Laser-assisted topical drug delivery by using a low-fluence fractional laser: Imiquimod and macromolecules

Journal of Controlled Release 153 (2011) 240–248

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Laser-assisted topical drug delivery by using a low-fluence fractional laser: Imiquimod and macromolecules Woan-Ruoh Lee a,b, Shing-Chuan Shen c, Saleh A. Al-Suwayeh d, Hung-Hsu Yang a, Cheng-Yin Yuan e, Jia-You Fang d,e,f,⁎ a

Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei 110, Taiwan Department of Dermatology, Taipei Medical University-Shuang Ho Hospital, Taipei 235, Taiwan Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan d Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia e Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan 333, Taiwan f Department of Cosmetic Science, Chang Gung Institute of Technology, Kweishan, Taoyuan, Taiwan b c

a r t i c l e

i n f o

Article history: Received 25 June 2010 Accepted 13 March 2011 Available online 22 March 2011 Keywords: Fractional laser Imiquimod Peptides Macromolecules Skin permeation

a b s t r a c t The aim of this study was to evaluate the ability of a low-fluence fractional erbium:yttrim-aluminum-garnet (Er:YAG) laser, with a wavelength of 2940 nm, for enhancing and controlling the skin permeation of imiquimod and macromolecules such as polypeptides and fluorescein isothiocyanate (FITC)-labeled dextran (FD). The in vitro permeation has been determined using a Franz diffusion cell, with porcine skin and nude mouse skin as the barriers. Hyperproliferative and ultraviolet (UV)-irradiated skins were also used as barrier models to mimic the clinical therapeutic conditions. Confocal laser scanning microscopy (CLSM) was used to examine the in vivo nude mouse skin uptake of peptide, FITC, and FD. Both in vitro and in vivo results indicated an improvement in permeant skin delivery by the laser. The laser fluence and number of passes were found to play important roles in controlling drug transport. Increases of 46- and 127-fold in imiquimod flux were detected using the respective fluences of 2 and 3 J/cm2 with 4 pulses. An imiquimod concentration of 0.4% from aqueous vehicle with laser treatment was sufficient to approximate the flux from the commercial cream with an imiquimod dose of 5% without laser treatment, indicating a reduction of the drug dose by 125fold. The enhancement of peptide permeation was size and sequence dependent, with the smaller molecular weight (MW) and more-hydrophilic entities showing greater enhancing effect. Skin permeation of FD with an MW of at least 150 kDa could be achieved with fractional laser irradiation. CLSM images revealed intense green fluorescence from the permeants after exposure of the skin to the laser. The follicular pathway was significant in laser-assisted permeation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The actinic keratosis (AK) refers to a sun-induced clinical erythematous lesion covered with scales that has a histological diagnosis consistent with squamous cell carcinoma (SCC) in situ. It has the potential to progress to invasive SCC [1]. Imiquimod is a toll-like receptor-7 agonist that was shown to boost the cutaneous immune response. It belongs to a novel class of topical immune response modifiers that was approved in 1997 by the US Food and Drug Administration (USFDA) for treating external genital and perianal warts [2]. A cream containing 5% imiquimod was subsequently approved by USFDA in 2004 as a topical treatment for AK [3]. There is a growing body of evidence to support the efficacy of imiquimod for benign diseases, ⁎ Corresponding author at: Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, Taoyuan 333, Taiwan. Tel.: + 886 3 2118800x5521; fax: + 886 3 2118236. E-mail address: [email protected] (J.-Y. Fang). 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.03.015

such as melanomas, basal cell carcinoma (BCC), and Bowen's disease [4– 6]. With local application, adverse reactions are the greatest concern with imiquimod, which were reported by approximately 50% of subjects in 2 treatment groups [7,8]. Local skin excoriation, moderate to severe pain, and pruritus are frequently reported [9]. Hence a reduction in the dose would lessen such adverse events. Recent advances in biotechnology have given rise to large numbers of macromolecules such as peptides/proteins and small interfering (si)RNA, which may have therapeutic and preventive potential for AK. T4 endonuclease V is an example which can be topically applied to treat AK [10,11]. However, the permeability of large hydrophilic molecules across the SC is extremely low [12]. Moreover, T4 endonuclease V also shows potential side effects of skin irritation [13]. Improved drug permeation is desirable to lessen the required dose or produce biologically significant activity in many cutaneous therapeutic approaches. The stratum corneum (SC) is the main barrier against drug permeation via the skin. Removal of the SC by mechanical abrasion, tape-stripping, or chemical treatment has been

