http://informahealthcare.com/lpr ISSN: 0898-2104 (print), 1532-2394 (electronic) J Liposome Res, Early Online: 1–9 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2014.999686
RESEARCH ARTICLE
Ethosomes for skin delivery of ropivacaine: preparation, characterization and ex vivo penetration properties Yingjie Zhai1, Rui Xu1, Yi Wang2, Jiyong Liu3, Zimin Wang2, and Guangxi Zhai1
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1
Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, China, 2Department of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai, China, and 3Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai, China Abstract
Keywords
Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, NaropinÕ Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86 ± 2.40 nm and drug loading of 8.27 ± 0.37%, EE of 68.92 ± 0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0 ± 11.5 mg cm2 at 12 h and 178.8 ± 7.1 mg cm2 at 0.5 h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration.
Ethosomes, ropivacaine, transdermal delivery, vesicle–skin interaction
Introduction Skin, the external barrier between body and the exterior environment, is conscientious in protecting the body from penetration of foreign matter into the body and drain of endogenous material. Delivering drug target to the skin itself or into circulation through skin was called topical/transdermal delivery route (Choi & Maibach, 2005). Due to the peculiar administration site and the absorptive pathway, the topical/ transdermal delivery route has several virtues (Zhai & Zhai, 2014; Zhai et al., 2014) compared to other administration pathways such as avoiding the first-pass effect, circumventing the enterohepatic circulation after oral administration, improving drug bioavailability and patient compliance as well as reducing side effects (Barry, 2001). Despite the aforementioned advantages of topical/transdermal delivery,
Address for correspondence: Guangxi Zhai, PhD, Professor, Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel: +86 53188382015. E-mail: [email protected]. Zimin Wang, PhD, Depatement of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Tel: +86 21-31161692. E-mail: [email protected]
History Received 24 July 2014 Revised 25 October 2014 Accepted 15 December 2014 Published online 27 January 2015
the major obstacle induced by the stratum corneum hinders the application of this delivery route to a large extent. The stratum corneum consists of 10–15 layers of dead corneocytes, is the upper barrier of the skin. These dead corneocytes are embedded within intercellular lipid matrix composed of ceramides, free fatty acids and cholesterol and jointed tightly by covalently bound lipids and cross-linked proteins and desmosomes (Van Smeden et al., 2014). Aims to overcome the permeation barrier and improve drug permeability, several efforts have been devoted (Subedi et al., 2010). One of the promising methods is the application of vesicular systems such as liposomes. In the early 1980s, Mezei & Gulasekharam (1980, 1982) first described the potential use of liposome for enhanced drug delivery to the skin. Since then, liposomes have been widely investigated for skin delivery due to their excellent biocompatibility (Akhtar, 2014; Touitou et al., 1994; Va´zquezGonza´lez et al., 2014). However, conventional liposome was found awkward in delivering drug into circulation through skin since they were mainly confined to the upper layer of the stratum corneum (Cereda et al., 2013; Duangjit et al., 2014). Novel liquid-state ethosomes were proposed to improve the penetration efficiency by the addition of ethanol within the liposome in the early 1990s. Ethosomes are non-invasive
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systems containing mainly of phospholipid (phosphatidylcholine; PC), ethanol at proper ratio range (20–45%) and water (Touitou et al., 2000). In comparison to rigid liposome, the phospholipids in ethosomes were packed loosely due to the presence of ethanol. Ethosomes were malleable, deformable vesicles which could be tailored for improved delivery of drugs target to or permeate through the skin more efficaciously. Particularly, ethanol could interact with the polar head group region of lipid molecules, leading to a reduction in the melting point of lipid within stratum corneum, thereby increase the fluidity and loosen the tight junction of stratum corneum (Mbah et al., 2014). On the other hand, ethosomes could squeeze through pores within stratum corneum by merits of high and self-optimizing deformability (Cevc et al., 1998). Thus, sizes of ethosomes up to 200–300 nm could penetrate in intact skin effortlessly. Up to now, numerous studies had demonstrated the superior ability of ethosome in enhancing penetration of both hydrophobic and hydrophilic molecules in vitro and in vivo. Jain et al (2007) evaluated the permeability of ethosomal formulation using lamivudine as model drug. The results indicated that the optimized ethosomal formulation showed 25 times higher transdermal flux (68.4 ± 3.5 mg/cm2/h) across the rat skin compared with that of lamivudine solution (2.8 ± 0.2 mg/cm2/h). Zhang et al. (2014) prepared psoralen-loaded ethosomes and evaluated the permeability in skin delivery. The results of ex vivo permeation study showed that the permeability of psoralen-loaded ethosomes was superior to that of rigid liposomes with the transdermal flux (38.89 ± 0.32 mg/cm2/h) and skin deposition (3.87 ± 1.74 mg/cm2), respectively, 3.50 and 2.15 times that achieved by rigid liposomes. Taken all together, ethosomes were proved competent as an efficient vesicle carrier for skin delivery. Ropivacaine, belongs to the family of the n-alkyl substituted pipecoloxylidide (McClure, 1996), is a novel long-acting local anesthetic. It was introduced into clinic in 1996 and proved less toxic on the cardiovascular system and central nervous system. Not only that, ropivacaine also showed considerable merit of a great separation of motor and sensory block, which was specifically beneficial in accelerating postoperative recovery (Chaykovska et al., 2014). Although ropivacaine is currently applied widely in clinic, a major drawback from the formulation point of view is that the application form NaropinÕ , which should be administrated intravenously, can cause patient non-convenience. Besides, adverse reactions like arrhythmia may occur as a consequence of over quick rate of intravascular injection (Leone et al., 2008). Therefore, developing convenient formulation intended for alerting the traditional intravascular injection form will make great sense. In this study, the potential of ethosome in delivering the hydrophobic ropivcaine through skin was evaluated using dorsal skin of mice as model. The ropivacaine-loaded ethosome was prepared and evaluated in terms of morphology, size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. Moreover, the ex vivo permeation study of ropivacaine-loaded ethosomes was performed to investigate their potential in delivering RPV transdermally. In addition, the vesicle–skin interaction was verified by histopathology study.
J Liposome Res, Early Online: 1–9
Materials and methods Materials Ropivacaine (RPV) was provided by Jinan Dexinjia Pharmaceutical Co. Ltd. (Jinan, China). Lipoid (soybean lecithin at 98% of phosphatidylcholine) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was provided by Beijing chemical reagent company (Beijing, China). Ethanol was obtained from Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). All other chemicals were of analytical purity and commercially available. Preparation of ropivacaine-loaded ethosomes The RPV-loaded ethosomes were prepared with a thin-film dispersion method (Figure 1) (Maitani et al., 1990). Briefly, phospholipon, cholesterol and RPV at certain amount were dissolved by 3 mL of ethanol within a 25 mL of roundbottomed flask. The organic phase was then removed by rotary evaporation under reduced pressure for 20 min to yield a drug-lipid film on the wall inside the flask. The flask was placed in a vacuum dryer for 12 h so as to make the residual ethanol volatilize thoroughly. The resulting drug–lipid film was then rehydrated with distilled water containing appropriate ethanol. Eventually, a homogeneous RPV-ethosomes dispersion was obtained from the supernatant after centrifugation at 4000 rpm for 10 min. In order to facilitate the differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study, the powder of RPV-ethosome was prepared by lyophilization. The formulation was firstly frozen at 20 C, and then continued to pre-freezed at 80 C for 24 h using an ultracold freezer (MDF-382E, SANYO, Japan). Lyophilization was carried out using a lyophilizer (LGJ0.5, Beijing Sihuan Instrument Company, China) at pressure of 0.1 mbar and temperature of 60 C for 48 h by adopting mannitol (5.0%, w/v) as cryoprotectant (Musmade et al., 2014). The obtained powders were collected and kept in desiccators for further analysis.
Figure 1. Preparation process of ethosomes by a thin-film dispersion method.
