The world-wide transdermal drug delivery market is quite large, but only a small number of agents have FDA approval. The primary reason for such limited development of this market is the difficulty in permeating the stratum corneum layer of human skin. Compressed lipid bilayers comprise the continuum portion of this layer, which is the primary barrier in skin. To overcome the barrier function of stratum corneum, various physical and chemical methods have been tested to increase the permeability of the stratum corneum to drugs. However, few have succeeded in delivering relevant agents at the appropriate flux levels without causing notable skin irritation or damage .
In our study, we developed a novel percutaneous delivery enhancing approach. Magainin peptide, our novel candidate of enhancer, is a 23-residue helical peptide that exhibits broad spectrum of antimicrobial activity properties. It has a net +4 charge and binds to negatively charged phospholipid membranes with the aid of electrostatic interactions, forming an amphiphilic helix and permeabilizing the bilayers . Magainin peptide was previously shown to disrupt vesicles which are made from lipid bilayer components representative of those found in human stratum corneum and this ability of magainin allows us to propose that magainin can increase skin permeability . Therefore, we tested the hypothesis that magainin, a peptide believed to form pores in bacterial cell membranes, can increase skin permeability by disrupting stratum corneum lipid structure. We further hypothesized that magainin's enhancement requires co-administration of a surfactant chemical enhancer to increase magainin penetration into the skin. In support of these hypotheses, synergistic enhancement of transdermal permeation can be observed with magainin peptide in combination of N-lauroyl sarcosine (NLS) in 50% ethanol-PBS solution.
2. Experimental methods
Intact epidermis was isolated from dermis using the heat separation method. A piece of excised skin was placed in a vertical Franz diffusion glass cell and the 2 wt% of NLS solution(N-Lauroyl Sarcosine in 50% ethanol solution) or NLS and 100 μM magainin peptide mixture solution is placed in the donor chamber and the Franz cell is kept in the refrigerator at 4 oC for 12 h. A fluorescent compound (1 mM fluorescein) or real drug model (1mM Granisetron) in PBS solution (pH from 5 to 11) is applied to the stratum corneum side of the skin and the recovery of fluorescein or Granisetron every 1hr for 5hr in the receiver fluid on the epidermal side of the skin sample is then measured by spectrofluorimetry or HPLC.
The exposure to a known surfactant chemical enhancer, NLS, in 50% ethanol solution increased in vitro skin permeability to fluorescein 15 fold and the combination of magainin and NLS-ethanol synergistically increased skin permeability 47 fold. In contrast, skin permeability was unaffected by exposure to magainin without co-enhancement by NLS-ethanol. (Figure 1.C)
Figure 1. (A) The change of transition midpoint temperature(Tm) measured by DSC (B) CH2 (i) asymmetric and (ii) symmetric stretching frequency measured by FT-IR (C) transdermal penetration amount of fluorescein with NLS and Magainin peptide in 50% ethanol solution
The exact mechanism of this synergistic effect has not been elucidated, so several characterization methods were applied to investigate the mechanism of synergistic enhancement. Two endothermic transition temperatures in DSC related to the lipid packing were decreased by about 10oC, which shows that NLS and magainin peptide disrupt and increase the fluidity of the stratum corneum lipid. (Fig.1a) The exposure of stratum corneum to NLS and magainin caused an increase in lipid related carbon-hydrogen stretching frequencies, which suggest an increase in the lipid chain disorder and fluidity. (Fig.1b) From wide angle XRD (Fig 2A), we observed that NLS and magainin peptide reduced the crystalline cholesterol peak and from small angle XRD (Fig 2B), ceramide peak (d=6.13nm) and cholesterol peak (d=3.38nm) is reduced by NLS and magainin peptide. These analyses showed that NLS-ethanol disrupted stratum corneum lipid structure and that the combination of magainin and NLS-ethanol disrupted stratum corneum lipids even further.
Figure 2. (a) Wide angle X-ray scattergram and (b) small angle X-ray scattergram of human stratum corneum treated with NLS or NLS/magainin peptide combination relative to the control
Finally, skin permeability was enhanced by changing the charge of magainin peptide via pH change. We modulated pH from 5 to 11 to change the magainin charge from positive to neutral, which decreased skin permeability to a negatively charged model drug (fluorescein) and increased skin permeability to a positively charged drug (granisetron). This suggests that an attractive interaction between the drug and magainin peptide improves transdermal flux.
Figure 3. Enhancement of transdermal (A) fluorescein and (B) granisetron delivery as a function of pH with NLS (˘) or magainin + NLS (£) in 50% ethanol.
This is the first study to use a pore-forming peptide as a skin penetration enhancer and the novel approach to use one percutaneous enhancer to increase the penetration of another percutaneous enhancer into the skin. It is suggested that antimicrobial peptides can be used as efficient skin permeability enhancers in conjunction with other enhancers. By changing the pH of the drug solution, we can also control the drug delivery amount.
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