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Design, synthesis of novel vesicular systems using turpentine as a skin permeation enhancer
Accepted Manuscript Design, synthesis of novel vesicular systems using turpentine as a skin permeation enhancer A. Behtash Oskuie, S.A. Nasrollahi, S. Nafisi PII:
S1773-2247(17)30708-6
DOI:
10.1016/j.jddst.2017.10.015
Reference:
JDDST 497
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 24 August 2017 Revised Date:
3 October 2017
Accepted Date: 16 October 2017
Please cite this article as: A. Behtash Oskuie, S.A. Nasrollahi, S. Nafisi, Design, synthesis of novel vesicular systems using turpentine as a skin permeation enhancer, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/j.jddst.2017.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Design, synthesis of novel vesicular systems
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using turpentine as a skin permeation enhancer
A. Behtash Oskuie1, S. A. Nasrollahi2, S. Nafisi1*
Nanodermatology Unit, Center for Research & Training in Skin Diseases & Leprosy, Tehran,
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Department of Chemistry, Central Tehran Branch, Islamic Azad University, Tehran, Iran
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University of Medical Sciences, Tehran, Iran
The authors declare no conflict of interests
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Correspondence: [email protected] Shohreh Nafisi, Prof ,
Department of Chemistry, Central Tehran Branch, IAU, Tehran, Iran Tel: +98 912 144 1462 Fax: +98 21 880 74907
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Addition of skin penetration enhancer in a formulation is the simplest and most common technique to improve transdermal permeation. We aimed to develop novel liposome and ethosome vesicular systems using turpentine as a skin penetration enhancer for improving
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fluconazole skin permeability.
Fluconazole was encapsulated in various liposomal and ethosomal formulations The prepared
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formulations were characterized for size, size distribution, zeta potential (Z), entrapment efficiency (EE %), drug content, in-vitro, ex-vivo skin permeation and stability studies. The
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vesicles were found spherical in structure as confirmed by Scanning Electron Microscopy (SEM). Fluconazole was successfully entrapped in liposomes and ethosomes with relatively uniform drug content and entrapment efficiency in the range of 90.84 to 91.0 %. Liposome formulation; F3 and ethosome formulation; F7 with the highest drug entrapment efficiency were obtained as optimized formulations. Cumulative percent drug release for liposome (F3) and
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ethosome (F7) formulations were 82.52 and 90.84%, respectively. In-vitro, ex-vivo and antifungal effect of drug loaded ethosomes and liposomes, and the effect of penetration enhancer (turpentene) were studied and compared with free drug. The formulations containing turpentene
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showed higher permeation characteristics and can be proposed as promising delivery systems for
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fluconazole topical delivery.
Keywords: Fluconazole; Turpentine; Skin permeation; liposome; ethosome
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Fluconazole, a synthetic antifungal agent belonging to the group of triazole has been used in treatment of oropharyngeal, esophageal, or vaginal and urinary tract infection as well as other serious systemic candidal infections. It is also effective against superficial fungal infections and
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dermatophytoses. Fluconazole is available commercially as cream, tablets and injections in spite of its well-known adverse effects including nausea, vomiting, bloating and abdominal discomfort. Accounting these problems, drug delivery technologies should be developed which
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reduce drug dosing frequency along with sustained or controlled release of the medicament as well as reduced systemic side effects [1-2]. Vesicular systems such as liposome and ethosome
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formulations have widely proposed to bypass the topical drug problems [3-6]. Currently liposomes are used as novel drug delivery systems due to their flexibility and clinical efficacy. They can encapsulate hydrophilic and lipophilic drugs and protect them from degradation. They also have affinity to keratinocytes membrane and can penetrate deeper into
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skin and hence give better absorption. Applied on the skin, liposomes may act as a solubilizing matrix for poorly soluble drugs, penetration enhancer as well as local depot at the same time diminishing drugs side effects. Topical liposome formulations can relatively be more effective
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and less toxic than conventional formulations [7-12]. Ethosomes, novel carrier systems used for drug delivery have higher penetration through
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biological membranes mainly skin. The lipid vesicles composed of phospholipids, and relatively high alcohol concentration are obtained by slight modification of liposomes. They can permeate more rapidly through skin layers and possess significantly higher transdermal flux in comparison to conventional liposomes [13-15]. Although, the exact ethosomes mechanism for higher permeation into deeper skin layers is not still clear, synergistic combination effects of
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phospholipids and ethanol in vesicular formulations have been suggested for deeper distribution and penetration [16-19]. To achieve successful transdermal drug delivery, enhancement of skin permeability is of
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prime concern. Recently several physical, electrical, chemical and biochemical techniques have been proposed to increase skin permeability. Amongst these techniques, permeability improvement by chemical methods is being most widely used owing to economic, simplicity and
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rapidity. Chemical permeation enhancers either improve the solubility or partition coefficient or increase drug diffusion across the skin. However, their toxicity and irritancy to skin has led to
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quest of natural skin penetration enhancers [20-25].
Terpenes, obtained from pistachio gum have been successfully used as penetration enhancers especially for lipophilic drugs. Structurally, terpenes consist of isoprene units (C5H8) can be classified as hydrocarbons, alcohols, ketones, and oxides. They offer advantages over many other
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penetration enhancers, in part because they are categorized as safe substances by the U.S. food and drug administration [26-27]. Turpentine, composed of terpenes has demonstrated skin penetration potential in various traditional applications such as herbal medicine. However, there
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is limited permeation study of pistachio gum (oil) on human skin [28-29]. Based on these premises, we developed novel nanovesicular carriers to improve fluconazole
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skin permeability. Fluconazole effectiveness was compared with the complexes of FLZliposome, FLZ-ethosome, FLZ-liposome-turpentine, and FLZ-ethosome-turpentine. Poly dispersity Index (PDI), zeta potential (Z), morphology, stability and drug content of the designed formulations were studied. Optimized formulations in terms of entrapment efficiency (EE %) were evaluated for their penetration profile, in-vitro, ex-vivo release and antifungal activity.
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I. MATERIALS and METHODS 1. Chemicals and reagents Fluconazole
was
received
from
Zahravi
Medical
Co.
(Tabriz,
Iran).
Soya
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phosphatidylcholine was from Lipoid Co. (Germany). Propylene glycol and polysorbate 80 were obtained from Merck (Germany). Cholesterol and agar were from Sigma Aldrich. Turpentine was received from Herbal Medicine Society (Tehran, Iran). Aspergillus niger was received from
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microbial bank of IROST (Tehran, Iran). All other chemicals were of analytical grade and used
2. Preparation of vesicular systems
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without further purification.
