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Invasomes composed of phosphatidylcholine, ethanol and terpenes [72]. One proposed mechanism of the penetration enhancing ability of elastic vesicles is that vesicles or vesicle constituents disorganise and disrupt intercellular lipid lamellae forming channel-like penetration pathways, through which drug molecules penetrate. The second proposed theory is that intact vesicles penetrate into SC through pre-existing channels with low penetration resistance, which is also supported by Cevc et al. Honeywell-Nguyen et al. demonstrated the partitioning of intact vesicles into deeper layers of SC. Terpenes, as constituents of invasomes, present potent penetration enhancers and they have been shown to enhance the percutaneous absorption of midazolam  [73], indomethacin, lorazepam, klonazepam [74], haloperidol [75], nicardipine, carbamazepine, tamoxifen [76] and many other drugs. Investigations employing differential scanning calorimetry and X-ray diffraction revealed that terpenes increase drug permeation by disrupting lipid packaging of SC and/or disturbing the stacking of the bilayers [77-79]. Ethanol, which has been also added to invasomes, presents an efficient penetration enhancer [80-83] and a synergistic effect of liposomes and ethanol has been described in the literature [84]. Invasomes could be a promising tool for delivering the highly hydrophobic photosensitizer temoporfin to the skin, which would be advantageous for the photodynamic therapy of cutaneous malignant (basal-cell carcinoma) or nonmalignant diseases [85].

Preparation of lipid vesicular system:
All the lipid vesicles were prepared by a conventional rotary evaporation method [86, 87]. Briefly, the appropriate weights of lipid or lipids were dissolved in methanol/chloroform solution (1:2, v/v) in round bottom flask. Thin lipid films were obtained by removing the organic solvents under vacuum condition (500 mbar 10 min, 200 mbar 10 min, 100 mbar 10 min, 35 mbar 1 hr) at a temperature more than transition temperature of lipid with a rotary evaporator.  The traces of solvent were removed from the deposited lipid film under vacuum overnight. The resulted dry lipid films on the inside wall of round bottom flask were hydrated and dispersed with different hydration systems like phosphate buffered saline corresponding to all formulations at room temperature. The obtained macroscopically homogenous solution was sonicated for totally 15 min in 3 cycles (5 min for each cycle and 5 min pause among these cycles) with a sonication ice-water bath. Then these suspensions were extruded through a series of 0.45, 0.22-μm polycarbonate membrane many times to produce liposomes of the desired size with the help of a Hamilton-Bonaduz extruder [88].

Characterization of different lipid vesicular systems:
Vesicles size and surface morphology:
Transmission electron microscope (TEM) was used as a visualizing aid for lipid vesicles. Scanning electron microscopy (SEM) is also conducted to characterize the surface morphology of the vesicles. The size distribution is determined, by Dynamic Light Scattering (DLS) technique using a computerized Autosizer inspection system. For vesicles size measurement, vesicular suspension is mixed with the appropriate buffer medium and a drop of vesicle dispersion is applied to a carbon film-covered copper grid and is stained with a 1% phosphotungstic acid. Then, samples are examined with a transmission electron microscope at an accelerating voltage of 80 kV. All particle sizes and zeta potentials are measured at room temperature. In order to evaluate the stability of ethosomes during storage, the size and zeta potential of vesicles are also monitored after being stored at 4 ?C [46].

Determination of the drug entrapment efficiency:
A centrifugation method was used to separate the incorporated drug in the free form. Vesicle suspensions were centrifuged. Following centrifugation, the supernatant and pellet were separated, and the concentration of drug was analyzed by high-performance liquid chromatography (HPLC) or spectrophotometrically [81].

Deformation index determination:
Comparative measurement of deformability of liposome bilayer with different penetration enhancers is generally carried out by extrusion measurement. The vesicle dispersion extruded at constant pressure through polycarbonate filters of definite pore size using an extrusion device. The deformability of vesicle is expressed in terms of deformation index (DI).

DI= j(d0/p)k (1/|d1 - d0| )

Where J is the amount of suspension recovered after extrusion, d0 and d1 are the mean diameter of vesicles before and after extrusion, p is the pore size of the membrane, and k is an amplification factor [34].

