The ablative fractional coagulation zone influences skin fluorescence intensities of topically applied test molecules-An in vitro study with fluorescence microscopy and fluorescence confocal microscopy.

BACKGROUND: Ablative fractional laser (AFL) increases uptake of topically applied skin agents. The coagulation zone (CZ) surrounding vertically ablated channels may influence uptake of drugs. OBJECTIVES: To investigate impact of CZ thickness on skin fluorescence intensities (FI) of a hydrophilic molecule by means of fluorescence microscopy (FM) and fluorescence confocal microscopy (FCM). Second, to compare FI of hydrophilic and lipophilic test molecules by FCM. STUDY DESIGN/METHODS AND MATERIALS: Microchannels with CZ thicknesses of 0, 20, and 80 µm were generated by microneedles or AFL (10,600 nm). Channels were 700 µm deep and number of channels kept constant per skin area. After 4 hours of incubation, FI induced by sodium fluorescein (NAF, hydrophilic, logarithmic partition-coefficient (logP) = -1.52, MW = 376.26) were quantified in both CZ and surrounding skin by FM (0-1,500 µm) and FCM (0-90 µm). FI of NAF and carboxyfluorescein (CAF, lipophilic, logP = 2.9, MW = 376.32) were compared by FCM. RESULTS: By FM, NAF-induced FI were higher in CZ than in surrounding skin (P ≤ 0.001). Highest NAF-FI were induced in skin pretreated with a thin CZ (CZ-20 µm), assessed by both FM and FCM and in particular, FI were higher than in skin pretreated with no CZ (CZ-0 µm) (FM P ≤ 0.041, FCM P < 0.012). Skin FI remained constant to a depth of 500 µm, which corresponded to approximate depth of microchannels (CZ-0 µm, CZ-20 µm, CZ-80 µm: 0-500 µm P ≥ 0.107). In accordance with FM data, FCM showed higher FI within CZ than in surrounding skin, but gradually decreased to zero at a depth of 90 µm. NAF-FI were higher than CAF-FI (P ≤ 0.036), and highest CAF-FI were induced by CZ-0 µm and CZ-20 µm compared to CZ-80 µm (P ≤ 0.009). CONCLUSIONS: The influence of the CZ thickness on skin FI differs between small hydrophilic and lipophilic test molecules. Results may have clinical relevance for laser-assisted drug delivery. Lasers Surg. Med. 51:68-78, 2019. © 2018 Wiley Periodicals, Inc.

The fractional laser-induced coagulation zone characterized over time by laser scanning confocal microscopy-A proof of concept study.

BACKGROUND: Ablative fractional laser (AFXL) is an acknowledged technique to increase uptake of topical agents in skin. Micro thermal ablation zones (MAZs) consist of ablated vertical channels surrounded by a coagulation zone (CZ). Laser scanning confocal microscopy (LSCM) images individual MAZs at 733 nm (reflectance confocal microscopy (RCM)). Further, LSCM can image sodium fluorescein (NaF) fluorescence with 488 nm excitation (fluorescence confocal microcopy (FCM)), a small hydrophilic test molecule (370 MW, log P -1.52), which may simulate uptake, bio-distribution and kinetics of small hydrophilic drugs. OBJECTIVES: To explore LSCM for combined investigations of CZ thickness and uptake, bio-distribution and kinetics of NaF in AFXL-exposed skin. STUDY DESIGNS/METHODS AND MATERIALS: Excised human abdominal skin samples were exposed to AFXL (15 mJ/microbeam, 2% density) and NaF gel (1000 µg/ml, 10 µl/cm2) in six repetitions, including untreated control samples. CZ thickness and spatiotemporal fluorescence intensities (FI) were quantified up to four hours after NaF application by RCM and FCM. Test sites were scanned to a depth of 200 µm, quantifying thickness of skin compartments (stratum corneum, epidermis, upper dermis), individual CZ thicknesses and FI in CZ and surrounding skin. RESULTS: RCM images established skin morphology to a depth of 200 µm. The CZ thickness measurements were feasible to a depth of 50 µm, and remained unchanged over time at 50 µm (P > 0.5). FI were detected to a depth of 160 µm and remained constant in CZ up to four hours after NaF application (15 minutes: 79 AU (73-92 AU), 60 minutes: 72 AU (58-82 AU), four hours: 78 AU (71-90 AU), P > 0.1). In surrounding skin, FI increased significantly over time, but remained lower than FI in CZ (15 minutes: 21 AU (17-22 AU), 60 minutes: 21 AU (19-26 AU), four hours: 42 (31- 48 AU), P = 0.03). AFXL-processed skin generated higher FI compared to non-laser processed skin in epidermis and upper dermis at 60 minutes and four hours (P = 0.03). CONCLUSIONS: By LSCM, assessment of the AFXL-induced CZ thickness was feasible to a depth of 50 µm, and assessment of FI from a small hydrophilic test molecule, NaF in CZ and surrounding skin feasible to a depth of 160 µm. Lasers Surg. Med. 50:70-77, 2018. © 2017 Wiley Periodicals, Inc.