2.1. Preparation of fluconazole liposomes
Different compositions of aqueous liposomal formulations were prepared by conventional
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lipid “Film hydration method” (Table 1). Lipid phase composed of phosphatidylcholine (200 mg), cholesterol (30 mg), polysorbate 80 (20 mg) and different turpentine concentrations (10, 20 and 30 mg) designated as F2, F3, F4 were dissolved in chloroform:methanol (2:1, V/V) together
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with fluconazole (20 mg) in a round bottom flask. Solvent was removed under reduced pressure in a rotary evaporator at 55 °C to obtain a thin film on the flask wall. The flask was kept
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overnight under vacuum to ensure the complete removal of residual solvent. The dry lipid film containing fluconazole was hydrated with 20 ml phosphate buffer solution (pH 7.4) at 60±2 °C. The dispersion was left undisturbed at room temperature for 2-3 h to allow complete swelling of the lipid film and to obtain vesicular dispersion. All the above-mentioned steps were performed under aseptic conditions and the glass-wares were sterilized by autoclaving. Phosphate buffer saline was passed through a 0.22 nm membrane filter, and the entire procedure was performed in
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a laminar flow hood. Fluconazole liposomes were separated from unentrapped drug by centrifugation at 13,000 rpm for 1 h and at 2 °C using cooling ultracentrifuge. The formed cake was washed twice each with 10 ml phosphate buffer saline and re-centrifuged for 1 h [30-33]. A
same drug and ethanol concentrations (Table 1).
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2.2. Preparation of fluconazole ethosomes
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liposome fluconazole suspension without turpentine (F1) was also prepared as a control in the
Ethosomes in different formulations were prepared by “Hot method” (Table 2).
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Phosphatidylcholine (200 mg), propylene glycol (20 mg) and fluconazole (20 mg) were dissolved in ethanol 96% (2.5 ml). Different concentrations of turpentine (10, 15, 20 mg) designated as F6, F7, F8 were added. To get nano-sized ethosomes, the preparation was sonicated by Probsonicator (SH 70 G, Bandlin Sonoplus UW 2070, Berlin, Germany) by stirring
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speed of 700 rpm at 4 ºC for 3 cycles of 5 min, with rest of 5 min between each cycle. The samples were centrifuged at 20,000 rpm for 30 min and evaluated for their entrapment efficiency [32]. An ethosome fluconazole suspension without turpentine (F5) was also prepared as a control
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in the same drug and ethanol concentrations (Table 2).
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3. Preparation of cream formulations In order to manufacturing base cream, lipid phase (containing glyceryl monostearate, mineral oil, butylated hydroxytoluene, Spermaceti and stearyl alcohol) was heated to 75 ºC. The aqueous phase (containing polysorbate 80, polysorbate 60, sorbic acid, potassium sorbate, sorbitol and distilled water) was added to the lipid phase at the same temperature under stirring (600 rpm) and cooled down to 25 ºC. Liposome and ethosome were added to the base cream under stirring. 6
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4. Characterization of drug formulations 4.1 Size, polydispersity index (PDI) and zeta potential (Z)
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Mean vesicle size and size distribution profile and zeta potential of liposomes and ethosomes were determined by using Malvern particle size analyzer model SM 2000, which follows Mie's theory of light scattering. The diluted visible suspension was added to sample dispersion unit and
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stirred at 2000 rpm in order to reduce the inter-particle aggregation; laser obscuration range was maintained between 10-20%. Average particle size was measured after performing the
5. Drug entrapment efficiency (EE %)
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experiment in triplicate.
Fluconazole associated with vesicular system was separated from unentrapped drug using centrifugation method. The vesicle suspension was centrifuged at 20,000 rpm for 1 h at a
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controlled temperature of 4 ºC. Supernatant containing unentrapped fluconazole was withdrawn and measured by UV spectrophotometrically at 260 nm against phosphate buffer saline (pH 7.4). The amount of fluconazole entrapped in vesicle was determined as follow [34-35]:
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EE % = [(Ct − Cf) / Ct] × 100
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Where Ct is concentration of total fluconazole and Cf is concentration of free fluconazole. 6. Distribution morphology Scanning electron microscopy was conducted to characterize the surface morphology of the selected liposome and ethosome formulations for which a drop of each formulation was mounted on clear glass stub, air dried and coated with polaron E 5100 Sputter coater (Polaron, UK) and visualized under SEM (JOEL-JSM-6510, Japan).
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7. Physical stability studies Physical stability tests were carried out to investigate vesicles aggregation and drug leakage from vesicles during storage. Prepared formulations were stored in transparent vials covered with
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a plastic cap at ambient temperature and 4 ºC. Physical stability of samples was evaluated by Z, mean vesicle size and EE % over 6 months.
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8. In-vitro drug release studies
In-vitro fluconazole permeation behavior from all liposome and ethosome formulations was
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investigated using cellophane membrane (0.45 µm). In brief, 20 mg of fluconazole and fluconazole liposome and ethosome formulations were suspended in phosphate buffer saline (1 ml, pH=7.4) in a glass cylinder having a length of 20 cm and a diameter of 2.5 cm. The cylinder was fitted before liposome suspension addition with a presoaked membrane [Spectra/Por
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membrane] and placed in a beaker (100 ml) containing 35 ml phosphate buffer saline (pH 7.4). The whole set was placed on a magnetic stirrer adjusted to a constant speed (150 rpm). Samples were collected every 1 h over a period of 10 h and assayed spectrophotometrically for drug
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content at 261 nm. Cumulative amount that permeated across the cellophane membrane was
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calculated and plotted against time. Results were the mean values of three runs [36].
9. Ex-vivo skin experiments Skin was obtained from patients undergoing abdomen reduction surgery, in Parsian Hospital. Skin slices were isolated with a dermatome from abdomen, and then stored at −18 ºC for at least 24 h. Skin permeation and uptake was determined using vertical Franz cell. Excised skin was rinsed with normal saline and aqueous ethanolic solutions. The skin was then sandwiched
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between two cells with stratum corneum side upwards. Receptor chamber was filled with 35 mL of normal saline solution. The cream test formulations 1% (0.5 g) was applied to skin surface (available diffusion area of 3.3 cm2). Receptor chamber content continuously stirred at 37 ºC was
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removed at appropriate intervals (1–6, 24 h) for drug concentration measurements and the cell was immediately refilled with fresh receptor solution. After 24 h, the application site was washed with normal saline solution to remove excess drug on skin surface. Skin was then cut with
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scalpel into small pieces; methanol was added (2.0 ml) for drug extraction. After 4 h stirring, the resulting suspension was centrifuged and supernatant assayed by UV analysis. Skin uptake was
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expressed as µg drug/cm2 of skin diffusion area [37].
9. Calculation of skin permeation parameters
Drug cumulative amount permeated per unit area was plotted as a function of time. Flux was
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calculated from linear portion slope. Drug permeability coefficient (????) across skin was calculated using relation derived from Fick’s first law of diffusion, which is expressed by the following equation:
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Kp = J/C
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Where J is flux and ?? is drug concentration in donor compartment.
