The aim of this study was to improve the transdermal permeation of Diclofenac sodium, a poorly water-soluble drug, employing conventional liposomes, ethosomes, and transfersomes. The prepared formulations had been characterized for the loaded drug amount and vesicle size. The prepared vesicular systems were incorporated into 1% Carbopol 914 gel, and a survey of
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs, which are used in both acute and chronic symptoms of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and dysmenorrhea treatment because of their analgesic, antipyretic, and anti-inflammatory roles. Their anti-inflammatory effect is due to cyclooxygenases inhibition and the consequent reduction of prostaglandin synthesis which leads to unfavorable side effects specifically on the stomach via systemic administration. Therefore, some NSAIDs are administered transdermally to achieve local or systemic effect as an alternative for oral and parenteral administration. Several formulation approaches have been developed for NSAID’s transdermal administration [
Diclofenac sodium (CAS 15307-79-6) was supplied by Alborz Company (Ghazvin, Iran). Disodium phosphate, monopotassium phosphate, ethanol, Carbopol 914, soya lecithin, cholesterol, and Span 80 were obtained from Merck Company (Darmstadt, Germany).
Several solvent systems have been developed to increase the solubility of active ingredients. These solvents must incorporate with substances having different lipophilicity degrees. In this study, reference hydroethanolic formulation was prepared at laboratory scale and at room temperature by dissolving Diclofenac sodium (1% w/w) in an ethanol : water (20 : 80) mixture. The appropriate quantity of Carbopol 914 powder was dispersed into hydroethanolic solution containing Diclofenac sodium (1% w/w) under constant stirring with magnetic stirrer and allowed to hydrate for 24 h at room temperature to swell. The dispersion was neutralized using triethanolamine (0.5% w/w).
Conventional liposomes are composed of phospholipid and cholesterol. The most common phospholipid is phosphatidylcholine obtained from soybean or egg yolk. In the present study, liposomes were prepared by a modified ethanol injection method. Briefly, phosphatidylcholine, cholesterol, and drug were dissolved in ethanol and injected slowly into the aqueous medium under mixing by homogenizer [
As well as conventional liposomes, a range of structurally similar vesicles have been developed, including ethosomes and transfersomes.
Similar to liposomes, ethosomes are composed of phospholipids but can contain 20–40% ethanol. Ethosomes were prepared by first dissolving the lipids and drug in ethanol, then adding the aqueous component slowly as a fine stream under mixing by homogenizer for 60 min [
Topical vesicular gel formulations were prepared incorporating vesicular dispersions containing drug (separated from the unentrapped drug) into the Carbopol gel under mechanical stirring.
A fixed concentration of Diclofenac sodium was used in all formulations to make the vehicles effect on percutaneous absorption comparable.
Mean particle size of vesicles was determined by photon correlation spectroscopy using Shimadzu particle size analyzer model SALD 2101 (Japan). Diluted liposome suspension was added to the sample dispersion unit while stirring at room temperature (in order to reduce the inter particle aggregation). The assay has been performed in triplicate.
The liposome-encapsulated Diclofenac sodium was separated from unentrapped drug by dialysis method [
Each experiment has been done in triplicate, and the data reported is the mean value.
Surface charge of drug-loaded vesicles was determined using Zetasizer (Malvern Instruments, Malvern, UK). Analysis time was kept 60 sec, and average zeta potential of the vesicles was determined.
Physical stability tests of the prepared vesicles were carried out to investigate the aggregation of vesicles and leakage of drug from them during storage. The prepared Diclofenac sodium vesicles were stored in transparent vials covered with plastic cap at ambient temperature and 4°C for three months. The physical stability was evaluated by mean vesicle size, EE%, and zeta potential measurement over a three-month period. Samples from each vesicle were withdrawn monthly, and the particle size, EE%, and zeta potential of the vesicles were measured as described previously.
Hairless rat skin was used for
This study was conducted in accordance with the Guide lines of the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health Publication no. 85-23, revised 1985).
