Fractional laser-assisted topical delivery of bleomycin quantified by LC-MS and visualized by MALDI mass spectrometry imaging

Abstract

Bleomycin exhibits antiproliferative effects desirable for use in dermato-oncology but topical use is limited by its 1415 Da molar mass. Ablative fractional laser (AFL)-assisted drug delivery has been shown to enhance drug uptake in skin. The aim of this study was with AFL to deliver bleomycin into skin, quantify uptake, and visualize biodistribution with mass spectrometry. In a Franz diffusion cell study, pig skin samples (n = 66) were treated with AFL (λ = 10,600 nm), 5% density, and 0, 5, 20, or 80 mJ/microbeam (mb) pulse energies before exposure to bleomycin for 0.5, 4, or 24 h. Bleomycin was quantified in biopsy cryosections at depths of 100, 500, and 1500 µm using high-performance liquid chromatography-mass spectrometry (LC-MS), and drug biodistribution was visualized for 80 mJ/mb samples by matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The pulse energies 5, 20, and 80 mJ/mb resulted in microscopic ablation zones (MAZs) reaching superficial, mid, and deep dermis respectively. Bleomycin was successfully delivered into the skin and deeper MAZs and longer exposure time resulted in higher skin concentrations. After 24 h, AFL exposure resulted in significant amounts of bleomycin throughout all skin layers (≥510 µg/cm³, p ≤ .002). In comparison, concentrations in intact skin exposed to bleomycin remained below limit of quantification. MALDI-MSI supported the quantitative LC-MS results by visualizing bleomycin biodistribution and revealing high uptake around MAZs with delivery into surrounding skin tissue. In conclusion, topical drug delivery of the large and hydrophilic molecule bleomycin is feasible, promising, and should be explored in an in vivo setting.

Keywords: Bleomycin; MALDI-MSI; drug delivery; fractional laser; skin cancer.

Figure 1.

Laser channel morphology and dimensions in HE-stained cryosections. (A) MAZ-Supf, depth: 195 µm (158–219 µm), width: 89 µm (80–101 µm), and coagulation zone: 34 µm (28–35 µm), n = 17 channels. (B) MAZ-Mid, depth: 602 µm (513–665 µm), width: 140 µm (118–160 µm), and coagulation zone: 47 µm (43–51 µm), n = 21 channels. (C) MAZ-Deep, depth: 1496 µm (1324–1599 µm), width: 251 µm (197–286 µm), and coagulation zone: 65 µm (60–76 µm), n = 21 channels. The coagulation-zones bordering the laser channels are visualized by more intense hematoxylin staining than the surrounding tissue. Dimensions are shown as median with interquartile range. Magnification ×40. MAZ-Supf: Microscopic ablation zone reaching superficial dermis; MAZ-Mid: Microscopic ablation zone reaching mid dermis; MAZ-Deep: Microscopic ablation zone reaching deep dermis.

Figure 2.

Bleomycin concentrations by exposure time, pulse energies, and skin depth. Boxplots of quantified bleomycin based on median and interquartile range with min/max whiskers for (A) 0.5 h, (B) 4 h, and (C) 24 h drug exposure time. Higher concentrations of bleomycin are seen closer to the skin surface. Higher pulse energies and longer drug exposure times result in higher concentrations of bleomycin. The y-axes are not standardized between diagrams. The dotted lines represent the limit of quantification. MAZ-Supf: Microscopic ablation zone reaching superficial dermis; MAZ-Mid: Microscopic ablation zone reaching mid dermis; MAZ-Deep: Microscopic ablation zone reaching deep dermis.

Figure 3.

Heatmap of enhancement ratios. Bleomycin concentration enhancement ratios achieved by increasing MAZ depth energy and/or extending the drug exposure time for the superficial, mid, and deep dermis, respectively. For each skin depth separately, the smallest MAZ and drug exposure time setting resulting in a median concentration above the limit of quantification (LOQ) is normalized to 1.00 as a base for enhancement ratios. Thus, values are normalized to Superficial dermis: 82 µg/cm³ (MAZ-Supf and 0.5 h), Mid dermis: 112.5 µg/cm³ (MAZ-Deep and 0.5 h), Deep dermis: 122.5 µg/cm³ (MAZ-Mid and 4 h). A darker hue of blue signifies a higher enhancement ratio. Crossed out cells signify values below LOQ. MAZ-Supf: Microscopic ablation zone reaching superficial dermis; MAZ-Mid: Microscopic ablation zone reaching mid dermis; MAZ-Deep: Microscopic ablation zone reaching deep dermis.

Figure 4.

Mass spectrometry imaging. MALDI-MSI of bleomycin B2 (m/z 1425.56323) with MAZ-Deep laser channels. (A) Vertically cut skin cryosection after 24 h of topical drug exposure. Laser channels are easily seen with high concentrations of bleomycin (yellow) in the coagulation zones and drug dissemination into the surrounding tissue. (B) Horizontally cut skin cryosections. A trend towards higher concentrations is seen along the x-axis of time, and lower down the y-axis of skin depth. The depicted intensity values for each image are based on maximum bleomycin detection within the individual skin sample and thus cannot be compared inter-individually. MALDI-MSI: Matrix assisted laser desorption/ionization mass spectrometry imaging; MAZ-Deep: Microscopic ablation zone reaching deep dermis.