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shown to enhance permeation. However, these approaches are limited due to a lack of control and reproducibility, and the potential to cause pain and irritation [14]. Much interest has been shown in the use of lasers for drug permeation enhancement because of their precise control on the ablated skin depth and good definition of the ablated range by the device [15–17]. In our previous study [18], a 3–5-day recovery of the SC was necessary after erbium:yttriumaluminum-garnet (Er:YAG) laser treatment for permeation enhancement. As a result of this slow recovery, interest in less-invasive methods has grown. A fractional laser is a relatively new device which places numerous microscopic zones of treatment in the skin surrounded by normal tissue [19]. Since this laser system resurfaces 5% to 20% of the skin at one time and does not cause full epidermal wounds, healing times are minimized [20]. Our previous study suggested that the SC integrity can be completely restored within 1 day after use of a fractional laser [21]. The aim of this work was to control and enhance imiquimod and macromolecular delivery, which are beneficial for treating AK and other skin diseases, via the skin using a fractional Er:YAG laser with minimal skin disruption. This work utilized an in vitro Franz cell to evaluate the skin permeation of imiquimod by laser treatment. The feasibility of the fractional laser for increasing topical macromolecule delivery such as peptides and dextrans was also assessed in the present study. Both porcine skin and nude mouse skin were employed as permeation barriers. Porcine skin is a good model for human skin in terms of hair density and blood vessels distribution [22]. Although nude mouse skin is thinner than the human skin, it is advantageous because of the limited variability among individuals and a similar follicle density to that of human skin [23]. Hyperproliferative and ultraviolet (UV)-irradiated skins were also used as barrier models. It is important because that the healthy skin is inadequate for evaluating the drug skin delivery for the diseased skin in actual clinical therapeutic situations. In the in vivo experiment, the uptake of peptides and dextrans in nude mouse skin was monitored by confocal laser scanning microscopy (CLSM). All laser fluences tested in this work used lower or comparable energies as compared to those utilized for clinical therapy (2–32 J/cm2) [24]. These lower fluences only partly ablated the SC layers without affecting viable skin. 2. Materials and methods 2.1. Materials Imiquimod (1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine) with a molecular weight (MW) and log P (octanol-water partition coefficient) of 240.3 and 2.7, respectively, was purchased from LKT Laboratories (St. Paul, MN, USA). Fluorescein isothiocyanate (FITC) and FITC-labeled dextran (FD) with average MWs of 4 (FD4), 20 (FD20), 40 (FD40), 70 (FD70), and 150 kDa (FD150) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Polypeptides of the form NH2Arg-Leu-Ala-COOH (peptide-1, MW 716), NH2-Pro-Arg-Leu-Leu-TyrSer-Trp-His-Arg-Ser-His-Arg-Ser-His-COOH (peptide-2, MW 2190),

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and NH2-Pro-Arg-Leu-Leu-Tyr-Ser-Trp-His-Leu-Trp-Tyr-Leu-Trp-TyrCOOH (peptide-3, MW 2354) were synthesized by Bio Basic (Markham, Canada). FITC was conjugated to the -NH2 terminus of the peptides. A cream containing 5% imiquimod (Aldara®) was provided by 3M Health Care (Loughborough, UK). 2.2. Fractional 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, called microscopic treatment zones (MTZs). Typical MTZs had a diameter of 250 μm. The area of one irradiation dot occupied 0.05 mm2. The dimension of the treatment area of the handpiece was 13 × 13 mm2. This dimension had 169 irradiation dots (13 × 13) for each pass of either 2 J/cm2 or 3 J/cm2. The coverage of the MTZs in this area was 5% for 1 pass (0.05 mm2 × 169/ 13 × 13 mm2). In this work, the skin received 1–6 passes at a fluence of 2 or 3 J/cm2. The operator rotated the handpiece through determined angles to produce various passes in a dispersive mode without overlapping the irradiation area of each pulse as shown in Fig. 1. 2.3. Animals The animal experiment protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University. Animals were housed and handled according to institutional guidelines. Food and water were given ad libitum. Pathogenfree pigs (1 week old) were supplied by the Animal Technology Institute Taiwan (Miaoli, Taiwan). Female nude mice (ICR-Foxn1nu strain) aged 8 weeks were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The total number of the pigs and nude mice used in this study was 8 and 44, respectively. 2.4. Induction of hyperproliferative and UV-irradiated skins Epidermal hyperproliferation was achieved using a tape-stripping technique as previously described [25]. The dorsal skin of nude mouse was stripped using cellophane tape (3M Scotch®) twice daily for 5 days. The mouse was fixed on a table without any anesthesia. The stripping was repeated 10 times during each session. After 5 days, the skin was monitored by examining the transepidermal water loss (TEWL) with an evaporimeter (Tewameter TM300, Courage and Khazaka, Köln, Germany). The skin was excised for the in vitro experiment until the TEWL values reached 8–10 g/m2/h. To obtain UVB-irradiated skin, a Bio-Spectra System Illuminator (Vilber Lourmat, France) was used to emit UVB at a wavelength of 312 nm. This method was modified from Moore et al. [26]. Briefly, UVB was supplied by UVB lamp, which delivered uniform irradiation at a distance of 10 cm from the animals. Mice were exposed to a single

Fig. 1. Gross observations of laser irradiation on thermal paper with different, numbers of passes treated with fractional laser at 3 J/cm2 for 1, 2, 4, and 6 passes (left to right). The operator rotated the square handpiece by different angles to produce multiple passes (2, 4, and 6 passes) in the dispersive mode. The occupied areas of the microscopic treatment zones (MTZs) for 1, 2, 4, and 6 passes compared to the handpiece area (13 × 13 mm2) were 5%, 10%, 20%, and 30%, respectively.