10. Antifungal activity
Selected formulations (F3 and F7) and blank formulation (without turpentine) were assayed for antifungal activity against the fungal strain Aspergillus niger, obtained from Microbial Bank of IROST (Tehran, Iran). The fungus was grown on Sabouraud’s agar plate at 25 ◦C. Fungal culture suspension was made in about 0.4 ml sterile water and stood for 20 min before
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transferring to solid agar medium where the strain was available in lyophilized form. Fungal culture suspension (0.1 ml) was mixed with 9.9 ml liquid broth (without agar) and inoculated for 24 h in an incubator at 25 ◦C. Inoculated liquid broth containing fungal culture suspension (1
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mL) was added to the sterile petri dishes of solidified agar growth medium. The inoculum was spread uniformly over the solid agar surface by clockwise and anticlockwise direction plate rotations - the formed well in the middle of the plates with the help of a sterile cork borer was
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filled with the formulations. For fungus growth, plates were incubated at 25 ◦C. Clear rings called inhibition zone were appeared around the dishes in 48 h. More effective formulations
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show larger inhibition zones. Antifungal activity was evaluated by measuring inhibition zone (in mm) of fungal growth surrounding the formulations after a week - the complete antifungal analysis was carried out in triplicates.
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11. Statistical analysis
Data analysis was carried out using Microsoft Excel 2010. Results are expressed as mean ± standard deviation (?? = 3). Statistically significant differences were determined using analysis of
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variance (ANOVA) with ?? < 0.05 as a minimal level of significance.
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II. RESULTS AND DISCUSSION Two types of vesicular systems, fluconazole-liposomes and fluconazole-ethosomes, and turpentine as a natural penetration enhancer are formulated and presented in Tables 1, 2. Different formulations were characterized for mean particle size, PDI, Z, EE%, distribution morphology, physical stability (Fig 1, Tables 3, 4). Optimized compositions were selected based on the highest entrapment efficiency amongst the formulations presented in Tables 1 and 2 and
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examined for in-vitro drug release studies, ex-vivo skin penetration experiments and antifungal activity (Figs 2, 3). Average size of FLZ-liposome and ethosome formulations were measured and presented in
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Table 3. Particle size of liposome formulations ranged from 68.8 to 367.0 nm with a relatively narrow size distribution (PDI<0.41), while the matching data for FLZ-ethosomes were 248 to 340 nm with a PDI<0.36 (Table 3). Average size of the liposome and ethosome formulations was
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greater than that of the vesicles with turpentine. Liposome and ethosome sizes are reduced by the addition of turpentene due to the presence of more turpentene in the outer layer than in the inner
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bilayer membranes. Decrease in liposomal and ethosomal sizes by turpentine can be explained with the same mechanism as reduction in liposomal size by surfactant addition [38-40]. Polydispersity index (PDI) were determined as a measurement of particle size homogeneity for the prepared liposome and ethosome formulations. PDI values for optimized formulations
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(F3 and F7) were 0.40 and 0.212, respectively (Table 3). Zeta potentials of all liposomes (-5.28 to -11.7) and the corresponding ethosomes were negative (-6.10 to -7.82). Z was found to be -10.40 and -7.51 mV for the optimized liposome
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(F3) and ethosome (F7) formulations, respectively (Table 3). Stability of formulations depends
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on the ingredients charges; ingredients with large negative or positive charge repel each other and do not come together and aggregate. Hence, ingredients with higher charges create more stable formulations. PC, a zwitterion compound with an isoelectric point between 6 and 7 has a negative charge at pH 7.4. Consequently, liposome and ethosome formulations show good physical and chemical stabilities due to the presence of PC which creates negative charge in their structure [39].
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Fluconazole entrapment efficiency in the vesicles ranged from 57.58 to 91.0 % for liposome and from 66.11 to 90.84 for ethosome formulations. The highest EE was observed for F3 (91.0 %) and F7 (90.84 %) containing 15 mg of turpentine. Optimized F3 and F7 formulations showed
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smallest particle sizes (68.82, 248.0 nm), respectively.
SEM results indicated small, spherical shape with a smooth surface for the optimized vesicles (Fig. 1). Obtained data were compatible with DLS results analysis regarding size confirming
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narrow size distribution of optimized liposomes and ethosomes (Table 3). F3 and F7 were examined for in-vivo, ex-vivo and antifungal activity studies.
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Physical stability study of the prepared vesicles showed lower percentage of fluconazole retained in formulations at room temperature rather than refrigerated temperature after 6 months. This may be due to increased lipid bilayer fluidity at higher temperatures which results in drug leakage. Vesicles were also reasonably stable in term of aggregation (Table 4).
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Figure 2 A, B shows the effect of different formulations on permeation profile of fluconazole. Results revealed that F3 (FLZ-liposome vesicle) and F7 (FLZ-ethosome vesicle) have the greatest drug release equal to 82.52 and 90.84% in 12 h, respectively. The highest
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amount of ex-vivo permeation was obtained for F3 and F7 formulations as well. These finding could be due to increased drug fluidity which is a consequence of synergic effect of polysorbate
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80, turpentine and alcohol (F3 and F7) [26, 40, 41]. Addition of polysorbate 80 and ethanol increased vesicle fluidity near the polar head group of the phospholipid bilayer. Results are compatible with other reports as well. It is hypothesized that polysorbate 80 turns its hydrophilic portion towards the phosphate group. Turpentine localizes near the end of phospholipid chain, consequently increases vesicle fluidity [37].
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Fluconazole cumulative amount permeated from ethosomal formulations containing ethanol and turpentene after 24 h is significantly higher than that of liposomes containing polysorbate 80 and turpentene (Fig. 3). Turpentene demonstrated a remarkable effect in release and skin
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permeation in all formulations comparing with blank formulation (F1).
Release experiments indicated controlled fluconazole release from all vesicle formulations, because of higher drug content and entrapment efficiency comparing with blank. Maximum
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release is also due to optimum polysorbate 80 concentration (20 %), because at this concentration, the surfactant molecule gets associated with phospholipid bilayer resulting in
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better drug partitioning and higher release from the vesicles [41-42].
Antifungul activity results in term of inhibition potency measured as zone diameter in mm are given in Table 5. All tested samples were found effective against Aspergillus niger following the order of F7>F3>blank. Significant differences were found between formulations; ethosome
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formulation (F7) containing turpentene and alcohol with inhibition zone of 32.6 mm was the most effective one. Although liposomal formulation (F3) containing turpentine showed high inhibition zone of 28.0 in comparison to blank formulation. Inhibition zone differences can be to greater fluconazole release rate from ethosome, which in turn is attributed to
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related
turpentene and alcohol content of the formulation. Turpentene like other unsaturated fatty acids
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increases skin permeation through disruption of stratum corneum ordered lipids. These changes promote fluidization of the lipids, simultaneously lower the lipid transition temperature, resulting in increased drug release and permeability [43]. ⃰
In the present study, novel fluconazole-loaded liposomes and ethosomes were successfully prepared. We found that the addition of penetration enhancer “turpentine” resulted in vesicles
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with smaller particle size and proper encapsulation efficiency suggesting that the novel vesicular systems can be considered as effective fluconazole carriers. Optimized vesicles (F3 and F7) containing 15 mg turpentene increased penetration up to 5 times comparing with fluconazole
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blank.
