For topical application of active agents various novel carrier systems were explored till date in the form of microparticles, nanoparticles, liposomes and cochleates, and so forth. Food and Drug Administration (FDA) has approved wide range of products like Retin-A, Carac cream, MicroPeel Plus, and oil control lotion due to improved efficacy and safety compared to the conventional topical drug delivery systems. Typically, such products form a highly concentrated layer of active ingredients on the skin which results in skin irritation and toxicity [
At present, the need of an exclusive delivery system has been required for the topically active sunscreen agents. Microsponge is sponge-like porous polymeric system in the range of 10–25
The ultraviolet (UV) rays responsible for sunburns increase the risk of basal cell carcinoma and malignant melanoma. These harmful UV radiations may be blocked by either absorption, reflection, or scattering of rays. The various actives were reported to avoid the exposure to harmful UV radiations. Based on the protective action, UV rays blocking agents are broadly divided into physical agents like zinc oxide and titanium dioxide and chemical agents like benzyl cinnamate and cinnamate derivatives, p-aminobenzoic acid, butyl methoxy dibenzoyl methane, and related compounds [
Oxybenzone is broad spectrum sunscreen agent widely used in the form of lotion and cream. It acts by absorbing entire UV-B radiations (280–320 nm) and mimics the energy conservation of law. The present oxybenzone loaded sunscreen formulations have been reported to cause skin irritation, photo allergic contact dermatitis, and systemic absorption [
This is the first attempt to investigate the feasibility of oxybenzone loaded microsponge gel for enhanced sunscreening efficiency with reduced skin irritation and toxicity. The objective of the present study was to formulate oxybenzone loaded ethyl cellulose (EC) microsponge by quasiemulsion solvent diffusion method by 32 factorial design. The optimized formulation was evaluated for physiochemical characterization, percentage yield, particle size, percent entrapment efficiency (%EE), drug release, surface topography, and differential scanning calorimetry (DSC). The optimized microsponge was incorporated into hydroxy propyl methyl cellulose (HPMC-K4M) gel. Further, prepared gel was evaluated for rheological characterization, skin irritation, minimal erythemal dose (MED), and sun protection factor (SPF) determination.
Oxybenzone was provided by Nulife Pharmaceuticals Ltd., Pune, as a gift sample. Ethyl cellulose-N10, hydroxy propyl methyl cellulose, was gifted by Emcure Pharmaceuticals Pvt. Ltd. Polyvinyl alcohol, dichloromethane, and methanol were purchased from Emerck (India) Ltd., Mumbai.
All the microsponge formulations were prepared using quasiemulsion solvent diffusion method. Required amount of oxybenzone (0.1% w/v) and EC (0.1–0.3% w/v) were dissolved in dichloromethane (DCM) (3–5 mL). The DCM solution was gradually added in 25 mL of aqueous solution of poly vinyl alcohol (PVA) (0.1% w/v) at room temperature with continuous magnetic stirring. Then the final mixture was filtered to separate formed microsponges and dried in the vacuum drier for 24 h.
A prior knowledge and understanding of the process variables under investigation led to preliminary experiments. Based on this preliminary data (not shown), the 32 factorial design was adopted to optimize the amount of EC and DCM as the independent variables. The particle size, percent drug entrapped, and the drug release were considered as dependent variables. The response surface graphs of the obtained results were also plotted. The coded and actual values are given in Table
Factorial design and characterization of experimental formulations.
Formulations |
% EE ± SD | % DC ± SD | % production yield | Particle size |
% DR ± SD |
---|---|---|---|---|---|
M1 (100, 3) | 92.3 ± 0.12 | 67.12 ± 0.42 | 50.14 ± 0.28 | 496 ± 0.12 | 20.08 ± 0.02 |
M2 (100, 5) | 93.7 ± 0.22 | 70.00 ± 0.56 | 59.76 ± 0.10 | 619 ± 0.02 | 21.08 ± 0.82 |
M3 (100, 7) | 98.6 ± 0.14 | 73.54 ± 0.82 | 73.54 ± 0.16 | 146 ± 0.32 | 22.28 ± 0.72 |
M4 (200, 3) | 90.1 ± 0.21 | 44.26 ± 0.42 | 44.10 ± 0.14 | 347 ± 0.42 | 12.19 ± 0.42 |
M5 (200, 5) | 96.3 ± 0.56 | 67.54 ± 0.32 | 67.76 ± 0.72 | 224 ± 0.52 | 12.88 ± 0.32 |
M6 (200, 7) | 98.5 ± 0.72 | 54.28 ± 0.22 | 55.22 ± 0.32 | 105 ± 0.62 | 13.74 ± 0.42 |
M7 (300, 3) | 95.3 ± 0.92 | 65.33 ± 0.29 | 65.11 ± 0.42 | 272 ± 0.02 | 12.23 ± 0.02 |
M8 (300, 5) | 96.7 ± 0.62 | 42.02 ± 0.12 | 42.78 ± 0.62 | 119 ± 0.28 | 12.94 ± 0.11 |
M9 (300, 7) | 96.9 ± 0.52 | 77.87 ± 0.92 | 77.56 ± 0.20 | 072 ± 0.77 | 13.76 ± 0.10 |
The following equation was obtained:
The optimized microsponge formulation was incorporated in 3% w/w HPMC base to obtain the gels.
