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• W2032926656 abstract "A multiphoton excitation–based fluorescence fluctuation spectroscopy method, Raster image correlation spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model fluorescent substances in excised human skin. In combination with structural information obtained by multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC). Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual-color fluorescently labeled liposomes—containing an amphiphilic fluorophore in the lipid bilayer and a hydrophilic fluorophore encapsulated in the liposome lumen—was measured using cross-correlation RICS. This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated) diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross-correlation below the skin surface, indicating that the penetration of intact liposomes is highly compromised by the skin barrier. A multiphoton excitation–based fluorescence fluctuation spectroscopy method, Raster image correlation spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model fluorescent substances in excised human skin. In combination with structural information obtained by multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC). Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual-color fluorescently labeled liposomes—containing an amphiphilic fluorophore in the lipid bilayer and a hydrophilic fluorophore encapsulated in the liposome lumen—was measured using cross-correlation RICS. This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated) diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross-correlation below the skin surface, indicating that the penetration of intact liposomes is highly compromised by the skin barrier. ATTO-647N 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine ATTO 647N Streptavidin cross-correlation Raster image correlation spectroscopy fluorescence correlation spectroscopy large unilamellar vesicle multiphoton excitation microscopy phosphate-buffered saline 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Rhodamine B Lissamine-rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine Raster image correlation spectroscopy stratum corneum tetramethylrhodamine dextran 3,000 MW Understanding the penetration properties of substances across biological barriers is vital for many areas of research. In the particular case of human skin, the characterization of structural and dynamical processes occurring across the tissue barrier is necessary for a better understanding of healthy and diseased tissue conditions. This information is furthermore required when designing successful transdermal drug delivery strategies (Cevc and Vierl, 2010Cevc G. Vierl U. Nanotechnology and the transdermal route: a state of the art review and critical appraisal.J Control Rel. 2010; 141: 277-299Crossref PubMed Scopus (460) Google Scholar; Konig et al., 2011Konig K. Raphael A.P. Lin L. et al.Applications of multiphoton tomographs and femtosecond laser nanoprocessing microscopes in drug delivery research.Adv Drug Deliv Rev. 2011; 63: 388-404Crossref PubMed Scopus (90) Google Scholar; Delouise, 2012Delouise L.A. Applications of nanotechnology in dermatology.J Invest Dermatol. 2012; 132: 964-975Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Most studies focused on measuring transdermal diffusion/penetration of drugs have been carried out using Franz cells (Ng et al., 2010Ng S.F. Rouse J.J. Sanderson F.D. et al.Validation of a static Franz diffusion cell system for in vitro permeation studies.AAPS PharmSciTech. 2010; 11: 1432-1441Crossref PubMed Scopus (172) Google Scholar) or tape stripping (Klang et al., 2012Klang V. Schwarz J.C. Lenobel B. et al.In vitro vs. in vivo tape stripping: validation of the porcine ear model and penetration assessment of novel sucrose stearate emulsions.Eur J Pharm Biopharm. 2012; 80: 604-614Crossref PubMed Scopus (102) Google Scholar). Franz cell experiments provide information on bulk diffusion through the skin but lack spatial resolution. Tape stripping (combined with an appropriate analytic method) measures the average penetration of substances at different depths in the skin (Marttin et al., 1996Marttin E. Neelissen-Subnel M.T. De Haan F.H. et al.A critical comparison of methods to quantify stratum corneum removed by tape stripping.Skin Pharmacol. 1996; 9: 69-77Crossref PubMed Scopus (83) Google Scholar) and, in some cases, may provide spatially resolved information (Lindemann et al., 2003Lindemann U. Wilken K. Weigmann H.J. et al.Quantification of the horny layer using tape stripping and microscopic techniques.J Biomed Opt. 2003; 8: 601-607Crossref PubMed Scopus (46) Google Scholar). Tape stripping is, however, inherently invasive, as the tissue must be destroyed to perform measurements. Furthermore, calibrating the amount of tissue removed is not trivial, as tape stripping can remove layers of the stratum corneum (SC) that originate from various depths because of furrows in the skin (van der Molen et al., 1997van der Molen R.G. Spies F. van ‘t Noordende J.M. et al.Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin.Arch Dermatol Res. 1997; 289: 514-518Crossref PubMed Scopus (122) Google Scholar). Alternatively, laser scanning confocal microscopy (van Kuijk-Meuwissen et al., 1998van Kuijk-Meuwissen M.E.M.J. Junginger H.E. Bouwstra J.A. Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study.Biochim Biophys Acta. 1998; 1371: 31-39Crossref PubMed Scopus (99) Google Scholar; Honeywell-Nguyen et al., 2004Honeywell-Nguyen P.L. Gooris G.S. Bouwstra J.A. Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo.J Invest Dermatol. 2004; 123: 902-910Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) or multiphoton excitation fluorescence microscopy (MPEM) (Kushner et al., 2007Kushner J.t. Kim D. So P.T. et al.Dual-channel two-photon microscopy study of transdermal transport in skin treated with low-frequency ultrasound and a chemical enhancer.J Invest Dermatol. 2007; 127: 2832-2846Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Carrer et al., 2008Carrer D.C. Vermehren C. Bagatolli L.A. Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study.J ControlRelease. 2008; 132: 12-20Crossref PubMed Scopus (87) Google Scholar) have been used to study transdermal penetration of fluorescently labeled substances in the intact tissue. These two techniques provide noninvasive, spatially resolved information of the probe’s fluorescence intensity distribution in the skin, allowing, e.g., a determination of the depth of penetration of the fluorescent compounds. At present, however, the data obtained with all the aforementioned methods do not provide information on the local diffusion properties of the applied substances while penetrating the skin. The objective of our work is to introduce a microscopy-based strategy that provides spatially resolved diffusion information of fluorescent substances at different depths within the skin tissue. The approach uses a combination of MPEM imaging and Raster image correlation spectroscopy (RICS) (Digman et al., 2005aDigman M.A. Brown C.M. Sengupta P. et al.Measuring fast dynamics in solutions and cells with a laser scanning microscope.Biophys J. 2005; 89: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, Digman et al., 2005bDigman M.A. Sengupta P. Wiseman P.W. et al.Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure.Biophys J. 2005; 88: L33-L36Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). RICS can extract dynamical information and the amount of fluorescent particles using the inherent time and spatial information contained in laser scanning fluorescence confocal images. Briefly, a diffusing fluorescent particle will move in the image and be detected in multiple pixels, leading to a characteristic spatial extent. Information on the spatial extent of the particles can be extracted from the images by performing a 2D spatial correlation analysis from a region of interest. By knowing the characteristics of the confocal laser scanning microscope, such as pixel dwell time, line time, and pixel size, it is possible to extract the diffusion and concentrations of fluorescently labeled particles. Whereas MPEM facilitates deep tissue imaging with a low extent of photobleaching and photodamage (Masters et al., 1998Masters B.R. So P.T. Gratton E. Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy.Ann NY Acad Sci. 1998; 838: 58-67Crossref PubMed Scopus (112) Google Scholar; Kushner et al., 2007Kushner J.t. Kim D. So P.T. et al.Dual-channel two-photon microscopy study of transdermal transport in skin treated with low-frequency ultrasound and a chemical enhancer.J Invest Dermatol. 2007; 127: 2832-2846Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Bloksgaard et al., 2012Bloksgaard M. Svane-Knudsen V. Sorensen J.A. et al.Structural characterization and lipid composition of acquired cholesteatoma: a comparative study with normal skin.Oto Neurotol. 2012; 33: 177-183Crossref PubMed Scopus (15) Google Scholar), RICS provides stacks of 2D maps of the fluorophore’s diffusion coefficient within the tissue (Vendelin and Birkedal, 2008Vendelin M. Birkedal R. Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy.Am J Physiol Cell Physiol. 2008; 295: C1302-C1315Crossref PubMed Scopus (55) Google Scholar; Gielen et al., 2009Gielen E. Smisdom N. vandeVen M. et al.Measuring diffusion of lipid-like probes in artificial and natural membranes by raster image correlation spectroscopy (RICS): use of a commercial laser-scanning microscope with analog detection.Langmuir. 