Ethosomes for skin delivery of ropivacaine
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DOI: 10.3109/08982104.2014.999686
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Encapsulation efficiency and drug loading
DSC analysis
In order to determine the encapsulation efficiency (EE%) and drug loading (DL%), the amount of entrapped RPV in RPVethosomes should be aforehand quantified. A Sephadex G-50 mini column centrifugation technique was adapted to separate the RPV-ethosomes from free RPV. Briefly, 0.5 mL of RPVethosomes formulation was placed onto a Sephadex G-50 mini column. The elution was conducted by adding 0.5 mL of distilled water and then centrifuging for 1 min at 500 rpm. The elution process was repeated for six times. Finally, the eluate containing RPV-ethosomes were collected and mixed with methanol to break down the structure of ethosomes. Thereafter, the amount of entrapped RPV was detected by HPLC. Aims to quantify the total amount of RPV in the formulation, another 0.5 mL of RPV-ethosome formulation was diluted directly by methanol and vortexed for 3 min to break down the structure of ethosomes and dissolve the drug. The EE% and DL% of RPVethosomes were calculated as follows.
The DSC analysis was conducted to study the thermal behavior of RPV-loaded ethosomes. Blank porcelain crucible was used as the standard reference material to calibrate the energy scale of the instrument (1/1600HT, METTLER, Switzerland). Approximately 5 mg of samples (RPV, mannitol, cholesterol, physical mixture and RPV-ethosome powder) was accurately weighed and placed into porcelain crucible. The analysis was performed from 30 to 400 C at a scanning rate of 10 C/min, under a constant nitrogen stream.
DL% ¼
Wloaded 100% Wtotal
EE% ¼
Wloaded 100% Wadded
In the formulas, Wloaded represents the amount of RPV entrapped in ethosomes; Wadded represents the amount of RPV in the formulation system. Wtotal stands for the total weight of all lipids in the formulation system. Optimization of the formulation Formulation optimization is very important for preparation because composition and preparation procedure can influence the physicochemical property of ethosomes. For a long time, researchers have been trying to use the techniques of experimental design to improve experimental works. In this study, uniform design (U13) was employed to optimize the formulation with EE% and DL% as the indexes. Observation of ethosome by transmission electron microscopy The micro-morphology of ethosomes was visualized using a transmission electron microscopy (TEM) with an accelerating voltage of 100 kV. Briefly, ethosome dispersion was spread on a microscopic carbon-coated grid followed by negatively stained with a 2% aqueous solution of phosphotungstic acid. The excess solution was removed by blotting. After drying, the specimen was viewed under the microscope at appropriate enlargement. Size distribution and zeta potential The size distribution and zeta potential of ethosomes were determined by dynamic light scattering (DLS) using a computerized inspection system (ZETASIZER, Nano-ZS, Malvern, UK). Prior to measurements, all samples were diluted to yield a suitable scattering intensity, if needed. Measurements were operated at a fixed angle of 90 . For each sample, the measurement was carried out in triplicate at 25 C and the average value was recorded.
XRD study The wide-angle X-ray scattering (WAXS) of different samples (RPV, mannitol, cholesterol, physical mixture and RPV-ethosome powder) was investigated by an X-ray diffractometer (D8, Advance, Bruker, Germany) with a Cu K radiation source fixed at 40 kV and 100 mA. The wavelength ˚ (0.154 nm). The XRD was perwas set at 1.5405 A formed from the initial 10 to the final 70 with a scanning rate at 1 /s. Ex vivo permeation study Full thickness dorsal skin excised from Kunming mice (male, 20 ± 2 g) was employed to evaluate the potential of ethosomes delivering RPV transdermally. The Kunming mice were supplied by Laboratory Animal Center of Shandong University and the experimental protocol was approved by the Ethics Committee of Shandong University. Prior to the study, the hair on the dorsal skin of animals was carefully trimmed short and removed by Na2S solution (8%, w/v) without scratching the skin surface. Then the mice were sacrificed and the dorsal skin was separated from the underlying connective tissue with a scalpel. The ex vivo skin permeation of RPV from RPV-ethosome formulations was studied using Franz diffusion cells with an effective permeation area of 3.14 cm2. Excised skin was mounted between the donor and receptor compartment with the epidermal side facing upward. Physiological saline (20 mL) containing 1% Tween 80 which was maintained at 32 ± 0.5 C using a water bath was used as receptor medium to meet the sink condition. Ethosomal formulation (0.7 mL) was applied to the epidermal surface of skin and covered with parafilm to avoid evaporation. At 0.5, 1, 2, 3, 6, 9 and 12 h time intervals, 0.5 mL of samples was taken from the receiver. Immediately following, 0.5 mL of fresh receptor medium was replaced to keep a stable receptor volume. The RPV concentration in the samples was analyzed by HPLC and the accumulative penetration amount (Qn) was calculated as follows:
Qn ¼
Cn V0 þ
Pn1 i¼1
Ci Vi
A
where Cn stands for the RPV concentration of the receiver medium at each sampling time, Ci for the drug concentration of the sample, A for the effective diffusion area (3.14 cm2), V0 and Vi stand for the volumes of the receiver solution (20 mL) and the sample (0.5 mL), respectively.
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Vesicle–skin interaction study by histopathology Histopathology study of skin was conducted to visualize the topical effect of RPV-ethosomes on skin (Zhai & Zhai, 2014; Zhai et al., 2014). RPV-ethosomes and RPV propylene glycol solution were applied to the excised rat skins for 12 h, respectively. Then, the treated skin pieces were collected within 10% formalin for tissue fixation for 24 h. After then, the specimen was washed and embedded in paraffin wax and vertically cut into 3–4 mm in thickness. Finally, the paraffin tissue sections were subjected to hematoxylin/eosin staining and examined using microscope. Histopathology study of untreated skin was also carried out as control.
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HPLC assays for ropivacaine In this study, RPV was quantified by reverse-phase HPLC using an Agilent 1200 system equipped with a UV monitor (Agilent G1314-60100). Separations were carried out using a BDS C18 column (4.6 250 mm, 5 mm). RPV was detected at 225 nm with a mobile phase containing a mixture of acetonitrile: phosphate buffer (60:40) at a flow-rate of 1.0 mL/min. The analysis method was evaluated through specificity, linearity, precision and accuracy. For the samples from ex vivo experiments, equal volume of acetonitrile was added to precipitate protein. Then the mixture was vortexed and centrifuged at 12 000 rpm for 10 min. The supernatant was collected and analyzed by HPLC. Statistical analysis The statistical significance of differences was determined using One-way analysis of variance (ANOVA). A value of p50.05 was considered to be significant.
Results and discussion Formulation optimization Uniform design was initially proposed by Fang (1980). It is capable of producing samples with high representativeness and accommodates the largest possible number of levels for each factor in the studied experimental domain (Liang et al., 2001). In this study, the uniform design (U13) was adopted to optimize the formulation variables with EE% and DL% as the indexes. The factors and results were shown in Table 1. The results showed that the EE% changed from 53.95% to 85.50% with the variation of independent variables, while the DL% sharply varied in a greater range from 1.70% to 8.08%. It indicated that the effect of the independent variables on DL% was greater than that on EE%. Therefore, the DL% was selected as a primary response indicator for the formulation optimization. The data was conducted by SPSS version 17.0 software (SPSS Inc., Chicago, IL; Wen et al., 2005) to get a regression equation. Y ¼ 0:042X1 þ 0:026X2 þ 0:123X3 þ 0:402X4 þ 0:002X1 X3 0:016X3 X4 0:049 In the formula, Y is the dependent variable (DL%), X1 (the amount of lecithin), X2 (the amount of cholesterol), X3 (the amount of RPV) and X4 (volume fraction of ethanol) are the independent variables.