Results revealed that liposomal and ethosomal fluidity correlated with skin penetration enhancement of entrapped drug. Increased system fluidity resulted in fluconazole penetration
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enhancement. Polysorbate 80 molecules, generally localized near the hydrophilic portion of the phospholipid bilayer, and turpentene accompanied with alcohol incorporated in drug
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compositions induce liposomal and ethosomal fluidity and show synergic fluconazole penetration enhancement. Present study shows that the novel liposome and ethosome formulations containing turpentine have potential to improve transdermal delivery and antifungal activity, reasonably. They can be considered as promising carriers to improve fluconazole
Acknowledgements
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permeability in a period of time.
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Authors appreciate Dr. M. Hadgi Yousefi, Surgeon of Parsian Hospital for providing abdominal skin sample and Department of Chemistry, Central Tehran Branch, Islamic Azad University for
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providing the facilities.
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drug delivery system, Pharma. Sci. Monitor 5 (2014) 298-312.
38. P. Minghetti, F. Cilurzo, A. Casiraghi, L. Motanari, Evaluation of ex vivo human skin permeation of genestein and daidzein. Drug Deliv. 13 (2006) 411-415.
39. T. Subongkot, T. Ngwhirunpat, Effect of liposomal fluidity on skin permeation of sodium fluorscein entrapped in liposomes, Int. J. Nanomed. 10 (2015) 4581-4592.
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40. L.M. Tasi, D.Z. Liu, W.Y. Chen. Microcalorimetric investigation of the interaction of polysorbate surfactants with unilamellar phosphatidylcholines liposomes. Colloids Surf. A. 213 (2003) 7–14. M. Cirri, P. Mura, Comparative study of
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41. M. Bragani, N. Mennini, F. Maestrrelli,
liposomes, transferosomes and ethosomes as carriers for improving topical delivery of celecoxib, Drug deliv. 19 (2012) 354-361.
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42. W. Rangsimawong, P. Opanasopit, T. Rojanarata, T. Ngawhirunpt, Terpene-containing
Bull. 37 (2014) 1936-1943.
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PEGylated liposome as transdermal carriers of a hydrophilic compound, Biol. Pharm.
43. M.U. Sakthi, D.R. Devi, V.N. Vedha Hari, Vesicular mode of drug delivery: a promising
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approach for anti-infective therapy, Int. J. chem. Sci. 12 (2014) 797-814.
Captions for Figures and Tables
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Table 1: The composition of FLZ-liposome formulations Table 2: The composition of FLZ-ethosome formulations Table 3: Size, PDI, Z and EE % of liposome and ethosome formulations
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Table 4: Stability of prepared vesicles during storage at 4 and 25 ºC for 6 months
Table 5: Inhibition zone of selected liposome (F3), ethosome (F7) formulations and blank fluconazole (without turpentine) against Aspergillus niger
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Fig. 1: Scanning electron microscope image of a) Optimized liposome formulation (F3) and b) Optimized ethosome formulation (F7).
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Fig. 2: In-vitro drug release profiles of a) F1-F4 of liposomal formulations b) F5-F8 of ethosomal formulations
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Fig. 3: Ex-vivo skin permeation of liposomal (F1, F3) and ethosomal (F5, F7) formulations
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Table 1 F2
F3
F4
20 200 30 20 -
20 200 30 20 10
20 200 30 20 15
20 200 30 20 20
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F1
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Composition (mg) Fluconazole Phosphatidylcholine Cholesterol Polysorbate 80 Turpentene
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Table 2 F2 20 200
F3 20 200
F4 20 200
20 2.5 -
20 2.5 10
20 2.5 15
20 2.5 20
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F1 20 200
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Composition Fluconazole (mg) Phosphatidylcholine (mg) Propyleneglycol (mg) Ethanol (ml) Turpentene (mg)
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0.30±0.02 0.30±0.02 0.40±0.02 0.41±0.02 0.206±0.03 0.318±0.01 0.212±0.05 0.364±0.10
Z (Mean±SD, mV) -11.7±1.06 -8.74±0.22 -10.40±0.58 -5.28±0.80 -6.10±0.51 -7.13±0.16 -7.51±0.22 -7.82±0.46
EE (Mean±SD, %) 57.78 ± 0.76 65.20±1.50 91.00±1.04 88.5±1.20 66.11±2.11 69.21±1.52 90.84±1.02 75.0±1.09
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Ethosomes
PDI (Mean±SD)
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Liposomes
F1 F2 F3 F4 F5 F6 F7 F8
Size (Mean ±SD, nm) 367.0 ±1.30 106.43±0.42 68.82±0.62 172.60±1.38 340.23±1.31 322.21±1.11 248.17±1.02 330.16±1.31
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Drug Formulation
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Table 3
Drug formulation
Size (Mean ±SD, nm) Initial After 6 months
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-
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Table 4
Liposome
F1 (4 ºC) (25 ºC) F2 (4 ºC) (25 ºC) F3 (4 ºC) (25 ºC)
Z (Mean ±SD, mV) Initial After 6 months
EE (Mean ±SD, %) Initial After 6 months
367±1.30 367±1.30
382±10.20 393±11.10
-11.7±1.06 -11.7±1.06
-12.2±2.10 -8.3±0.90
57.78 ± 0.76 57.78 ± 0.76
50.80±2.30 40.90±1.31
106.43±0.42 106.43±0.42
126.0±0.63 136.0±.72
-8.74±0.22 -8.74±0.22
-9.2±1.20 -4.37±3.10
65.20±1.50 65.20±1.50
59.23±1.03 53.32±1.13
68.82±0.62 68.82±0.62
77.0±0.52 98.0±0.22
-10.40±0.58 -10.40±0.58
-9.5±0.80 -8.6±1.10
91.00±1.20 91.00±1.20
81.70±1.12 74.62±5.20
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182.0±1.88 214.0±1.75
-5.28±0.80 -5.28±0.80
-5.0±3.20 -4.21±2.50
88.50±1.04 88.50±1.04
83.21±3.20 80.43±8.60
340.0±1.31 340.0±1.31
350.0±2.01 372.0±2.63
-6.10±0.51 -6.10±0.51
-6.0±1.90 -5.7±1.20
66.11±2.11 66.11±2.11
61.15± 1.60 54.32±2.50
322±1.11 322±1.11
330.0±1.18 342.0±2.81
-7.13±0.16 -7.13±0.16
-6.8±2.9 -5.6±3.1
69.21±1.52 69.21±1.52
64.1±4.60 61.2±4.20
248±1.31 248±1.31
259.0±1.43 285.0±2.72
-7.51±0.22 -7.51±0.22
-7.0±0.78 -5.4±1.30
90.84±1.09 90.84±1.09
86.00±7.10 79.33±5.10
330.0±1.02 330.0±1.02
340.0±2.2 353.0±1.86
-7.82±0.46 -7.82±0.46
-7.2±0.86 -6.3±0.52
75.0±1.02 75.0±1.02
70.32±2.20 67.4±9.20
Blank 18±0.57
F3 28.0±1.0
F7 32.6±0.6
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Drug Inhibition Zone (Mean±SD, mm)
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Table 5
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Ethosome
F5 (4 ºC) (25 ºC) F6 (4 ºC) (25 ºC) F7 (4 ºC) (25 ºC) F8 (4 ºC) (25 ºC)
172.6±1.38 172.6±1.38
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F4 (4 ºC) (25 ºC)
Figure 1
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Figure 2
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Figure 3
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2256.9 2000
952.6 1000
786 421.2
500
0 F3
F5
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F1
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1500
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Cumulative amount of drug permeated (µg/cm2)
2500
5
F7
S1773-2247(17)30708-6
DOI:
10.1016/j.jddst.2017.10.015
Reference:
JDDST 497
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 24 August 2017 Revised Date:
3 October 2017
Accepted Date: 16 October 2017
