Woan Ruoh Lee

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

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/cm(2) 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.

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

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/cm(2)) 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.

Topical delivery of methotrexate via skin pretreated with physical enhancement techniques: Low-fluence erbium:YAG laser and electroporation

The high hydrophilicity and molecular weight of methotrexate (MTX) make it difficult to deliver via the skin route for treating psoriasis or rheumatoid arthritis. The objective of this study was to enhance and optimize the skin permeation of MTX using two physical techniques: an erbium:yttrium‐aluminum‐garnet (Er:YAG) laser and electroporation.

Enhancement of topical small interfering RNA delivery and expression by low-fluence erbium:YAG laser pretreatment of skin.

RNA interference (RNAi) is rapidly becoming an important tool that is advancing research with therapeutic aims. It is necessary to develop efficient ways of guiding small interfering RNA (siRNA) to targeted tissues to induce an RNAi effect. Herein, we report on an active method for delivering macromolecular siRNA and its plasmid vector into the skin, using erbium:YAG (Er:YAG) laser pretreatment. The amount of siRNA transported through nude mouse skin was determined with an in vitro Franz diffusion assembly. Confocal laser scanning microscopy (CLSM) was used to examine the in vivo uptake of siRNA and the vector by the skin. The stratum corneum was partially ablated with the low-fluence laser. The results of in vitro experiments indicated a significant improvement in siRNA permeation with laser exposure, which showed a 2.4- to 10.2-fold increase compared with the nontreated group depending on the fluence used (1.2-1.7 J/cm(2)). A photomechanical wave generated by filtering the laser irradiation was sufficient to enhance siRNA permeation by 5-fold. CLSM revealed intense green fluorescence from naked siRNA within the epidermis and upper dermis after laser pretreatment, producing a 3.5-fold enhancement compared with the control. The green signal intensity in 1.7 J/cm(2)-treated skin was 4.2-fold higher than that in intact skin after the in vivo topical application of the siRNA expression vector. The increased signal was mainly in the dermis. This noninvasive, precisely controlled technique for siRNA therapy provides an efficient way to deliver siRNA and its vector into the skin.

Skin pretreatment with an Er:YAG laser promotes the transdermal delivery of three narcotic analgesics

Because of their low oral bioavailabilities and short half-lives, it may be more feasible to administer narcotic analgesics via the skin. However, this delivery method is limited by the low permeability of the stratum corneum (SC). The aim of this study was to enhance the transdermal delivery of three narcotic drugs, including morphine, nalbuphine, and buprenorphine, with an erbium:yttrium–aluminum–garnet (Er:YAG) laser pretreatment. In an in vitro pig skin permeation experiment, Er:YAG laser pretreatment of the skin produced a 10~35-fold enhancement in drug permeation that was dependent on the laser fluence and the narcotic analgesic used. The permeation of morphine and nalbuphine showed higher enhancement with Er:YAG laser treatment as compared to that of buprenorphine. This may have been due to the higher lipophilicity and molecular mass of buprenorphine than the other two narcotic drugs. A photomechanical wave was generated by filtering laser radiation through a polystyrene target. The experimental results showed that a single photomechanical wave was sufficient to enhance morphine permeation by sevenfold. This enhancement was significantly lower than that produced by direct laser irradiation, indicating the predominant mechanism of SC ablation by the Er:YAG laser for transdermal drug delivery.

Visualizing laser-skin interaction in vivo by multiphoton microscopy

Recently, multiphoton microscopy has gained much popularity as a noninvasive imaging modality in biomedical research. We evaluate the potential of multiphoton microscopy for monitoring laser-skin reaction in vivo. Nude mouse skin is irradiated with an erbium:YAG laser at various fluences and immediately imaged by a multiphoton microscope. The alterations of cutaneous nonlinear optical properties including multiphoton autofluorescence and second-harmonic generation associated with laser irradiation are evaluated morphologically and quantitatively. Our results show that an erbium:YAG laser at a low fluence can selectively disrupt the stratum corneum, and this alteration may account for the penetration enhancing effect of laser-assisted transcutaneous drug delivery. At a higher fluence, the zone of tissue ablation as well as the disruption of the surrounding stratum corneum, keratinocytes, and dermal extracellular matrix can be better characterized by multiphoton microscopy as compared with conventional histology. Furthermore, the degree of collagen damage in the residual thermal zone can be quantified by second-harmonic generation signals, which have significant difference between control skin, skin irradiated with a 1.5-, 8-, and 16-J/cm2 erbium:YAG laser (P<0.05). We show that multiphoton microscopy can be a useful noninvasive imaging modality for monitoring laser-skin reaction in vivo.

