| Research BackgroundAt present there is a emergency request on a novel transdermal local anesthetic pharmaceutical preparation which has many properties such an rapid onset time, effective action, low side effect and long enough duration for surgical operation time to eliminate the pain which is caused by various medical treatments such as skin puncturation, surgical intervention, laser cosmetology and so on. As so far there has been many correlated transdermal local anesthetic pharmaceutical preparations such as Eutectic Mixture of Local Anesthetics (EMLA) cream and other local anesthetic agents such as tincture, gel, and cream. All of the aboved preparations can not achieve the aim of the safe and effective because of the obvious adverse reaction or bad transdermal permeation.The biggest obstruct of drug delivery via skin is the natural defence of the skin—stratum comeum (SC). There have been many methods to increase the drug delivery through cutaneo including physical and chemical approaches, but the aboved approaches exist some limiations and shortages, such as chemical penetrating enhancer can interfere the structure of skin, and some noval physical transdermal devices or methods like microacupuncture needl have the defects of expensive cost and the skin nocuity which can not be avoided. Therefore, the study of a noval transdermal carrier plays a vital role on the investigations of Transdermal drug delivery systems (TDDS). The carrier will play a crucial part in the local anesthetic agent delivery through the skin. Ethosome, as a novel vesicular structure, is made of phosphatide, cholesterin, ethanol, and water, which is the liposome with high concentration ethanol. Ethosomes have the properties of stable thermodynamics, small size, high entrapment efficiency, strong penetration, and the good tolerance. So, in the precondition of safe utilization, ethosome as a local anesthetic carrier, will be enhance the transdermal permeation to achieve a safe and rapid effectiveness.This investigation plans to choose a amide local anesthetical agent—lidaocaine, which has many properties such as rapid onset time, strong anesthetic action, extensive diffusibility, good safety, and no obvisous vasodilatation, and to prepare the noval transdermal local anesthetic pharmaceutical preparation—lidocaine ethosome. The full-scale analyses and evaluation will be performed with regard to the physicochemical properties, which includes morphous, particle diameter, entrapment efficiency and stability, transdermal permeation, safety, and pharmacodynamics and so on.ObjectiveTo determine the formulation of lidocaine ethosome; to analyze and evaluate the morphous, particle diameter, entrapment efficiency, stability, transdermal permeation and its correlated influencing factors, safety, and pharmacodynamics. The research achivements will supply a noval local anesthetic pharmaceutical preparation to clinical treatment, which will have many properties such as good safety, rapid local anesthetic action and long enough duration for various operations.Method1. Use L9 (34) orthogonal test to determine optimal formulation of 5% lidocaine ethosome:The quality percentage concentration of absolute ethanol, egg phosphatidyl choline, and cholesterol were chosen to be the consideration factors. And the entrapment efficiency was chosen to be the index of 5% lidocaine ethosome in order to select the optimal formulation;2. The preparation of 5% lidocaine ethosome and liposome①The preparation of 5% lidocaine ethosome by injection-sonication-filter method:Egg phosphatidyl choline and cholesterol which were exactly weighed were dissolved along with lidocaine in absolute ethanol. Ultrapure water was added slowly in a fine stream with constant mixing with vortex mixer until all the materials completely lysised. And then, the system was sonicated for 2 min with pulses of 105w by Ultrasonic Processor. The final preparation was obtained after process of being filtered using 0.22μm filter;②The preparation of 5% lidocaine liposome by film-dispersing method:The same amount of egg phosphatidyl choline, cholesterol and lidocaine were also prepared using film-dispersing method as follows:the egg phosphatidyl choline, cholesterol and 5% w/w lidocaine were dissolved in absolute ethanol in a pear-shaped flask, and the flask was then attached to a rotary evaporator under vacuum until a thin lipid film on the inner wall of the flask was formed. The film was dispersed with water and hydrated completely. The system was then sonicated with the method described in ethosome preparation;3. Calculate the loading efficiency and encapsulation efficiency of lidocaine ethosome using dialysis technique and HPLC①To calculate the loading efficiency of lidocaine formulations:add 50μl sample to 10 ml measuring flask to the line with methanol, completely mix, then be sonicated for 10 min, finally for High-performance Liquid Chromatography (HPLC);②The encapsulation efficiency was determined by dialysis technique:dialysis