J Liposome Res, Early Online: 1–9
The regression equation provided an insight into the effect of the different independent variables on Y (p50.05). The larger the optimized value (Y) was, the better the formulation was. In overall consideration of better stability and higher DL%, an optimized combination was selected as follows, the amount of RPV, lecithin, cholesterol was 14, 90, 10, respectively, and the volume percentage of ethanol was 40%. The results of the verification test showed that the DL% of the optimized RPV-ethosomes was 8.27 ± 0.37%, verifying a certain predictability of the regression equation as compared to the predicted value of 7.80%. Besides, the EE% of the optimized RPV-ethosomes was 68.92 ± 0.29%. The improvement in DL% may be related to the presence of ethanol in ethosomes since it may allow for better solubility of RPV, improving the distribution of RPV within the inner aqueous phase and the lipid bilayers of the vesicle (Li et al., 2012). Physicochemical characterization of RPV-ethosomes The RPV-loaded ethosomal delivery system was characterized by size distribution and zeta potential, visualization under TEM. As shown in Figure 2(a), the size distribution of RPV-loaded ethosomes, as measured by dynamic light scattering (DLS), showed narrow unimodal peak with polydispersity index (PDI) of 0.201, indicating that the prepared ethosome was relatively homogenous in size distribution. Size was critical to topical drug delivery systems, since it was reported that vesicles smaller than 300 nm were able to deliver the drug into deeper layers of the skin (Verma et al., 2003). The average diameter of ethosomes obtained in our study was 73.86 ± 2.40 nm, indicating that the formulation had potential for delivering drug through the skin. The charge of the vesicular dispersion has an influence on the stability of the formulation. The higher zeta potential was, the better the long-term stability was, as electrostatic repulsion between vesicles could avoid aggregation. The zeta potential of the obtained RPV-loaded ethosomal delivery system was 12.9 mV (Figure 2b), which could forecast a good stability for the formulation. The electronegativity may be related to the presence of 40% ethanol since it was reported that the addition of 30% ethanol induced a transition in the charge of the vesicles from positive (+4.6 ± 0.2 mV) to negative (4.3 ± 0.2 mV; Touitou et al., 2000). Concretely,
Table 1. The factors and results of the U13 Uniform Design. Factors
Results
Groups
X1 (mg)
X2 (mg)
X3 (mg)
X4 (%)
DL%
EE%
1 2 3 4 5 6 7 8 9 10 11 12
100 100 100 200 200 200 300 300 300 400 400 400
5 10 10 15 20 20 5 5 10 15 15 20
15 25 15 25 15 25 10 20 10 20 10 20
35 25 40 30 25 35 30 40 35 25 40 30
5.18 ± 0.25 4.00 ± 0.11 8.08 ± 0.22 3.22 ± 0.09 3.22 ± 0.17 4.67 ± 0.33 2.03 ± 0.64 4.02 ± 0.10 1.90 ± 0.06 3.26 ± 0.13 1.70 ± 0.07 2.96 ± 0.23
53.95 ± 1.02 61.25 ± 0.81 59.4 ± 0.34 54.05 ± 0.71 69.5 ± 1.32 67.55 ± 0.33 64.65 ± 0.54 70.45 ± 0.79 62.05 ± 1.41 85.50 ± 1.47 80.20 ± 0.91 77.10 ± 1.37
Ethosomes for skin delivery of ropivacaine
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DOI: 10.3109/08982104.2014.999686
the change of zeta potential was attributed to the net negative charge and facilitation of ionization conferred by ethanol on the vesicle surface (Mbah et al., 2014). As illustrated in Figure 1, ethanol molecules distributed within the inner aqueous phase and the lipid bilayers of the vesicle. The terminal hydroxyls of ethanol molecules distributed within the lipid bilayers of the vesicle may stretch to the outer medium phase due to the hydrophilic group. Hydrogen bond may occur among the hydroxyl groups in the ethanol molecules or between ethanol molecules and water molecules, which would favor a high net negative charge and facilitation of ionization. The prepared RPV-ethosome dispersion was bluish and transparent in appearance (Figure 3a), whereas the system of crude RPV dispersed in water at the same drug concentration was suspension with some precipitation of drug (Figure 3b). When examined under TEM, the RPV-ethosomes appeared as unilamellar vesicles (Meng et al., 2014) with a predominantly spherical shape without aggregation among vesicles (Figure 3c). The size of the vesicles obtained from the TEM decreased little compared to that obtained by DLS (73.86 ± 2.40 nm). There may be two reasons. For one thing, it is difficult to stain the outer hydrophilic surface of the ethosome (Mi et al., 2014), for another, the negatively stained RPV-ethosomes were allowed to dry for contrast enhancement prior to the TEM measures, the dehydration process may lead to a shrink to the hydrophilic surface. The results of physicochemical characterization showed the RPVethosomes were prepared successfully and an improvement of the dispersibility of RPV was achieved after formulated to RPV-ethosomes.
Figure 2. The size (a) and Zeta potential (b) of the RPV-loaded ethosomes.
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DSC analysis In order to inspect the changes of crystalline structure compared with the raw materials (Luan et al., 2013), the DSC analysis was conducted. Figure 4 displays the DSC thermograms of the raw materials, their physical mixture (RPV, mannitol, cholesterol and lecithin) and RPV-ethosome freezedried powder. It was evident that each raw material exhibited particular endothermic peaks. Particularly, the endothermic peak of pure RPV was at 148.67 C (a), and that of mannitol was at 168.04 C (b). The characteristic peaks of RPV were present in the DSC curves of physical mixture (d), just with a slight shift (141.69 C), meaning that the initial crystalline form of the drug was retained. Conversely, no endothermic peak corresponding to the fusion of RPV was observed in the curve of RPV-ethosome freeze-dried powder (e). This comparison between (d) and (e) suggested that the crystalline form of RPV was unchanged when RPV was physically mixed with the excipients, but transformed into non-crystalline state when encapsulated into ethosomes. XRD study The XRD study was employed to analyze whether the crystalline structure of RPV in ethosome was changed or not and the results were shown in Figure 5. It was evident from
Figure 3. Photographic images of RPV-loaded ethosomes (a) and RPV suspension (b). (c) represents the morphology of RPV-loaded ethosomes obtained from the optimized formulation.
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Figure 4. DSC analysis thermograms: (a) ropivacaine; (b) mannitol; (c) cholesterol; (d) the physical mixture of RPV-ethosomes; (e) RPVethosomes freeze-dried powder.
J Liposome Res, Early Online: 1–9
Figure 6. Ex vivo permeation profile of RPV-ethosomes (g) and RPV propylene glycol () by using excised mice skin as model (mean ± SD; n ¼ 5).
Figure 7. Ex vivo permeation profile of RPV-ethosomes and RPV propylene glycol at 0.5, 6 and 12 h. *p50.05. Figure 5. X-ray diffraction spectra: (a) ropivacaine; (b) cholesterol; (c) mannitol; (d) the physical mixture of RPV-ethosome; (e) RPVethosome freeze-dried powder.
Figure 5 that RPV showed a strong diffraction peak at 10.68 (a), while the cholesterol showed a characteristic diffraction peak at 14.21 (b). The RPV diffraction peak was also present in the curve of the physical mixture (d), which proved that the crystal form of RPV was the same with that of the pure RPV powder. In contrast, the characteristic diffraction peak of RPV disappeared in the curve of RPV-ethosome freeze-dried powder (e), indicating a disordered crystalline state of RPV in ethosome. These results proved that RPV had been successfully incorporated into the lipid matrix of ethosomes in amorphous or disordered state. Ex vivo permeation study The ex vivo permeation study of RPV from ethosomes or propylene glycol solution on the full-thickness mice skin was
carried out by using Franz diffusion cells with an effective permeation area of 3.14 cm2. The accumulative penetration amount (Qn) of RPV from the experimental ethosomal formulation or propylene glycol solution was shown in Figure 6. It was evident that ethosome-mediated delivery significantly increased the transdermal flux of RPV with a value of 360.5 ± 21.4 mg cm2 at 12 h. Nevertheless, the transdermal flux of RPV from propylene glycol solution as control was only 141.3 ± 13.5 mg cm2 at 12 h. This tendency indicated the potential of ethosomes in substantially delivering RPV across skin was considerable, relative to the control propylene glycol solution. Particularly, aims to summarize the permeation manner of RPV ethosomes in a more intuitive way, the Qn of RPV from ethosomal formulation and propylene glycol solution at 0.5, 6 and 12 h was magnified purposely and displayed in Figure 7.