In vitrodrug release (flux studies):
The permeation of DS-bearing Transfersomes through an artificial cellophane membrane was performed in Franz-type diffusion cells with a diffusion area of 1.77cm2 [90]. The receptor medium was phosphate buffer which was constantly stirred at 100rpm with a small magnetic bar. The receptor compartment was maintained at 37±0.2 ?C by a circulating water jacket. An equivalent amount was placed in the donor compartment. Samples were withdrawn from the receptor compartment via the sampling port at different time intervals to 24 h, and immediately replaced with an equal volume of fresh receptor solution. All samples were analyzed for drug content spectrophotometrically or HPLC. The obtained data were kinetically treated to determine the order of release. The flux at 24 h (J) was assessed and the release profile curves were constructed for all formulae [60].

I = Amount of permeated drug/ Time x Area of release membrane

In vivo, ex vivopermeation study:
The assessment of percutaneous permeation of molecules is one of the main steps in the initial design and later in the evaluation of dermal or transdermal drug delivery systems. The literature reports numerous ex vivo, in vitro and in vivo models used to determine drug skin permeation profiles and kinetic parameters, some studies focusing on the correlation of the data obtained using these models with the dermal/transdermal absorption in humans. In- vitro permeation studies to clinical performance, presenting various experimental models used in dermal/transdermal research, including the use of excised human or animal skin, cultured skin equivalents and animals (rat, mice, rabbit, pig etc.) [91].

Visualization of skin penetration using confocal laser scanning microscopy (CLSM):
CLSM has provided a significant tool with which to visualize skin structure and the localization of fluorescent probes within the tissue. Because of its nondestructive nature, and the fact that little or no sample preparation is necessary, CLSM offers a reasonably faithful representation of reality with few artifacts. The potential to recreate three-dimensional visualization of the tissue is another significant advantage relative to other microscopic techniques. CLSM provides complementary image information to that obtained from other conventional microscopic and histological methods. The major contribution of CLSM in the topical/transdermal field to-date have been mechanistic, particularly in terms of revealing preferred penetration pathways following, the use of different delivery technologies. Nevertheless, there remain important limitations of CLSM. First, only a restricted range of fluorophores are available for imaging and attachment of these markers, for example, to a dry of interest may change significantly the permeability behaviour (rate, extent, route of transport, etc.). Second, the technique reveals at best only semi-quantitative information, and no approach to calibrate the fluorescence intensities observed has yet been demonstrated. Third, the images obtained are static views of reality captured at a particular point in time; thus, does a strongly fluorescent region imply a key pathway through which a large fraction of the total transport is occurring, or does it suggest an area where the fluorophore has become tightly bound and/or immobilized [92].

The ability of lipid vesicles consisting of components other than phospholipids and cholesterol or their semisynthetic derivatives [93] to enhance the encapsulation of bioactive substances provides new promising perspectives for establishing new, efficient and stable carriers for drug delivery. The potential of lipid vesicles for transdermal iontophoretic drug delivery was first reported by Vutla et al. (1996) [94].  Some studies were followed, investigating such approach, using either traditional or deformable liposomes [95-97]. The exact benefits remain unclear, as a result of paucity of reported investigations; however, it appears that combining iontophoresis with lipid vesicles facilitates drug delivery to deeper layers of skin including enhanced trans-follicular delivery. Studies will continue to further investigate such a promising approach.

A new lipid vesicular system, the propylene glycol-embodying liposomes (PG-liposomes), composed of phospholipid, propylene glycol and water, was recently introduced, developed and investigated, as a carrier for skin delivery of drugs. PG-liposomes of the local anesthetic, cinchocaine, showed high entrapment efficiency and were relatively more stable than other liposomal formulations, including deformable liposomes and ethosomes. In vivo skin deposition studies, carried out using rabbit dorsal skin, indicated that cinchocaine PG-liposomes were superior, as carriers for skin delivery, relative to other liposomal formulations. PG-liposomes showed improved skin deposition, even relative to deformable liposomes and ethosomes, suggesting that PG-liposomes, recently developed, may have promising future as carriers for skin delivery of drugs [47]. Another approach that could increase the photoprotective effect against UV radiation comprises targeted delivery of α tocoperol into the deeper skin layers and across the cell membranes. For this purpose, ethosomal vitamin E compositions were designed and tested [98, 99]. Ethosomes are phospholipid soft vesicles composed of safe and natural components approved for pharmaceutical and cosmetic use.



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