bag (MWC:8000-14000) was cut to the length of 10 cm before the test begun. Seal one end of the bag, and then 2ml of vesicular sample was added into the bag, finally close the other end of the bag. Put the bag with sample into the beaker which contained 250 ml 2% ethanol water solution. The system was put on the magnetic stirrer under mixing at 60rpm. After 9 hours equilibrium dialysis, withdraw the outer solution of the bag for HPLC to test the free lidocaine of the vesicle;4. Observe the morphology of ethosomal by Transmission Electronic Microscopy (TEM)A drop of the sample solution was placed on a copper grid, and the material excess was removed with a filter paper. A 3% phosphotungstic acid solution was dropped onto the grid. The excess of staining solution was removed. After drying the specimen was viewed under the microscope at 120 k-fold enlargements at an accelerating voltage of 120 kV;5. Determine the size of ethosome by ZetasizerThe measurement was done at a temperature of 25±1℃.The intensity of the laser light scattered by the samples was detected at an angle of 90°with a photomultiplier. The sample (2 ml) was added to the test glass without any dilution for visualization;6. Use Franz diffusion cell to performe the in vitro permeation studies of 5% lidocaine ethosome, liposome, and ethanol water solution, compare and analyze the influences of licoaine formulation on ethosome transdermal permeationThe skin of Sprague-Daeley (SD) strain rats was used for permeation.Rats weighing 250-300 g were anesthetized by carbrital peritoneal injection, and the hair of test animals were carefully trimmed short with electric razor. The abdominal skin was separated from the underlying connective tissue. The excised skin was placed on aluminum foil, and the dermal side of the skin was gently teased off for any adhering fat and/or subcutaneous tissue.The in vitro skin permeation of lidocaine formulations was studied using Franz diffusion cell with an effective permeation area and receptor cell volume of 2.92cm2 and 7 ml, respectively. The temperature was maintained at 37±0.5℃. The receptor compartment was constantly stirred by magnetic stirrer at 300 rpm. The stratum comeum of the skin faced the supplying cell. Ethosomal formulation, liposomal formulation, ethanol water solution and the blank ethosomal formulation, liposomal formulation and ethanol water solution (1.5ml, respectively) were applied to the epidermal surface of skin. Samples (0.4ml) were withdrew through the sampling port of the diffusion cell at 0.5-,1-,2-,3-,6-,9-, and 11 hour time intervals and analyzed by high-performance liquid chromatography assay. An equal volume of fresh 2% ethanol water solution was replaced into the receptor compartment after each sampling;7. Determine 5% lidocaine ethosome stability via the changes of temperatures and timesThe 5% lidocaine ethosome system were deposited at various temperatures (4±1℃,25±1℃and 37±1℃) for 60 days. And the ethosome system was kept in sealed vials. Samples were withdrew periodically and analyzed for the drug content by HPLC to evaluate their stability;8. Use the method of local infiltration on the back skin of guinea pigs to compare the activity of the 5% lidocaine ethosome, liposome, and ethanol water solutionIn vivo efficiency was evaluated on male guinea pigs and by employing the pinprick test for accessing the local anesthetic activity. The hairs on the back of the guinea pigs were shaven with an electric razor with a rest period of 24h to recover from any skin injury that might have occurred during shaving of the skin. A single dose each of accurately amount all these formulations was applied on the 3 cm2 area to the shaven back of guinea pig. A needle was used to induce the shivering of the skin. Response was checked immediately when those formulations were applied at every minute in the first ten minutes, and then 15,20,25,30,40,50,60,80,100, and 120min. The same method was used when the drugs were removed after the drugs were administrated on the back skin of the rats for 1 hour. But the interval time was 10,20,40,60,80,100, and 120 min after the removal of drugs;9. To measure the safety of lidocaine ethosome usage by naked eye observation and tissue pathology of the guinea pigs back skin which was treated by 5% lidocaine ethosomeThe male guinea pigs (250-300g), which were treated as above in the study of the in vivo local anesthetics activity of lidocaine vesicles, were chosen to monitor the induced erythema and edema following the topical application of the forrmulation. The skins of the guinea pigs was separated to integrated and unintegrated skin. The unintegrated skin was made by the needle prick. The interval time to observe the skin appearances was 1,24,48h and 72h after removal the drug which was administrated for 24h. The pathological manifestations were also observed at the defined interval. The erythema was accounted score 0 for no erythema,1 for light erythema,2 for obvious erythema,3 for moderate to serious erythema, and 4 for prunosus erythema and escharosis. The edema was accounted score 0-4 for no edama, light edema, moderate edema (only edema outline), and serious edema, respectively. The pathological observation indexes includes the changes of stratum comeum structure, and the dermal tissue inflammation changes such as visible heterophil granulocyte infiltration, vasodilation hyperemia, inflammatory cell infiltration around the blood vessel, vasculitides, dermal oedema, or proliferation and fibrosis of fibrolast and so on;10. Statistical treatment①PSS13.0 was used to analyze statistically all datas;.