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DOI: 10.3109/08982104.2014.999686
Ethosomes for skin delivery of ropivacaine
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Figure 8. Histopathology photomicrographs of untreated skin (a), skin treated with RPV propylene glycol solution (b) and RPV-ethosomes (c).
As shown, the Qn of RPV from ethosomal formulation was relatively high (178.8 ± 7.1 mg cm2) even at 0.5 h, almost 19-folds that from propylene glycol group (p50.05). It suggested that ethosomes could interact with the skin rapidly and penetrate in a high-efficient, quick way. Primarily, the colloidal properties of ethosome should be responsible for the improved accumulative penetration amount (Qn) and the high-efficient penetration rate. The above reported effects could be ascribed to the favorable flexibility and the penetration enhancement function of the ethosomes. The presence of ethanol could increase flexibility and improve deformability of the vesicles, which might allow ethosome to squeeze through skin channels in a self-adapting manner and thus carry RPV across the intact skin (Sinico & Fadda, 2009). This proposed that ethosome could facilitate drug transport by a fast partitioning in the stratum corneum, thereby carrying the encapsulated RPV into stratum corneum. Except for the drug carrier function, ethosome might play a role as penetration enhancers. Ethanol as good solvent might increase the solubility of RPV in the vehicle and accordingly, the permeation of ethanol could improve RPV partitioning into the membrane by altering the solubility properties of the tissue (Megrab et al., 1995). Besides, ethanol was able to extract some of the lipid fraction from the stratum corneum, disturb the organization of the lipid bilayer of stratum corneum, creating the improved RPV flux through the skin
if applied for prolonged times (El Maghraby et al., 2001). Not only that, some components of ethosomes, particularly lecithin, could fuse or mix with skin lipids by disturbing the lamellar arrangement of the lipids within stratum corneum, or even achieve lipid exchange (Sinico et al., 2005). Taken together, either was the main or the only mechanism of action for the improved skin permeability of RPV from ethosome. A possible synergistic effect conducted by the two sides seems to be the contributor. Vesicle–skin interaction study In order to investigate whether ethosomes affected the structure of stratum corneum or not, tissue sections of the skin were stained with hematoxylin/eosin. Histopathological images were shown in Figure 8. In control group (Figure 8a), a compact stratum corneum with tightly conjugated adjacent corneocyte layer could be observed visually. Application of RPV propylene glycol solution changed the tight junction slightly (Figure 8b). However, as shown in Figure 8(c), it was evident that the treatment of RPV-ethosomes increased the gap among adjacent cells. Besides, the stratum corneum appeared swollen, with total thickness increased in Figure 8(c). The results of the histopathology study of skin indicated that ethosomal formulation could significantly affect the upper histology of skin.
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There may be two mechanisms that underlie the topical effect of RPV-loaded ethosomes on skin. First, a colloidal film formed by RPV-ethosomes covered on the skin surface might significantly reduce or prevent skin dehydration and provide better wetting function to the skin and loosen the barrier properties (Lv et al., 2009). As for the ethosome itself, ethanol might penetrate into the stratum corneum and loosen the ‘‘bricks-mortar’’ structure of stratum corneum. Besides, some components like lecithin could fuse the skin lipids by disturbing their lamellar arrangement (Sinico et al., 2005). The synergistic effects of combination of phospholipids and ethanol were suggested to be responsible for the vesicle–skin interaction (Touitou et al., 2001).
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Conclusion The application of ethosome as a vesicular carrier could overcome the limitation of low permeability of stratum corneum barrier. In this study, RPV-loaded ethosomes were prepared and evaluated as a vesicular carrier for skin delivery. The optimized RPV-loaded ethosomes showed unilamellar vesicle structure with the mean particle size of 73.86 ± 2.40 nm and DL% of 8.27 ± 0.37%, EE% of 68.92 ± 0.29%, respectively. The DSC analysis and XRD study revealed the amorphous state of RPV in ethosomes, indicating RPV was successfully incorporated into ethosomes. The results of the ex vivo permeation study indicated that the ethosome-mediated delivery increased the transdermal flux of RPV in a highefficient, rapid way compared with propylene glycol solution as control. The histopathology study proved that the interaction between ethosome and skin could loosen the ‘‘bricks-mortar’’ structure of stratum corneum barrier. In summary, our study was the first report in developing RPV-loaded ethosome for skin delivery and the results demonstrated that ethosomal delivery systems were superior in delivering RPV through skin.
Declaration of interest The authors declare no conflicts of interest. This work is supported by grants from Shanghai Municipality Science and Technology commission (12nm0500700, 11DZ1971400) and the National Nature Science Foundation (No.81171766, No.81373896).
References Akhtar N. (2014). Vesicles: a recently developed novel carrier for enhanced topical drug delivery. Curr Drug Deliv 11:87–97. Barry BW. (2001). Novel mechanisms and devices to enable successful transdermal drug delivery. Eur J Pharm Sci 14:101–14. Cereda CMS, Franz-Montan M, da Silva CMG. (2013). Transdermal delivery of butamben using elastic and conventional liposomes. J Liposome Res 23:228–34. Cevc G, Gebauer D, Stieber J. (1998). Ultraflexible vesicles, Transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin. Biochim Biophys Acta 1368:201–15. Chaykovska L, Blohme´ L, Mayer D. (2014). Paraincisional subcutaneous infusion of ropivacaine after open abdominal vascular surgery shows significant advantages. Ann Vasc Surg 28:837–44. Choi MJ, Maibach HI. (2005). Elastic vesicles as topical/transdermal drug delivery systems. Int J Cosmet Sci 27:211–21. Duangjit S, Obata Y, Sano H. (2014). Comparative study of novel ultradeformable liposomes: menthosomes, transfersomes and
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liposomes for enhancing skin permeation of meloxicam. Biol Pharm Bull 37:239–47. El Maghraby GMM, Williams AC, Barry BW. (2001). Skin delivery of 5-fluorouracil from ultradeformable and standard liposomes in-vitro. J Pharm Pharmacol 53:1069–77. Fang KT. (1980). The uniform design: application of numbertheoretic methods in experimental design. Acta Math Appl Sin 3: 363–72. Jain S, Tiwary AK, Sapra B. (2007). Formulation and evaluation of ethosomes for transdermal delivery of lamivudine. AAPS Pharm Sci Tech 8:249–57. Leone S, Di Cianni S, Casati A. (2008). Pharmacology, toxicology, and clinical use of new long acting local anesthetics, ropivacaine and levobupivacaine. Acta Biomed 79:92–105. Liang Y, Fang K, Xu Q. (2001). Uniform design and its applications in chemistry and chemical engineering. Chemomet Intel Lab Syst 58: 43–57. Li G, Fan Y, Fan C. (2012). Tacrolimus-loaded ethosomes: physicochemical characterization and in vivo evaluation. Eur J Pharm Biopharm 82:49–57. Luan JJ, Zhang DR, Hao LL. (2013). Design and characterization of Amoitone B-loaded nanostructured lipid carriers for controlled drug release. Drug Deliv 20:324–30. Lv QZ, Yu AH, Xi YW. (2009). Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery. Int J Pharm 372:191–8. Maitani Y, Nakagaki M, Nagai T. (1990). Surface potential of liposomes with entrapped insulin. Int J Pharm 64:89–98. Mbah CC, Builders PF, Attama AA. (2014). Nanovesicular carriers as alternative drug delivery systems: ethosomes in focus. Expert Opin Drug Deliv 11:45–59. McClure JH. (1996). Ropivacaine. Br J Anaesth 76:300–7. Megrab NA, Williams AC, Barry BW. (1995). Oestradiol permeation across human skin, silastic and snake skin membranes: the effects of ethanol/water cosolvent systems. Int J Pharm 116: 101–12. Meng S, Chen Z, Yang L, et al. (2014). High-efficient nano-carrier gel systems for testosterone propionate skin delivery. Pharm Dev Technol [Epub ahead of print]. Mezei M, Gulusekharam V. (1980). Liposomes, a selective drug delivery system for the topical route of administration. Life Sci 26:1473–7. Mezei M, Gulusekharam V. (1982). Liposomes, a selective drug delivery system for the topical route of administration: gel dosage form. J Pharm Pharmacol 34:473–4. Mi P, Kokuryo D, Cabral H. (2014). Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating Gd-DTPA for contrast enhanced MRI diagnosis of solid tumors. J Control Release 174:63–71. Musmade KP, Trilok M, Dengale SJ. (2014). Development and validation of liquid chromatographic method for estimation of naringin in nanoformulation. J Pharm 2014:1–8. Sinico C, Manconi M, Peppi M. (2005). Liposomes as carriers for dermal delivery of tretinoin: in vitro evaluation of drug permeation and vesicle–skin interaction. J Control Release 103:123–36. Sinico C, Fadda AM. (2009). Vesicular carriers for dermal drug delivery. Expert Opin Drug Deliv 6:813–25. Subedi RK, Oh SY, Chun MK. (2010). Recent advances in transdermal drug delivery. Arch Pharm Res 33:339–51. Touitou E, Junginger HE, Weiner ND. (1994). Liposomes as carriers for topical and transdermal delivery. J Pharm Sci 83:1189–203. Touitou E, Dayan N, Bergelson L, et al. (2000). Ethosomes-novel vesicular carriers: characterization and delivery properties. J Control Release 65:403–18. Touitou E, Godin B, Dayan N. (2001). Intracellular delivery mediated by an ethosomal carrier. Biomaterials 22:3053–9. Van Smeden J, Janssens M, Gooris GS. (2014). The important role of stratum corneum lipids for the cutaneous barrier function. BBA Mol Cell Biol 1841:295–313. Va´zquez-Gonza´lez ML, Bernad R, Calpena AC. (2014). Improving ex vivo skin permeation of non-steroidal anti-inflammatory drugs: enhancing extemporaneous transformation of liposomes into planar lipid bilayers. Int J Pharm 461:427–36. Verma DD, Verma S, Blume G. (2003). Particle size of liposomes influences dermal delivery of substances into skin. Int J Pharm 258: 141–51.
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Wen S, Zhang T, Tan T. (2005). Optimization of the amino acid composition in glutathione fermentation. Process Biochem 40:3474–9. Zhai YJ, Yang XY, Zhao LL. (2014). Lipid nanocapsules for transdermal delivery of ropivacaine: in vitro and in vivo evaluation. Int J Pharm 471:103–11.
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Zhai YJ, Zhai GX. (2014). Advances in lipid-based colloid systems as drug carrier for topic delivery. J Control Release 193:90–9. Zhang YT, Shen LN, Wu ZH. (2014). Comparison of ethosomes and liposomes for skin delivery of psoralen for psoriasis therapy. Int J Pharm 471:449–52.
RESEARCH ARTICLE
Ethosomes for skin delivery of ropivacaine: preparation, characterization and ex vivo penetration properties Yingjie Zhai1, Rui Xu1, Yi Wang2, Jiyong Liu3, Zimin Wang2, and Guangxi Zhai1
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1
Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, China, 2Department of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai, China, and 3Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai, China Abstract
Keywords
Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, NaropinÕ Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86 ± 2.40 nm and drug loading of 8.27 ± 0.37%, EE of 68.92 ± 0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0 ± 11.5 mg cm2 at 12 h and 178.8 ± 7.1 mg cm2 at 0.5 h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration.
Ethosomes, ropivacaine, transdermal delivery, vesicle–skin interaction
Introduction Skin, the external barrier between body and the exterior environment, is conscientious in protecting the body from penetration of foreign matter into the body and drain of endogenous material. Delivering drug target to the skin itself or into circulation through skin was called topical/transdermal delivery route (Choi & Maibach, 2005). Due to the peculiar administration site and the absorptive pathway, the topical/ transdermal delivery route has several virtues (Zhai & Zhai, 2014; Zhai et al., 2014) compared to other administration pathways such as avoiding the first-pass effect, circumventing the enterohepatic circulation after oral administration, improving drug bioavailability and patient compliance as well as reducing side effects (Barry, 2001). Despite the aforementioned advantages of topical/transdermal delivery,
Address for correspondence: Guangxi Zhai, PhD, Professor, Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China. Tel: +86 53188382015. E-mail: [email protected]. Zimin Wang, PhD, Depatement of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Tel: +86 21-31161692. E-mail: [email protected]
History Received 24 July 2014 Revised 25 October 2014 Accepted 15 December 2014 Published online 27 January 2015
the major obstacle induced by the stratum corneum hinders the application of this delivery route to a large extent. The stratum corneum consists of 10–15 layers of dead corneocytes, is the upper barrier of the skin. These dead corneocytes are embedded within intercellular lipid matrix composed of ceramides, free fatty acids and cholesterol and jointed tightly by covalently bound lipids and cross-linked proteins and desmosomes (Van Smeden et al., 2014). Aims to overcome the permeation barrier and improve drug permeability, several efforts have been devoted (Subedi et al., 2010). One of the promising methods is the application of vesicular systems such as liposomes. In the early 1980s, Mezei & Gulasekharam (1980, 1982) first described the potential use of liposome for enhanced drug delivery to the skin. Since then, liposomes have been widely investigated for skin delivery due to their excellent biocompatibility (Akhtar, 2014; Touitou et al., 1994; Va´zquezGonza´lez et al., 2014). However, conventional liposome was found awkward in delivering drug into circulation through skin since they were mainly confined to the upper layer of the stratum corneum (Cereda et al., 2013; Duangjit et al., 2014). Novel liquid-state ethosomes were proposed to improve the penetration efficiency by the addition of ethanol within the liposome in the early 1990s. Ethosomes are non-invasive
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systems containing mainly of phospholipid (phosphatidylcholine; PC), ethanol at proper ratio range (20–45%) and water (Touitou et al., 2000). In comparison to rigid liposome, the phospholipids in ethosomes were packed loosely due to the presence of ethanol. Ethosomes were malleable, deformable vesicles which could be tailored for improved delivery of drugs target to or permeate through the skin more efficaciously. Particularly, ethanol could interact with the polar head group region of lipid molecules, leading to a reduction in the melting point of lipid within stratum corneum, thereby increase the fluidity and loosen the tight junction of stratum corneum (Mbah et al., 2014). On the other hand, ethosomes could squeeze through pores within stratum corneum by merits of high and self-optimizing deformability (Cevc et al., 1998). Thus, sizes of ethosomes up to 200–300 nm could penetrate in intact skin effortlessly. Up to now, numerous studies had demonstrated the superior ability of ethosome in enhancing penetration of both hydrophobic and hydrophilic molecules in vitro and in vivo. Jain et al (2007) evaluated the permeability of ethosomal formulation using lamivudine as model drug. The results indicated that the optimized ethosomal formulation showed 25 times higher transdermal flux (68.4 ± 3.5 mg/cm2/h) across the rat skin compared with that of lamivudine solution (2.8 ± 0.2 mg/cm2/h). Zhang et al. (2014) prepared psoralen-loaded ethosomes and evaluated the permeability in skin delivery. The results of ex vivo permeation study showed that the permeability of psoralen-loaded ethosomes was superior to that of rigid liposomes with the transdermal flux (38.89 ± 0.32 mg/cm2/h) and skin deposition (3.87 ± 1.74 mg/cm2), respectively, 3.50 and 2.15 times that achieved by rigid liposomes. Taken all together, ethosomes were proved competent as an efficient vesicle carrier for skin delivery. Ropivacaine, belongs to the family of the n-alkyl substituted pipecoloxylidide (McClure, 1996), is a novel long-acting local anesthetic. It was introduced into clinic in 1996 and proved less toxic on the cardiovascular system and central nervous system. Not only that, ropivacaine also showed considerable merit of a great separation of motor and sensory block, which was specifically beneficial in accelerating postoperative recovery (Chaykovska et al., 2014). Although ropivacaine is currently applied widely in clinic, a major drawback from the formulation point of view is that the application form NaropinÕ , which should be administrated intravenously, can cause patient non-convenience. Besides, adverse reactions like arrhythmia may occur as a consequence of over quick rate of intravascular injection (Leone et al., 2008). Therefore, developing convenient formulation intended for alerting the traditional intravascular injection form will make great sense. In this study, the potential of ethosome in delivering the hydrophobic ropivcaine through skin was evaluated using dorsal skin of mice as model. The ropivacaine-loaded ethosome was prepared and evaluated in terms of morphology, size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. Moreover, the ex vivo permeation study of ropivacaine-loaded ethosomes was performed to investigate their potential in delivering RPV transdermally. In addition, the vesicle–skin interaction was verified by histopathology study.
