The Microneedles Array Technology Enhances The RhEGF Percutaneous Penetration

ObjectiveThe epidermal growth factor,as one of the earliest discovered cell growth factor,has been widely applied to various kinds of cosmetics.Because of the skin barrier function,most of the epidermal growth factor in the cosmetics can't percutaneous penetration.The purpose of this paper is to research through the microneedle technology,improve the epidermal growth factor transdermal absorption,thereby increasing its physiological effects,in order to achieve satisfactory cosmetic effect,but also for other macromolecular drug percutaneous penetration provides a good method.Materials and methods1.After shaving hair,taking the full skin,the skin before and after pruning the subcutaneous and the skin after microneedle acupuncture to stain with HE,observing the skin bleeding and the subcutaneous pruning level by eyes,observing the integrity of the epidermis,the pruning level and the depth of needling histologically.2.The rats were divided into 5 groups: normal saline group,10.0mg / L rhEGF group,microneedle + 1.0mg / L rhEGF group,microneedle + 10.0mg / L rhEGF group.,Microneedle +20.0 mg / L rhEGF group.The content of EGF in the solution was determined by rhEGF ELISA KIT method.The cumulative permeation and cumulative permeability of the drugs were calculated at 0.5h,1h,2h,4h,6h and 8h after the application.Confirmed that the use of microneps after rhEGF obtained a good penetration effect.3.Three rats weighing about 200 g were weighed and cultured for 24 h,and intraperitoneally injected with chloral hydrate(0.003 m L · g-1)for anesthesia.A pair of symmetrical parts on both sides of the spine of the rats were drawn with a diameter of about 0.015 m,and the left and right sides were treated with a microneedle array.After 1 min treatment with 5 N pressure,3 were stained with gentian violet,photographed and visualized Erythema and edema appear and cuticle recovery.4.Gentian violet staining,gross and histological observation: SD rats treated by operation 3,with gentian violet staining,photographed,visual observation and record whether the skin with erythema and edema and cuticle recovery.All the layers were cut with HE staining and histological changes were observed.5.Calcein dyeing experiment: covering with the calcein cotton ball,dyeing 10 min in dark place,removing the calcein cotton,then using saline solution and alcohol cotton to wipe 2 times alternately.Taking full thickness skin and triming the subcutaneous tissue,then putting the skin on microscope slides and observing under the fluorescence microscope with the excitation wavelength is 490 nm and the emission wavelength is 515 nm,recording the number of spots,as well as obtaining fluorescence image.6.10 SD rats were prepared,weighing about 200 ± 30 g,divided into 2 groups,each group of5,according to the operation of 3 treatment.The rats in the first group were given 10.0mg / L rhEGF group.The left side was labeled with 0.5h,1h and 2h from the lateral side to the tail,and the right was labeled 4h,6h and 8h from the beginning to the end.Drugs to the designated time point after cutting the skin full layer and subcutaneous meat membrane are pulp.The second group was the microneedle + 10.0mg / L rhEGF group.The left side was marked with 0.5h,1h,and 2h from the lateral side to the tail.The right side was marked 4h,6h and 8h from the beginning to the end.Micrometer 5N pressure,the immediate external application of 10.0mg / L rhEGF until the specified time,take smear parts of the skin full layer,the whole layer of skin and subcutaneous meatus layers were separated,respectively,homogenate,using rhEGF ELISA KIT determination of skin and subcutaneous meat Membrane rhEGF concentration.7.The data of EGF of skin and dartos were expressed as mean±SD.The statistical analysis was calculated by SPSS19.0,all experiment groups were comparied with each other comprehensively by using SNK-q test(n = 5,P <0.05).Results1.Rats on both sides of the spine hair shaved clean,no scars and bleeding points,feeding 24 h,the stratum corneum intact,skin barrier structure is sound.After pruning to retain the dermis,no subcutaneous muscle and adipose tissue.Micro-needle array acupuncture after rat skin,the stratum corneum is destroyed,the micro-needle penetrates into the depth of the dermis.Over time,the rat skin gradually healed,in 12 h ~ 16 h skin healing,cuticle integrity recovery.2.The rhEGF group had better transdermal effect compared with rhEGF group.After administration of 0.5h in the normal saline group can be measured trace EGF,indicating the normal presence of EGF in the skin.RhEGF group,micro-needle + 1.0mg / L rhEGF group,micro-needle + 10.0mg / L rhEGF group,microneedle + 20.0mg / L rhEGF group compared with the normal saline group can be measured rhEGF P <0.05.RhEGF group,microgel + 1.0mg / LrhEGF group,micro-needle + 10.0mg / L rh EGF group,microneedle + 20.0mg / L rhEGF group with time migration rhEGF compared with the last time point Increased throughput,P <0.05.The penetration rate of rhEGF group was 10.0mg / L rhEGF group compared with 10.0mg / L rhEGF group.The permeation rate of rh EGF group was 10.0mg / L rhEGF group at 2h,4h,6h and 8h respectively.Of 9.485,19.989,12.768,6.994 times(P <0.05).The transdermal dose per unit time and the transdermal rate per unit time reached the peak at 2 ~ 4h unit time,and the cumulative transdermal volume and cumulative transdermal rate of each group were significantly higher than those of the control group(P <0.05)Time is slowly increasing.The cumulative transdermal rate of each group was less than 1%.3.Microneedle acupuncture 8h still visible gentian violet residue on the stratum corneum,16 h after the basic lack of gentian violet residue.It is suggested that the healing time of micropores is8 ~ 16 h.4.Calcium chlorophyll staining: micrometer treatment of skin microporous healing process,calcein through the amount of reduction,fluorescent spot smaller,12 h when the weak visible,16 h can not see the fluorescent spot.5.Trauma healing histological observation: roller microneedle treatment of skin barrier damage,deep dermal superficial,with the healing time,microporous shrinkage,smaller size.To16 h,the micropores were covered with proliferating epidermal cells,and the skin barrier was basically restored.6.The content of EGF in the skin tissue of the micro-needle + 10.0mg / L group and 10.0mg/ L group was higher than that in the normal skin at 2h,4h,6h and 8h(P <0.05).The content of EGF in the skin of the micro-needle + 10.0mg / L group and 10.0mg / L group reached the peak at 4 h,which was 1.92 times of normal skin(P> 0.05).7.10.0 mg / L rh EGF group,microneedle + 10.0 mg / L rh EGF group was much higher than normal skin(P <0.05)at most time points.The content of EGF in the tissue of micelles + 10.0mg /L rhEGF group was higher than that in 10.0mg / L rhEGF group(P <0.05).Conclusions1.After the microneedle treatment,the amount of skin in the skin and the myometrium increased and reached the physiological concentration of promoting growth,but the cumulative infiltration was less than 1%.2.Microneedle caused by micro trauma healing within 16 h,the skin barrier function recovery.3.The content of EGF in the skin tissue of the micro-needle + 10.0mg / L group and the10.0mg / L group was higher than that of the normal skin at 2h,4h,6h and 8h,and reached the physiological effect of EGF.