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UVB dose of 150 mJ/cm2 for 1 min every day for 7 days and then killed after the last exposure. Based on a programmable microprocessor, the Bio-Spectra system constantly monitored the UV light emission. 2.5. Skin histological examination After laser irradiation, hyperproliferation, and UV treatment, the skin was histologically examined. Each porcine skin specimen was dehydrated using ethanol, embedded in paraffin wax, and stained with hematoxylin and eosin for examining morphology. The inflammatory response of nude mouse skin was verified by cyclooxygenase (COX)-2 staining by the method described previously [27]. For each skin sample, three different sites were examined and evaluated under light microscopy (Eclipse 4000, Nikon, Tokyo, Japan). The digital photomicrographs were then processed with Adobe PhotoDeluxe (Adobe Systems, San Jose, CA, USA). 2.6. In vitro skin permeation The diffusion cell used in the in vitro experiment was a Franz diffusion assembly. A piece of excised porcine dorsal skin or nude mouse dorsal skin was mounted with the SC side facing towards the donor compartment. After pretreatment with the laser, the skin surface was wiped with a cotton wool swab several times. The receptor compartment (5.5 ml) was filled with 20% ethanol in pH 7.4 citrate-phosphate buffer and neat pH 7.4 buffer for imiquimod and macromolecules, respectively. The donor phase was filled with 0.5 ml of vehicles with permeants. The donor concentration of imiquimod in 20% propylene glycol/pH 7.4 buffer was 0.1% (w/v). The concentrations of peptides and FD in pH 7.4 buffer were 500 and 120 μM, respectively. The available diffusion circle radius of the cell was 0.5 cm, which gave a diffusion area of the cell of 0.79 cm2. The stirring rate and temperature of the receptor were respectively kept at 600 rpm and 37 °C. At standard intervals, 300μl aliquots of receptor medium were withdrawn and immediately replaced by an equal volume of fresh receptor solution. Samples of imiquimod were analyzed using a high-performance liquid chromatographic (HPLC) method. A fluorescence spectrometer was used to quantify the permeated amounts of FITC-labeled macromolecules. The HPLC system for imiquimod included an L-2130 pump, an L2200 sample processor, and an L-2400 UV–visible detector all from Hitachi (Tokyo, Japan). A 25-cm-long, 4-mm inner diameter stainless RP-18 column (Merck, Darmstadt, Germany) was used as the stationary phase. The mobile phase was a methanol:acetonitrile:water:triethylamine (18:27:53:2) mixture at a flow rate of 1 ml/min. The UV–visible detector was set at 224 nm. The macromolecules were analyzed using a fluorescence spectrometer (F-2500, Hitachi) at an excitation wavelength of 494 nm and emission wavelength of 520 nm.

2.8. Statistical analysis The statistical analysis of differences between various treatments was performed using unpaired Student's t-test. A 0.05 level of probability was taken as the level of significance. 3. Results 3.1. Skin histological examination As depicted in Fig. 1, one pass of the fractional laser occupied 5% of the exposed thermal paper area. The distance between the dots was 1000 μm. The increase in the irradiated pass increased the occupied region from 5% (1 pass) to 30% (6 passes). Porcine skin was exposed to the fractional laser at 3 J/cm2 to determine the effects on the skin integrity. In a control experiment, porcine skin underwent no treatment to breach the barrier function of the SC prior to the application of drugs. Optical microscopy indicated no observable damage to the whole skin in the untreated group as shown in Fig. 2A. No observable disruption of the skin surface treated by the laser was seen with the naked eye. Under light microscopy, fractional treatment of the tissue achieved partial removal of the SC as indicated by the arrow on the left side of Fig. 2B. The arrow indicates the margin between the laser dot and the surrounding normal tissue. All of the SC layers remained in the normal zone. 3.2. In vitro skin permeation of imiquimod The in vitro porcine skin permeation of imiquimod without and with laser application was investigated. Fig. 3 illustrates imiquimod

2.7. In vivo skin permeation The dorsal skin of a nude mouse was first treated by irradiation with a fractional laser. Then a glass cylinder with an available area of 0.785 cm2 was placed on the dorsal skin with glue (Instant Super Glue®, Kokuyo, Osaka, Japan). Medium at 0.2 ml of FITC-labeled molecules the same as in vitro experiment was added to the cylinder. The administration duration was 2 h. After excising the skin on which the vehicle had been applied, the skin surface was washed 10 times with a cotton cloth immersed in water. The skin samples were examined for fluorescence signals by CLSM. The skin thickness was optically scanned at about 10-μm increments through the z-axis of a Leica TCS SP2 confocal microscope (Wetzlar, Germany). In total, 10 x,y-sections from the skin surface were combined to show the confocal fluorescence image of the skin. The separate x,y-sections at each z-level were also examined. Optical excitation was carried out with excitation a 484 nm, and the fluorescence emission was detected at 515–700 nm.

Fig. 2. Histological examination of porcine skin stained with hematoxylin and eosin with (A) no treatment (control group) and (B) fractional laser treatment at 3 J/cm2 (Magnification ×100). The arrow indicates the margin between the microscopic treatment zones (MTZs) and the surrounding normal tissue.

W.-R. Lee et al. / Journal of Controlled Release 153 (2011) 240–248

2

0 J/cm (control) 2 J/cm 2 x 1 2 J/cm 2 x 2 2 J/cm 2 x 4 2 J/cm 2 x 6

12 10

Flux (mean±SD) 0.19±0.05

8 6 0.10±0.04 4

3.3. Modulation of imiquimod dose for the laser-treated modality

0.05±0.01 0.04±0.02 0.002±0.003 0

10

20

30

40

Time (h)

B 30 0 J/cm 2 (control) 3 J/cm 2 x 1 3 J/cm 2 x 2 3 J/cm 2 x 4 3 J/cm 2 x 6

25 20

Flux (mean±SD) 0.46±0.11

15

0.29±0.03

10

3.4. In vitro skin permeation of peptides and FD

0.17±0.06 0.13±0.02 5 0.002±0.003

0 0

10

20

30

40

Time (h) Fig. 3. In vitro cumulative amount-time profiles of the topical delivery of imiquimod by fractional laser treatment of porcine skin at fluences of (A) 2 and (B) 3 J/cm2 with different numbers of passes. Each value represents the mean ± SD (n = 4 from four pig donors).

