The Mechanism Of Transdermal Delivery Of Nanoemulsion And The Study Of Lidocaine Nanoemulsion As An Local Anesthesia Dosage Form

BACKGROUNDA new skin local anesthesia praeparatum which can provides fast and valid effect, low side effect and maintains the operation time is urgently needed in clinic. It will be used to satisfied the analgesia demands of skin superficial traumatic occlusion pain caused by skin penetrative examination, skin surgery operation and skin laser cosmetology. While those dosage forms such as EMLA cream, tincture, gel and cream can't achieve the ideal anesthesia goal of safety and effect because of their obvious side effects or low transdermal penetrative efficiency.Nanoemulsion (NE) has many features such as easy be prepared, stability, obvious solubilization, antibosis and peculiar transdermal feature, so it has been becoming one hot spot of TDDS researchs. The main reason to prevent nanoemulsion applied on skin because of its formula, especially the surfactants reconstituents are trend to induce irritation to skin, while some related researches have pointed out that many surfactants are closely with the singular transdermal efficiency of nanoemulsion. So those researches should be carried out such as reasonable reconstituents like nonionic wetting agents mixed with other reconstituents according to fixed proportions, then its transdermal mechanism is researched systematically such as what reconstituents is the main factor in transdermal penetration progress and its effect mechanism, how about the effect rule of the nanoemulsion effect on skin and the relative evaluation between its transdermal permeation efficiency and irritation, etc. Those researches will be fundament to explore new nanoemulsion transdermal praeparatum based on this nanoemulsion formula. The researches at present trended to compare the penetration efficiency between nanoemulsion with other praeparatums., but the transdermal mechanism and the main role between skin penetration and its irritation still have not been researched systematically by now.Based on the researches of the transdermal mechanism which formula has been identified, the anesthesia drug lidocaine which has many features such as strong diffusion ability, long time of therapy, safety with low irritation was used and new nanoemulsion preparing artwork which can lower the surfactants content in nanoemulsion was explored, then the stability, quality control, penetration ability, skin irritation and transdermal mechanism of nanoemulsion were researched systematically, then new skin local anesthesia drug with fast and ideal effect, low skin irritation, without sensitization and good effect is desired to be prepared successfully.OBJECTIVETo definite the reconstituents of nanoemulsion formula, optimize the preparation artwork and analyze the physico-chemical properties of blank nanoemulsion, fluorescein OB-nanoemulsion and lidocaine nanoemulsion. To analyze the nanoemulsion effect on the skin ultrastructure and find the main factor in nanoemulsion and explore its mechanism. To investigate the penetration ability, path and dynamic distribution role in skin of animal in vivo. To analyze the quality and stability of lidocaine nanoemulsion. To compare the penetration ability ofnanoemulsion with other preparation. To evaluate the skin irritation and the mainfactor of nanoemulsion through cytology experiment and living animal skin test; Toanalyze the drug action of nanoemulsion initially. In these studies, we aim to clarifythe transdermal effect mechanism of nanoemulsion and to evaluate lidocainenanoemulsion.METHODS一,Formula Definition of blank nanoemulsion, fluorescein nanoemulsion andlidocaine nanoemulsion and investigation of their physico-chemical properties.The reconstituents of nanoemulsion formula were identified, pseudoternary phase diagrams and origin 7.0 software were used to identify the fittest Km value (surfactant/co-surfactant) of blank nanoemulsion, fluorescein nanoemulsion, lidocaine nanoemulsion and their ratio composition of reconstituents, the drop size, distribution, zeta electronic potential will be observed through Malwen Zetasizer, the shape of nanoemulsion will be observed through electronic microscope and the system type will be determined through staining method.二,The skin penetration mechanism research of blank nanoemulsion.The mice were divided into blank nanoemulsion, negative control and blank control teams. The abdominal skin of mice treated with different drugs for 2 and 6h respectively, then the skins were dehydrated by 70～100% alcohol, part of skins were dyed by CO₂ and sprayed with colloidal gold particles, then those skins were observed through SEM. The other skins were made into ultrathin section and were double-dyed by uranyl acetate and lead citrate, then skins were observed through TEM. Then stratum corneum structure changes of those skins treated with different praeparatums were compared with each other and the prickle cell distances among them were analyzed with Image Pro 5.0 software. 