusion factor F is also because optimal BPO-SLN dispersion has a particle size in the nanometer range (283 ± 7.39), and thus possesses a large specific surface area and associated adhesive properties when compared with marketed product [44] .

for 90 days at 40 ± 2°C/75 ± 5% was 61.05 ± 2.89%, with a slight decrease that occurred in comparison with 64.61 ± 2.38% on the day of preparation with no significant difference (p < 0.05). Ex vivo skin permeation & deposition of BPO

Stability studies

Optimum BPO-SLN showed good stability over a period of 90 days (Table 5) . The varying temperature (i.e., 25 ± 2°C/60 ± 5% RH and 40 ± 2°C/75 ± 5%) over a period of 90 days had no significant effect on the particle features such as size, zeta potential and PI (p < 0.05). The EE of optimum BPO-SLN stored 100

Marketed product BPO-SLN dispersion BPO-SLN gel

90

% BPO

80 70 60 50 40 30 20 10 0 1

2

3

Figure 6. Comparison of the benzoyl peroxide levels from ex vivo skin permeation studies (at 6 h). (1) BPO remained over the skin, (2) BPO deposited in the skin, and (3) BPO permeated in receptor chamber. Values represent mean ±standard deviation; n = 3. BPO: Benzoyl peroxide.

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Therapeutic Delivery (2014) 5(6)

Drug permeation in the receptor chamber, drug deposition in the skin and drug remaining on the skin was carried out with the objective to determine the ‘drugenriched core’ effect of optimal BPO-SLN dispersion and BPO-SLN gel in contrast with marketed product. After 6 h, amount of drug deposited in the skin was 57.02 ± 2.07% and 41.10 ± 1.79% from optimal BPOSLN dispersion and BPO-SLN gel in contrast with 18.96 ± 1.18% from marketed product (Figure 6) . This increased deposition of BPO was attributed to uniformity of small particle size, high skin occlusion factor F and ‘drug-enriched core’ features of developed BPOSLN system (both BPO-SLN dispersion and BPOSLN gel). As depicted in Figure 6, >70% BPO from marketed product remained on the skin, in contrast with 40% and nearly 60% BPO from optimal BPOSLN dispersion and BPO-SLN gel, respectively. Such significant difference (p < 0.05) was mainly attributed to the nanometeric particle size of developed BPOSLN system. The fact that nanometer-sized particles can make closer contact with superficial junctions of corneocytes clusters and furrows present between corneocytes islands favors accumulation for several hours, allowing sustained BPO release [45] . The presence of 2.8 and 1.3% BPO from optimal BPO-SLN dispersion and BPO-SLN gel, respectively, in the receptor medium could be attributed to the presence of non-

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ionic surfactant such as Tween 80 used for stabilization of SLN. Such surfactants enhance drug permeation only to a minor extent [46] . According to these results, many authors have observed the same feature using SLN [47–49] . SLNs therefore represent a highly effective carrier for topical BPO preparations, where controlled release and skin targeting is desired. Draize skin irritation test

A major disadvantage associated with the BPO therapy is skin irritation, which strongly limits its utility and acceptability by the patients. Ideally, the delivery system of BPO should be able to diminish or abolish Control

Marketed product

Research Article

the erythematic episodes. However, most of the currently marketed conventional dosage forms, such as creams, are not able to reduce the irritation caused by topical application of BPO. It was foreseen that encapsulation of BPO in SLN would lead to controlled release of BPO, thus reducing skin irritation. Draize patch test is a reliable method and the results obtained from this study can be linked to results obtained in humans. The results (MES) of Draize skin irritation test are depicted in Figure 7. At 24 h of test, the results revealed that the BPO-SLN gel did not show any sign erythema (score 0) in contrast with the marketed product (score 2). Moreover, steady state increase in BPO-free SLN

BPO-SLN gel

Score 0

Score 0

Score 0

Score 0

Score 2

Score 0

Score 0

Score 0

Score 2

Score 0

Score 0

Score 0

Score 3

Score 0

Score 1

72 h

48 h

24 h

Initial

Score 0

Figure 7. Photographs of Draize skin irritation test carried out on albino rabbits. Control (Group 1); marketed product (Group 2), BPO-free SLN (Group 3) and BPO-SLN gel (Group 4). Score: minimum erythemal score. BPO-SLN gel: Batch F3 dispersion converted to gel 0.25% gel. Marketed product: Benzac AC 2.5%, Galderma®. BPO: Benzoyl peroxide; SLN: Solid lipid nanoparticle.

