Many types of skin surgery, such as curettage of molluscum, laser cosmetic surgery, and electrosurgery, require topical anesthetics with a rapid onset and long duration of activity. Direct injection of lidocaine, the most commonly used local anesthetic, has the advantages of instant onset and simple administration. Direct injection is also painful and may be poorly tolerated, especially in children. Anatomic marks, which are very important in facial surgery, may be altered by direct injection. Percutaneous delivery systems for lidocaine base, such as ointment, tincture, and liposomes, have been developed for clinical use,1 but each has drawbacks.
Ethosomes are an innovative vesicular delivery system, with advantages that include thermodynamic stability, small particle size, high loading efficiency (LE), and high encapsulation efficiency.2 Ethosomal formulations have been used for the administration of several drugs, including finasteride and matrine.3,4 Due to their deformability, ethosomes can effectively penetrate the epidermis, and even into deeper layers of the skin.5–7
We hypothesized that lidocaine base in an ethosomal formulation may be superior to the existing percutaneous preparations of lidocaine base, especially in thermodynamic stability, onset time, and duration. We therefore prepared 5% (w/w) lidocaine base ethosomes and measured their size, LE, encapsulation efficiency, and percutaneous penetrating efficiency in vitro, their effectiveness in vivo, and their ability to irritate the skin of guinea pigs.
Preparation of Lidocaine Base Formulations
The orthogonal test L9 (34) table was used to design the formulations of lidocaine base ethosomes. The vesicular system was composed of 3% to 5% (w/w) egg phosphatidyl choline, 0.15% to 0.2% (w/w) cholesterol, 35% to 45% (w/w) anhydrous ethanol, drug (5% lidocaine base, w/w), and ultrapure water. The egg phosphatidyl choline and cholesterol were dissolved in anhydrous ethanol with lidocaine base, followed by the slow addition of ultrapure water and vortexing at 1500 rpm for 10 minutes (Haimen Instruments, Jiangsu, China). The mixture was sonicated with a pulse of 105 W for 2 minutes in an Ultrasonic Processor (Cole-Parmer Instruments, Vernon Hills, IL) and filtered through a 0.22-μm filter. The lidocaine base ethosomes were stored in sealed vials at 25°C ± 1°C.
As a control, lidocaine base liposomes containing the same amounts of egg phosphatidyl choline, cholesterol, and drug (5% lidocaine base, w/w) as mentioned earlier were prepared using a film-dispersing method.8 We also prepared a second control formulation consisting of lidocaine base hydroethanolic solution containing 5% (w/w) lidocaine base and 35% (w/w) anhydrous ethanol.
Characterization of Lidocaine Base Ethosomes
A 50-μL aliquot of ethanol base ethosomes was added to a 10-mL measuring flask, which was subsequently filled with methanol to the calibration tail, followed by sonication for 10 minutes. The amount of lidocaine base in the flask was determined by a high performance liquid chromatography (HPLC) analytic method, and the LE was calculated as:
where Wtest is the amount of lidocaine base determined by HPLC and Wtotal is the theoretical amount of lidocaine base in the 50-μL sample.
Encapsulation efficiency was measured as previously described.9 Briefly, a dialysis bag with a molecular weight cutoff of 8000 to 14,000 Da was precut into 10-cm lengths. One end of each dialysis bag was sealed, and 2-mL lidocaine base ethosomes were added. The other end of each dialysis bag was sealed, and the bags were placed in a beaker containing 250 mL 2% (w/w) anhydrous ethanol and mixed with a magnetic stirrer (Yangyinpu Instrument Ltd., Shanghai, China) at the rate of 60 rpm. The concentration of lidocaine base in the solution outside the bag was analyzed every hour by HPLC, until equilibrium was reached. Encapsulation efficiency was calculated as:
where Wfree is the concentration of lidocaine base in the solution outside the bag, and Wtotal is the theoretical concentration of lidocaine base.