accumulation kinetics following topical delivery of 0.1% imiquimod. For each permeation study, the linear ascent of the curve was used to determine the flux (μg/cm2/h) as summarized behind the curves in Fig. 3. These curves corresponded well to pseudo zero-order kinetics. Permeation was measured as a function of the laser fluence and number of pulses. As a result, imiquimod showed very low permeability without laser irradiation. Exposure to 1 pulse of the laser was sufficient to enhance imiquimod flux by factors of 25 and 65 over the untreated group (p b 0.05) at fluences of 2 and 3 J/cm2, respectively. Exposure to higher fluences or numbers of passes further increased imiquimod permeation in a controlled manner, with 6 passes at 3 J/cm2 exhibiting the greatest flux (0.46 μg/cm2/h, p b 0.05). The skin delivery of imiquimod irradiated by 2 passes was comparable (p N 0.05) to that by 1 pass for both fluences tested. The fluxes of imiquimod via porcine skin and nude mouse skin are compared in Table 1. Intensities of 2 and 3 J/cm2 with 4 passes were selected for this comparison. Data from the untreated group revealed that nude mouse skin was more permeable than porcine skin, as previously known. The enhancing effects of laser treatment are calculated in Table 1 Table 1 The fluxes (μg/cm2/h) and enhancement ratios (ER) of imiquimod via porcine skin and nude mouse skin by treatment of fractional laser. Fluence

Porcine skin Flux (μg/cm2/h)

2

0 J/cm 2 J/cm2 × 4 3 J/cm2 × 4

0.002 ± 0.003 0.1 ± 0.04 0.3 ± 0.03

Nude mouse skin ER – 46 127

Flux (μg/cm2/h) 0.1 ± 0.03 5.2 ± 1.1 6.5 ± 1.5

The drug permeation from the 5% imiquimod cream (Aldara®) was examined in an in vitro experiment as shown in Fig. 4. Imiquimod revealed a flux of 0.79 μg/cm2/h. This value can fulfill therapeutic benefits in a clinical status. We tried to attain this flux value for the lasertreated group by lowering the imiquimod concentration in the donor. A 3-J/cm2 fluence with 4 pulses was used to irradiate nude mouse skin. Five concentrations (0.005%–0.04%) of imiquimod were added to the donor vehicle for the investigation. Fig. 4 illustrates that the flux increased following an increase in the amount of imiquimod applied. A concentration of 0.04% in the presence of laser treatment reached a flux similar to that of imiquimod permeation from Aldara® (p N 0.05).

Next, we addressed the skin transport efficacy of macromolecules by laser application. Since the in vivo studies were to be performed with these macromolecules, nude mouse was utilized as both the in vitro and in vivo animal models for macromolecules. Values of the peptide flux (pmol/cm2/h) and ER with laser treatment are given in Table 2. Peptide-1, a polypeptide with 3 amino acid units, showed a low passive flux of 6.9 pmol/cm2/h. The permeation of peptide-1 with laser treatment (3 J/cm2 for 4 passes) was superior (p b 0.05) to that of the untreated group with an enhancement of 310-fold. The addition of amino acid units from 3 (peptide-1) to 14 (peptide-2) led to an increment (p b 0.05) of passive transport via intact skin. The peptide with a greater MW (peptide-2) produced a lower enhancement compared to the smaller one (peptide-1), giving an ER value of 29. The effect of the amino acid sequence on the skin delivery of peptides by the fractional laser was also examined. The -COOH terminus of the His-Ser-Arg-His-Ser-Arg sequence of peptide-2 was 1.0

0.8

2

2 0

Cumulative amount (µg/cm2)

in terms of the enhancement ratio (ER). Upon irradiation at 2 J/cm2, the fractional laser increased the imiquimod flux via porcine skin and nude mouse skin by factors of 46- and 40-fold, respectively. The increase in laser fluence (3 J/cm2) led to a greater enhancement of the flux. A 127fold enhancement of the imiquimod flux was observed in porcine skin. Although a 3-J/cm2 intensity could further enhance imiquimod permeation via nude mouse skin, the ER was significantly lower compared to that of porcine skin (51 vs. 127).

Imiquimod flux (µg/cm /h)

Cumulative amount (µg/cm2)

A 14

243

*

* 0.4

*

0.2

0.0

Al

3

da

ra

(5

%

)

ER – 40 51

Enhancement ratio (ER) was flux of laser-pretreated group/flux of non-treatment group. Each value represents the mean ± SD (n = 4).

*

0.6

3

J/

cm 2 x4

3

J/

(0

cm 2 x

.0

4

3

J/

cm 2 x

(0

.0

4%

)

4

3

J/

cm 2 x

(0

.0

3%

)

4

J/

cm 2 x

(0

)

4

(0

.0

.0

2%

1%

)

05

%

)

Fig. 4. Comparison of imiquimod fluxes via nude mouse skin with passive transport of imiquimod 5% cream (Aldara®) and laser-assisted permeation of imiquimod from 20% propylene glycol/pH 7.4 buffer at different drug concentrations. Each value represents the mean ± S.D. (n = 4). *, p b 0.05 as compared to the flux value of imiquimod from Aldara® cream.

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Table 2 The fluxes (pmol/cm2/h) and enhancement ratios (ER) of peptides via nude mouse skin by treatment of fractional laser. Peptide type

0 J/cm2

3 J/cm2 × 4

ER

Peptide-1 Peptide-2 Peptide-3

6.9 ± 1.9 18.8 ± 8.7 1.8 ± 0.6

2133.2 ± 996.7 551.1 ± 71.8 7.8 ± 3.3

310 29 4

Enhancement ratio (ER) was flux of laser-pretreated group/flux of non-treatment group. Each value represents the mean ± SD (n = 5).