Percentage yield is determined by calculating the initial weight of raw materials and the weight of microsponge. Percentage yield was calculated by using the following formula:
The mean particle size of the microsponge dispersion was determined by polarising microscope (model number E600POL). The scale was used which was numbered from one to ten microns, each unit corresponding to one micron.
For the analysis of entrapped oxybenzone, microsponge was dispersed in methanol to release the entrapped drug. The unentrapped oxybenzone was separated from the microsponge by centrifugation. For this, the dispersion was transferred into Eppendorf tube and centrifuged at 20,000 rpm for 1 hr at 4°C. The free drug remained in supernatant while entrapped drug was retained in the pellet. The supernatant, with subsequent dilution and filtration, was then analyzed for drug concentration spectrophotometrically at 287 nm. The percent entrapment efficiency (%EE) was calculated as the following equation:
To know the rate and extent of drug release from microsponge, dissolution of oxybenzone loaded microsponge was studied using USP dissolution test apparatus (USP XXIII). Accurately weighed samples of microsponge equivalent to 25 mg of oxybenzone were placed in 900 mL of phosphate buffer pH 7.4 and were subjected for dissolution with a paddle speed of 150 rpm at 37 ± 0.5°C. Aliquots (5 mL) were withdrawn at 5 min initially and then at hourly intervals up to 8 hours and assayed spectrophotometrically at 287 nm. The percentage of drug released at various time intervals was calculated and plotted against time [
Surface topography of the selected optimized formulation was characterised using scanning electron microscopy (SEM). Freshly prepared microsponge samples were mounted on the aluminium stub and coated with gold-palladium layer by autofine coater (Joel, JFC, Tokyo, Japan) and analyzed with a scanning electron microscope (Joel, JSM-6360, Tokyo, Japan) operated at a 10 kV acceleration.
DSC studies were carried out using Mettler-Toledo DSC 821e instrument equipped with an intercooler (Mettler-Toledo, Switzerland). Indium and zinc standards were used to calibrate the DSC temperature and enthalpy scale. The samples were hermetically sealed in aluminium crucibles and heated at a constant rate of 10°C/min over a temperature range of 25–300°C. Inert atmosphere was maintained by purging nitrogen gas at flow rate of 50 mL/min.
Rheological measurements of the microsponge loaded gel and blank gel were performed using a controlled stress rheometer (Viscotech Rheometer, Rheological Instruments AB, Lund, Sweden). Data analysis was done with stress rheological basic software, version 5.0. A cone and plate geometry was used with 25 mm diameter and cone of 1.0° [
In creep recovery, samples were subjected to a fixed stress from LVR for 100 s and then allowed to recover. The creep compliance,
Skin irritation test of optimized oxybenzone loaded microsponge gel (M9) was compared with the marketed and placebo gel. The present study was employed in the three groups of rats (
The 0.5 g of each test product was placed on each area (25 × 25 mm) for 30 minutes. Finally treated skin area of rats was washed off by tap water. Scoring of the erythema was performed at 24 and 72 hours and both the treated and controlled sites were covered and wrapped by cotton bandage. Reactions on skin were measured after 24 hr and 72 hr in form of erythema. The mean erythemal scores were recorded (ranging from 0 to 4) according to Draize scale [
The study was performed using Wistar rat model. This model is suitable for the study because of the photochemical changes taking place in rat skin after UV exposure. Before conducting actual SPF testing, a study was carried out to determine MED with respect to time of unprotected skin and protected skin of Wistar rat. This was carried out on total of nine rats. They were divided into three different groups each comprising three rats and maintained separately. A solar simulator high pressure mercury vapour lamp (Osram Ultra Vitalex of 300 w) was used as a UV light source. First group of rats was kept directly under the solar simulator lamp and was sampled after every one minute. The rats in the second and third group were applied with marketed sunscreen lotion and oxybenzone loaded microsponge gel, respectively, and the same sampling procedure was followed. The presence of reddening (erythema) of skin was noted after 3 hr of completion of study.