三,Visualization and quantitation research of skin permeation of nanoemulsion in vivo.Male wistar rats were divided into three teams of nanoemulsion, tincture, IPM and Labrasol/Plurol Oleique solution which containing the same concentration of OB, then the blank control of nanoemulsion and tincture were set. Drugs were applied on skins before skins were moved from the rat for 0.5 and 2h, then skins were made into frozen sections, which containing epidermis, upper dermis, lower dermis and skin appendages (hair follicle, sweat glands and sebaceous glands), then the frozen sections were scanned and analyzed through CLSM. The fluorescein in skins at different times were visualized, analyzed and collected through software installed in the CLSM machine, the fluorescein intensity in skins were quantitative analyzed and compared with each other.å››,The quality control and stability research of lidocaine nanoemulsion.The lidocaine nanoemulsion analysis condition and method through HPLC were researched, the quality control study of lidocaine nanoemulsion was carried out through HPLC and those indexes of illumination, temperature, air exposure and accelerated test of lidociane nanoemulsion were analyzed.五,The transdermal experiment research of lidocaine nanoemulsion in vitro.Improved Franz diffusion cell and rat skins ex vivo as transdermal model were explored, HPLC was used to determined the lidocaine content in the receptive liquid. The accumulative transdermal absorption quality Q values and skin penetration ratios of 5% lidocaine nanoemulsion, 5% lidocaine gel and 5% lidocaine tincture were compared with and the permeation process type of lidocaine nanoemulsion was analyzed.å…­,The skin irritation experiment research of lidocaine nanoemulsion.(一) The irration of lidocaine nanoemulsion to Hacat cell was determined through MTT method, the IC₅₀ values of lidocaine nanoemulsion and its main reconstituentswere calculated and the main factor which induced irritation were analyzed.(二) Federal hazardous substances act (FHSA) was used to evaluated the irration oflidocaine nanoemulsion to integrated and un-integrated skins of rabbits.(三) The histology changes of lidocaine nanoemulsion to integrated and un-integratedskins were researched through histopathology七,The initial drug action experiment research of lidocaine nanoemulsion.The initial drug action experiment research of lidocaine nanoemulsion was carried out through the back skin of cavia infiltrating method and compared the drug action with that of EMLA.RESULTS一,Formula definition of blank nanoemulsion, OB fluorescein nanoemulsion and lidocaine nanoemulsion and investigation of their physico-chemical properties.pseudoternary phase diagrams was explored to definite the best fittest Km values of blank nanoemulsion, fluorescein nanoemulsion and lidocaine nanoemulsion are the same value of 3, the IPM:water:S/C ratios of nanoemulsion, fluorescein nanoemulsion and lidocaine nanoemulsion are 14%:36%:50%, 13%:40%:47% and 15%:40%:45%, the lidocaine content in lidocaine nanoemulsion is 5%. The average particle diameters of three nanoemulsions are 28.8, 65.4 and 29.8nm, the electric potential are 1.9, 2.6 and 3.2mv. The nanoemulsion drop shows their appearance are Spherical shape and belong to multi-disperses system, the types of three nanoemulsion are O/W types.二,The skin penetration mechanism research of blank nanoemulsionThe result of SEM shows that compared with the control of sodium chloride solution, the skin stratum corneum surface become tough and closed barrier is destroyed, the distances between prickle cells are enlarged and phenomenon of obvious striped fragments of stratum corneum can be observed. The result of TEM shows that skins treated with nanoemulsion and sodium chloride solution for 2 and 6h, the distance values between prickle cells were 2.69±0.08μm and 1.21±0.09μm, nanoemulsion enlarged the space between prickle cells more wider than that of sodium chloride solution(F=2278.400, P=0.000), the stratum corneum of nanoemulsion thinningz degree and layers space extension are more obvious, some junctions of prickle cells are breakdown.三,Visualization and quantitation research of skin permeation of nanoemulsion in vivo.After nanoemulsion and tincture have penetrated skin for 0.5h, the fluorescein degrees of nanoemulsion and tincture in the epidermis(F=2273.95, P=0.000), upper dermis(F=157092.5, P=0.000) and lower dermis(F=417890.3, P =0.000) have obvious difference, while the fluorescein in the skin appendages have quiet difference(F=0.394, P=0.539). After tincture, nanoemulsion, IPM and Labrasol/Plurol Oleique solution have penetrated skin for 2h, the fluorescein in the epidermis(F=9095.790, P=0.000), upper dermis(F=5058.656, P=0.000) and lower dermis(F=11040.87, P=0.000) have obvious difference, while the fluorescein in the skin appendages have quiet difference(F=0.337, P=0.799), the fluorescein of that four matters in the skin appendages of 0.5 and 2h have quiet difference(F=1.046, P=0.402). So the skin penetrating ability of nanoemulsion no only depend on one or some reconstituents but the whole system consists of all reconstituents. The result shows that the transdermal efficiency of nanoemulsion is more stronger than that of tincture, The hair follicle, sweat glands and sebaceous glands should play some important roles in the transdermal progress of praeparatums, the skin stratum corneum treated with nanoemulsion and Labrasol/Plurol Oleique solution are thinner than that of normal skin. å››,The quality control and stability research of lidocaine nanoemulsion.The color spectrum can be satisfied with the analysis demand of nanoemulsion and the specificity of HPLC can also satisfied the demand. The lidocaine content in nanoemulsion is 49.87±0.45μg·mL^-1, the time stability of nanoemulsion is ideal but if it is put in a place with highlight, high-temperature(>60℃) or low-temperature(<4℃), the nanoemulsion will be instability, so lidocaine nanoemulsion should be preserved in a place away form light and with common temperature.