②Multiple comparison of group means: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;③The orthogonal design was performed by using orthogonal design assistantⅡV3.1;④The repeated meaures analysis of variance was used to analyze the repeated measure datas;⑤The curve was obtained through Graphpad Prism 5.0 software;⑥All reported data are mean±.E.M. Statistical significance was considered to be granted at P<0.05.Results1. From the orthogonal test, the optimal formulation was composed of 35% absolute ethanol,5% egg phoshpatidyl choline,0.2% cholesterol,5% lidocaine, and 49.8% ultrapure water; the sequence of the range of these three factors from maximum to minimum was egg phosphatidyl choline, absolute ethanol, and cholesterol, respectively, which indicated that the egg phosphatidyl choline has the biggest effect on the formulation of ethosome, followed by absolute ethanol and cholesterol. The loading efficiency of these formulations was 95.0±0.06%(n=3), and entrapment efficiency was 51.44±3.75%(n=7).2. The loading efficiency and the entrapment efficiency of lidocaine liposome were 98.85±0.24%(n=4),and 46.20±1.50%(n=7) respectively.3. The amount of drug outside the dialysis bags during the determination of the entrapment efficiency and in the receptor compartment throughout the in vitro skin permeation experiments was determined by HPLC assay using methanol:0.0257M ammonium acetate (70:30 vol/vol) mixture as mobile phase delivered at 0.8 ml/min by LA-20A vp pump. A ten-microliter injection was eluted in C-8 column (4.6×250mm,5μm) and the temperature of the column was 30℃. The column eleunt was monitored at 270 nm using PDA UV detector.4. The examination of the prepared formulation by TEM revealed the predominance of spherical-shaped vesicles.5. The diameter particle was determined by Zetasizer. The size of the optimal formula is 31.33±2.63 nm, and the polydispersity index (PI) was 0.42±0.21.6. Repeated measure analysis of the absorbed dose of 5% lidocaine ethosome, liposome, and hydroethanol show:①The analysis results of repeated and interaction effect show:there were significant differences among the three formulations (F=110.970, P<0.001); there were significant differences among times (F=369.059, P<0.001); there were significant interaction effect between fomulations and skin permeation time (F=179.226, P<0.001);②Multiple comparison of the permeation accumulation dose of lidocaine ethosome, liposome,and hydreoethanolic solution show:the datas were not satisfied for homoscedasticity(F=71.654, P<0.001), so we used Welch to analyze. There was statistical significance between the three groups (F=19.001, P<0.001), and Dunnett's was used for multiple comparison.There was significant difference between lidocaine ethosome and liposome (P=0.001), there were statistical significance between lidocaine ethosome and hydreoethanol solution (P<0.001), and there were also statistical significance between lidocaine liposome hydreoethanol solution (P=0.0049);.③To regress the accumulated values (0.5-1lh) of lidocaine ethosome against time get linear equation:Q=421.664t-187.120 (R2=0.912, F=340.198,P<0.001), the permeation rate was421.7±22.86μg·cm-2·h-1;④To regress the accumulated values (0.5-11h) of lidocaine liposome against time get linear equation:Q=135.435t-73.837(R2=0.918, F=368.175, P<0.001), the permeation rate was 135.435±7.058μg·cm-2·h-1;⑤To regress the accumulated values (0.5-11h) of lidocaine hydroethanol solution against time get linear equation:Q=46.832t-0.750 (R2=0.735, F=91.753, P=0.000),,the permeation rate was 46.832±4.889ug·cm-2·h-1;⑥The skin permeation rate of licoaine was 3.113,and 9.004 times higher than that of lidocaine liposome,hydroethanol solution, respectively.7. Repeated mesure analysis of the negative reactions when lidocaine ethosome, liposome, hydroethanol solution acted on the integrated skin of guinea pigs show:①The analysis results of repeated and interaction effect show:there were significant differences among the three formulations(F=14.918, P<0.001); there were significant differences among times (F=863.566, P<0.001); there were no significant interaction effect between fomulations and skin permeation time (F=2.401, P=0.101);②Multiple comparison of the negative reaction when lidocaine ethosome, liposome, and hydreoethanolic solution were used on the back skin of guinea pigs show:the datas were satisfied for