| Research BackgroundSkin is the largest organ of the human, the stratum corneum (SC), so-called "brick and mortar" model, is composed of layers of flattened corneocytes filled with the fibrous protein keratin and embedded in a multilamellar lipidic material containing ceramides, cholesterol and free fatty acids, which forms the narual barrier for extetnal material to be delivered into.At present, many clinical topical formulations are hydrophilic drug, such as 5- aminolevulinic acid, diclofenac sodium, etc. Even though prepared as a cream, gel and other conventional transdermal formulations, it is still not enough for percutaneous penetration, so that large clinical treatment discounted. In order to reduce the barrier effect of the stratum corneum, numerous techniques have been employed to improve transdermal or topical delivery for local or systemic therapy. These include the use of chemical penetration enhancers, adjustment of chemical potential of the drug, microneedls, iontophoresis, electroporation or sonophoresis. However, these approaches are subjected to some serious limitations. For instance, physical approaches are mostly painful and expensive while chemical permeation enhancers are known to cause permanent skin damage.With the development of materials science and nanotechnology, the novel transdermal drug delivery system has become an important way to solve above problems. Among them, as a novel nano-carrier for transdermal penetration, ethosomes mainly contain phospholipids, alcohol (ethanol and isopropyl alcohol) in relatively high concentration and water, which have been demonstrated that have the properties of thermodynamics stability, small size, high entrapment efficiency, powerful penetration, and the good biocompatibility. Therefore, it obtains wide attention in the field of the Transdermal drug delivery system (TDDS).Unlike conventional liposomes, ethosomes were shown to permeate through the stratum corneum barrier and were reported to possess significantly higher transdermal flux in comparison to liposomes. Beside these, the ethosomal carrier was also capable of providing an efficient intracellular delivery of both hydrophilic and lipophilic molecules and the permeation of an antibiotic peptide. However, it is still not clear of the exact mechanism of enhanced skin penetration by ethosomes and the permeation process of ethosome in the skin. While, to understand the knowledge of skin penetration pathways and vesicle-skin interactions in and through the skin is a prerequisite for the development and optimization of ethosomes for transdermal drug delivery.Confocal laser scanning micrography (CLSM) is a good optical analysis technology to visualize the fluorescent compounds distribution in the skin from different formulations. With the help of fluorescent tracer, we can observe the distribution of the drug formulation itself or the loaded drug in the skin in real time, in vivo, especially in the hair follicle, sebaceous and sweat glands, it is very important of these knowledge to study the drug that for topical application and target to hair follicle, moreover, we can use the software to quantitatively assign and statistical analysis of the fluorescence of the unit area, the amount of percutaneous penetration of the carrier, penetration depth of the skin, time performance, distribution and elimination of laws and other data discrepancies, it is also expected to establish a new method of skin pharmacokinetic study.In the present study we use rhodamine B mimics the hydrophilic drugs and NBD-PC label the Egg PC to tract the PC distribution in the skin. We prepare and determine the optimal formulation of the Rhodamine B ethsomes, and to study its physicochemical properties. Using the confocol laser scanning micrograhy to visualize the penetration process of the rhodamine B ethosomes following non-occlusively applied onto the rat abdominal skin, in vivo, and to investigate of the mechanism for ethosomes promote transdermal penetration of hydrophilic drug. Meanwhile, we use TEM to observe the ultrastructual changes after the interaction between ethosomes and rat skin.ObjectiveTo prepare the rhodamine B ethosoems and study its physicochemical properties; To determine the mechanism for ethosoems promote skin penetration of hydrophilic drug and the ultrastructural changes of the skin after treated with ethosoems, thus, to provide detailed experimental data and build a solid theoretical foundation for the subsequent research of the transdermal formulations of the ethosoems.Method1. Use L9 (34) orthogonal test to determine optimal formulation of 0.02% Rhodamine Bethosome:The quality percentage concentration of egg phosphatidyl choline, the volume fraction of absolute ethanol and untrasound time were chosen to be the consideration factors. And the particle size was chosen to be the index of 0.02% Rhodamine B ethosome in order to select the optimal formulation.2. The preparation of 0.02% Rhodamine B ethosome and liposome2.1The preparation of 0.02% Rhodamine B ethosome by injection-sonication method:Briefly, Egg PC which was exactly weighed by analytical balance was added into a glass vial and solubilized with ethanol.The glass vial was sealed up completely and connected with a tube to a syringe system to allow the addition of double-distilled water and to avoid ethanol evaporation as far as possible. Following the solubilization of lipid, rhodamine B aqueous