replaced by the sequence, Tyr-Trp-Leu-Tyr-Trp-Leu, to form a morelipophilic peptide (peptide-3). In order to confirm this hypothesis, a molecular modeling software (Discovery Studio® version 2.0, Accelrys Inc., San Diego, USA) was used to calculate partition coefficient (log P). The predicted log P was −3.5, −7.9, and 4.6 for peptide-1, peptide-2, and peptide-3, respectively. A very low permeability of peptide-3 via intact skin (1.8 pmol/cm2/h) was detected. This was because this lipophilic peptide could not completely be dissolved in the pH 7.4 buffer vehicle. We did not try to incorporate any cosolvents or organic solvents in the vehicle since they may have caused denaturation of the peptides. In order to compare the permeation of various peptides, the donor concentration remained identical (500 μM) for all peptides examined. Laser exposure of 3 J/cm2 only increased peptide-3 flux by 4-fold, which was far less than those of peptide-1 and peptide-2. In order to further evaluate the influence of the MW of the macromolecules on the skin delivery by the fractional laser, a series of FDs with various MWs from 4 to 150 kDa was used as model permeants. The small, hydrophilic FITC (MW 389) was also employed as a permeant in the in vitro study for comparison with FD as summarized in Table 3. FITC exhibited a higher passive flux of 25.8 pmol/cm2/h compared to FD because of its smaller size. No or negligible permeation was detected for FD transport in the untreated control group. Application of the fractional laser substantially increased the delivery of FD. The laser produced 165and 49-fold increases in skin permeation of FITC and FD4, respectively, compared to the control. It is clear that the laser pulses elevated the flux of FD10 from 0 to 12.6 pmol/cm2/h. The laser also increased the permeation of FD with MWs greater than FD10 (FD20–FD150) although the enhancement was lower than that of FD10. There was no significant difference (pN 0.05) in the fluxes of FD20, FD40, FD70, and FD150 with use of the laser.

Compared to the control group with no treatment (Fig. 5A), Fig. 5B shows a representative example of microscopic images of vertical skin sections with hyperproliferative induction. COX-2 can be an indicator of skin inflammation and disruption [29]. It can be seen that the tapestripping technique increased the epidermal thickness by 2-fold. A significant COX-2 expression, as seen in epidermis for the dark-brown staining, was observed in epidermal cells of the dorsal skin of mice after hyperproliferative induction. Fig. 4C depicts images of the skin after UVB exposure. UVB treatment was effective in inducing inflammatory responses including erythema and edema formation. Skin samples isolated from the treated area revealed an epidermal thickening in response to UVB-induced injury in these animals. The expression of COX-2 was higher with than without UVB irradiation. UVB irradiation also increased the epidermal thickness by 2-fold.

3.5. In vitro skin permeation via hyperproliferative and UV-irradiated skin In this study, nude mouse skin was developed which simulated AK in order to evaluate imiquimod permeation for comparison with normal skin. It was confirmed that the tape-stripping method can induce epidermal hyperplasia. On the other hand, UVB irradiation is the best-studied causative agent in the pathogenesis of AK [28].

Table 3 The fluxes (pmol/cm2/h) and enhancement ratios (ER) of FITC-labeled dextran (FD) via nude mouse skin by treatment of fractional laser. FD type

0 J/cm2

3 J/cm2 × 4

ERa

FITC FD4K FD10K FD20K FD40K FD70K FD150K

25.8 ± 24.3 0.4 ± 0.3 0 0 0 0 0

4257.9 ± 1215.3 17.8 ± 4.9 12.6 ± 6.8 1.4 ± 0.6 2.4 ± 0.8 1.3 ± 0.5 0.8 ± 0.4

165 49 –b – – – –

Each value represents the mean ± SD (n = 4). a Enhancement ratio (ER) was flux of laser-pretreated group/flux of non-treatment group. b –, not determined.

Fig. 5. Histological examination of nude mouse skin stained with cyclooxygenase (COX)-2 with (A) no treatment (control group); (B) hyperproliferative treatment by the tape-stripping technique; and (C) UV irradiation (Magnification ×400). Arrows indicate the thickness of the stratum corneum (SC) and epidermis.

W.-R. Lee et al. / Journal of Controlled Release 153 (2011) 240–248 Table 4 The fluxes (pmol/cm2/h) and enhancement ratios (ER) of imiquimod via hyperproliferative and UVB-irradiated skin by treatment of fractional laser. Skin type

0 J/cm2

3 J/cm2 × 4

ER

Normal skin Hyperproliferative skin UVB-irradiated skin

0.1 ± 0.03 0.2 ± 0.1 0.2 ± 0.1

6.5 ± 1.5 4.5 ± 0.6 2.8 ± 1.1

51 24 18

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among the imiquimod permeation rates from the three skin types. The hyperproliferative and UV-irradiated skins generally exhibited lower drug permeation (p b 0.05) compared to normal skin in the lasertreated group. The ER values for hyperproliferative skin and UVirradiated skin treated by the laser were 24 and 18, respectively. This enhancement was less than that of normal skin.

Enhancement ratio (ER) was flux of laser-pretreated group/flux of non-treatment group. Each value represents the mean ± SD (n = 4).

3.6. In vivo skin permeation

Table 4 compares imiquimod fluxes in normal skin, hyperproliferative skin produced by stripping, and UV-irradiated skin. The skin without laser exposure showed no significant difference (p N 0.05)

Skin permeation experiments were also conducted on in vivo nude mouse skin using the laser at 3 J/cm2 and 4 pulses. Fig. 6 shows confocal images obtained from control and laser-treated skin samples

Fig. 6. Confocal laser scanning microscopic (CLSM) micrographs of nude mouse skin after the in vivo topical administration of permeants via the skin. (A) Blank control of the skin with water treatment; (B) Non-treatment group with FITC administration; (C) fractional laser treatment at 3 J/cm2 with 4 passes and FITC administration; (D) non-treatment group with peptide-2 administration; (E) fractional laser treatment at 3 J/cm2 with 4 passes and peptide-2 administration; (F) non-treatment group with FD4 administration; (G) fractional laser treatment at 3 J/cm2 with 4 passes and FD4 administration; (H) non-treatment group with FD150 administration; (I) fractional laser treatment at 3 J/cm2 with 4 passes and FD150 administration. The skin specimen was viewed by CLSM at ~10-μm increments through the Z-axis. The images are a combination of 10 fragments from the skin surface.