homoscedasticity (F=1.638, P=0.199).There was statistical significance between the three groups (F=7.696, P=0.001), and LSD method was used for multiple comparison.There was significant difference between lidocaine ethosome and liposome (P=0.004), there were statistical significance between lidocaine ethosome and hydreoethanol solution (P<0.001), but there were no statistical significance between lidocaine liposome hydreoethanol solution (P=0.443);Repeated mesure analysis of the negative reactions after lidocaine ethosome, liposome, hydroethanol solution acted on the integrated skin of guinea pigs for 1h and then removal these three drugs show:①The analysis results of repeated and interaction effect show:there were significant differences among the three formulations (F=226.925, P=0.000); there were significant differences among times (F=106.462, P=0.000); there were significant interaction effect between fomulations and skin permeation time(F=3.335, P=0.040);②ultiple comparison of the negative reaction when lidocaine ethosome, liposome, and hydreoethanolic solution were used on the back skin of guinea pigs for 1h and then removal the drugs show:the datas were satisfied for homoscedasticity (F=1.6382.323, P=0.102).There was statistical significance between the three groups (F=128.489, P<0.001), and LSD method was used for multiple comparison. There was significant difference between lidocaine ethosome and liposome(P<0.001), there were statistical significance between lidocaine ethosome and hydreoethanol solution (P<0.001), but there were no statistical significance between lidocaine liposome hydreoethanol solution (P<0.001);8. The stability profiles of lidocaine loaded ethosomal formulations evaluated for entrapment efficiency of drug at various temperatures suggested the storage of the novel formulation.①nalysis of covariance was used to analyze the stability of lisocaine ethosome. Exclusion the effect of basic entrapment efficiency of 60 days ago on that of 60 days later, there was significant difference among different temperatures (F=58.190, P<0.001), and basic entrapment efficiency of 60 days ago was no statistical significance against that of 60 days later;②The data did not satisfy homoscedasticity (F=33.496, P＜0.001), so use Welch method to analyze. There were statistical significance (F=205.640, P<0.001);③Use Dunnett's T3 method to compare the entrapment efficiency of lidociane ethosome.And there was significant difference between under the conditions of 4±1℃and 37±1℃against the entrapment efficiecy of 60 days ago (P＜0.001, P= 0.048, respectively), but there was no statistical significance under the condition of 25±1℃against the entrapment efficiecy of 60 days ago (P=1). The drug leakage of lidocaine under the condition of 4±1℃was maximum, followed by 37±1℃and 25±1℃minimum.9. Measurement of erythema and edema scores upon exposure of hairless guinea pigs skin to lidocaine ethosome revealed that there was no significant erythoma and edema. While lidocaine ethosome treated on the integrated and unintegrated skin for 1h, the basic structure of skin epiderm was still complete, and the stratum comeum was changed to be thinner. At the meantime, the stratum comeum of unintegrated skin was damaged at some degree. While for 24h, the basic structure of skin epiderm was still complete, and the stratum comeum of unintegrated skin was recovered at some degree. While for 48h, the structure of skin epiderm was integrated, and the recovery of the stratum comeum of unintegrated skin was visible. While for 72h, the stratum comeum of both kinds of skin was as same as the normal skin. At the time of 1,24,48, and 72h, there was no visible heterophil granulocyte infiltration, vasodilation hyperemia, inflammatory cell infiltration around the blood vessel, vasculitides, dermal oedema, or proliferation and fibrosis of fibrolast and so on.Conclusion1.5% lidocaine ethosomes have a spherical structure with homogeneous size distribution, small size diameter, small polydispersity index, high entrapment efficiency, and stable thermodynamics under room temperature;2. The study of in vitro transdermal permeation reveals that the permeation efficiency of 5% lidocaine ethosome was much faster than 5% lidocaine liposome and ethanol water solution; the main fact in the formulation to affect the transdermal delivery of ethosome was ethanol, and the co-operation of ethonal, egg phosphatidyl choline and the skin;3. The study of in vivo activity of local anesthetic demonstrate that 5% lidocaine has a shorter onset time and longer duration of local anesthetic than that of 5%lidocaine liposome and ethanol water solution;4. The appearance and the pathological manifestation of the skin which was treated by 5%lidocaine ethosome instruct that 5%lidocaine ethosome has a satisfactory safety for skin usage;5.5% lidocaine ethosome as a novel local anesthetic pharmaceutical preparation will be much worth to investigating in the future. |
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