solution was added slowly (200μLmin-1) to obtain the ethosomal colloidal suspensions in a constant stirring condition (700 r·min-1) with a magnetic stirrer. Mixing was continued for an additional 5 minutes after the drug pour into completely. The final preparation was obtained after processes of being sonicated 5 minutes of 150W by Ultrasonic probe at 4℃.2.2The preparation of 0.02% Rhodamine B liposome by film-dispersing method:In a typical procedure, egg PC for final concentration of 2% w/v was added into a round-bottomed flask and dissolved in a small amount of chloroform.The organic solvent was removed by rotary evaporation vacuum at 60℃ until a thin lipid flim on the wall of a round-bottomed flask, was obtained.The resulting lipid film was kept under vacuum overnight in order to eliminate the traces of organic solvent. The deposited lipid film was then hydrated with 0.02%w/v rhodamine B aqueous solution by rotation (60 rpm) for 30min at room temperature. Finally, liposomal suspensions were sonicated 5 minutes of 150W by Ultrasonic probe at 4℃ for particle homogenization.2.3 The preparation of NBD-PC labeled 0.03%Rhodamine B loaded ethosomes:The fluorescent probe, NBD-PC, was dissolved in the ethanol with the egg phosphatidyl choline. The ratio of PC to NBD-PC was 100:1 M. The other procedure followed the process described above.2.4 Separation of non-entrapped Rhodamine B from NBD-PC labeled Rhodamine B loaded ethosomes:A filtration technique using Amicon Ultra-4 centrifugal devices (Millipore Corporation, USA) was utilized to separate non-entrapped rhodamine B from NBD-PC labeled Rhodamine B loaded ethosomes. The NBD-PC labeled Rhodamine B loaded ethosomes (4 mL) was added into an ultrafiltration tube, with a cut-off molecular weight of 3,000 Da, and then the mixture was centrifuged at 12,000x g, at 4℃, for 1 hour. The filtrate was removed, and then the retentate tube was inverted in a new collection tube, to collect the entrapped NBD-PC labeled Rhodamine B loaded ethosomes via centrifugation at 1,000×g, at 4℃, for 5 minutes. The entrapped NBD-PC labeled Rhodamine B loaded ethosomes obtained was immediately used in the skin penetration experiments.3. Size and Zeta Potential analysis by Dynamic Light Scattering (DLS):The particle size and zeta potential of ethosomes and liposomes were determined using a laser diffraction technique on a Zetasizer at 25℃ with a heliumneon laser at 630nm.Before measurements, the vesicular suspension was diluted with appropriate medium. That means 35% ethanol solution and water were used to dilute ethosomes and liposomes, respectively. The determination was repeated three times per sample.4.The morphology of the ethosomes visualized by Transmission electron microscopy:The morphology of the ethosomes was observed using TEM. The sample was diluted with 35% ethanol solution, then placed in sonicator bath for 30 minutes. The sample was then dropped onto a formvar-coated copper grid, and than a 3% phosphotungstic acid solution was dropped onto the grid. The excess of staining solution was removed using filter paper. Afterwards, the sample was observed under the microscope at an accelerating voltage of 80 kV.5. Determine 0.02% rhodamine B ethosome stability via the changes of temperatures and timesThe 0.02% rhodamine B ethosome system was stored at various temperatures (4±1℃ and 25±1℃) for 90 days. And the ethosome system was kept in sealed vials. Samples were withdrew periodically and analyzed for the partical size by Zetasizer to evaluate its stability;6.The in vivo penetration study of the rhodamine B ethosomes:Sprague-Dawley strain rats were devided into four groups (n=6) by random table and were anesthetized by intraperitoneal injection of choloral hydrate during the in vivo percutaneous study. The abdominal fur was shaved off(< 2mm) with a manual shaver carefully to avoid hurting the application site and washed with normal saline. We put a flat glass cylinder that served as drug pools with an area of 0.64 cm2 onto the rat abdominal skin using cyanoacrylate adhesive. During the experiment, the application site and the preparations were kept level.200 μL of the different formulations was added into the drug pool covered with parafilm to avoid any evaporation process, and the glass cylinders were kept protected from light during the permeation study. The designated application period of three rats of every group is 1,4and 8h, while, the excess formulations on the other three rats of every group were removed after administrated for 8 hours and the interval time was 1,4and 8h after the removal of formulations. The rats were humanely sacrificed at the different designated application period. The abdominal skin that had been in contact with the test formulations was peeled off and immediately washed at least 3 times with normal saline and the subcutaneous tissue and fat were removed by a pair of scissors and scalpel. The tissue samples were cryo-sliced by a cryostat immediately and visualized under confocal laser scanning microscopy (CLSM).7. The in vivo study of the mechanism of the enhanced skin penetration by ethosomes:Sprague-Dawley strain rats were devided into three groups (n=3) by random table and were anesthetized by intraperitoneal injection of choloral hydrate during the in vivo percutaneous study. The abdominal fur was shaved off(< 2mm) with a manual shaver carefully to avoid hurting the application site and washed with normal