Vesicular Size and Range
The vesicular size of lidocaine base ethosomes was determined using a Zetasizer (3000HAS, Malvern Instrument, London, UK) at 25°C ± 1°C. A 2-mL aliquot of lidocaine base ethosomes was added to a glass test tube without dilution for visualization, and the intensity of laser light scattered by samples was detected at a 90° angle with a photomultiplier. The vesicular size range of lidocaine base ethosomes was described by the parameter of polydispersity.
The stability of lidocaine base ethosomes was evaluated by comparing encapsulation efficiency values determined on days 0 and 60 after ethosomes preparation, the preparations having been sealed in vials at 25°C ± 1°C.
In Vitro Percutaneous Penetration
All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Southern Medical University, Guangzhou, Guangdong, China. Healthy Sprague-Dawley rats, weighing 250 to 300 g, were anesthetized by peritoneal injection of carbrital. The abdominal hairs on each rat were carefully trimmed (<1 mm) with an electric razor, and the abdominal skin was carefully dissected from the underlying connective tissue with a scalpel. The excised skin was placed on aluminum foil. Any adhering fat or subcutaneous tissue was gently removed from the dermal side of the skin.
The in vitro percutaneous penetrating capability of lidocaine base ethosomes was evaluated using Franz-type diffusion cells, with an effective permeability area of 2.92 cm2 and a receptor of 7 mL. The receptor compartment was filled with 7 mL physiologic saline containing 2% (w/w) anhydrous ethanol at 37°C ± 0.5°C, and the solution was constantly stirred by a magnetic stirrer at 300 rpm. Lidocaine base preparations of ethosomes, liposomes, and hydroethanolic solution and their blank preparations (1.5 mL each) were applied onto the epidermal surface of the skin. Samples of 0.4 mL were withdrawn through the sampling port of the diffusion cell at 0.5, 1, 2, 3, 6, 9, and 11 hours after each sampling, and an equal volume of physiologic saline containing 2% anhydrous ethanol was added to the receptor compartment.
HPLC Determination of Lidocaine Base
The amount of lidocaine base in the receptor compartment of Franz cells was determined using a model LC-20A liquid chromatographic system (Shimadzu, Kyoto, Japan). Methanol and 0.0257 M ammonium acetate (70: 30, v/v) were used as the mobile phase and delivered by a pump at 0.8 mL/min. Each 10 µL aliquot of sample was eluted by the mobile phase in a C-8 column (4.6 × 250 mm, 5 μm, Shimadzu) at 30°C and was monitored using a UV detector at a wavelength of 270 nm.
In Vivo Effectiveness
The in vivo effectiveness of lidocaine base ethosomes was evaluated by pinprick tests on guinea pigs. Guinea pig skin has an instinctive response to pinprick, as shown by the appearance of a shivering reflex. Application of local anesthetic prevents this response. Observers were blinded to the treatment of experimental animals.
Guinea pigs weighing 250 to 300 g and with a normal response to pinprick were randomly divided into 4 groups containing 6 pigs each. The hair on the back of each guinea pig was shaved with an electric razor. The remaining fuzz was cleared by a depilatory 24 hours before the assay. Aliquots (200 µL each) of lidocaine base ethosomes, liposomes, and hydroethanolic solution were applied to the skin. The area of skin covered by drugs was 3 cm2. The degree of shivering reflex response induced by acupuncture was recorded after 30, 40, 50, 60, 80, 100, and 120 minutes. The durations of anesthetic of 3 lidocaine base preparations (ethosomes, liposomes, and hydroethanolic solution) were compared. The shivering reflex response of guinea pigs treated with drugs for 1 hour was recorded at 10, 20, 40, 60, 80, 100, and 120 minutes. As a control, we also used the respective blank preparations (no lidocaine base).