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate

Key Term Time-kill assay: Rate at which concentrations of an anti-microbial agent(s) kill a bacterial isolate.

skin irritation was observed at 48 h (score 2) and 72 h (score 3) for marketed product. The increased skin irritation in the case of marketed product observed in Draize test is directly correlated with the results of ex vivo skin permeation study (Figure 6) . At 6 h, 1.3% of BPO from BPO-SLN gel was found in receptor medium, in contrast with 8.4% of BPO from marketed product (an almost sevenfold increase), so the observed increase in skin irritation was mainly attributed to the greater amount of BPO diffusing into the dermis from marketed product. Thus, BPO-SLN gel demonstrated remarkable advantage over marketed product in improving skin tolerability of BPO, indicating its potential in improving topical delivery of BPO and patient acceptance. Antibacterial activity

The effect of an optimal BPO-SLN on bacterial and fungal growth was studied by incorporating increasing concentrations of BPO-SLN in nutrient and sabouraud agar plates inoculated with 107 CFU/ml from different bacterial, as well as fungal strains. The results of MICs revealed that optimal BPO-SLN dispersion and BPO-SLN gel exhibited the strongest antibacterial activities in comparison with marketed product against all tested species associated with acne vulgaris (Table 6) . This activity is mainly attributed to the fast internalization of BPO-SLN from cell walls of bacteria [50] , which leads to oxidation of bacterial proteins and thereby inhibits protein and nucleotide synthesis, metabolic pathways and mitochondrial activity. Subsequently experiments were conducted to determine

zone of inhibition of optimal BPO-SLN dispersion, BPO-SLN gel and marketed product against different species, and the results revealed that optimal BPOSLN dispersion shows the strongest inhibitory activity against P. acne, with a inhibition zone of 32 ± 0.61 mm (Table 6) in comparison with BPO-SLN gel and marketed product. However, the marketed product shows a relatively higher zone of inhibition against fungi C. albicans (11 ± 1.02) and A. niger (16 ± 1.26) in comparison with optimal BPO-SLN dispersion and BPO-SLN gel (Table 6) . Time-kill assay

The time-kill curves of optimal BPO-SLN dispersion, BPO-SLN gel and marketed product against P. acne, performed in broth as well as on the surface of porcine skin, are depicted in Figure 8A & B, respectively. The data are presented in terms of the log10 CFU/ml reduction and plotted graphically against time (h) of sample removal. At each time point, samples were assessed in triplicate; the final CFU/ ml value was an average (mean) of the three readings. Figure 8A depicts that optimal BPO-SLN dispersion and BPO-SLN gel at a concentration of 20 μg/ml is immediately bactericidal, causing more than 2.5-log reduction (p < 0.05) after 1 h of exposure time in contrast with marketed product (0.5-log reduction), and almost 5-log reduction in contrast with 3-log reduction of marketed product at 4 h. Also, in a porcine skin model (Figure 8B) both BPO-SLN systems (0.25%) show a significant reduction (p < 0.05) in bacterial population in comparison with marketed preparation (2.5%) at 2 h. These results agree with data in reports published elsewhere [51] . The results of this study suggest that developed BPO-SLN system has strong bactericidal activity

Table 6. Minimum inhibitory concentration and zone of inhibition of optimal benzoyl peroxide-solid lipid nanoparticle dispersion, benzoyl peroxide-solid lipid nanoparticle gel and marketed product against various bacteria and fungi. Organism

MIC (μg/ml) ± SD

Zone inhibitions (mm) ± SD

Optimal BPO-SLN dispersion

BPO-SLN gel

Marketed product

Optimal BPO-SLN BPO-SLN gel dispersion

Marketed product

Staphylococcus aureus

2.13 ± 0.10

2.51 ± 0.16

3.19 ± 0.21

25 ± 0.11

24 ± 0.17

20 ± 0.28

Escherichia coli

1.23 ± 0.21

1.26 ± 0.12

2.64 ± 0.10

20 ± 0.23

19 ± 0.54

17 ± 0.91

Pseudomonas aeruginosa 2.02 ± 0.03

2.12 ± 0.21

3.03 ± 0.53

15 ± 0.09

15 ± 0.25

12 ± 0.93

Candida albicans

1.02 ± 0.17

1.18 ± 0.26

2.58 ± 0.28

09 ± 1.26

10 ± 0.14

11 ± 1.02

Aspergillus niger

1.12 ± 0.11

1.03 ± 0.32

2.29 ± 0.09

11 ± 0.37

14 ± 0.16

16 ± 1.26

Propionibacterium acnes

2.09 ± 0.12

2.15 ± 0.19

3.41 ± 0.25

32 ± 0.61

31 ± 0. 91

25 ± 0.48

Values represent mean ±SD; n = 3. Optimal BPO-SLN dispersion: Batch F3. BPO-SLN gel: Batch F3 dispersion converted to 0.25% gel. Marketed product: Benzac AC 2.5%, Galderma®. BPO-SLN: Benzoyl peroxide-solid lipid nanoparticle; MIC: Minimum inhibitory concentration; SD: Standard deviation.