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after topical administration of FITC, peptide-2, FD4, and FD150. A combination of 10 x,y-sections (about 100 μm) of horizontal sectioning from the skin surface is shown in this figure. No or negligible fluorescence signal was observed in the profiles of skin treated with blank aqueous solution without permeants (Fig. 6A), suggesting a limited interference of the fluorescence from the skin itself. As depicted in Fig. 6B, a negligible fluorescence signal was observed in the FITC profiles of skin with no laser irradiation. The confocal image of FITC showed an intense green fluorescence after laser exposure (Fig. 6C). Some green filaments (circles in Fig. 6C) can be seen in this image. We consider them to be hair follicles. The same phenomenon was detected for the passive transport of peptide-2 (circles in Fig. 6D). The peptide showed moderate fluorescence in the skin with passive diffusion. This result is consistent with the in vitro permeation experiment, which indicated the highest flux among the polypeptides. Peptide-2 fluorescence was more intense when the area of skin had previously been treated with the fractional laser (Fig. 6E). There was a significant increase in the fluorescence intensity of skin exposed to the laser compared to the control for FD4 and FD150 (Fig. 6F–I). The fractional laser induced greater FD4 than FD150 accumulation, which was identical to the in vitro results. It is clear that FD4 and FD150 molecules were largely retained in hair sheaths and hair shafts after laser irradiation (circles in Fig. 6G, I). In order to further explore the skin distribution of the permeants within the laser-treated skin, the separate x,y-sections obtained at each z-level of the skin by CLSM imaging are demonstrated in Fig. 7. The skin was optically scanned at 10-μm increments for 10 x,ysections from the surface of skin (from left to right). Since the thickness of nude mouse SC and epidermis is about 11 and 18 μm, respectively [18], the first three sections could be characterized as SC and epidermal layers. As can be seen in Fig. 7A, only a little green signal is observed in the first two segments. Since the SC was ablated by the laser, the first two sections were possibly the epidermal layers. The fluorescence signal was able to reach the upper and middle dermis with intense mark. After a significant accumulation in the upper dermis, the intensity of the green fluorescence gradually faded

away with an increase in the skin depth. A same phenomenon was detected for the other permeants, including peptide-2 and FDs (Fig. 7B to D). The signal from the combined imaging in Fig. 6 was proved to be from the dermal layers but not from the epidermis. It is noticeable that the green signal from FD5 was absent in both epidermis and the upper dermis, as there is negligible fluorescence in the first four segments (Fig. 7C). Most of FD5 signal was concentrated in some depths of dermis. 4. Discussion The long duration of imiquimod therapy is inconvenient for patients and affects compliance. One way to shorten the treatment duration is to increase the frequency of treatments [3]. However, this nearly always produces local adverse reactions, leading to the discontinuation of therapy. Hence new methods for enhancing imiquimod permeation to reduce the dose are urgently needed. The fractional laser at the fluences used for skin rejuvenation (high fluence) causes small, spatially limited zones of disruption within skin tissues due to local energy deposition [20]. The superficial nature of the laser-induced ablation with low fluences can contribute to the safety and rapid restoration of the SC's integrity. The fractional laser is a promising new modality on which the present work was based, and it produced a consistent level of imiquimod permeation enhancement with significantly reduced skin alterations. As can be seen in the histological profiles, the fractional laser ablated a portion of the SC layers with no influence on the surrounding normal areas. As the area of the treatment is very small, lateral migration of corneocytes occurs rapidly, which leads to the complete re-epithelialization of the SC within 1 day [21]. With the fractional approach, the zone of ablation is concentrated within the MTZ, and the surrounding viable tissue acts as a reservoir of stem cells, growth factors, and inflammatory cells that quickly respond to the injury and facilitate rapid recovery [30]. Full epidermal healing occurs within 1 day even after clinical procedures using higher fluences (2– 32 J/cm2) [24].

Fig. 7. Confocal laser scanning microscopic (CLSM) micrographs of nude mouse skin after the in vivo topical administration of permeants via the laser-treated skin (3 J/cm2 with 4 passes) by a separate x,y-sections at each z-level from the skin surface (from left to right). (A) FITC; (B) peptide-2; (C) FD4; and (D) FD150. The skin specimen was viewed at 10-μm increments through the z-axis.