Please cite this article as: A. Behtash Oskuie, S.A. Nasrollahi, S. Nafisi, Design, synthesis of novel vesicular systems using turpentine as a skin permeation enhancer, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/j.jddst.2017.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Design, synthesis of novel vesicular systems
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using turpentine as a skin permeation enhancer
A. Behtash Oskuie1, S. A. Nasrollahi2, S. Nafisi1*
Nanodermatology Unit, Center for Research & Training in Skin Diseases & Leprosy, Tehran,
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2
Department of Chemistry, Central Tehran Branch, Islamic Azad University, Tehran, Iran
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University of Medical Sciences, Tehran, Iran
The authors declare no conflict of interests
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Correspondence: [email protected] Shohreh Nafisi, Prof ,
Department of Chemistry, Central Tehran Branch, IAU, Tehran, Iran Tel: +98 912 144 1462 Fax: +98 21 880 74907
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Addition of skin penetration enhancer in a formulation is the simplest and most common technique to improve transdermal permeation. We aimed to develop novel liposome and ethosome vesicular systems using turpentine as a skin penetration enhancer for improving
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fluconazole skin permeability.
Fluconazole was encapsulated in various liposomal and ethosomal formulations The prepared
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formulations were characterized for size, size distribution, zeta potential (Z), entrapment efficiency (EE %), drug content, in-vitro, ex-vivo skin permeation and stability studies. The
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vesicles were found spherical in structure as confirmed by Scanning Electron Microscopy (SEM). Fluconazole was successfully entrapped in liposomes and ethosomes with relatively uniform drug content and entrapment efficiency in the range of 90.84 to 91.0 %. Liposome formulation; F3 and ethosome formulation; F7 with the highest drug entrapment efficiency were obtained as optimized formulations. Cumulative percent drug release for liposome (F3) and
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ethosome (F7) formulations were 82.52 and 90.84%, respectively. In-vitro, ex-vivo and antifungal effect of drug loaded ethosomes and liposomes, and the effect of penetration enhancer (turpentene) were studied and compared with free drug. The formulations containing turpentene
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showed higher permeation characteristics and can be proposed as promising delivery systems for
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fluconazole topical delivery.
Keywords: Fluconazole; Turpentine; Skin permeation; liposome; ethosome
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Fluconazole, a synthetic antifungal agent belonging to the group of triazole has been used in treatment of oropharyngeal, esophageal, or vaginal and urinary tract infection as well as other serious systemic candidal infections. It is also effective against superficial fungal infections and
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dermatophytoses. Fluconazole is available commercially as cream, tablets and injections in spite of its well-known adverse effects including nausea, vomiting, bloating and abdominal discomfort. Accounting these problems, drug delivery technologies should be developed which
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reduce drug dosing frequency along with sustained or controlled release of the medicament as well as reduced systemic side effects [1-2]. Vesicular systems such as liposome and ethosome
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formulations have widely proposed to bypass the topical drug problems [3-6]. Currently liposomes are used as novel drug delivery systems due to their flexibility and clinical efficacy. They can encapsulate hydrophilic and lipophilic drugs and protect them from degradation. They also have affinity to keratinocytes membrane and can penetrate deeper into
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skin and hence give better absorption. Applied on the skin, liposomes may act as a solubilizing matrix for poorly soluble drugs, penetration enhancer as well as local depot at the same time diminishing drugs side effects. Topical liposome formulations can relatively be more effective
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and less toxic than conventional formulations [7-12]. Ethosomes, novel carrier systems used for drug delivery have higher penetration through
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biological membranes mainly skin. The lipid vesicles composed of phospholipids, and relatively high alcohol concentration are obtained by slight modification of liposomes. They can permeate more rapidly through skin layers and possess significantly higher transdermal flux in comparison to conventional liposomes [13-15]. Although, the exact ethosomes mechanism for higher permeation into deeper skin layers is not still clear, synergistic combination effects of
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phospholipids and ethanol in vesicular formulations have been suggested for deeper distribution and penetration [16-19]. To achieve successful transdermal drug delivery, enhancement of skin permeability is of
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prime concern. Recently several physical, electrical, chemical and biochemical techniques have been proposed to increase skin permeability. Amongst these techniques, permeability improvement by chemical methods is being most widely used owing to economic, simplicity and
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rapidity. Chemical permeation enhancers either improve the solubility or partition coefficient or increase drug diffusion across the skin. However, their toxicity and irritancy to skin has led to
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quest of natural skin penetration enhancers [20-25].
Terpenes, obtained from pistachio gum have been successfully used as penetration enhancers especially for lipophilic drugs. Structurally, terpenes consist of isoprene units (C5H8) can be classified as hydrocarbons, alcohols, ketones, and oxides. They offer advantages over many other
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penetration enhancers, in part because they are categorized as safe substances by the U.S. food and drug administration [26-27]. Turpentine, composed of terpenes has demonstrated skin penetration potential in various traditional applications such as herbal medicine. However, there
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is limited permeation study of pistachio gum (oil) on human skin [28-29]. Based on these premises, we developed novel nanovesicular carriers to improve fluconazole
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skin permeability. Fluconazole effectiveness was compared with the complexes of FLZliposome, FLZ-ethosome, FLZ-liposome-turpentine, and FLZ-ethosome-turpentine. Poly dispersity Index (PDI), zeta potential (Z), morphology, stability and drug content of the designed formulations were studied. Optimized formulations in terms of entrapment efficiency (EE %) were evaluated for their penetration profile, in-vitro, ex-vivo release and antifungal activity.
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I. MATERIALS and METHODS 1. Chemicals and reagents Fluconazole
was
received
from
Zahravi
Medical
Co.
(Tabriz,
Iran).