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Materials and methods Materials Ropivacaine (RPV) was provided by Jinan Dexinjia Pharmaceutical Co. Ltd. (Jinan, China). Lipoid (soybean lecithin at 98% of phosphatidylcholine) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was provided by Beijing chemical reagent company (Beijing, China). Ethanol was obtained from Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). All other chemicals were of analytical purity and commercially available. Preparation of ropivacaine-loaded ethosomes The RPV-loaded ethosomes were prepared with a thin-film dispersion method (Figure 1) (Maitani et al., 1990). Briefly, phospholipon, cholesterol and RPV at certain amount were dissolved by 3 mL of ethanol within a 25 mL of roundbottomed flask. The organic phase was then removed by rotary evaporation under reduced pressure for 20 min to yield a drug-lipid film on the wall inside the flask. The flask was placed in a vacuum dryer for 12 h so as to make the residual ethanol volatilize thoroughly. The resulting drug–lipid film was then rehydrated with distilled water containing appropriate ethanol. Eventually, a homogeneous RPV-ethosomes dispersion was obtained from the supernatant after centrifugation at 4000 rpm for 10 min. In order to facilitate the differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study, the powder of RPV-ethosome was prepared by lyophilization. The formulation was firstly frozen at 20 C, and then continued to pre-freezed at 80 C for 24 h using an ultracold freezer (MDF-382E, SANYO, Japan). Lyophilization was carried out using a lyophilizer (LGJ0.5, Beijing Sihuan Instrument Company, China) at pressure of 0.1 mbar and temperature of 60 C for 48 h by adopting mannitol (5.0%, w/v) as cryoprotectant (Musmade et al., 2014). The obtained powders were collected and kept in desiccators for further analysis.
Figure 1. Preparation process of ethosomes by a thin-film dispersion method.
Ethosomes for skin delivery of ropivacaine
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DOI: 10.3109/08982104.2014.999686
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Encapsulation efficiency and drug loading
DSC analysis
In order to determine the encapsulation efficiency (EE%) and drug loading (DL%), the amount of entrapped RPV in RPVethosomes should be aforehand quantified. A Sephadex G-50 mini column centrifugation technique was adapted to separate the RPV-ethosomes from free RPV. Briefly, 0.5 mL of RPVethosomes formulation was placed onto a Sephadex G-50 mini column. The elution was conducted by adding 0.5 mL of distilled water and then centrifuging for 1 min at 500 rpm. The elution process was repeated for six times. Finally, the eluate containing RPV-ethosomes were collected and mixed with methanol to break down the structure of ethosomes. Thereafter, the amount of entrapped RPV was detected by HPLC. Aims to quantify the total amount of RPV in the formulation, another 0.5 mL of RPV-ethosome formulation was diluted directly by methanol and vortexed for 3 min to break down the structure of ethosomes and dissolve the drug. The EE% and DL% of RPVethosomes were calculated as follows.
The DSC analysis was conducted to study the thermal behavior of RPV-loaded ethosomes. Blank porcelain crucible was used as the standard reference material to calibrate the energy scale of the instrument (1/1600HT, METTLER, Switzerland). Approximately 5 mg of samples (RPV, mannitol, cholesterol, physical mixture and RPV-ethosome powder) was accurately weighed and placed into porcelain crucible. The analysis was performed from 30 to 400 C at a scanning rate of 10 C/min, under a constant nitrogen stream.
DL% ¼
Wloaded 100% Wtotal
EE% ¼
Wloaded 100% Wadded
In the formulas, Wloaded represents the amount of RPV entrapped in ethosomes; Wadded represents the amount of RPV in the formulation system. Wtotal stands for the total weight of all lipids in the formulation system. Optimization of the formulation Formulation optimization is very important for preparation because composition and preparation procedure can influence the physicochemical property of ethosomes. For a long time, researchers have been trying to use the techniques of experimental design to improve experimental works. In this study, uniform design (U13) was employed to optimize the formulation with EE% and DL% as the indexes. Observation of ethosome by transmission electron microscopy The micro-morphology of ethosomes was visualized using a transmission electron microscopy (TEM) with an accelerating voltage of 100 kV. Briefly, ethosome dispersion was spread on a microscopic carbon-coated grid followed by negatively stained with a 2% aqueous solution of phosphotungstic acid. The excess solution was removed by blotting. After drying, the specimen was viewed under the microscope at appropriate enlargement. Size distribution and zeta potential The size distribution and zeta potential of ethosomes were determined by dynamic light scattering (DLS) using a computerized inspection system (ZETASIZER, Nano-ZS, Malvern, UK). Prior to measurements, all samples were diluted to yield a suitable scattering intensity, if needed. Measurements were operated at a fixed angle of 90 . For each sample, the measurement was carried out in triplicate at 25 C and the average value was recorded.
XRD study The wide-angle X-ray scattering (WAXS) of different samples (RPV, mannitol, cholesterol, physical mixture and RPV-ethosome powder) was investigated by an X-ray diffractometer (D8, Advance, Bruker, Germany) with a Cu K radiation source fixed at 40 kV and 100 mA. The wavelength ˚ (0.154 nm). The XRD was perwas set at 1.5405 A formed from the initial 10 to the final 70 with a scanning rate at 1 /s. Ex vivo permeation study Full thickness dorsal skin excised from Kunming mice (male, 20 ± 2 g) was employed to evaluate the potential of ethosomes delivering RPV transdermally. The Kunming mice were supplied by Laboratory Animal Center of Shandong University and the experimental protocol was approved by the Ethics Committee of Shandong University. Prior to the study, the hair on the dorsal skin of animals was carefully trimmed short and removed by Na2S solution (8%, w/v) without scratching the skin surface. Then the mice were sacrificed and the dorsal skin was separated from the underlying connective tissue with a scalpel. The ex vivo skin permeation of RPV from RPV-ethosome formulations was studied using Franz diffusion cells with an effective permeation area of 3.14 cm2. Excised skin was mounted between the donor and receptor compartment with the epidermal side facing upward. Physiological saline (20 mL) containing 1% Tween 80 which was maintained at 32 ± 0.5 C using a water bath was used as receptor medium to meet the sink condition. Ethosomal formulation (0.7 mL) was applied to the epidermal surface of skin and covered with parafilm to avoid evaporation. At 0.5, 1, 2, 3, 6, 9 and 12 h time intervals, 0.5 mL of samples was taken from the receiver. Immediately following, 0.5 mL of fresh receptor medium was replaced to keep a stable receptor volume. The RPV concentration in the samples was analyzed by HPLC and the accumulative penetration amount (Qn) was calculated as follows:
Qn ¼
Cn V0 þ
Pn1 i¼1
Ci Vi
A
where Cn stands for the RPV concentration of the receiver medium at each sampling time, Ci for the drug concentration of the sample, A for the effective diffusion area (3.14 cm2), V0 and Vi stand for the volumes of the receiver solution (20 mL) and the sample (0.5 mL), respectively.