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Skin delivery aspects of benzoyl peroxide-loaded solid lipid nanoparticles for acne treatment 

B

8

8

7

7

6

6

Log CFU/ml

Log CFU/ml

A

5 4 3

5 4 3

2

2

1

1

0

0

2

1

3 4 Time (h)

5

6

Control Optimal BPO-SLN dispersion BPO-SLNgel Marketed preparation

Research Article

0 0

1

2

3 4 Time (h)

5

6

Control Optimal BPO-SLN dispersion BPO-SLNgel 2 Marketed preparation

Figure 8. The time-kill curves of optimal benzoyl peroxide-solid lipid nanoparticle dispersion, benzoyl peroxidesolid lipid nanoparticle gel and marketed product against Propionibacterium acne in 6 h. (A) Performed in broth, (B) performed on the surface of porcine skin. The values represent mean ±standard deviation; n = 3. Optimal BPOSLN dispersion: Batch F3. BPO-SLN gel: Batch F3 dispersion converted to 0.25% gel. Marketed product: Benzac AC 2.5%, Galderma®. BPO-SLN: Benzoyl peroxide-solid lipid nanoparticle.

against P. acne in contrast with marketed product. The improved bactericidal activity is mainly attributed to the better interactions of the lipid nanoparticles with the bacterial cell wall, resulting in increased skin deposition, controlled release profile and improved skin targeting efficacy of the developed BPO-SLN system in contrast with marketed product. Conclusion BPO-SLNs were prepared using Precirol ATO 5 as lipid matrix, Tween 80 as a surfactant and DDW as a dispersion medium using the solvent evaporation method. Pivotal parameters like a drug to lipid ratio, surfactant concentration and sonication amplitude were optimized by 23 full factorial design to obtain optimal particle size and highest % EE. The zeta potential of -7.66 ± 0.94 mV was attributed to the partial hydrolysis of surfactant and long chain length of lipid. Crystallographic studies confirm the low degree of crystallinity of BPO-SLN. TEM studies revealed spherical particles and the formation of a ‘drug-enriched core’ type of SLN. Results of stability testing showed that optimal BPO-SLN dispersion was stable for 90 days at varying temperatures. Ex vivo permeation studies demonstrated lower permeation rate, which led to increased skin drug deposition and, eventually, reduced risk of skin irritation of BPO-SLN in contrast with a marketed product. The strong antibacterial activity was due to the uni-

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formity of small particle size, large increase in surface area as a result of nanonization, high occlusion factor F resulting in increased skin deposition, controlled release, and improved skin targeting efficacy of the developed BPO-SLN system. In brief, the originality of the current study was that the statistically designed and developed BPO-SLN system was investigated and exhibited strong antibacterial activity against a wide spectrum of bacteria (anaerobic and aerobic) and fungi. Moreover, it is the first time that BPOloaded SLNs were evaluated by time-kill assay (performed in BHI broth as well as on the surface of porcine skin). Future perspective BPO is a first-line treatment for topical acne vulgaris, with potent bactericidal activity against P. acne. Our strategy involving factorial design and encapsulation of BPO in SLN would be a smart choice not only for BPO delivery, but also for other classes of topical antiacne agents. Considering the results of the current study, directions for future work include design and development of combination SLNs (i.e., BPO + retinoids or antibiotics) through to our current strategy to target two or more pathogenic factors of acne. Such combination products, which are complimentary and synergistic in their mode of action, simplify the patient’s lifestyle in terms of reducing notorious side effects and dosing frequency.

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Research Article  Pokharkar, Mendiratta, Kyadarkunte, Bhosale & Barhate Acknowledgements The authors would like to thank Gattefosse and Lubrizol for providing gift samples of the excipients.

conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Financial & competing interests disclosure The authors are grateful for financial support from All India Council for Technical Education, New Delhi (F. No. 8023/BOR/ RID/ RPD-126/2009-10 Dt. 31/03/2010). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial

Ethical conduct of research The study protocol was approved by Institutional Animal Ethical Committee (IAEC) constituted as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals), Government of India.

Executive Summary Background • A facile method to prepare stable solid lipid nanoparticles (SLNs) of benzoyl peroxide (BPO) was designed and developed to reduce the various notorious side effects associated with BPO.

Preparation of BPO-SLNs • With the aim to prepare a ‘drug-enriched core’ type of SLN, the lipid with higher BPO solubility, that is, Precirol ATO 5, was eventually selected, thereby resulting in higher percentage enscapulation efficiency (%EE) of BPO-SLNs. • The lipid:drug ratio and Tween 80 concentrations proved to be key contributing factors that affected the particle size and %EE of BPO-SLNs.

Crystallographic investigations • Studies revealed that crystalline BPO was dissolved and embedded in lipid matrix.

In vitro occlusion test • High occlusion factor F value of BPO-SLN gel was mainly attributed to nanometric particle size and high lipid concentration.

Ex vivo skin permeation & deposition of BPO • BPO-SLNs demonstrated lower permeation rate, resulting in increased skin drug deposition and, eventually, reduced risk of skin irritation in contrast with marketed BPO products.

Draize skin irritation test • The developed BPO-SLN gel system demonstrated remarkable advantages over marketed product in improving the skin tolerability of BPO.

Antibacterial activity • BPO-SLNs exhibited strong antibacterial activity against all species tested.

Time-kill assay • The developed BPO-SLN system has better interactions with the bacterial cell wall, hence it resulted in strong bactericidal activity against Propionibacterium acne, in contrast with marketed product.

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