Mechanism of the synergic effects of calcium chloride and electroporation on the in vitro enhanced skin permeation of drugs

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Abstract

We have already reported the substantial synergic effects of CaCl2 and electroporation (EP) on in vitro skin permeation of calcein and FITC dextrans. In the present paper, we investigated the mechanisms for these effects by considering changes in lamellar structure and barrier recovery time of the biggest skin barrier, the stratum corneum, by this combined treatment. The change in skin lamellar structure was evaluated by lipid mobility in the stratum corneum using ATR–FTIR, calcein release from stratum corneum-lipid liposomes (SCLL), in vitro skin permeation of calcein and transepidermal water loss (TEWL). The ATR–FTIR measurement, in vitro skin permeation and changes in TEWL were also used for examining the barrier recovery time.

The CH stretching band of skin lipids produced with EP was blue-shifted when compared to that without EP. Asymmetric CH stretching was highest with EP in CaCl2 solution. Little release of calcein was observed from SCLL without EP, whereas higher releases were observed after EP with or without NaCl or CaCl2. Particularly high calcein release (>20%) was observed over 60 min with EP in CaCl2 solution. The in vitro permeation study of calcein was conducted through excised hairless rat skin that was pretreated with EP before skin excision. Permeation rate was highest in skin excised immediately after in vivo EP, and this rate decreased with time after EP treatment. TEWL recovered to control levels within 2 h after EP in distilled water or NaCl solution, whereas high TEWL was maintained after EP in CaCl2 solution.

These results suggest that at least lamellar destruction of stratum corneum must be related to the enhanced skin permeation of drugs by the combination of CaCl2 and EPF. On the other hand, a prolonged enhancing effect on the skin permeation of calcein by this combination may be due to a high lamellar destruction and/or delayed barrier repair of stratum corneum.

Introduction

Topical administration of therapeutic drugs to improve the quality of life (QOL) of patients has numerous advantages. It is difficult, however, to efficiently deliver effective doses of therapeutic drugs through the skin into systemic circulation because the stratum corneum, the uppermost layer of skin, acts as large barrier to the ingress of exogenous materials, such as therapeutic drugs, and the egress of endogenous compounds, such as water. The stratum corneum has a thickness of 10–15 μm and consists of keratinocytes surrounded by an extracellular milieu of lipids organized to have multilamellar bilayer structures. One must overcome these intercellular lipid bilayers in order to use skin as an effective application site for drugs. Various investigations have already been conducted in an effort to improve skin permeation of drugs into or through the stratum corneum using several techniques; application of chemical enhancers [1], sonophoresis [2], iontophoresis [3], use of prodrugs [4] and most recently, electroporation (EP) [5].

Electroporation is thought to increase skin permeation of drugs by creating new pores or pathways in the stratum corneum. We previously reported that the penetration-enhancing effects of electroporation were closely related to the electric field produced in the stratum corneum [6]. We also reported that electrolytes in the drug solution had a substantial effect on the skin permeation of calcein and FITC dextrans in in vitro permeation experiments using excised hairless rat skin [7], [8]. Interestingly, large molecular FITC dextran (MW=35,600) was able to permeate the skin when a combination of CaCl2 and electroporation was used [8].

In the present study, the mechanisms of transdermal enhancement by combined treatment of CaCl2 and electroporation was investigated. To this end, the synergic effects were examined by measuring changes in skin lamellar structure and barrier repair time. Changes in the skin lamellar structure were evaluated based on lipid motility in the stratum corneum using ATR–FTIR and the release of calcein from liposomes composed of stratum corneum lipid (SCLL), in vitro skin permeability of calcein and measurement of transepidermal water loss (TEWL). In contrast, barrier recovery was evaluated by ATR–FTIR, in vitro skin permeability and TEWL.

Section snippets

Materials

Calcein sodium (MW=623, z=−4) was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). NaCl, CaCl2 and EDTA-2Na were obtained from Wako (Osaka, Japan). Phosphate-buffered saline (PBS) was purchased from Takara Shuzo (Osaka). Ceramide (Type 2), palmitic acid, cholesterol and cholesteryl sulfate were obtained from Sigma Aldrich (St. Louis, MO, USA). Other reagents were of analytical grade and were used without further purification.

Animals

Male hairless rats (HWY strain, 8–10 weeks old, 200–250 g in body

ATR–FTIR experiments

Table 1 shows the ATR–FTIR spectra for skin immediately (0 h), 6 h and 24 h after in vivo electroporation. CH bond stretching can be seen between 2800 and 3000 cm−1. Asymmetric stretching peaks (∼2920 cm−1) were broader and blue-shifted following electroporation. CH asymmetric stretching vibrations without electroporation (control study) were observed at 2918.1 cm−1, whereas those immediately after electroporation in distilled water, NaCl or CaCl2 solution were observed at 2918.9, 2920.9 or

Discussion

The mechanism responsible for the synergic effects of CaCl2 and electroporation was examined by measuring changes in the skin lamellar structure and barrier repair time. Skin lamellar structure changes were examined by evaluating motility of stratum corneum lipids using ATR–FTIR and release of calcein from SCLL, in vitro skin permeability of calcein and TEWL. The ATR–FTIR, in vitro skin permeability and TEWL were also used for examining the barrier recovery after electroporation.

Motility of

Conclusion

Electroporation with CaCl2 solution altered the barrier function of skin, and the effect on the skin permeation was maintained for a few hours after the combined treatment. The maintained effect may be due to the high destroying effect on the skin barrier or delaying effect on the recovery of barrier function of stratum corneum. Each contribution was not cleared yet. However, this combination of CaCl2 and electroporation markedly increased the skin permeation of high and low molecular weight

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