Soya
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phosphatidylcholine was from Lipoid Co. (Germany). Propylene glycol and polysorbate 80 were obtained from Merck (Germany). Cholesterol and agar were from Sigma Aldrich. Turpentine was received from Herbal Medicine Society (Tehran, Iran). Aspergillus niger was received from
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microbial bank of IROST (Tehran, Iran). All other chemicals were of analytical grade and used
2. Preparation of vesicular systems
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without further purification.
2.1. Preparation of fluconazole liposomes
Different compositions of aqueous liposomal formulations were prepared by conventional
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lipid “Film hydration method” (Table 1). Lipid phase composed of phosphatidylcholine (200 mg), cholesterol (30 mg), polysorbate 80 (20 mg) and different turpentine concentrations (10, 20 and 30 mg) designated as F2, F3, F4 were dissolved in chloroform:methanol (2:1, V/V) together
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with fluconazole (20 mg) in a round bottom flask. Solvent was removed under reduced pressure in a rotary evaporator at 55 °C to obtain a thin film on the flask wall. The flask was kept
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overnight under vacuum to ensure the complete removal of residual solvent. The dry lipid film containing fluconazole was hydrated with 20 ml phosphate buffer solution (pH 7.4) at 60±2 °C. The dispersion was left undisturbed at room temperature for 2-3 h to allow complete swelling of the lipid film and to obtain vesicular dispersion. All the above-mentioned steps were performed under aseptic conditions and the glass-wares were sterilized by autoclaving. Phosphate buffer saline was passed through a 0.22 nm membrane filter, and the entire procedure was performed in
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a laminar flow hood. Fluconazole liposomes were separated from unentrapped drug by centrifugation at 13,000 rpm for 1 h and at 2 °C using cooling ultracentrifuge. The formed cake was washed twice each with 10 ml phosphate buffer saline and re-centrifuged for 1 h [30-33]. A
same drug and ethanol concentrations (Table 1).
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2.2. Preparation of fluconazole ethosomes
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liposome fluconazole suspension without turpentine (F1) was also prepared as a control in the
Ethosomes in different formulations were prepared by “Hot method” (Table 2).
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Phosphatidylcholine (200 mg), propylene glycol (20 mg) and fluconazole (20 mg) were dissolved in ethanol 96% (2.5 ml). Different concentrations of turpentine (10, 15, 20 mg) designated as F6, F7, F8 were added. To get nano-sized ethosomes, the preparation was sonicated by Probsonicator (SH 70 G, Bandlin Sonoplus UW 2070, Berlin, Germany) by stirring
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speed of 700 rpm at 4 ºC for 3 cycles of 5 min, with rest of 5 min between each cycle. The samples were centrifuged at 20,000 rpm for 30 min and evaluated for their entrapment efficiency [32]. An ethosome fluconazole suspension without turpentine (F5) was also prepared as a control
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in the same drug and ethanol concentrations (Table 2).
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3. Preparation of cream formulations In order to manufacturing base cream, lipid phase (containing glyceryl monostearate, mineral oil, butylated hydroxytoluene, Spermaceti and stearyl alcohol) was heated to 75 ºC. The aqueous phase (containing polysorbate 80, polysorbate 60, sorbic acid, potassium sorbate, sorbitol and distilled water) was added to the lipid phase at the same temperature under stirring (600 rpm) and cooled down to 25 ºC. Liposome and ethosome were added to the base cream under stirring. 6
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4. Characterization of drug formulations 4.1 Size, polydispersity index (PDI) and zeta potential (Z)
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Mean vesicle size and size distribution profile and zeta potential of liposomes and ethosomes were determined by using Malvern particle size analyzer model SM 2000, which follows Mie's theory of light scattering. The diluted visible suspension was added to sample dispersion unit and
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stirred at 2000 rpm in order to reduce the inter-particle aggregation; laser obscuration range was maintained between 10-20%. Average particle size was measured after performing the
5. Drug entrapment efficiency (EE %)
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experiment in triplicate.
Fluconazole associated with vesicular system was separated from unentrapped drug using centrifugation method. The vesicle suspension was centrifuged at 20,000 rpm for 1 h at a
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controlled temperature of 4 ºC. Supernatant containing unentrapped fluconazole was withdrawn and measured by UV spectrophotometrically at 260 nm against phosphate buffer saline (pH 7.4). The amount of fluconazole entrapped in vesicle was determined as follow [34-35]:
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EE % = [(Ct − Cf) / Ct] × 100
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Where Ct is concentration of total fluconazole and Cf is concentration of free fluconazole. 6. Distribution morphology Scanning electron microscopy was conducted to characterize the surface morphology of the selected liposome and ethosome formulations for which a drop of each formulation was mounted on clear glass stub, air dried and coated with polaron E 5100 Sputter coater (Polaron, UK) and visualized under SEM (JOEL-JSM-6510, Japan).
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7. Physical stability studies Physical stability tests were carried out to investigate vesicles aggregation and drug leakage from vesicles during storage. Prepared formulations were stored in transparent vials covered with
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a plastic cap at ambient temperature and 4 ºC. Physical stability of samples was evaluated by Z, mean vesicle size and EE % over 6 months.
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8. In-vitro drug release studies
In-vitro fluconazole permeation behavior from all liposome and ethosome formulations was
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investigated using cellophane membrane (0.45 µm). In brief, 20 mg of fluconazole and fluconazole liposome and ethosome formulations were suspended in phosphate buffer saline (1 ml, pH=7.4) in a glass cylinder having a length of 20 cm and a diameter of 2.5 cm. The cylinder was fitted before liposome suspension addition with a presoaked membrane [Spectra/Por
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membrane] and placed in a beaker (100 ml) containing 35 ml phosphate buffer saline (pH 7.4). The whole set was placed on a magnetic stirrer adjusted to a constant speed (150 rpm). Samples were collected every 1 h over a period of 10 h and assayed spectrophotometrically for drug
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content at 261 nm. Cumulative amount that permeated across the cellophane membrane was
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calculated and plotted against time. Results were the mean values of three runs [36].
9. Ex-vivo skin experiments Skin was obtained from patients undergoing abdomen reduction surgery, in Parsian Hospital. Skin slices were isolated with a dermatome from abdomen, and then stored at −18 ºC for at least 24 h. Skin permeation and uptake was determined using vertical Franz cell. Excised skin was rinsed with normal saline and aqueous ethanolic solutions. The skin was then sandwiched
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between two cells with stratum corneum side upwards. Receptor chamber was filled with 35 mL of normal saline solution. The cream test formulations 1% (0.5 g) was applied to skin surface (available diffusion area of 3.3 cm2). Receptor chamber content continuously stirred at 37 ºC was
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removed at appropriate intervals (1–6, 24 h) for drug concentration measurements and the cell was immediately refilled with fresh receptor solution. After 24 h, the application site was washed with normal saline solution to remove excess drug on skin surface. Skin was then cut with
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scalpel into small pieces; methanol was added (2.0 ml) for drug extraction. After 4 h stirring, the resulting suspension was centrifuged and supernatant assayed by UV analysis. Skin uptake was
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expressed as µg drug/cm2 of skin diffusion area [37].
