Controlled intra- and transdermal protein delivery using a minimally invasive Erbium:YAG fractional laser ablation technology

Abstract

The aim of the study was (i) to investigate the feasibility of using fractional laser ablation to create micropore arrays in order to deliver proteins into and across the skin and (ii) to demonstrate how transport rates could be controlled by variation of poration and formulation conditions. Four proteins with very different structures and properties were investigated – equine heart cytochrome c (Cyt c; 12.4kDa), recombinant human growth hormone expressed in Escherichia coli (hGH; 22kDa), urinary follicle stimulating hormone (FSH; 30kDa) and FITC-labelled bovine serum albumin (FITC-BSA; 70kDa). The transport experiments were performed using a scanning Er:YAG diode pumped laser (P.L.E.A.S.E.®; Precise Laser Epidermal System). The distribution of FITC-BSA in the micropores following P.L.E.A.S.E.® poration was visualised by using confocal laser scanning microscopy (CLSM). Porcine skin was used for the device parameter and CLSM studies; its validity as a model was confirmed by subsequent comparison with transport of Cyt c and FITC-BSA across P.L.E.A.S.E.® porated human skin. No protein transport (deposition or permeation) was observed across intact skin; however, P.L.E.A.S.E.® poration enabled total delivery after 24h of 48.2±8.9, 8.1±4.2, 0.2±0.1 and 273.3±30.6μg/cm2 for Cyt c, hGH, FSH and FITC-BSA, respectively, using 900 pores/135.9cm2. Calculation of permeability coefficients showed that there was no linear dependence of transport on molecular weight ((1.6±0.3), (0.1±0.05), (0.08±0.03) and (0.9±0.1)×10−3cm/h, for Cyt c, hGH, FSH and FITC-BSA, respectively); indeed, a U-shaped curve was observed. This suggested that molecular weight was not a sufficiently sensitive descriptor and that transport was more likely to be determined by the surface properties of the respective proteins since these would govern interactions with the local microenvironment. Increasing pore density (i.e. the number of micropores per unit area) had a statistically significant effect on the cumulative permeation of both Cyt c (at 100, 150, 300 and 600pores/cm2, permeation was 11.2±2.4, 15.3±11.8, 33.8±10.5 and 51.2±15.8 4μg/cm2, respectively) and FITC-BSA (at 50, 100, 150 and 300pores/cm2, it was 58.5±15.3, 132.6±40.0, 192.7±24.4, 293.3±76.5μg/cm2, respectively). Linear relationships were established in both cases. However, only the delivery of FITC-BSA was improved upon increasing fluence (53.3±22.5, 293.3±76.5, 329.6±11.5 and 222.1±29.4μg/cm2 at 22.65, 45.3, 90.6 and 135.9J/cm2, respectively). The impact of fluence – and hence pore depth – on transport will depend on the relative diffusivities of the protein in the micropore and in the ‘bulk’ epidermis/dermis. Experiments with Cyt c and FSH confirmed that delivery was dependent upon concentration, and it was shown that therapeutic delivery of the latter was feasible. Cumulative permeation of Cyt c and FITC-BSA was also shown to be statistically equivalent across porcine and human skin. In conclusion, it was demonstrated that laser microporation enabled protein delivery into and across the skin and that this could be modulated via the poration parameters and was also dependent upon the concentration gradient in the pore. However, the role of protein physicochemical properties and their influence on transport rates remains to be elucidated and will be explored in future studies.