2009; 25: 5209-5218Crossref PubMed Scopus (52) Google Scholar). Furthermore, to discriminate between colocalization and joint diffusion of molecules, we use cross-correlation RICS (CC-RICS) to detect whether two different molecular species with two different fluorescent colors diffuse together. Our study consists of two main parts. First, we obtained diffusion maps of fluorescent substances with different physical properties (amphiphilic or hydrophilic) within human skin SC. Second, we applied our approach to investigate the integrity of liposomes during transdermal penetration experiments into excised skin. Liposomes, generally large unilamellar vesicles (LUVs), are often used in the cosmetics industry and have been suggested as vehicles to transport and deliver drugs through the skin (Blume et al., 1996Blume G. Cevc G. Schatzlein A. Novel corticosteroidal dermatics based on the ultradeformable drug carriers, transfersomes.in: 23rd international symposium on controlled release of bioactive materials proceedings, Controlled Release Society. 1996: 713-714Google Scholar; Cevc, 1997Cevc G. Drug delivery across the skin.Expert Opin Investig Drugs. 1997; 6: 1887-1937Crossref PubMed Scopus (107) Google Scholar, Cevc, 2003Cevc G. Transdermal drug delivery of insulin with ultradeformable carriers.Clin Pharmacokinet. 2003; 42: 461-474Crossref PubMed Scopus (140) Google Scholar, Cevc, 2004Cevc G. Lipid vesicles and other colloids as drug carriers on the skin.Adv Drug Deliv Rev. 2004; 56: 675-711Crossref PubMed Scopus (573) Google Scholar; van Kuijk-Meuwissen et al., 1998van Kuijk-Meuwissen M.E.M.J. Junginger H.E. Bouwstra J.A. Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study.Biochim Biophys Acta. 1998; 1371: 31-39Crossref PubMed Scopus (99) Google Scholar). Multiple studies have shown that deformable LUVs, the so-called Transfersomes, function as superior transdermal penetration enhancers (Cevc et al., 1995Cevc G. Schatzlein A. Blume G. Transdermal drug carriers—basic properties, optimization and transfer efficiency in the case of epicutaneously applied peptides.J Control Release. 1995; 36: 3-16Crossref Scopus (260) Google Scholar). Although different techniques have been applied to document the penetration of the liposomes into the skin, such as transmission electron microscopy (Bouwstra and Honeywell-Nguyen, 2002Bouwstra J.A. Honeywell-Nguyen P.L. Skin structure and mode of action of vesicles.Adv Drug Deliv Rev. 2002; 54: S41-S55Crossref PubMed Scopus (226) Google Scholar; Honeywell-Nguyen et al., 2004Honeywell-Nguyen P.L. Gooris G.S. Bouwstra J.A. Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo.J Invest Dermatol. 2004; 123: 902-910Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Honeywell-Nguyen et al., 2006Honeywell-Nguyen P.L. Groenink H.W.W. Bouwstra J.A. Elastic vesicles as a tool for dermal and transdermal delivery.J Liposome Res. 2006; 16: 273-280Crossref PubMed Scopus (33) Google Scholar), tape stripping (Honeywell-Nguyen et al., 2004Honeywell-Nguyen P.L. Gooris G.S. Bouwstra J.A. Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo.J Invest Dermatol. 2004; 123: 902-910Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), laser scanning confocal microscopy, and MPEM (van Kuijk-Meuwissen et al., 1998van Kuijk-Meuwissen M.E.M.J. Junginger H.E. Bouwstra J.A. Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study.Biochim Biophys Acta. 1998; 1371: 31-39Crossref PubMed Scopus (99) Google Scholar; Cevc et al., 2002Cevc G. Schatzlein A. Richardsen H. Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements.Biochim Biophys Acta. 2002; 1564: 21-30Crossref PubMed Scopus (289) Google Scholar; Alvarez-Roman et al., 2004Alvarez-Roman R. Naik A. Kalia Y.N. et al.Visualization of skin penetration using confocal laser scanning microscopy.Eur J Pharm Biopharm. 2004; 58: 301-316Crossref PubMed Scopus (216) Google Scholar; Carrer et al., 2008Carrer D.C. Vermehren C. Bagatolli L.A. Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study.J ControlRelease. 2008; 132: 12-20Crossref PubMed Scopus (87) Google Scholar; Simonsson et al., 2011Simonsson C. Madsen J.T. Graneli A. et al.A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations.Toxicol Appl Pharmacol. 2011; 252: 221-227Crossref PubMed Scopus (19) Google Scholar), there is still no conclusive answer to the question of how the liposomes facilitate transdermal penetration of drugs (Cevc et al., 1998Cevc G. Gebauer D. Stieber J. et al.Ultraflexible vesicles, transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin.Biochim Biophys Acta. 1998; 1368: 201-215Crossref PubMed Scopus (428) Google Scholar, Cevc et al., 2002Cevc G. Schatzlein A. Richardsen H. Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements.Biochim Biophys Acta. 2002; 1564: 21-30Crossref PubMed Scopus (289) Google Scholar; van Kuijk-Meuwissen et al., 1998van Kuijk-Meuwissen M.E.M.J. Junginger H.E. Bouwstra J.A. Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study.Biochim Biophys Acta. 1998; 1371: 31-39Crossref PubMed Scopus (99) Google Scholar; El Maghraby et al., 2006El Maghraby G.M.M. Williams A.C. Barry B.W. Can drug-bearing liposomes penetrate intact skin?.J Pharm Pharmacol. 2006; 58: 415-429Crossref PubMed Scopus (194) Google Scholar; Bahia et al., 2010Bahia A.P.C.O. Azevedo E.G. Ferreira L.A.M. et al.New insights into the mode of action of ultradeformable vesicles using calcein as hydrophilic fluorescent marker.Eur J Pharm Sci. 2010; 39: 90-96Crossref PubMed Scopus (41) Google Scholar). By using a combination of CC-RICS (Choi et al., 2011Choi C.K. Zareno J. Digman M.A. et al.Cross-correlated fluctuation analysis reveals phosphorylation-regulated paxillin-FAK complexes in nascent adhesions.Biophys J. 2011; 100: 583-592Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and MEFM imaging, we demonstrate that the ability to measure spatially resolved joint diffusion of two-color fluorescently labeled liposomes provides new information about the structural evolution of these carriers during transdermal penetration experiments. Diffusion measurements of different fluorescent hydrophilic probes ([9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride (RhB), ATTO 647N Streptavidin (ATTO-647N-STREP), and tetramethylrhodamine dextran 3,000MW (TMR-DEX)) and amphiphilic probes (Lissamine-rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (RhB-PE) and ATTO-647N 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (ATTO-647N-PE)) were performed in human skin SC using RICS. The average diffusion coefficients of these probes at a depth of 2–4μm from the skin surface obtained by analyzing the whole image frame for several measurements (see details in the Materials and Methods section) were as follows: RhB, 0.72±0.10μm2s−1; RhB-PE, 0.83±0.15μm2s−1; ATTO-647N-STREP, 0.27±0.12μm2s−1; TMR-DEX, 0.78±0.09μm2s−1; and ATTO-647N-PE, 0.34±0.2μm2s−1. In particular, the diffusion values for the hydrophilic probes RhB, ATTO-647N-STREP, and TMR-DEX substantially differ from that observed in phosphate-buffered saline (PBS) buffer (420±40, 74±4, and 122±10μm2s−1, respectively, measured by fluorescence correlation spectroscopy (FCS)). Figure 1 shows how the diffusion data can be processed to obtain spatially resolved diffusion maps. Figure 1a shows a representative fluorescence intensity image, whereas Figure 1b shows a diffusion map of ATTO-647N-STREP of the central area of Figure 1a, obtained 2μm under the SC surface. In Figure 1c a typical spatial correlation of a fluorescent dye together with the fit of the data is shown. The diffusion map presented in Figure 1b is constructed by merging the spatially resolved diffusion information on top of the corresponding fluorescence intensity image (see Materials and Methods section). From the images, it is clear that the diffusion is heterogeneous across the volume sampled and that there is no clear correlation between fluorescence intensity and diffusion. Before applying the double-labeled LUVs or transfersomes to the skin, diffusion experiments were performed in buffer using both cross-correlation-FCS and CC-RICS. This was carried out with the aim to characterize the colloidal dispersions of LUV/transfersome obtained after preparation/purification (see Materials and Methods section), i.e., to characterize their size distribution, efficiency of membrane labeling (RhB-PE or ATTO647N-PE), and encapsulation of the hydrophilic probes (ATTO-647N-STREP or TMR-DEX). Figure 2a shows typical CC-RICS data for RhB-PE/ATTO-647N-STREP-labeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) LUVs in PBS. The large cross-correlation observed between the two different fluorescent probes indicates that they diffuse together. Representative RICS and CC-RICS data of a batch of POPC LUVs are presented in Supplementary Figure S1a–c online. For this batch, the diffusion coefficients measured were as follows: ATTO-647N-STREP, 3.70±0.20μm2s−1; RhB-PE, 3.20±0.20μm2s−1 and 3.20±0.20μm2s−1 for the cross-correlation. The slow diffusion observed for ATTO-647N-STREP (much slower than the 74±4μm2s−1 of the free ATTO-647N-STREP in PBS buffer) confirms that the ATTO-647N-STREP is indeed encapsulated in the lumen of the vesicles. Control experiments showed that the addition of the surfactant Triton X-100 (final concentration 0.1%) resulted in a total loss of cross-correlation between the two fluorescent probes, suggesting, as expected, the breakup of the vesicles. In addition, it was observed that the diffusion coefficient of the hydrophilic probe increased to the value of the free probe in buffer (see Supplementary Figure S2b online). All these results show that the LUVs are