In Vivo Cutaneous Irritancy
The potential for skin irritation (erythema and swelling) of lidocaine base ethosomes was evaluated as described.10 Cutaneous irritation by lidocaine base ethosomes was evaluated in male white guinea pigs, which were housed in an air-conditioned room (25°C). The hair on the back of each animal was trimmed 24 hours before the test. The skin on 6 animals was damaged by pinpricks, whereas the skin on 6 other animals was left intact. All animals were treated with 200-μL lidocaine base ethosomes for 24 hours. The skin was scored for erythema and swelling 1, 24, 48, and 72 hours after application. As a negative control, guinea pigs were treated with normal saline. Erythema was scored as 0 to 3, with 0 indicating no erythema, 1 indicating very slight erythema (barely perceptible; light pink in color), 2 indicating well-defined erythema (dark pink in color), and 3 indicating moderate to severe erythema (light red in color). Swelling was scored as 0 to 3, indicating no, light, moderate, and serious swelling, respectively. Skin treated with lidocaine base ethosomes was also examined histopathologically.
All quantitative data were expressed as mean ± SD and analyzed using SPSS software (version 13.0, SPSS Inc, Chicago, IL). The stability of lidocaine base ethosomes at 0 and 60 days was compared using independent sample t tests; 95% confidence intervals (CIs) were also calculated. Transdermal flux, onset time, and duration of lidocaine base ethosomes, liposomes, and hydroethanolic solution were compared by using repeated-measures analysis of variance. When the spherical assumption was violated, the Greenhouse–Geisser correction was applied. The Bonferroni correction was used in multiple comparisons if the data met the homogeneity of variance; otherwise, we used the Dunnett T3 test. A P value <0.05 was considered statistically significant.
The formulation of lidocaine base ethosomes was determined by the maximum encapsulation efficiency, as described.11 The R values in Table 1 indicate that encapsulation efficiency was affected most by egg phosphatidyl choline (ingredient B), followed by ethanol (ingredient A) and cholesterol (ingredient C). The 5% (w/w) lidocaine base ethosomes were prepared with 5% (w/w) lidocaine base, 5% (w/w) egg phosphatidyl choline, 0.2% (w/w) cholesterol, 35% (w/w) ethanol, and ultrapure water, to yield the maximum encapsulation efficiency.
The mean LE of 5% (w/w) lidocaine base ethosomes was 95.0% ± 0.06% (n = 6). As determined by the Zetasizer, the mean particle diameter of these ethosomes was 31.33 ± 2.63 nm (n = 6), and their mean polydispersity index was 0.42± 0.21. The mean encapsulation efficiency of lidocaine base ethosomes was 52.96% (95% lower CI, 51.73%). After storage at 25°C ± 1°C for 60 days, the mean encapsulation efficiency was 52.84% (95% lower CI, 52.47%). Equal variance was not assumed (F = 120.060, P < 0.001). The mean encapsulation efficiency at 0 and 60 days did not differ significantly (95% CI, −1.12% to 1.34%; P = 0.833).
The cumulative transdermal penetration profiles of the 3 investigated formulations are shown in Figure 1. The sphericity assumption was violated (Mauchly W = 0.000, P < 0.001), so the Greenhouse–Geisser correction was used to correct the violation (ε = 0.402), resulting in a significant group × time interaction (F = 158, P < 0.001). We found that the percutaneous flux of lidocaine base in vitro differed significantly (F = 121, P < 0.001) among the 3 preparations, being significantly higher from ethosomes than from liposomes (95% corrected CI, 1129–1818 µg/(cm2·h); P < 0.001) and from hydroethanolic solution (95% corrected CI, 1468–2157 µg/(cm2·h); P < 0.001). However, it did not show the significant difference between liposomes and hydroethanolic solution (95% corrected CI, −6.21 to 683 µg/(cm2·h); P = 0.055).
The negative responses of guinea pig in the pinprick test to lidocaine base from ethosomes, liposomes, and hydroethanolic solution are shown in Figure 2. The sphericity assumption was violated (Mauchly W = 0.014, P < 0.001), so the Greenhouse–Geisser correction was used to correct the violation (ε = 0.545), resulting in a significant group × time interaction (F = 2.769, P = 0.019). The negative responses induced by lidocaine base ethosomes were significantly greater than those for lidocaine base liposomes (95% corrected CI, 0.52–2.67 times; P = 0.003) and hydroethanolic solution (95% corrected CI, 1.14–3.27 times; P < 0.001). However, there was no significant difference between lidocaine base liposomes and hydroethanolic solution (95% corrected CI, −0.45 to 1.69 times; P = 0.422).