The
During the preliminary study, concentration of EC and DCM which would produce nonaggregating, nonregimenting, and porous microsponge dispersion was determined. The predicted concentration of EC and DCM was decisive in the preparation and stabilization of microsponge. EC was added to provide structural integrity and PVA was added as an emulsifying agent. The concentration of DCM showed influence on EE and porosity. The preliminary data suggested that the concentration of PVA required was 100 mg. This was kept constant. Altering the concentration of EC and DCM caused pronounced effect on microsponge dispersion.
The concentration of PVA and oxybenzone was kept constant. The effects of EC and DCM on the microsponge size, entrapment efficiency, and
The size of the microsponge ranges from 72 ± 0.77 to 619 ± 0.02
According to (
Response surface plots of (a) particle size, (b) entrapment efficiency, and (c) drug release.
The negative value of
The interaction term
It is general observation that increase in the polymer ratio increases the EE. The reason for increase in EE with high polymer ratios is reduced diffusion rate of drug solution from concentrated polymeric solutions into external phase. This provides more time for the droplet formation and may improve the yield of microsponge and entrapment efficiency. The equation was generated by fitting the observed coefficient in (
According to results of EE as shown in (
The positive value of
The interaction term
The second most important parameter which needs to be monitored during microsponge preparation for its best performance was the release of the drug from the microsponge. The oxybenzone should be released slowly over the extended period of time to modulate low concentration gradient for skin transport. Therefore, multiple regression analysis for the drug release as per the factorial design revealed the good fit
As per (
The positive value of interaction term
Formulation | Kinetic model |
|
---|---|---|
M3 | Peppas equation | 0.97 ± 0.09 |
M6 | Higuchi equation | 0.96 ± 0.12 |
M9 | Higuchi equation | 0.95 ± 0.11 |
Skin irritation test.
Animal number | Reaction | Placebo gel (24 h) | Placebo gel (72 h) | M9 gel (24 h) | M9 gel (72 h) | Marketed lotion (24 h) | Marketed lotion (72 h) |
---|---|---|---|---|---|---|---|
1 | Erythema | 0 | 0 | 0 | 0 | 1 | 1 |
2 | Erythema | 0 | 0 | 1 | 0 | 2 | 1 |
3 | Erythema | 1 | 0 | 1 | 0 | 1 | 1 |
4 | Erythema | 0 | 0 | 1 | 0 | 1 | 1 |
Primary irritation index (PII ) | — | 1/8 = 0.125 | 3/8 = 0.375 | 9/8 = 1.12 | |||
— | Negligible irritation | Negligible irritation | Slight irritation |
(a) Comparative
The SEM of microsponge was shown in Figure
Microsponge with surface pore size.
As shown in Figure
DSC plots of (a) pure drug and (b) formulation.
The optimized microsponge formulation (M9) was incorporated in 3% w/w HPMC base to obtain the gels. Initially required amount of HPMC was added to water and kept overnight for complete hydration of polymer chains. Microsponge dispersion was added to the hydrated HPMC solution to obtain a final concentration of 2.5% w/w of oxybenzone. The prepared gel was used for drug deposition study.
The microsponge gel showed creep recovery of 74.05% compared to 65.11% of blank gel. The same results were depicted in Figure
Creep recovery of blank and drug loaded microsponge gel.
Skin irritation test of optimized oxybenzone loaded microsponge gel (M9) was compared with the marketed and placebo gel. Erythema with score of 0–2 was observed on all rats. After 72 hours, disappearance of all erythema was observed from test areas applied with placebo gel and microsponge gel as shown in Table
The time required for the development of erythema for the unprotected rats was 7 min. The rats protected by the marketed formulations showed 140 mins for the development of erythema. The microsponge gel showed the 170 min which was more as compared to marketed formulation. This result was maybe observed due to the controlled release of drug from the microsponge loaded gel which helped to avoid excessive deposition of drug.
The SPF determination of optimized oxybenzone loaded microsponge gel (M9) was compared with the marketed gel. The microsponge gel showed SPF 25 as compared to SPF 20 of the marketed lotion. This was maybe due to slower release of drug from the microsponge gel which provides prolonged retention of oxybenzone. This was reflected in the comparison with marketed lotion where the oxybenzone loaded microsponge gel was found to be more photoprotective. Thus, as shown in Figure
Protected skin applied with M9 microsponge gel.
The present study concluded successful preparation and optimization of oxybenzone loaded microsponge gel with the enhanced sunscreening efficiency.
The authors declare that there is no conflict of interests regarding the publication of this paper.