研究目的
To investigate the impact of coagulation zone (CZ) thickness on skin fluorescence intensities (FI) of topically applied hydrophilic and lipophilic test molecules using fluorescence microscopy and fluorescence confocal microscopy, and to compare the FI between these molecules.
研究成果
The coagulation zone thickness influences skin fluorescence intensities differently for hydrophilic and lipophilic molecules, with highest intensities for hydrophilic NAF in thin CZ and for lipophilic CAF in no or thin CZ. This suggests CZ-dependent drug uptake mechanisms, potentially optimizing laser-assisted drug delivery for different drug types.
研究不足
The study was conducted in vitro, used only one time point (4 hours), and FCM had limited detection depth (up to 90 μm) and field of view. Physicochemical factors of test molecules and translation to clinical settings need further research.
1:Experimental Design and Method Selection:
The study used an in vitro design with pig skin samples to generate microchannels with different CZ thicknesses (0, 20, 80 μm) using microneedles or an ablative fractional laser (AFL). Fluorescence microscopy (FM) and fluorescence confocal microscopy (FCM) were employed to quantify fluorescence intensities (FI) of sodium fluorescein (NAF, hydrophilic) and carboxyfluorescein (CAF, lipophilic) after 4 hours of incubation.
2:Sample Selection and Data Sources:
Skin samples were excised from the flanks of seven Danish landrace/Yorkshire pigs, stored at -80°C, and thawed before use. Test areas measured 1x1 cm.
3:List of Experimental Equipment and Materials:
Equipment included microneedles (540 derma roller), AFL system (Ultrapulse, DeepFX handpiece, Lumenis Inc.), fluorescence microscope (Olympus IX70), CCD camera (ORCA-R2, Hamamatsu Corp.), confocal microscope (Vivascope 1500, Mavig), infrared thermometer (RS-1327), and software (ImageJ, SPSS, Prism GraphPad). Materials included sodium fluorescein, carboxyfluorescein (Sigma-Aldrich), hydro alcoholic gel (LEO Pharma), and saline.
4:Experimental Procedures and Operational Workflow:
Microchannels were generated with specified CZ thicknesses and depth. Test molecules were applied topically and incubated for 4 hours. Skin sections were prepared at various depths for FM imaging, and confocal scans were performed. FI were measured in regions of interest (ROI) for CZ and surrounding skin, with auto-fluorescence subtracted where necessary.
5:Data Analysis Methods:
Non-parametric statistics (Mann-Whitney and Wilcoxon tests) were used with Bonferroni correction. Data were analyzed in SPSS and graphed in Prism GraphPad.
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CCD camera
ORCA-R2
Hamamatsu Corp.
Capture high-resolution images for fluorescence microscopy.
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carboxyfluorescein
5(6)-carboxyfluorescein
Sigma-Aldrich
Lipophilic test molecule for fluorescence imaging to compare with sodium fluorescein.
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microneedle
540 derma roller
Denmark
Generate microchannels with no coagulation zone (CZ-0 mm) in skin samples.
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ablative fractional laser
Ultrapulse, DeepFX handpiece
Lumenis Inc.
Generate microchannels with coagulation zones of specified thicknesses (CZ-20 mm and CZ-80 mm) in skin samples.
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fluorescence microscope
Olympus IX70
Olympus Denmark A/S
Acquire fluorescence and light microscopy images of skin sections at various depths.
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confocal microscope
Vivascope 1500 multi laser
Mavig
Perform reflectance and fluorescence confocal microscopy imaging of skin samples.
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infrared thermometer
RS-1327
Taiwan
Measure skin surface temperature to ensure consistency during experiments.
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sodium fluorescein
Sigma-Aldrich
Hydrophilic test molecule for fluorescence imaging to study drug uptake.
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gel
hydro alcoholic gel no. 130-345-06-01P
LEO Pharma
Vehicle for diluting and applying test molecules to skin samples.
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