五,The transdermal experiment research of lidocaine nanoemulsion in vitro.The accumulative transdermal absorption quality Q values of 5% lidocaine nanoemulsion, gel and tincture have obvious difference(F=11742.52, P=0.000), nanoemulsion>gel >tincture. The skin penetrating rate of nanoemulsion, gel and tincture are 155.64±7.48,63.29±4.20 and 48.58±3.13μg·cm^-2·h^-1, the apparent skin penetrating rate of nanoemulsion is 2.46 and 3.20 times than that of gel and tincure, and the permeation process of nanoemuslion follows zero order release kinetics.å…­,The skin irritation experiment research of lidocaine nanoemulsion. (一) The inhibition rates between lidocaine nanoemulsion and Hacat cell have no obvious difference(P=0.878), while the inhibition rates among nanoemulsion, SDS, Labrasol and Oleique have obvious difference(P=0.000), the inhibition rate to Hacat cell of Labrasol is higher than that of nanoemulsion, SDS and Oleique. The IC₅₀ value of lidocaine nanoemulsion is obviously higher than that of SDS(P=0.049), while the IC₅₀ value of Labrasol is obviously lower than that of lidocaine nanoemulsion(P=0.004), Plurol Oleique(P=0.031) and SDS(P=0.001).(二) Lidocaine nanoemulsion and blank nanoemulsion have no irritation to skin of living animal. After skins were treated with lidocaine nanoemulsion, the results of histopathology shows that the epidermis is integrated and there have no such phenomenons of neutrophilic granulocyte infiltration, vasodilatation and Vascular engorgement, inflammatory cell infiltration around blood vessel, vasculitides, dermis edema and fibrocyte hyperplasia or fibrosis.七,The initial drug action experiment research of lidocaine nanoemulsion.The result shows that the negative reaction of integrated skin group and un-integrated skin group of cavia treated with nanoemulsion, EMLA and Sodium Chloride have no obvious difference(P=0.000), the negative reaction number of nanoemulsion is more than that of EMLA, while the negative reaction number of EMLA is more than that of sodium chloride(P=0.000). After integrated skin treated with drugs for 1h and un-integrated skin treated with drugs for 0.5h, the negative reaction number between lidocaine nanoemulsion and EMLA have no obvious difference(P=0.502), , while the negative reaction numbers of them are higher that of sodium chloride group(P=0.000).CONCLUSION.一,Pseudoternary phase diagrams combining with Origin softeware can be explored to optimize the formula of nanoemuslion and definite the reconstituents ratio; The drop size ,distribution , shape and system type can be observed through Maerwen Zetasizer combining with electron microscope; The physico-chemical properties of blank nanoemulsion, fluorescein nanoemulsion and lidocaine nanoemulsion is satisfied.二,The favourable skin penetration ability lies the whole system composed with all reconstituents, besides the hydration function of system, the surfactant of Labrasol and Plurol Oleique play a important role of striping skin stratum corneum to promote skin penetration, also, the hair follicle, sweat glands and sebaceous glands play some important roles in the skin penetration of preparations. 三,The formation, features and the skin penetration ability of nanoemulsion depend on category, as the surfactants is the main factor to induce irritation to skin, so it is the most important point to choose safe and valid surfactants.å››,Lidocaine nanoemulsion has good stability, but it should be preserved in a place away from light and with common temperature. The skin penetration ability of lidocaine nanoemulsion surpass than that of tincture and gel, the permeation process of nanoemuslion follows zero order release kinetics.五,The main skin irritation factor of lidocaine nanoemulsion is the Labrasol reconstituent, but the irritation of lidocaine nanoemulsion is lower than that of SDS which has been widely used in shampoo and detergent and has been regarded as the positive control of slight skin irritation, so it should be safe to apply the lidocaine nanoemulsion on skin, it will not induce irritation to skin and make the skin tissue pathology changed.å…­,The effect time of lidocaine nanoemulsion act on integrated and un-integrated skin is faster than that of EMLA, while the anesthesia maintaining time has no obvious difference with EMLA, So the result shows that the pharmacodynamics of lidocaine nanoemulsion is better than that of EMLA cream.