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Vesicle–skin interaction study by histopathology Histopathology study of skin was conducted to visualize the topical effect of RPV-ethosomes on skin (Zhai & Zhai, 2014; Zhai et al., 2014). RPV-ethosomes and RPV propylene glycol solution were applied to the excised rat skins for 12 h, respectively. Then, the treated skin pieces were collected within 10% formalin for tissue fixation for 24 h. After then, the specimen was washed and embedded in paraffin wax and vertically cut into 3–4 mm in thickness. Finally, the paraffin tissue sections were subjected to hematoxylin/eosin staining and examined using microscope. Histopathology study of untreated skin was also carried out as control.
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HPLC assays for ropivacaine In this study, RPV was quantified by reverse-phase HPLC using an Agilent 1200 system equipped with a UV monitor (Agilent G1314-60100). Separations were carried out using a BDS C18 column (4.6 250 mm, 5 mm). RPV was detected at 225 nm with a mobile phase containing a mixture of acetonitrile: phosphate buffer (60:40) at a flow-rate of 1.0 mL/min. The analysis method was evaluated through specificity, linearity, precision and accuracy. For the samples from ex vivo experiments, equal volume of acetonitrile was added to precipitate protein. Then the mixture was vortexed and centrifuged at 12 000 rpm for 10 min. The supernatant was collected and analyzed by HPLC. Statistical analysis The statistical significance of differences was determined using One-way analysis of variance (ANOVA). A value of p50.05 was considered to be significant.
Results and discussion Formulation optimization Uniform design was initially proposed by Fang (1980). It is capable of producing samples with high representativeness and accommodates the largest possible number of levels for each factor in the studied experimental domain (Liang et al., 2001). In this study, the uniform design (U13) was adopted to optimize the formulation variables with EE% and DL% as the indexes. The factors and results were shown in Table 1. The results showed that the EE% changed from 53.95% to 85.50% with the variation of independent variables, while the DL% sharply varied in a greater range from 1.70% to 8.08%. It indicated that the effect of the independent variables on DL% was greater than that on EE%. Therefore, the DL% was selected as a primary response indicator for the formulation optimization. The data was conducted by SPSS version 17.0 software (SPSS Inc., Chicago, IL; Wen et al., 2005) to get a regression equation. Y ¼ 0:042X1 þ 0:026X2 þ 0:123X3 þ 0:402X4 þ 0:002X1 X3 0:016X3 X4 0:049 In the formula, Y is the dependent variable (DL%), X1 (the amount of lecithin), X2 (the amount of cholesterol), X3 (the amount of RPV) and X4 (volume fraction of ethanol) are the independent variables.
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The regression equation provided an insight into the effect of the different independent variables on Y (p50.05). The larger the optimized value (Y) was, the better the formulation was. In overall consideration of better stability and higher DL%, an optimized combination was selected as follows, the amount of RPV, lecithin, cholesterol was 14, 90, 10, respectively, and the volume percentage of ethanol was 40%. The results of the verification test showed that the DL% of the optimized RPV-ethosomes was 8.27 ± 0.37%, verifying a certain predictability of the regression equation as compared to the predicted value of 7.80%. Besides, the EE% of the optimized RPV-ethosomes was 68.92 ± 0.29%. The improvement in DL% may be related to the presence of ethanol in ethosomes since it may allow for better solubility of RPV, improving the distribution of RPV within the inner aqueous phase and the lipid bilayers of the vesicle (Li et al., 2012). Physicochemical characterization of RPV-ethosomes The RPV-loaded ethosomal delivery system was characterized by size distribution and zeta potential, visualization under TEM. As shown in Figure 2(a), the size distribution of RPV-loaded ethosomes, as measured by dynamic light scattering (DLS), showed narrow unimodal peak with polydispersity index (PDI) of 0.201, indicating that the prepared ethosome was relatively homogenous in size distribution. Size was critical to topical drug delivery systems, since it was reported that vesicles smaller than 300 nm were able to deliver the drug into deeper layers of the skin (Verma et al., 2003). The average diameter of ethosomes obtained in our study was 73.86 ± 2.40 nm, indicating that the formulation had potential for delivering drug through the skin. The charge of the vesicular dispersion has an influence on the stability of the formulation. The higher zeta potential was, the better the long-term stability was, as electrostatic repulsion between vesicles could avoid aggregation. The zeta potential of the obtained RPV-loaded ethosomal delivery system was 12.9 mV (Figure 2b), which could forecast a good stability for the formulation. The electronegativity may be related to the presence of 40% ethanol since it was reported that the addition of 30% ethanol induced a transition in the charge of the vesicles from positive (+4.6 ± 0.2 mV) to negative (4.3 ± 0.2 mV; Touitou et al., 2000). Concretely,
Table 1. The factors and results of the U13 Uniform Design. Factors
Results
Groups
X1 (mg)
X2 (mg)
X3 (mg)
X4 (%)
DL%
EE%
1 2 3 4 5 6 7 8 9 10 11 12
100 100 100 200 200 200 300 300 300 400 400 400
5 10 10 15 20 20 5 5 10 15 15 20
15 25 15 25 15 25 10 20 10 20 10 20
35 25 40 30 25 35 30 40 35 25 40 30
5.18 ± 0.25 4.00 ± 0.11 8.08 ± 0.22 3.22 ± 0.09 3.22 ± 0.17 4.67 ± 0.33 2.03 ± 0.64 4.02 ± 0.10 1.90 ± 0.06 3.26 ± 0.13 1.70 ± 0.07 2.96 ± 0.23
53.95 ± 1.02 61.25 ± 0.81 59.4 ± 0.34 54.05 ± 0.71 69.5 ± 1.32 67.55 ± 0.33 64.65 ± 0.54 70.45 ± 0.79 62.05 ± 1.41 85.50 ± 1.47 80.20 ± 0.91 77.10 ± 1.37
Ethosomes for skin delivery of ropivacaine
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DOI: 10.3109/08982104.2014.999686
the change of zeta potential was attributed to the net negative charge and facilitation of ionization conferred by ethanol on the vesicle surface (Mbah et al., 2014). As illustrated in Figure 1, ethanol molecules distributed within the inner aqueous phase and the lipid bilayers of the vesicle. The terminal hydroxyls of ethanol molecules distributed within the lipid bilayers of the vesicle may stretch to the outer medium phase due to the hydrophilic group. Hydrogen bond may occur among the hydroxyl groups in the ethanol molecules or between ethanol molecules and water molecules, which would favor a high net negative charge and facilitation of ionization. The prepared RPV-ethosome dispersion was bluish and transparent in appearance (Figure 3a), whereas the system of crude RPV dispersed in water at the same drug concentration was suspension with some precipitation of drug (Figure 3b). When examined under TEM, the RPV-ethosomes appeared as unilamellar vesicles (Meng et al., 2014) with a predominantly spherical shape without aggregation among vesicles (Figure 3c). The size of the vesicles obtained from the TEM decreased little compared to that obtained by DLS (73.86 ± 2.40 nm). There may be two reasons. For one thing, it is difficult to stain the outer hydrophilic surface of the ethosome (Mi et al., 2014), for another, the negatively stained RPV-ethosomes were allowed to dry for contrast enhancement prior to the TEM measures, the dehydration process may lead to a shrink to the hydrophilic surface. The results of physicochemical characterization showed the RPVethosomes were prepared successfully and an improvement of the dispersibility of RPV was achieved after formulated to RPV-ethosomes.
Figure 2. The size (a) and Zeta potential (b) of the RPV-loaded ethosomes.