9. Calculation of skin permeation parameters
Drug cumulative amount permeated per unit area was plotted as a function of time. Flux was
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calculated from linear portion slope. Drug permeability coefficient (????) across skin was calculated using relation derived from Fick’s first law of diffusion, which is expressed by the following equation:
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Kp = J/C
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Where J is flux and ?? is drug concentration in donor compartment.
10. Antifungal activity
Selected formulations (F3 and F7) and blank formulation (without turpentine) were assayed for antifungal activity against the fungal strain Aspergillus niger, obtained from Microbial Bank of IROST (Tehran, Iran). The fungus was grown on Sabouraud’s agar plate at 25 ◦C. Fungal culture suspension was made in about 0.4 ml sterile water and stood for 20 min before
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transferring to solid agar medium where the strain was available in lyophilized form. Fungal culture suspension (0.1 ml) was mixed with 9.9 ml liquid broth (without agar) and inoculated for 24 h in an incubator at 25 ◦C. Inoculated liquid broth containing fungal culture suspension (1
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mL) was added to the sterile petri dishes of solidified agar growth medium. The inoculum was spread uniformly over the solid agar surface by clockwise and anticlockwise direction plate rotations - the formed well in the middle of the plates with the help of a sterile cork borer was
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filled with the formulations. For fungus growth, plates were incubated at 25 ◦C. Clear rings called inhibition zone were appeared around the dishes in 48 h. More effective formulations
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show larger inhibition zones. Antifungal activity was evaluated by measuring inhibition zone (in mm) of fungal growth surrounding the formulations after a week - the complete antifungal analysis was carried out in triplicates.
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11. Statistical analysis
Data analysis was carried out using Microsoft Excel 2010. Results are expressed as mean ± standard deviation (?? = 3). Statistically significant differences were determined using analysis of
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variance (ANOVA) with ?? < 0.05 as a minimal level of significance.
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II. RESULTS AND DISCUSSION Two types of vesicular systems, fluconazole-liposomes and fluconazole-ethosomes, and turpentine as a natural penetration enhancer are formulated and presented in Tables 1, 2. Different formulations were characterized for mean particle size, PDI, Z, EE%, distribution morphology, physical stability (Fig 1, Tables 3, 4). Optimized compositions were selected based on the highest entrapment efficiency amongst the formulations presented in Tables 1 and 2 and
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examined for in-vitro drug release studies, ex-vivo skin penetration experiments and antifungal activity (Figs 2, 3). Average size of FLZ-liposome and ethosome formulations were measured and presented in
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Table 3. Particle size of liposome formulations ranged from 68.8 to 367.0 nm with a relatively narrow size distribution (PDI<0.41), while the matching data for FLZ-ethosomes were 248 to 340 nm with a PDI<0.36 (Table 3). Average size of the liposome and ethosome formulations was
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greater than that of the vesicles with turpentine. Liposome and ethosome sizes are reduced by the addition of turpentene due to the presence of more turpentene in the outer layer than in the inner
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bilayer membranes. Decrease in liposomal and ethosomal sizes by turpentine can be explained with the same mechanism as reduction in liposomal size by surfactant addition [38-40]. Polydispersity index (PDI) were determined as a measurement of particle size homogeneity for the prepared liposome and ethosome formulations. PDI values for optimized formulations
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(F3 and F7) were 0.40 and 0.212, respectively (Table 3). Zeta potentials of all liposomes (-5.28 to -11.7) and the corresponding ethosomes were negative (-6.10 to -7.82). Z was found to be -10.40 and -7.51 mV for the optimized liposome
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(F3) and ethosome (F7) formulations, respectively (Table 3). Stability of formulations depends
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on the ingredients charges; ingredients with large negative or positive charge repel each other and do not come together and aggregate. Hence, ingredients with higher charges create more stable formulations. PC, a zwitterion compound with an isoelectric point between 6 and 7 has a negative charge at pH 7.4. Consequently, liposome and ethosome formulations show good physical and chemical stabilities due to the presence of PC which creates negative charge in their structure [39].
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Fluconazole entrapment efficiency in the vesicles ranged from 57.58 to 91.0 % for liposome and from 66.11 to 90.84 for ethosome formulations. The highest EE was observed for F3 (91.0 %) and F7 (90.84 %) containing 15 mg of turpentine. Optimized F3 and F7 formulations showed
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smallest particle sizes (68.82, 248.0 nm), respectively.
SEM results indicated small, spherical shape with a smooth surface for the optimized vesicles (Fig. 1). Obtained data were compatible with DLS results analysis regarding size confirming
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narrow size distribution of optimized liposomes and ethosomes (Table 3). F3 and F7 were examined for in-vivo, ex-vivo and antifungal activity studies.
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Physical stability study of the prepared vesicles showed lower percentage of fluconazole retained in formulations at room temperature rather than refrigerated temperature after 6 months. This may be due to increased lipid bilayer fluidity at higher temperatures which results in drug leakage. Vesicles were also reasonably stable in term of aggregation (Table 4).
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Figure 2 A, B shows the effect of different formulations on permeation profile of fluconazole. Results revealed that F3 (FLZ-liposome vesicle) and F7 (FLZ-ethosome vesicle) have the greatest drug release equal to 82.52 and 90.84% in 12 h, respectively. The highest
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amount of ex-vivo permeation was obtained for F3 and F7 formulations as well. These finding could be due to increased drug fluidity which is a consequence of synergic effect of polysorbate
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80, turpentine and alcohol (F3 and F7) [26, 40, 41]. Addition of polysorbate 80 and ethanol increased vesicle fluidity near the polar head group of the phospholipid bilayer. Results are compatible with other reports as well. It is hypothesized that polysorbate 80 turns its hydrophilic portion towards the phosphate group. Turpentine localizes near the end of phospholipid chain, consequently increases vesicle fluidity [37].
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Fluconazole cumulative amount permeated from ethosomal formulations containing ethanol and turpentene after 24 h is significantly higher than that of liposomes containing polysorbate 80 and turpentene (Fig. 3). Turpentene demonstrated a remarkable effect in release and skin
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permeation in all formulations comparing with blank formulation (F1).
Release experiments indicated controlled fluconazole release from all vesicle formulations, because of higher drug content and entrapment efficiency comparing with blank. Maximum
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release is also due to optimum polysorbate 80 concentration (20 %), because at this concentration, the surfactant molecule gets associated with phospholipid bilayer resulting in
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better drug partitioning and higher release from the vesicles [41-42].