The anesthetic duration of the 3 lidocaine base formulations is shown in Figure 3. Because the sphericity assumption was violated (Mauchly W = 0.077, P = 0.041), the Greenhouse–Geisser correction was used to correct the violation (ε = 0.515), resulting in a significant group × time interaction (F = 3.538, P = 0.005). The duration of anesthesia was significantly longer for lidocaine base ethosomes than for liposomes (95% corrected CI, 2.25–4.23 times; P < 0.001) and hydroethanolic solution (95% corrected CI, 3.32–5.30 times; P < 0.001) and was significantly longer for lidocaine base liposomes than for hydroethanolic solution (95% corrected CI, 0.08–2.06 times; P = 0.032). We found no evidence of erythema or swelling of the skin. The lack of cutaneous irritation was confirmed by histopathologic examination.
Over the past 150 years, percutaneous preparations of anesthetics have evolved from simple solutions to creams, ointments, gels, liposomes, and sophisticated patches.12 These formulations are characterized by slow onset and short duration of action. Ethosomes were shown to act as carriers of drugs13 and to promote their percutaneous penetration.14,15 Among the agents used to date in ethosomal preparations are cannabidiol, azelaic acid, and bacitracin. Ethosomal preparations have many excellent characteristics, including small particle size, good encapsulation efficiency stability, rapid percutaneous penetration, and low irritation,16–18 suggesting that ethosomes could be used as percutaneous carriers of lidocaine base.
Although ethanol can increase membrane fluidity of ethosomes and improve their stability,19 an ethanol concentration >45% (w/w) has been shown to decrease the value of encapsulation efficiency, perhaps because the membrane may leak in the presence of high concentrations of ethanol.20 Thus, ethanol concentrations in ethosomes should be controlled within a certain range. Encapsulation efficiency may also be affected by many other factors, including changes in the proportions of ethosomal ingredients, as shown in the orthogonal test. We found that membrane dialysis was more appropriate than ultracentrifugation in the determination of ethosomal encapsulation efficiency, perhaps because the kinetic energy created during ultracentrifugation could destroy the structure of the ethosomes, leading to leakage of drug from the ethosomal membrane.21
The percutaneous flux of lidocaine base was much higher from ethosomes than from liposomes and hydroethanolic solution. Ethanol may enhance penetration through stratum corneum lipids22 and effectively promote the mobility of the ethosomal membrane. The role of ethanol in percutaneous penetration was confirmed by our in vivo experiments, which showed that the onset time of ethosomes was shorter than that of liposomes containing the same ingredients except for ethanol. In addition, ethosomes can be stored in the skin, making the anesthetic duration of ethosomes longer than that of the other 2 lidocaine base preparations.
Because the structure and function of skin are complex, the results obtained from in vivo experiments may be more accurate than those from in vitro assays. Therefore, the irritation and effectiveness of drugs on skin have been generally evaluated by animal experiments in vivo, despite their subjective interpretation. Objectivity may be enhanced by using a sufficient number of experimental animals, by evaluations performed by ≥2 observers blinded to treatment, and, for skin irritation, by histopathologic examination.
Our results confirm previous findings that the high encapsulation efficiency, stability, and excellent percutaneous penetration of drugs by ethosomes are due to the presence of ethanol. Further research should include the use of other types of phospholipids and short-chain alcohols to prepare ethosomes and to determine whether these ethosomes further enhance percutaneous penetration of drugs. Ethosomes are promising drug carriers for topical administration of local anesthetics, especially when a rapid onset of effect is desired.
Name: Xiaoliang Zhu, PhD.
Contribution: This author helped write the manuscript.
Attestation: Xiaoliang Zhu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Fuli Li, MM.
Contribution: This author helped write the manuscript.
Attestation: Fuli Li has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Xuebiao Peng, MD.
Contribution: This author helped write the manuscript.
Attestation: Xuebiao Peng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Kang Zeng, MM.
Contribution: This author helped write the manuscript.
Attestation: Kang Zeng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Steven L. Shafer, MD.
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