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

https://doi.org/10.1016/j.jconrel.2011.03.015Get rights and content

Abstract

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 125-fold. 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.

Graphical abstract

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.

  1. Download : Download full-size image

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, such as melanomas, basal cell carcinoma (BCC), and Bowen's disease [4], [5], [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 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], [16], [17]. In our previous study [18], a 3–5-day recovery of the SC was necessary after erbium:yttrium-aluminum-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.

Section snippets

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 NH2-Arg-Leu-Ala-COOH (peptide-1, MW 716), NH2

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

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

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

References (47)

  • T.M. Chiu et al.

    In vitro and in vivo anti-photoaging effects of an isoflavone extract from soybean cake

    J. Ethnopharmacol.

    (2009)
  • R.J. Caniglia

    Erbium:YAG laser resurfacing

    Facial Plast. Surg. Clin. North Am.

    (2004)
  • A. Naik et al.

    Transdermal drug delivery: overcoming the skin barrier function

    Pharm. Sci. Technol. Today

    (2000)
  • F.J. Verbaan et al.

    Assembled microneedle arrays enhance the transport of compounds varying over a large range of molecular weight across human dermatomed skin

    J. Control. Release

    (2007)
  • B.D. Bath et al.

    Scanning electrochemical microscopy of iontophoretic transport in hairless mouse skin. Analysis of the relative contributions of diffusion, migration, and electroosmosis to transport in hair follicles

    J. Pharm. Sci.

    (2000)
  • J.Y. Fang et al.

    Transdermal delivery of macromolecules by erbium:YAG laser

    J. Control. Release

    (2004)
  • A. Davidson et al.

    Transdermal drug delivery by coated microneedles: geometry effects on effective skin thickness and drug permeability

    Chem. Eng. Res. Des.

    (2008)
  • Y.G. Bachhav et al.

    Effect of controlled laser microporation on drug transport kinetics into and across the skin

    J. Control. Release

    (2010)
  • H. Schöfer

    Evaluation of imiquimod for the therapy of external genital and anal warts in comparison with destructive therapies

    Br. J. Dermatol.

    (2007)
  • M. Wuest et al.

    Induction of the members of Notch pathway in superficial basal cell carcinomas treated with imiquimod

    Arch. Dermatol. Res.

    (2007)
  • L.I. Harrison et al.

    Pharmacokinetics and safety of imiquimod 5% cream in the treatment of actinic keratoses of the face, scalp, or hands and arms

    Arch. Dermatol. Res.

    (2004)
  • E. Stockfleth et al.

    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.

    (2007)
  • S. Salasche et al.

    A review of imiquimod 5% cream for the treatment of various dermatological conditions

    Clin. Exp. Dermatol.

    (2003)
  • Cited by (103)

    • Laser-Assisted Drug Delivery

      2023, Advances in Cosmetic Surgery
    • Laser irradiation of ocular tissues to enhance drug delivery

      2021, International Journal of Pharmaceutics
    • Laser-Assisted Drug Delivery

      2020, Advances in Cosmetic Surgery
    View all citing articles on Scopus
    View full text