Erbium: YAG laser resurfacing increases skin permeability and the risk of excessive absorption of antibiotics and sunscreens: The influence of skin recovery on drug absorption

While laser skin resurfacing is expected to result in reduced barrier function and increased risk of drug absorption, the extent of the increment has not yet been systematically investigated. We aimed to establish the skin permeation profiles of tetracycline and sunscreens after exposure to the erbium:yttrium-aluminum-garnet (Er:YAG) laser during postoperative periods. Physiological and histopathological examinations were carried out for 5 days after laser treatment on nude mice. Percutaneous absorption of the permeants was determined by an in vitro Franz cell. Ablation depths varied in reaching the stratum corneum (10 μm, 2.5 J/cm²) to approach the epidermis (25 μm, 6.25 J/cm²) and upper dermis (40 μm, 10 J/cm²). Reepithelialization evaluated by transepidermal water loss was complete within 2-4 days and depended on the ablation depth. Epidermal hyperplasia was observed in the 40-μm-treated group. The laser was sufficient to disrupt the skin barrier and allow the transport of the permeants into and across the skin. The laser fluence was found to play an important role in modulating skin absorption. A 25-μm ablation depth increased tetracycline flux 84-fold. A much smaller enhancement (3.3-fold) was detected for tetracycline accumulation within the skin. The laser with different fluences produced enhancement of oxybenzone skin deposition of 3.4-6.4-fold relative to the untreated group. No penetration across the skin was shown regardless of whether titanium dioxide was applied to intact or laser-treated skin. However, laser resurfacing increased the skin deposition of titanium dioxide from 46 to 109-188 ng/g. Tetracycline absorption had recovered to the level of intact skin after 5 days, while more time was required for oxybenzone absorption. The in vivo skin accumulation and plasma concentration revealed that the laser could increase tetracycline absorption 2-3-fold. The experimental results indicated that clinicians should be cautious when determining the dose for postoperative treatment.

Erbium–Yttrium–Aluminum–Garnet Laser Irradiation Ameliorates Skin Permeation and Follicular Delivery of Antialopecia Drugs

Alopecia usually cannot be cured because of the available drug therapy being unsatisfactory. To improve the efficiency of treatment, erbium-yttrium-aluminum-garnet (Er-YAG) laser treatment was conducted to facilitate skin permeation of antialopecia drugs such as minoxidil (MXD), diphencyprone (DPCP), and peptide. In vitro and in vivo percutaneous absorption experiments were carried out by using nude mouse skin and porcine skin as permeation barriers. Fluorescence and confocal microscopies were used to visualize distribution of permeants within the skin. Laser ablation at a depth of 6 and 10 μm enhanced MXD skin accumulation twofold to ninefold depending on the skin barriers selected. DPCP absorption showed less enhancement by laser irradiation as compared with MXD. An ablation depth of 10 μm could increase the peptide flux from zero to 4.99 and 0.33 μg cm(-2) h(-1) for nude mouse skin and porcine skin, respectively. The laser treatment also promoted drug uptake in the hair follicles, with DPCP demonstrating the greatest enhancement (sixfold compared with the control). The imaging of skin examined by microscopies provided evidence of follicular and intercellular delivery assisted by the Er-YAG laser. Besides the ablative effect of removing the stratum corneum, the laser may interact with sebum to break up the barrier function, increasing the skin delivery of antialopecia drugs. The minimally invasive, well-controlled approach of laser-mediated drug permeation offers a potential way to treat alopecia. This studys findings provide the basis for the first report on laser-assisted delivery of antialopecia drugs.

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.

BACKGROUND Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail. OBJECTIVE Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction. METHODS Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy. RESULTS Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations. CONCLUSIONS Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo.

Non-ablative fractional laser assists cutaneous delivery of small- and macro-molecules with minimal bacterial infection risk.

Use of the ablative laser has been approved to enhance topical drug penetration. Investigation into the usefulness of the non-ablative laser for assisting drug delivery is very limited. In this study, we explored the safety and efficacy of the non-ablative fractional erbium:glass (Er:glass) laser as an enhancement approach to promote drug permeation. Both pig and nude mouse skins were employed as transport barriers. We histologically examined the skin structure after laser exposure. The permeants of 5-aminolevulinic acid (ALA), imiquimod, tretinoin, peptide, dextrans and quantum dots (QD) were used to evaluate in vitro and in vivo skin passage. The fractional laser selectively created an array of photothermal dots deep into the dermis with the preservation of the stratum corneum and epidermis. The barrier function of the skin could be recovered 8-60h post-irradiation depending on the laser spot densities. The application of the laser caused no local infection of Staphylococcus aureus and Pseudomonas aeruginosa. Compared to intact skin, ALA flux was enhanced up to 1200-fold after laser exposure. The penetration enhancement level by the laser was decreased following the increase of permeant lipophilicity. The skin accumulation of tretinoin, an extremely lipophilic drug, showed only a 2-fold elevation by laser irradiation. The laser promoted peptide penetration 10-fold compared to the control skin. Skin delivery of dextrans with a molecular weight (MW) of at least 40kDa could be achieved with the Er:glass laser. QD with a diameter of 20nm penetrated into the skin with the assistance of the non-ablative laser. The confocal microscopic images indicated the perpendicular and lateral diffusions of dextrans and nanoparticles via laser-created microscopic thermal zones. Controlled Er:glass laser irradiation offers a valid enhancement strategy to topically administer the permeants with a wide MW and lipophilicity range.