saline. We put a flat glass cylinder that served as drug pools with an area of 0.64 cm2 onto the rat abdominal skin using cyanoacrylate adhesive. During the experiment, the application site and the preparations were kept level.200μL of the NBD-PC labeled rhodanmine B loaded ethosomes was added into the drug pool covered with parafilm to avoid any evaporation process, and the glass cylinders were kept protected from light during the permeation study. The designated application period is 1,4and 8h. The rats were humanely sacrificed at the different designated application period. The abdominal skin that had been in contact with the test formulations was peeled off and immediately washed at least 3 times with normal saline and the subcutaneous tissue and fat were removed by a pair of scissors and scalpel. Half of the tissue samples were cryo-sliced by a cryostat immediately and visualized under confocal laser scanning microscopy (CLSM), the other half of the tissue samples were ultrathin-sliced using an ultramicrotome and visualized with Transmission electron microscopy (TEM).8.To prepare cryo-sllice for CLSM observation:The Sprague-Daeley strain rat abdominal skin treated with kinds of formulations was divided to observe rhodamine B and NBD-PC distributions in the cross section using a cryostat.Each tissue sample from various time periods was mounted with a sufficient amount of OCT compound onto a metal sample holder and then transferred to a cryomicrotome and frozen at-20℃.The frozen skin was sectioned into 8 μm slices before being placed on adhesion slides, then mounted with glycerinum and covered with a cover slip. The penetration of fluorescent probes was assessed by confocal laser scanning microscopy immediately.9. To prepare ultrathin-slice for TEM observation:The Sprague-Daeley strain skin samples treated with NBD-PC labeled rhodanmine B loaded ethosomes were investigated for ultrastructural changes using TEM observation. Normal skin was used as a control.The skin samples were cut into a size of 2×4mm2 and fixed overnight (at 4℃) with 2.5% glutaraldehyde (by volume), and 1% osmium tetroxide (weight to volume) in 0.1 M PBS for 2 hours. After fixation, the samples were dehydrated in a range of ethanol solutions (35%,50%,70%,95%, and 100%) and infiltrated with Spurr’s resin. The resin-embedded samples were incubated at 70℃ for 8 hours. Ultrathin sections were cut using an ultramicrotome using a diamond knife, collected on copper grids, and intensified with uranium acetate and lead citrate. The samples were visualized using TEM.10. Confocal Laser Scanning Microscopy Study:The cross-sections of the rat skin from various time periods were assessed by confocal laser scanning microscopy (CLSM). All optical sections were recorded with the same settings. Rhodamine B was excited with the 568 nm laser line from a Kr laser, and the fluorescent emission signals are represented by a red colour, NBD-PC were excited with the 488-nm laser line from an argon laser, and the fluorescent emission signals are represented by a green colour. Images were acquired using a 30x objective lens at which the laser could scan through the tissue.The fluorescence intensity and area of each image were analyzed by Image-Pro Plus. The fluorescence intensity and area of each image was plotted against time.At the same time, the images were corrected by the auto fluorescence of the rat skin, with the black level setting.11.Data analysis and statistics:The significance of the differences between different formulations was tested using the Student t-test and analysis of variance (ANOVA). If our dates were fit for test of homogeneity of variances, we dealed them with LSD. If our dates were not fit for it, we analyzed all datas through Welch and Dunnett’s T3. The differences are considered statistically significant when p< 0.05. The statistical analysis using SPSS13.0 and the reported data are mean ±S.E.M. The plot was drawn by Origin 7.5 software.Results1.According to this orthogonal test, the optimal prescription of the 0.02% Rhodamine B ethosomes composed of 2%(w/v) egg phosphatidyl choline,35%(v/v) ethanol,0.02%(w/v)Rhodamine B. The sequence of influent the particle size of the three factors was absolute ethanoL, egg phosphatidyl choline and ultrasound time, respectively. The particle size of the optimal prescription was 113.5±2.3nm (n=3).2. The morphology of the 0.02% Rhodamine B ethosomes observed via TEM was predominant of spherical-shaped vesicles.3. The particle size and zeta potentials of the optimal prescription were determined by Zetasizer were 113.5±2.3nm,-19.5±2.3mV, respectively, with the PDI of 0.132±0.035.