Fractional laser-assisted drug uptake: Impact of time-related topical application to achieve enhanced delivery.

BACKGROUND AND OBJECTIVE: Ablative fractional laser (AFXL) is acknowledged to increase uptake of topically applied agents in skin. AFXL channels gradually close over time, which may impair this capability. The time frame for applying a drug after AFXL exposure remains to be established. The aim of this study, was to investigate the importance of time-related topical application after AFXL exposure and to relate resultant uptake in skin with AFXL channel morphology and skin integrity. STUDY DESIGN/MATERIALS AND METHODS: Buttock skin of healthy volunteers (n = 11) was exposed to 10,600 nm fractional CO2 laser using 5% density, 120 µm beam diameter, 15 mJ pulse energy. Sodium fluorescein (NaF) a small, hydrophilic molecule (370 MW, log P = -1.52) was applied under standardized conditions at specific time points after laser exposure (0, 2, 5, 10, 30, 60, 90 minute, 6, 24, and 48 hours). Fluorescence photography collected fluorescence images up to 180 minute after NaF application. Optical coherence tomography (OCT) assessed AFXL channel dimensions and transepidermal water loss (TEWL) estimated loss of skin integrity. RESULTS: Fluorescence intensities (FI) were significantly elevated when NaF was applied up to 6 hours after laser exposure compared to non-laser-processed skin (median FI 1947 arbitrary units [interquartile range 1,246-3,560] versus 1,004 [350-1,538], P < 0.02). The highest FI occurred when NaF was applied within 30 minute after laser exposure and similar FI were reached for applications at 0, 2, 5, 10, and 30 minute after AFXL exposure (0 minute: 3,866 [3,526-4,575], 30 minute: 3,775 AU [3,070-4,484], P > 0.1). NaF application later than 30 minute after AFXL exposure resulted in gradually decreasing FI, becoming similar to intact skin when applied at 24-48 hours after AFXL exposure (P > 0.2). OCT images demonstrated that AFXL channels closed over time (100% [100-100%] open up to 30 minute, 75% [4-86%] at 6 hours and 3% [0-15%] at 24-48 hours after AFXL exposure). TEWL measurements proved loss of skin integrity up to 6 hours after AFXL exposure, while integrity was similar in laser-exposed and non-laser-exposed skin at 24-48 hours. CONCLUSIONS: The time frame to maintain enhanced drug delivery sustained for several hours after AFXL exposure, corresponding to channel morphology and loss of skin integrity. Lasers Surg. Med. 49:348-354, 2017. © 2016 Wiley Periodicals, Inc.

Spatiotemporal closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy.

BACKGROUND AND OBJECTIVE: Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) offer high-resolution optical imaging of the skin, which may provide benefit in the context of laser-assisted drug delivery. We aimed to characterize postoperative healing of ablative fractional laser (AFXL)-induced channels and dynamics in their spatiotemporal closure using in vivo OCT and RCM techniques. STUDY DESIGN/MATERIALS AND METHODS: The inner forearm of healthy subjects (n = 6) was exposed to 10,600 nm fractional CO2 laser using 5 and 25% densities, 120 µm beam diameter, 5, 15, and 25 mJ/microbeam. Treatment sites were scanned with OCT to evaluate closure of AFXL-channels and RCM to evaluate subsequent re-epithelialization. RESULTS: OCT and RCM identified laser channels in epidermis and upper dermis as black, ablated tissue defects surrounded by characteristic hyper-and hyporeflective zones. OCT imaged individual laser channels of the entire laser grid, and RCM imaged epidermal cellular and structural changes around a single laser channel to the depth of the dermoepidermal junction (DEJ) and upper papillary dermis. OCT images visualized a heterogeneous material in the lower part of open laser channels, indicating tissue fluid. By OCT the median percentage of open channels was evaluated at several time points within the first 24 hours and laser channels were found to gradually close, depending on the used energy level. Thus, at 5 mJ/microbeam, 87% (range 73-100%) of channels were open one hour after laser exposure, which declined to 27% (range 20-100%) and 20% (range 7-93%) at 12 and 24 hours after laser exposure, respectively. At 25 mJ/microbeam, 100% (range 100-100%) of channels were open 1 hour after laser exposure while 53% (range 33-100%) and 40% (range 0-100%) remained open at 12 and 24 hours after exposure. Median depth and width of open channels decreased over time depending of applied energy. RCM verified initial re-epithelialization from day 2 for all energy levels used. Morphology of ablation defects by OCT and RCM corresponded to histological assessments. CONCLUSIONS: OCT and RCM enabled imaging of AFXL-channels and their spatiotemporal closure. Laser channels remained open up to 24 hours post laser, which may be important for the time perspective to deliver topical substances through AFXL channels.