At the end of the permeation experiments, the skin was carefully removed from the Franz cell and the remaining formulation on the skin surface was swabbed and washed first with PBS pH 7.4 and then with methanol. The procedure was repeated twice to ensure that no traces of formulation were left onto skin surface. The permeation area of the skin was excised, weighed, and then cut into small pieces to extract the drug content present in skin with ethanol. The resulting solutions were centrifuged (1500 rpm), and the Diclofenac sodium levels were measured and expressed as percent of initially applied drug.
The cumulative amount of drug permeated per unit area was plotted as a function of time. The flux was calculated from the slope of the linear portion. The permeability coefficient
Data analysis was carried out using Microsoft Excel 2010. Results are expressed as mean ± standard deviation
The mean size of prepared conventional liposomes and ethosomes was
Physical stability study of the prepared vesicles showed higher percentage of drug retained in formulations stored at refrigerated than at room temperature after three months. This may be due to higher fluidity of lipid bilayers at higher temperatures resulting in higher drug leakage. Analysis of drug leakage study data revealed that vesicular gel was significantly more stable than vesicles suspension, and also both types of formulations were significantly more stable at refrigerated temperature than at room temperature (Table
Composition, particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency percent (EE%) of prepared vesicles.
Formulation | Phospholipid (mg/mL) | Cholesterol (mg/mL) | Span 80 (mg/mL) | Particle size (nm) | PDI | Zeta potential (mV) | EE% |
---|---|---|---|---|---|---|---|
Liposome | 100 | 30 | — | 152 ± 21.3 | 0.37 | −39.2 ± 3.9 | 42.61 ± 3.62 |
Ethosome | 100 | 30 | — | 202 ± 20.6 | 0.34 | −41.2 ± 2.1 | 51.72 ± 4.36 |
Transfersome | 100 | 30 | 30 | 145 ± 6.6 | 0.11 | −38.9 ± 4.1 | 46.73 ± 5.21 |
Stability of prepared vesicles during storage at 4°C and 25°C for three months.
Formulation (code) | Particle size (nm) | Zeta potential (mV) | EE% | |||
---|---|---|---|---|---|---|
Initial | After 3 months | Initial | After 3 months | Initial | After 3 months | |
Liposomal dispersion (F3) | ||||||
4°C | 152 ± 21.3 | 193 ± 23.6 | −39.2 ± 3.9 | −40.2 ± 2.1 | 42.61 ± 3.62 | 36.2 ± 1.9 |
25°C | 152 ± 21.3 | 219 ± 16.4 | −39.2 ± 3.9 | −36.1 ± 0.9 | 42.61 ± 3.62 | 32.1 ± 2.6 |
Ethosomal dispersion (F4) | ||||||
4°C | 202 ± 20.6 | 235 ± 30.1 | −41.2 ± 2.1 | −42.3 ± 1.2 | 51.72 ± 4.36 | 44.6 ± 1.8 |
25°C | 202 ± 20.6 | 266 ± 8.6 | −41.2 ± 2.1 | −39.2 ± 3.1 | 51.72 ± 4.36 | 39.9 ± 3.2 |
Transfersomal dispersion (F5) | ||||||
4°C | 145 ± 6.6 | 189 ± 9.5 | −38.9 ± 4.1 | −36.2 ± 1.1 | 46.73 ± 5.21 | 42.3 ± 0.9 |
25°C | 145 ± 6.6 | 223 ± 19.2 | −38.9 ± 4.1 | −39.2 ± 0.8 | 46.73 ± 5.21 | 35.2 ± 1.1 |
Liposomal gel (F6) | ||||||
4°C | 152 ± 21.3 | 169 ± 10.2 | −39.2 ± 3.9 | −39.9 ± 3.2 | 42.61 ± 3.62 | 39.5 ± 1.0 |
25°C | 152 ± 21.3 | 182 ± 12.0 | −39.2 ± 3.9 | −37.2 ± 2.5 | 42.61 ± 3.62 | 38.2 ± 2.6 |
Ethosomal gel (F7) | ||||||
4°C | 202 ± 20.6 | 221 ± 16.2 | −41.2 ± 2.1 | −40.2 ± 1.9 | 51.72 ± 4.36 | 46.8 ± 2.4 |
25°C | 202 ± 20.6 | 230 ± 20.0 | −41.2 ± 2.1 | −41.1 ± 1.2 | 51.72 ± 4.36 | 44.1 ± 1.7 |
Transfersomal gel (F8) | ||||||
4°C | 145 ± 6.6 | 186 ± 9.2 | −38.9 ± 4.1 | −35.2 ± 2.9 | 46.73 ± 5.21 | 43.6 ± 2.6 |
