| [Background and Objective] Transdermal drug delivery system (TDDS) is an ideal route for drug administration with broad market prospects in the world. In recent years, a number of chemical and physical techniques have been developed to enhance biological macromolecules transport across the skin, ranging from chemical enhancers, nanotechnology, iontophoresis, ultrasound, electroporation, microneedles to thermal or radiofrequency ablation, and so on. However, when these innovative methods overcame the skin barrier and realize effective TDDS, skin was also irritated or damaged to various degrees. So the key challenge in TDDS field is to realize sufficient transport while impairing skin barrier in a reversible and safe manner. In this study, we chose fractional laser (Er:YAG,2,940nm, SCITON ProFractionalTM), a specialized skin resurfacing device, as a percutaneous absorption promoter by generating micro-thermal treatment zones in the skin. ProFractional was chosen for the advantages of little damage, rapid skin reconstruction and well practical control. Meanwhile, TRITC-conjugated Goat Anti-Mouse IgG (T-IgG), a water-soluble fluorescent antibody with Molecular Weight (MW) of150kDa, was served as a model biomacromolecule in this TDDS study. We evaluated the capability of ProFractional for transdermal delivery of T-IgG both in vitro and in vivo.[Methods] The dorsal skin of SD baby rats (3-5d) were chosen as skin model, and classical Franz diffusion cells was used to investigate the laser-assisted delivery of T-IgG across murine skin in vitro. The effects of laser fluence (0,12.5,25, and37.5J/cm2), pore density (0;5%,100pores/cm2;10%,210pores/cm2), as well as antibody concentration (0,0.5,1and2ug/ul) on T-IgG cumulative permeation dynamics, were also evaluated in2h,4h,6h,8h,20h and24h. The activity of T-IgG post-delivery in vitro for24h was further evaluated by Western blot. Meanwhile, the skin permeation of T-IgG in vivo at each time point (2h,4h,6h and24h) was visualized by confocal laser scanning microscopy (CLSM) and cryo-section from coronal and vertical plane, respectively. The fluorescence distribution in murine skin was evaluated to explore the transdermal route of T-IgG.[Results] Pathological results showed that the thickness of stratum corneum, viable epidermis, dermis, subcutaneous tissue and entire skin of SD rat (3-5d) was about20-40,30-50,90-150,80-100, and300-400μm, respectively. When murine skin was treated with fractional laser at different fluence (12.5,25, and37.5J/cm2), micro-channels were found in pathological paragraphs with different depth about50,100and150μm, respectively.Cumulative delivery of T-IgG across murine skin in vitro was0.282±0.013(2h),0.353±0.018(4h),0.430±0.029(6h),0.522±0.027(8h),0.746±0.108(20h) and0.748±0.083μg/cm2(24h) in intact skin group (laser non-treatment), which was significantly different to that in followed groups with fractional laser treatment (two-way ANOVA,P<0.001), but of no significance to PBS control group (the transport was mostly nonspecific protein in skin)(P>0.05), with0.213±0.021(2h),0.278±0.011(4h),0.342±0.039(6h),0.402±0.048(8h),0.581±0.032(20h) and0.665±0.027μg/cm2(24h), respectively.When laser fluence increased from12.5to37.5J/cm2, cumulative T-IgG delivery increased accordingly in24h. The cumulative T-IgG delivery in three fluence groups (12.5,25and37.5J/cm2,5%pore density,2ug/ul T-IgG15μl), was0.755±0.142vs.1.211±0.139vs.1.192±0.372(2h, P>0.05),1.769±0.419vs.2.345±0.398vs.2.157±0.411(4h, P>0.05),2.430±0.275vs.2.795±0.621vs.3.152±0.927(6h,P>0.05),2.841±0.295vs.3.189±0.454vs.4.299±1.629(8h, P>0.05),4.179±0.873vs.6.224±0.273vs.7.985±1.613(20h, P<0.001) and4.688±1.413vs.6.367±0.215vs.8.330±1.876μg/cm2(24h, P<0.001), respectively. When compared the effect of fluence at12.5to37.5J/cm2, total T-IgG delivery was significantly different at later stage (20h and24h), but of no significance at the first8h by two-way ANOVA.When the fluence was fixed at25J/cm2and pore density increased from5%to10%pores/cm2, the cumulative delivery of T-IgG was0.610±0.034vs.1.211±0.139(2h, P<0.05),1.128±0.077vs.2.345±0.398(4h, P<0.001),1.789±0.087vs.2.795±0.621(6h, P<0.001),2.124±0.115vs.3.189±0.454(8h, P<0.001),4.997±0.335vs.6.224±0.273(20h, P<0.001) and6.217±0.198vs.6.367±0.215p.g/cm2(24h,P>0.05), respectively. The cumulative delivery of T-IgG increased obviously within8h. Then the flux decreased gradually after8h and reach equilibrium at24h.When the fluence and pore density were fixed (25J/cm2,10%), increasing T-IgG concentration in the donor compartment from0.5to2μg/ul resulted in significant enhancement of T-IgG delivery across murine skin in whole24h, with0.656±0.266vs.1.211±0.139(2h, P<0.001),0.791±0.146vs.2.345±0.398(4h, P<0.001),0.998±0.188vs.2.795±0.621(6h, P<0.001),1.107±0.167vs.3.189±0.454(8h, P<0.001),1.914±0.666vs.6.224±0.273(20h, P<0.001) and2.439±0.804vs.6.367±0.215μg/cm2(24h,P<0.001), respectively. Increasing T-IgG concentration resulted in significant enhancement of T-IgG delivery in whole24h. Furthermore, the biological activity of T-IgG post-delivery for24h also retained to combine with murine IgG, which confirmed by western blot.After administration of T-IgG (2μg/ul,15μl) to intact murine skin in vivo, there was no red fluorescence signal either in a CLSM photograph or a cryo-section after2h,4h,6h and24h. Followed administration of T-IgG to laser treated murine skin for2h in vivo, the red fluorescence signal of T-IgG was negligible either in a CLSM photograph or a cryo-section too. But the fluorescence signal was then found in the micro-channels left by fractional laser in4h, and grew more intense and diffused around micro-channels after6h. Followed T-IgG administration for24h, T-IgG migrated about150μm depth in CLSM photograph and reached the dermis of SD rats, the fluorescence signal was diffused in whole skin, with the brightest signal in hair follicles. Meanwhile, most micro-channels shown clearly in first6h were healed and closed, and only few fuzzy micro-channels were found in both CLSM photograph and cryo-section in24h.[Conclusion] Fractional laser (Er:YAG) ablation is a safe, effective and controllable method for transdermal delivery of intact antibody with MW of150kDa. Furthermore, antibody transport is able to be controlled in the early phase, later stage and the whole period by adjusting pore density, laser fluence and antibody concentration, respectively. The micro-channels left by fractional photo-thermal effects are the transdermal pathway of how the fractional laser promotes antibody delivery. This safe and controllable approach will provide theoretical basis and research direction for noninvasive diagnosis and biological therapy. |
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