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DSC analysis In order to inspect the changes of crystalline structure compared with the raw materials (Luan et al., 2013), the DSC analysis was conducted. Figure 4 displays the DSC thermograms of the raw materials, their physical mixture (RPV, mannitol, cholesterol and lecithin) and RPV-ethosome freezedried powder. It was evident that each raw material exhibited particular endothermic peaks. Particularly, the endothermic peak of pure RPV was at 148.67 C (a), and that of mannitol was at 168.04 C (b). The characteristic peaks of RPV were present in the DSC curves of physical mixture (d), just with a slight shift (141.69 C), meaning that the initial crystalline form of the drug was retained. Conversely, no endothermic peak corresponding to the fusion of RPV was observed in the curve of RPV-ethosome freeze-dried powder (e). This comparison between (d) and (e) suggested that the crystalline form of RPV was unchanged when RPV was physically mixed with the excipients, but transformed into non-crystalline state when encapsulated into ethosomes. XRD study The XRD study was employed to analyze whether the crystalline structure of RPV in ethosome was changed or not and the results were shown in Figure 5. It was evident from
Figure 3. Photographic images of RPV-loaded ethosomes (a) and RPV suspension (b). (c) represents the morphology of RPV-loaded ethosomes obtained from the optimized formulation.
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Figure 4. DSC analysis thermograms: (a) ropivacaine; (b) mannitol; (c) cholesterol; (d) the physical mixture of RPV-ethosomes; (e) RPVethosomes freeze-dried powder.
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Figure 6. Ex vivo permeation profile of RPV-ethosomes (g) and RPV propylene glycol () by using excised mice skin as model (mean ± SD; n ¼ 5).
Figure 7. Ex vivo permeation profile of RPV-ethosomes and RPV propylene glycol at 0.5, 6 and 12 h. *p50.05. Figure 5. X-ray diffraction spectra: (a) ropivacaine; (b) cholesterol; (c) mannitol; (d) the physical mixture of RPV-ethosome; (e) RPVethosome freeze-dried powder.
Figure 5 that RPV showed a strong diffraction peak at 10.68 (a), while the cholesterol showed a characteristic diffraction peak at 14.21 (b). The RPV diffraction peak was also present in the curve of the physical mixture (d), which proved that the crystal form of RPV was the same with that of the pure RPV powder. In contrast, the characteristic diffraction peak of RPV disappeared in the curve of RPV-ethosome freeze-dried powder (e), indicating a disordered crystalline state of RPV in ethosome. These results proved that RPV had been successfully incorporated into the lipid matrix of ethosomes in amorphous or disordered state. Ex vivo permeation study The ex vivo permeation study of RPV from ethosomes or propylene glycol solution on the full-thickness mice skin was
carried out by using Franz diffusion cells with an effective permeation area of 3.14 cm2. The accumulative penetration amount (Qn) of RPV from the experimental ethosomal formulation or propylene glycol solution was shown in Figure 6. It was evident that ethosome-mediated delivery significantly increased the transdermal flux of RPV with a value of 360.5 ± 21.4 mg cm2 at 12 h. Nevertheless, the transdermal flux of RPV from propylene glycol solution as control was only 141.3 ± 13.5 mg cm2 at 12 h. This tendency indicated the potential of ethosomes in substantially delivering RPV across skin was considerable, relative to the control propylene glycol solution. Particularly, aims to summarize the permeation manner of RPV ethosomes in a more intuitive way, the Qn of RPV from ethosomal formulation and propylene glycol solution at 0.5, 6 and 12 h was magnified purposely and displayed in Figure 7.
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DOI: 10.3109/08982104.2014.999686
Ethosomes for skin delivery of ropivacaine
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Figure 8. Histopathology photomicrographs of untreated skin (a), skin treated with RPV propylene glycol solution (b) and RPV-ethosomes (c).
As shown, the Qn of RPV from ethosomal formulation was relatively high (178.8 ± 7.1 mg cm2) even at 0.5 h, almost 19-folds that from propylene glycol group (p50.05). It suggested that ethosomes could interact with the skin rapidly and penetrate in a high-efficient, quick way. Primarily, the colloidal properties of ethosome should be responsible for the improved accumulative penetration amount (Qn) and the high-efficient penetration rate. The above reported effects could be ascribed to the favorable flexibility and the penetration enhancement function of the ethosomes. The presence of ethanol could increase flexibility and improve deformability of the vesicles, which might allow ethosome to squeeze through skin channels in a self-adapting manner and thus carry RPV across the intact skin (Sinico & Fadda, 2009). This proposed that ethosome could facilitate drug transport by a fast partitioning in the stratum corneum, thereby carrying the encapsulated RPV into stratum corneum. Except for the drug carrier function, ethosome might play a role as penetration enhancers. Ethanol as good solvent might increase the solubility of RPV in the vehicle and accordingly, the permeation of ethanol could improve RPV partitioning into the membrane by altering the solubility properties of the tissue (Megrab et al., 1995). Besides, ethanol was able to extract some of the lipid fraction from the stratum corneum, disturb the organization of the lipid bilayer of stratum corneum, creating the improved RPV flux through the skin
if applied for prolonged times (El Maghraby et al., 2001). Not only that, some components of ethosomes, particularly lecithin, could fuse or mix with skin lipids by disturbing the lamellar arrangement of the lipids within stratum corneum, or even achieve lipid exchange (Sinico et al., 2005). Taken together, either was the main or the only mechanism of action for the improved skin permeability of RPV from ethosome. A possible synergistic effect conducted by the two sides seems to be the contributor. Vesicle–skin interaction study In order to investigate whether ethosomes affected the structure of stratum corneum or not, tissue sections of the skin were stained with hematoxylin/eosin. Histopathological images were shown in Figure 8. In control group (Figure 8a), a compact stratum corneum with tightly conjugated adjacent corneocyte layer could be observed visually. Application of RPV propylene glycol solution changed the tight junction slightly (Figure 8b). However, as shown in Figure 8(c), it was evident that the treatment of RPV-ethosomes increased the gap among adjacent cells. Besides, the stratum corneum appeared swollen, with total thickness increased in Figure 8(c). The results of the histopathology study of skin indicated that ethosomal formulation could significantly affect the upper histology of skin.
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Y. Zhai et al.
There may be two mechanisms that underlie the topical effect of RPV-loaded ethosomes on skin. First, a colloidal film formed by RPV-ethosomes covered on the skin surface might significantly reduce or prevent skin dehydration and provide better wetting function to the skin and loosen the barrier properties (Lv et al., 2009). As for the ethosome itself, ethanol might penetrate into the stratum corneum and loosen the ‘‘bricks-mortar’’ structure of stratum corneum. Besides, some components like lecithin could fuse the skin lipids by disturbing their lamellar arrangement (Sinico et al., 2005). The synergistic effects of combination of phospholipids and ethanol were suggested to be responsible for the vesicle–skin interaction (Touitou et al., 2001).
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Conclusion The application of ethosome as a vesicular carrier could overcome the limitation of low permeability of stratum corneum barrier. In this study, RPV-loaded ethosomes were prepared and evaluated as a vesicular carrier for skin delivery. The optimized RPV-loaded ethosomes showed unilamellar vesicle structure with the mean particle size of 73.86 ± 2.40 nm and DL% of 8.27 ± 0.37%, EE% of 68.92 ± 0.29%, respectively. The DSC analysis and XRD study revealed the amorphous state of RPV in ethosomes, indicating RPV was successfully incorporated into ethosomes. The results of the ex vivo permeation study indicated that the ethosome-mediated delivery increased the transdermal flux of RPV in a highefficient, rapid way compared with propylene glycol solution as control. The histopathology study proved that the interaction between ethosome and skin could loosen the ‘‘bricks-mortar’’ structure of stratum corneum barrier. In summary, our study was the first report in developing RPV-loaded ethosome for skin delivery and the results demonstrated that ethosomal delivery systems were superior in delivering RPV through skin.
Declaration of interest The authors declare no conflicts of interest. This work is supported by grants from Shanghai Municipality Science and Technology commission (12nm0500700, 11DZ1971400) and the National Nature Science Foundation (No.81171766, No.81373896).
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