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There was a linear relationship between imiquimod permeation and the number of laser passes used. Moreover, increased skin permeability was associated with increased fluence. This suggests that the fractional laser can precisely control drug permeation via the skin. The depth and area irradiated by the laser may be directly responsible for the increased drug transport. Ablation of the SC layers and the photomechanical wave (PW) are possible mechanisms of how the fractional laser promotes drug delivery. The Er:YAG laser emits light to ablate the SC with a minimal residual thermal effect [31], hence the heating mechanism of this laser can be neglected. The PW is a broadband, unipolar, compressive wave. It induces expansion of the lacunar spaces within the highly tortuous intercellular domains of the remaining SC. The PW can transiently permeabilize the SC and facilitate drug delivery into the skin [32]. Another possibility is the ability of the Er:YAG laser to decrease water retention within the skin. This laser emits light with a 2940-nm wavelength which is highly absorbed by water [33]. Imiquimod is basically a lipophilic molecule [34]. The loss of water from the skin may increase the lipophilicity of the SC, thus increasing the lipophilic drug affinity to the SC. This can partly explain the relationship of higher permeation with higher fluences and numbers of passes. Further study is necessary to explore this dehydration mechanism. The MW and lipophilicity are among the physicochemical parameters commonly used to describe variations in skin transport. The increase in the molecular size and lipophilicity result in a reduced enhancement effect by laser irradiation. The larger molecular masses of the peptides may have impeded their permeation via laser-treated skin, although a portion of the SC had been ablated. As peptides/proteins increase in size, they are more likely to have tertiary and quaternary structures. The more-complex structures of peptides may have difficulty penetrating into the skin even though it has been laser-treated. Another possibility is that the SC is not the sole significant contributor to resistance for peptides. Viable skin appears to be responsible for some bulk of the resistance. It was noted that the passive diffusion of peptide2 was higher than that of peptide-1 with a lower MW. The increase in MW of peptides contributes to an increase in the peptide charge and functional moiety. The molecular modeling Discovery Studio® had shown 3 and 11 pKa values for peptide-1 and peptide-2, respectively. This indicates abundant charges of the peptides with greater molecular volume. The probability of interaction with the transport pathway also increases with an increasing MW [35]. This may increase the probability that peptides of a larger size can permeate the skin. The experimental results demonstrated that peptide transport by the laser is highly sequence specific (peptide-2 vs. peptide-3). The permeation of the more-hydrophilic peptide was more effective compared to the less-hydrophilic one once the SC was partially removed by the laser. A permeant must pass through the thicker and more-hydrophilic viable skin, and the permeation resistance decreases the skin delivery of lipophilic permeants [36]. Since the viable epidermis/dermis was not affected by the laser, it was still a permeation barrier for lipophilic molecules after laser irradiation. The results of skin permeation of the peptides suggest that the molecular size is important for delivery efficiency induced by the fractional laser. A significant delivery of all peptides used in this study occurred with laser treatment. The cutoff value of the MW by the laser was further examined using FD as the macromolecular model. FD can act as a model permeant for peptides/proteins because of its molecular homogeneity, hydrophilic properties, and higher level of stability [37]. The results of passive diffusion showed that FD with an MW of N10 kDa demonstrated no permeation via nude mouse skin. This indicates that the inherent barrier property of nude mouse skin still existed for FD macromolecules. The large size of FD precluded its transport via intact skin to an appreciable extent. The permeation of FD was enhanced by the fractional laser. A cutoff limit of molecular size was not reached since significant delivery of all FDs detected in this work occurred with laser treatment. This suggests that the skin delivery of macromolecules of at least 150 kDa can be achieved with the fractional laser. A previous study

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[38] found that microneedles, a physical technique based on the formation of mechanically produced conduits through the SC by an array of small needles, can produce enhancement of FD permeation with an MW of 72 kDa. Lombry et al. [39] indicated that transdermal/topical delivery of FD at 40 kDa can be achieved by skin electroporation, another physical approach to create micropores within intercellular lipids of the SC. The fractional laser opens up the opportunity to deliver macromolecules with great MW (4–150 kDa) into the skin which is an attractive route for some therapeutic and preventive entities such as peptides, oligonucleotides, siRNA, and vaccines. Hyperproliferative and UV-irradiated skin was induced to be permeation barriers in the in vitro imiquimod delivery experiment due to their resemblance to AK. The results from the histological observations confirmed the successful induction of these skin types. This induction did not change the passive transport of imiquimod without laser treatment. However, a reduction in the enhancement effect of drug permeation via laser-treated skin was observed for the diseased skin. The SC is a predominant barrier for imiquimod transport. According to the histological images, the SC showed insignificant changes after induction. The permeation across the SC was the rate-limiting step for imiquimod regardless of which skin type was examined, resulting in similar drug fluxes in a condition without laser treatment. As some SC layers were ablated by the laser, the drug delivery was relatively low in the skin with a thicker epidermis because of the creation of longer pathways through which the drug had to pass. Much past research utilized healthy skin to assess drug permeation, and the results from such studies might not be adequate for predicting the skin targeting ability of a drug in disordered skin. The skin model used in this study would be helpful for resolving such a deficiency. Since the fractional laser greatly enhanced the skin permeation of drugs, the applied dose could be reduced to achieve similar therapeutic benefits. A commercial cream with 5% imiquimod showed a flux of ~8 μg/ cm2/h. Imiquimod at 0.04% with laser treatment on skin reached a similar flux to the cream although the drug vehicle differed between the two conditions. The imiquimod dose required by laser-assisted permeation was 125-fold lower than that by passive transport using cream. This reduction can decrease the possibility of causing skin irritation which is frequently observed during imiquimod therapy. Treatment cost calculations exhibited that imiquimod is the most expensive treatment for AK among four USFDA-cleared therapies including photodynamic therapy, 5-fluorouracil, diclofenac, and Aldara® [40]. As imiquimod is expensive, reducing the required dose is also a notable advantage. Compared to the control in the in vivo study, increased green fluorescence in skin treated with the fractional laser could clearly be seen with FITC, the peptide, and FD. The ablation of SC could improve the imaging depth of CLSM since the SC is a major limitation for obtaining the deeper tissue imaging due to the light scattering properties. The confocal images demonstrate the importance of hair follicles on the permeants investigated. Hair follicles are increasingly being recognized as an important route of entry for drug skin delivery [41]. There is evidence that peptides/protein delivery can be facilitated by the transfollicular route [12]. Hair follicles are separate entities within the skin. A hair is basically composed of keratin, the same as the constituent of the SC. The laser may propagate its ablation effect around the follicles. The transfollicular pathway opened up by the laser may be essential for peptide delivery. This route is especially important for the skin delivery of macromolecules with large MWs and high charges [42]. Subsequently, the transfollicular pathway is likely to be a significant route for peptides and FD delivery enhanced by the fractional laser. The importance of this route was also elucidated with a conventional Er:YAG laser [43]. Skin permeability can also be enhanced through the use of microneedles. The action of the microneedle technique on the skin resembles the mechanism of the fractional laser. Arranged in an array, there are micron-scale projections that penetrate through the SC with some microscopic zones surrounded by normal tissue [44]. The

