Fractional laser as a tool to enhance the skin permeation of 5-aminolevulinic acid with minimal skin disruption: A comparison with conventional erbium:YAG laser

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Abstract

The aim of this study was to examine the in vitro skin delivery and in vivo protoporphyrin IX (PpIX) accumulation of topically applied 5-aminolevulinic acid (ALA) enhanced by a fractional laser pretreatment. This was achieved by applying an array of microscopic treatment zones (MTZ) to the skin by ablation of superficial stratum corneum in a determined area. Re-epithelialization determined by transepidermal water loss was completed within 1 day after fractional laser irradiation. The conventional laser used in comparison showed more severe skin disruption and a greater recovery duration of 2 days. The in vitro ALA permeation was measured using a Franz cell apparatus, with nude mouse skin and porcine skin as the permeation barriers. The efficacy of the enhancement was determined as a function of various laser fluences (2 and 3 J/cm2) and number of passes (1–6 passes). The flux of ALA via laser-treated nude mouse skin was 27–124-fold higher than that across intact skin. A 3–260-fold increase in ALA flux was detected by using the porcine skin as the permeation barrier. The skin permeation was also investigated in a model of hyperproliferative skin obtained by repeated tape stripping. The results showed that the hyperproliferative skin was more permeable to ALA in comparison to the normal skin. The in vivo localization of PpIX in nude mouse skin was imaged using confocal laser scanning microscopy. As expected, an intense red fluorescence was observed in the lower epidermis and upper dermis after fractional laser irradiation. The penetration depth was also increased by the laser. The safety and efficacy of enhancing ALA permeation were demonstrated by using the fractional laser at low fluences.

Graphical abstract

Fractional erbium:YAG laser ablation on stratum corneum for enhancing 5-aminolevulinic acid permeation via the skin with less skin disruption in comparison to the conventional laser.

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Introduction

The range of medical and surgical laser applications is expanding rapidly. Laser skin resurfacing is an effective treatment option for many patients with cutaneous photodamage, wrinkles, rhytides, and acne scarring [1], [2]. For this kind of procedure, skin remodeling is initiated by controlled ablation to the skin. Resurfacing lasers at low fluence can also promote drug delivery via the skin through precise control of stratum corneum (SC) removal [3], [4], [5]. Despite these benefits, the epidermal ablation affected by these procedures can result in a skin irritation, prolonged erythema, and an extended postoperative recovery period [6]. As a result of these risks, interest in less invasive methods of effective skin treatment has grown. Fractional laser treatment is a relatively new procedure accomplished by the placement of numerous microscopic zones of damage in the skin surrounded by islands of normal tissue [7]. Since this laser system resurfaces the skin 5% to 20% at one time and does not cause full epidermal wounds; healing time is minimized [8], [9]. Our previous study suggested that the erbium:yttrim–aluminum–garnet (Er:YAG) laser can effectively enhance and control topical/transdermal drug delivery [10], [11], [12], [13]. Under these conditions, the skin had recovered to a normal status 3–5 days after Er:YAG laser treatment. The aim of this work was to assess the skin disruption and feasibility of the fractional laser for enhancing topical drug delivery. A conventional Er:YAG laser was utilized for comparison.

The model drug used in this study was 5-aminolevulinic acid (ALA), a metabolic precursor to protoporphyrin IX (PpIX) in the heme biosynthetic pathway. Subsequent irradiation of photodynamic therapy (PDT) leads to singlet oxygen production and free radicals, causing cellular damage and tissue necrosis [14]. It has been reported that ALA-PDT is useful for treating superficial skin cancers, actinic keratoses, psoriasis, cutaneous T-cell, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC) [15], [16]. Because ALA is a polar compound, the permeability across the skin is low, making it difficult to achieve the desired targets in the skin tissues [17], [18]. Several methodological proposals addressed this problem through the use of iontophoresis, micro-needles, permeation enhancers, and ester prodrugs [19], [20]. A 2–400-fold increase in ALA permeation can be detected by using different approaches. The laser ablation of SC may be useful for ALA for increasing skin permeation characteristics.

In the present study, the structural and ultrastructural alteration of the skin after fractional laser treatment was examined by gross observation, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Lasers with various wavelengths is recently designed to produce fractional mode, including Er glass laser (1540 nm), Er-doped fiber laser (1550 nm), Er:YAG laser (2940 nm), and CO2 laser (10,600 nm) [6], [7], [8]. A fractional Er:YAG laser (MCL 30 Dermablate) was selected to enhance and control ALA permeation in this work. The safety of the laser and recovery of skin barrier was examined within physiologic parameters by measuring transepidermal water loss (TEWL). This study used an in vitro Franz cell to evaluate the skin permeation of ALA by laser treatment. Both nude mouse skin and porcine skin were used as the permeation barriers in the present study. Moreover, hyperproliferative skin induced by a tape-stripping method was used as skin barrier model for drug permeation in order to mimic an actual clinical therapy situation [21]. In the in vivo study, the distribution of PpIX in nude mouse skin was monitored using confocal laser scanning microscopy (CLSM). All laser fluences tested in this work used lower energies than those utilized in clinical situations.

Section snippets

Laser assembly

The fractional Er:YAG laser (MCL 30 Dermablate, Asclepion Laser Technologies, Jena, Germany) has a wavelength of 2940 nm and a pulse duration of 400 μs. An articulated arm was used to deliver the laser beam onto the skin surface. The handpiece was able to create microscopic columns of skin ablation, namely, microscopic treatment zones (MTZ). Typical MTZs had a diameter of 250 μm. The occupied area of one irradiation dot was about 0.05 mm2. The dimension of the treatment area of the handpiece was 13 ×

Structural and ultrastructural examination of the skin

Nude mouse skin was exposed to the laser to determine the effects on the skin structure. The fluences used to enhance ALA permeation were 2 and 3 J/cm2. Under these low energies, no observable disruption of skin surface could be seen by the naked eyes. A higher fluence of 10 J/cm2 was irradiated on the skin surface so as to see the gross appearance of fractional laser dots photoed by a digital camera. At this fluence, as shown in the bottom of Fig. 1A, the skin was visibly disrupted. One pass of

Discussion

The permeability of hydrophilic ALA via intact skin is always low [16], [17], making it difficult to achieve desired therapeutic benefits. The Er:YAG laser is an ablative tool for the SC capable of precise control. Although the SC-stripping technique can also remove the SC, the area and depth of the treated SC cannot be precisely modulated; its safety and recovery ability are also questionable. Er:YAG laser can ablate the SC in a controlled manner by modulating the irradiated energy and area [5]

Conclusion

In conclusion, ALA permeation can be effectively enhanced by ablating the SC with a low-fluence fractional laser. A correlation between in vitro ALA flux and in vivo PpIX accumulation in skin has been demonstrated. The disruption to skin tissue by the fractional Er:YAG laser was limited and transient. The skin barrier function recovered to a normal status within 1 day. On the basis of the present results it may be suggested that the fractional laser is a potential enhancement method for skin

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