25°C | 145 ± 6.6 | 196 ± 11.1 | −38.9 ± 4.1 | −38.9 ± 3.1 | 46.73 ± 5.21 | 40.3 ± 1.1 |
Equal amounts of Diclofenac sodium from different formulations, including solution, dispersion, and Carbopol gel incorporated form, were applied on the skin surface in donor compartment to make a comparison among their drug penetration ability through rat skin. Amount of Diclofenac sodium permeated through excised mouse skin over 24 h was plotted versus time. Figure
The mechanism of release kinetics was evaluated by fitting the permeation data to the zero-order and Higuchi diffusion models. All permeation profiles of vesicular dispersions and gels fit well into the Higuchi diffusion model (
Table
Permeated amount of Diclofenac sodium at 24 h, flux, permeability coefficient, and residual drug remaining in the skin as percent of initially applied drug for the formulations. Results are revealed as mean ± standard deviation (
Formulation (code) | Permeated amount at 24 h (µg/cm2) | Flux (µg/cm2/h) | Permeability coefficient |
Residual drug (%) |
---|---|---|---|---|
Hydroethanolic solution (F1) | 84.3 ± 2.3 | 3.36 ± 0.68 | 0.336 ± 0.06 | 0.89 ± 0.26 |
Carbopol gel (F2) | 176.6 ± 6.5 | 7.17 ± 1.69 | 0.717 ± 0.16 | 2.32 ± 0.95 |
Liposomal dispersion (F3) | 421.2 ± 35.6 | 16.32 ± 2.35 | 1.632 ± 0.23 | 4.32 ± 1.12 |
Ethosomal dispersion (F4) | 952.6 ± 95.3 | 37.77 ± 6.2 | 3.777 ± 0.6 | 10.58 ± 2.01 |
Transfersomal dispersion (F5) | 1026.6 ± 63.8 | 37.87 ± 3.15 | 3.781 ± 0.32 | 12.26 ± 3.25 |
Liposomal gel (F6) | 786.0 ± 86.3 | 29.13 ± 5.24 | 2.913 ± 0.52 | 12.62 ± 2.68 |
Ethosomal gel (F7) | 2256.9 ± 68.3 | 91.69 ± 6.51 | 9.169 ± 0.65 | 23.9 ± 1.86 |
Transfersomal gel (F8) | 2405.5 ± 110.6 | 100.57 ± 3.61 | 10.057 ± 0.36 | 21.32 ± 2.63 |
It is clear that the cumulative amount of drug permeated across rat skin after 24 h for both hydroethanolic solution and Carbopol gel (
Liposomes as bimolecular phospholipid bilayers are capable of encapsulating hydrophobic, hydrophilic, and amphiphilic drugs. Liposomes by diffusing into the stratum corneum, disrupting the bilayer fluidity in the stratum corneum, loosening the lipid structure of the stratum corneum, and providing impaired barrier function of these layers to the drug act as penetration enhancers. Moreover, some studies reported that phospholipids in liposomes may mix with the stratum corneum lipids creating a lipid-enriched environment. This lipid enriched layer in the skin is preferred by lipophilic drugs, resulting in enhanced skin uptake. In some cases, phospholipids themselves can increase the solubility of lipophilic drugs such as Diclofenac sodium [
Transfersomes are vesicles composed of phospholipids, with 10–25% surfactant and 3–10% ethanol. Transfersomes up to 500 nm can squeeze to penetrate the stratum corneum barrier spontaneously. It has been demonstrated that as transfersomes due to their elasticity and high deformable structure could reach deeper dermal tissues and even the systemic circulation, they ensure higher skin permeation than conventional liposomes. When drugs remain strongly associated with the vesicles, elastic vesicles can be used to transfer drugs rapidly into the deeper layers of the stratum corneum, and subsequently administered drugs can permeate into the viable epidermis. Therefore, elastic vesicles have superior characteristics compared to rigid conventional vesicles [
The comparative evaluation of
The authors report no conflict of interests.