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microneedles are inserted into the skin at a depth of the viable epidermis [45]. The fractional laser at low fluence only affects the superficial skin structure. This laser can propagate its ablation effect with no need to touch the skin, thus avoiding the possibility of infection from the device. The partial removal of the SC and the fractional effect are relatively safe with its application. The drawbacks of the laser being a large device with a high price cannot be ignored. These disadvantages of the laser may be resolved by employing novel products designed as portable lasers [46,47]. This is expected to promote the application of laser-assisted drug delivery in the near future. 5. Conclusions High levels of drug permeation are desirable to lessen the drug dose or produce significant effect for cutaneous therapy. This study showed that a low-fluence fractional laser can efficiently deliver topically applied imiquimod and macromolecules via the skin under both in vitro and in vivo conditions. Both the fluence and pulse number are critical parameters for efficient permeant transfer by this method. The fractional laser can be used to deliver molecules with a MW of up to 150 kDa. A comparison of drug permeation with commercial cream indicated that the laser can reduce the applied imiquimod dose with similar therapeutic efficiencies. This obviously has implications for clinical practice, as lower application doses would mean fewer adverse events and lower costs. The fractional laser investigated in this work used lower energies than those normally utilized for resurfacing aims. Only a limited layer and area of the SC is removed using this laser treatment. As the SC rapidly regenerates, the skin should recover within a short duration after laser treatment. The potential of this technique certainly merits further investigations. References [1] G. Hadley, S. Derry, R.A. Moore, Imiquimod for actinic keratosis: systematic review and meta-analysis, J. Invest. Dermatol. 126 (2006) 1251–1255. [2] H. Schöfer, Evaluation of imiquimod for the therapy of external genital and anal warts in comparison with destructive therapies, Br. J. Dermatol. 157 (Suppl. 2) (2007) 52–55. [3] N. Swanson, W. Abramovits, B. Berman, J. Kulp, D.S. Rigel, S. Levy, Imiquimod 2.5% and 3.75% for the treatment of actinic keratoses: results of two placebo-controlled studies of daily application to the face and balding scalp for two 2-week cycles, J. Am. Acad. Dermatol. 62 (2010) 582–590. [4] S. Metcalf, N. Crowson, M. Naylor, R. Haque, R. Cornelison, Imiquimod as an antiaging agent, J. Am. Acad. Dermatol. 56 (2007) 422–425. [5] M. Wuest, R. Dummer, M. Urosevic, Induction of the members of Notch pathway in superficial basal cell carcinomas treated with imiquimod, Arch. Dermatol. Res. 299 (2007) 493–498. [6] J. Anwar, D.A. Wrone, A. Kimyai-Asadi, M. Alam, The development of actinic keratosis into invasive squamous cell carcinoma: evidence and evolving classification schemes, Clin. Dermatol. 22 (2004) 189–196. [7] L.I. Harrison, S.L. Skinner, T.C. Marbury, M.L. Owens, S. Kurup, S. McKane, R.J. Greene, Pharmacokinetics and safety of imiquimod 5% cream in the treatment of actinic keratoses of the face, scalp, or hands and arms, Arch. Dermatol. Res. 296 (2004) 6–11. [8] E. Stockfleth, W. Sterry, M. Carey-Yard, J. Bichel, Multicentre, open-label study using imiquimod 5% cream in one or two 4-week courses of treatment for multiple actinic keratoses on the head, Br. J. Dermatol. 157 (Suppl. 2) (2007) 41–46. [9] S. Salasche, S. Shumack, A review of imiquimod 5% cream for the treatment of various dermatological conditions, Clin. Exp. Dermatol. 28 (2003) 1–3. [10] D.B. Yarosh, M.T. Canning, D. Teicher, D.A. Brown, After sun reversal of DNA damage: enhancing skin repair, Mutat. Res. 571 (2005) 57–64. [11] B. Berman, S. Amini, W. Valins, S. Block, Pharmacotherapy of actinic keratosis, Exp. Opin. Pharmacother. 10 (2009) 3015–3031. [12] Y. Chen, Y. Shen, X. Guo, C. Zhang, W. Yang, M. Ma, S. Liu, M. Zhang, L.P. Wen, Transdermal protein delivery by a coadministered peptide identified via phage display, Nat. Biotechnol. 24 (2006) 455–460. [13] S.O. Francis, M.J. Mahlberg, K.R. Johnson, M.E. Ming, R.P. Dellavalle, Melanoma chemoprevention, J. Am. Acad. Dermatol. 55 (2006) 849–861. [14] J.A. Mikszta, J.B. Alarcon, J.M. Brittingham, D.E. Sutter, R.J. Pettis, N.G. Harrey, Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery, Nat. Med. 8 (2002) 415–419. [15] M. Ogura, S. Sato, K. Nakanishi, M. Uenoyama, T. Kiyozumi, D. Saitoh, T. Ikeda, H. Ashida, M. Obara, In vivo targeted gene transfer in skin by the use of laser-induced stress waves, Lasers Surg. Med. 34 (2004) 242–248. [16] C. Gómez, Á. Costela, I. Carcía-Moreno, F. Llanes, J.M. Teijón, D. Blanco, Laser treatments on skin enhancing and controlling transdermal delivery of 5fluorouracil, Lasers Surg. Med. 40 (2008) 6–12.

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