Antifungul activity results in term of inhibition potency measured as zone diameter in mm are given in Table 5. All tested samples were found effective against Aspergillus niger following the order of F7>F3>blank. Significant differences were found between formulations; ethosome
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formulation (F7) containing turpentene and alcohol with inhibition zone of 32.6 mm was the most effective one. Although liposomal formulation (F3) containing turpentine showed high inhibition zone of 28.0 in comparison to blank formulation. Inhibition zone differences can be to greater fluconazole release rate from ethosome, which in turn is attributed to
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turpentene and alcohol content of the formulation. Turpentene like other unsaturated fatty acids
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increases skin permeation through disruption of stratum corneum ordered lipids. These changes promote fluidization of the lipids, simultaneously lower the lipid transition temperature, resulting in increased drug release and permeability [43]. ⃰
In the present study, novel fluconazole-loaded liposomes and ethosomes were successfully prepared. We found that the addition of penetration enhancer “turpentine” resulted in vesicles
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with smaller particle size and proper encapsulation efficiency suggesting that the novel vesicular systems can be considered as effective fluconazole carriers. Optimized vesicles (F3 and F7) containing 15 mg turpentene increased penetration up to 5 times comparing with fluconazole
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blank.
Results revealed that liposomal and ethosomal fluidity correlated with skin penetration enhancement of entrapped drug. Increased system fluidity resulted in fluconazole penetration
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enhancement. Polysorbate 80 molecules, generally localized near the hydrophilic portion of the phospholipid bilayer, and turpentene accompanied with alcohol incorporated in drug
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compositions induce liposomal and ethosomal fluidity and show synergic fluconazole penetration enhancement. Present study shows that the novel liposome and ethosome formulations containing turpentine have potential to improve transdermal delivery and antifungal activity, reasonably. They can be considered as promising carriers to improve fluconazole
Acknowledgements
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permeability in a period of time.
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Authors appreciate Dr. M. Hadgi Yousefi, Surgeon of Parsian Hospital for providing abdominal skin sample and Department of Chemistry, Central Tehran Branch, Islamic Azad University for
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providing the facilities.
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Captions for Figures and Tables
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Table 1: The composition of FLZ-liposome formulations Table 2: The composition of FLZ-ethosome formulations Table 3: Size, PDI, Z and EE % of liposome and ethosome formulations
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Table 4: Stability of prepared vesicles during storage at 4 and 25 ºC for 6 months
Table 5: Inhibition zone of selected liposome (F3), ethosome (F7) formulations and blank fluconazole (without turpentine) against Aspergillus niger
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Fig. 1: Scanning electron microscope image of a) Optimized liposome formulation (F3) and b) Optimized ethosome formulation (F7).
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Fig. 2: In-vitro drug release profiles of a) F1-F4 of liposomal formulations b) F5-F8 of ethosomal formulations
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Fig. 3: Ex-vivo skin permeation of liposomal (F1, F3) and ethosomal (F5, F7) formulations
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Table 1 F2
F3
F4
20 200 30 20 -
20 200 30 20 10
20 200 30 20 15
20 200 30 20 20
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F1
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Composition (mg) Fluconazole Phosphatidylcholine Cholesterol Polysorbate 80 Turpentene
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Table 2 F2 20 200
F3 20 200
F4 20 200
20 2.5 -
20 2.5 10
20 2.5 15
20 2.5 20
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F1 20 200
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Composition Fluconazole (mg) Phosphatidylcholine (mg) Propyleneglycol (mg) Ethanol (ml) Turpentene (mg)
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0.30±0.02 0.30±0.02 0.40±0.02 0.41±0.02 0.206±0.03 0.318±0.01 0.212±0.05 0.364±0.10
Z (Mean±SD, mV) -11.7±1.06 -8.74±0.22 -10.40±0.58 -5.28±0.80 -6.10±0.51 -7.13±0.16 -7.51±0.22 -7.82±0.46
EE (Mean±SD, %) 57.78 ± 0.76 65.20±1.50 91.00±1.04 88.5±1.20 66.11±2.11 69.21±1.52 90.84±1.02 75.0±1.09
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Ethosomes
PDI (Mean±SD)
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Liposomes
F1 F2 F3 F4 F5 F6 F7 F8
Size (Mean ±SD, nm) 367.0 ±1.30 106.43±0.42 68.82±0.62 172.60±1.38 340.23±1.31 322.21±1.11 248.17±1.02 330.16±1.31
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Drug Formulation
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Table 3
Drug formulation
Size (Mean ±SD, nm) Initial After 6 months
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Table 4
Liposome
F1 (4 ºC) (25 ºC) F2 (4 ºC) (25 ºC) F3 (4 ºC) (25 ºC)
Z (Mean ±SD, mV) Initial After 6 months
EE (Mean ±SD, %) Initial After 6 months
367±1.30 367±1.30
382±10.20 393±11.10
-11.7±1.06 -11.7±1.06
-12.2±2.10 -8.3±0.90
57.78 ± 0.76 57.78 ± 0.76
50.80±2.30 40.90±1.31
106.43±0.42 106.43±0.42
126.0±0.63 136.0±.72
-8.74±0.22 -8.74±0.22
-9.2±1.20 -4.37±3.10
65.20±1.50 65.20±1.50
59.23±1.03 53.32±1.13
68.82±0.62 68.82±0.62
77.0±0.52 98.0±0.22
-10.40±0.58 -10.40±0.58
-9.5±0.80 -8.6±1.10
91.00±1.20 91.00±1.20
81.70±1.12 74.62±5.20
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182.0±1.88 214.0±1.75
-5.28±0.80 -5.28±0.80
-5.0±3.20 -4.21±2.50
88.50±1.04 88.50±1.04
83.21±3.20 80.43±8.60
340.0±1.31 340.0±1.31
350.0±2.01 372.0±2.63
-6.10±0.51 -6.10±0.51
-6.0±1.90 -5.7±1.20
66.11±2.11 66.11±2.11
61.15± 1.60 54.32±2.50
322±1.11 322±1.11
330.0±1.18 342.0±2.81
-7.13±0.16 -7.13±0.16
-6.8±2.9 -5.6±3.1
69.21±1.52 69.21±1.52
64.1±4.60 61.2±4.20
248±1.31 248±1.31
259.0±1.43 285.0±2.72
-7.51±0.22 -7.51±0.22
-7.0±0.78 -5.4±1.30
90.84±1.09 90.84±1.09
86.00±7.10 79.33±5.10
330.0±1.02 330.0±1.02
340.0±2.2 353.0±1.86
-7.82±0.46 -7.82±0.46
-7.2±0.86 -6.3±0.52
75.0±1.02 75.0±1.02
70.32±2.20 67.4±9.20
Blank 18±0.57
F3 28.0±1.0
F7 32.6±0.6
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Drug Inhibition Zone (Mean±SD, mm)
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Table 5
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Ethosome
F5 (4 ºC) (25 ºC) F6 (4 ºC) (25 ºC) F7 (4 ºC) (25 ºC) F8 (4 ºC) (25 ºC)
172.6±1.38 172.6±1.38
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F4 (4 ºC) (25 ºC)
Figure 1
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Figure 3
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2256.9 2000
952.6 1000
786 421.2
500
0 F3
F5
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F1
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1500
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Cumulative amount of drug permeated (µg/cm2)
2500
5
F7