(n=3)4. The stability profiles of rhodamine B ehosomes evaluated for partical size stored at various temperatures after 90 days. There was no significant difference under the condition of 25±1℃ (P=0.126), but under the conditions of 4±1℃, there were significant differences (P=0.000) compared with the partical size of rhodamine B ethosomes measured 90 days ago.5.The fluorescent area and mean fluorescent intensity represent the penetration depth and amount of the different formulations after application on the rats skin, respectively. At 1h, there are bright red fluorescence in the stratum corneum and the upper hair follicles of the skin from ethosomal system, and the fluorescent area and mean fluorescent intensity significantly (P=0.000)higher than the other three formulations. While, there are no significant difference between rhodamine B liposomes and hydroethanolic solution (P=0.908, P=0.863). At 4h, the red fluorescence from the ethosomes penetrate the dermis and deeper hair follicles of the skin, and the fluorescent area and mean fluorescent intensity significantly (P=0.000) higher than the other three formulations. And there are also significant difference between rhodamine B liposomes and hydroethanolic solution(P=0.002, P=0.001). At 8h, the red fluorescence of the ethosomes in the dermis of the skin brighter than at 4h, and the fluorescent area and mean fluorescent intensity significantly (P=0.000)higher than the other three formulations. When the excess formulations removed from the application location after 8 hours, the red fluorescence from all formulations gradually decreased with the increases of the time. One hour after removed the drugs, the red fluorescence of the ethosomes in the dermis of the skin is very weak, but the fluorescent area significantly (P=0.000, P=0.001, P=0.000) higher than the other three formulations, as well as the mean fluorescent intensity (P=0.000). Four hours later, the red fluorescence of the ethosomes in the dermis disappeared, and within the deeper hair follicles also very weak, while, the fluorescent area significantly (P=0.000, P=0.002, P=0.000) higher than the other three formulations, as well as the mean fluorescent intensity (P=0.000). Eight hours later, there are only weak red fluorescence seen in the stratum corneum of the ethosomes, and the fluorescent area significantly (P=0.000) higher than the other three formulations, as well as the mean fluorescent intensity (P=0.000, P=0.002, P=0.000).6. We use CLSM to observe the rhodamine B and NBD-PC distribution in the rat abdominal skin after the application of NBD-PC labeled Rhodamine B loaded ethosomes in vivo. At 1 hour, we can observe bright green fluorescence in the upper hair follicles, hair shaft center and weak green fluorescence in the stratum corneum, while, the red fluorescence are weak in the stratum corneum and hair follicles and no red fluorescence in the center of the hair shaft Four hours later, there are bright linear green fluoresceence in the stratum corneum and deeper hair follicles, as well as in the center of the hair shaft.In addition to distributing in the stratum corneum and hair follicles of the red fluorescence, it can also be seen in the viable epidermis with weak intensity. Eight hours later, the green fluorescence still in the stratum corneum and hair follicles, while, the red fluorescence can penetrate into viable epidermis and dermis with strong intensity and diffuse around the hair follicles with weak intensity.7. The ultrastructural changes of the rat skin after treated with NBD-PC labeled Rhodamine B loaded ethosomes observed via TEM. From the TEM images of the normal rat skin, we can observe that corneodesmosomes connecting flattened cornified cells in the stratum corneum to one another, as well as to keratinocyte cells in the stratum granulosum, hence, the whole structure of the stratum corneum is dense. After the rats skin treated with NBD-PC labeled Rhodamine B loaded ethosomes for 4 hours. Since the hydration effect of the double-distilled water result in corneocyte swelling, while, the upper stratum corneum had broader-shaped corneucytes than in the lower stratum cormeum, the intercellular spaces were wider those of the normal skin, because the corneodesmosomes that connected the corneocytes between each stratum corneum layer were degraded. Moreover, there are many disintegrated particles and intact particles in the intercellular spaces of the stratum cormeum, but not in the stratum granulosum. In viable epidermis, desmosomes and hemidesmosomes, which connect stratum basale and papillary dermis, were normal.Conclusions1.0.02% Rhodamine B ethosoems have a spherical-shaped structure with narrow size distribution, small size diameter, small polydispersity index, negative zeta potential, and have a good stability when stored at 25±1℃.2. The study of in vivo transdermal permeation indicates that the permeation depth and the amount of 0.02% Rhodamine B ethosome was much better than 0.02% Rhodamine B liposome, hydroethanolic solution and water solution with a shorter lag time. Moreover,0.02% Rhodamine B ethosome has a longer deposition time than 0.02% Rhodamine B liposome, hydroethanolic solution and water solution that have a sustained-release effect when percutaneous penetration3. Part of the ethosomes rapidly distribute to the follicle area and penetrate into the deep of the hair follicle and around the hair follicles when transdermal drug delivery, and the rest of ethosomes penetrate into the stratum corneum through intercellular and transcellular pathways.4. Ethosomes can act as a complete carrier penetrate into the stratum corneum and hair follicles during percutaneous administration, and then disintegration after interact with the lipids in the stratum corneum and hair follicles, phospholipids after disintegration staying in the stratum corneum and hair follicles area act as Penetration enhancer, rather than penetrate into the viable epidermis and dermis, thereafter, the drug loaded in the ethosomes penetrate into the viable epidermis and dermis alone. Meanwhile ethosomes can form a lipid membrane on the surface of the stratum corneum that has a role in the packet. |
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