suitable for the experiments in skin. Download .pdf (.51 MB) Help with pdf files Supplementary Information Similar results were obtained for the colloidal dispersions of LUVs and transfersome labeled with ATTO-647N-PE and TMR-DEX. Supplementary Figure S2a online shows typical FCS autocorrelation curves and the cross-correlation curve for a batch of transfersomes labeled with ATTO-647N-PE with encapsulated TMR-DEX. The measured G0 values (the amplitude of the 2D spatial autocorrelation that are inversely proportional to the number of fluorescent particles in the observation volume) are nearly the same for the ATTO-647N-PE-labeled transfersomes, the encapsulated TMR-DEX, and for their cross-correlation. This result indicates that the encapsulated TMR-DEX (on average one per liposome) and the ATTO-647N-PE molecules (∼5 per liposome) experience joint diffusion and are seen as one entity, i.e., a fluorescent particle whose fluctuations are correlated. The diffusion coefficients measured in buffer (using both FCS and RICS) for the LUVs/transfersomes were in a range of 3–4μm2s−1 for the membrane dye, the hydrophilic dye, and the cross-correlation. This diffusion information corresponds to diffusing objects with a size distribution (vesicle diameter) of ∼120±20nm. This size distribution is in good agreement with the size of transfersomes used in previous transdermal drug delivery experiments (Cevc et al., 1998Cevc G. Gebauer D. Stieber J. et al.Ultraflexible vesicles, transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin.Biochim Biophys Acta. 1998; 1368: 201-215Crossref PubMed Scopus (428) Google Scholar; van Kuijk-Meuwissen et al., 1998van Kuijk-Meuwissen M.E.M.J. Junginger H.E. Bouwstra J.A. Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study.Biochim Biophys Acta. 1998; 1371: 31-39Crossref PubMed Scopus (99) Google Scholar; Cevc and Vierl, 2010Cevc G. Vierl U. Nanotechnology and the transdermal route: a state of the art review and critical appraisal.J Control Rel. 2010; 141: 277-299Crossref PubMed Scopus (460) Google Scholar). The diffusion of RhB-PE/ATTO-647N-STREP-labeled LUVs was measured by RICS and CC-RICS in the SC of excised human skin. On the surface of corneocyte clusters, the average diffusion was 0.47±0.25μm2s−1 for RhB-PE and 0.29±0.10μm2s−1 for ATTO-647N-STREP. However, a few of the measurements showed diffusion values of ∼2.5±1.0μm2s−1, indicating the existence of freely diffusing vesicles on the skin surface, and in about half of the measurements it was possible to observe a very weak cross-correlation between the fluorescent signals of the two probes. The G0 value of the RhB-PE was found to be ∼10 times smaller than the G0 value measured for ATTO-647N-STREP, a situation different from that observed in buffer, suggesting a change in the characteristics of the sample. Specifically, the lower G0 values of RhB-PE indicate that there are more RhB-PE molecules than ATTO-647N-STREP in the observation volume, suggesting vesicle rupture. The diffusion coefficient of the cross-correlation was 0.98±0.70μm2s−1; however, the values had a wide distribution and varied considerably from 0.33 to 2.5μm2s−1 (see Supplementary Figure S1d–f online.) Taken together, this information indicates the presence of a large fraction of fragmented vesicles coexisting with a small fraction of intact vesicles on the SC surface. The measurements were repeated at a depth of 4–6μm from the surface of the skin (see Supplementary Figure S1g–i online). The average diffusion coefficients for RhB-PE and ATTO647N-STREP were 0.66±0.08 and 0.27±0.12μm2s−1, respectively. Again, the G0 value of the RhB-PE was found to be ∼10 times smaller than the G0 value of the ATTO-647N-STREP. As indicated above, this result reflects that there is a higher concentration of RhB-PE molecules than ATTO-647N-STREP in the observation volume compared with that observed in buffer, suggesting rupture of the vesicles. Interestingly, these experiments show a total lack of cross-correlation (see Figure 2b). Similar results were found for ATTO-647N-PE/TMR-DEX-labeled POPC LUVs, where again a total lack of cross-correlation was observed at a depth of 4–6μm from the surface of the skin (Figure 3). In addition, no measurable differences in the diffusion coefficients were observed between experiments performed with high and low concentrations of LUVs (see Materials and Methods section), suggesting that the diffusion process is independent of the concentration of lipids within the concentration range explored (Figure 3). No measurable differences were observed between the 1- and 5-hour labeling for any of the samples (liposomes or transfersomes). Therefore, the presented data are representative of both conditions. Diffusion experiments were also carried out using ATTO-647N-PE/TMR-DEX-labeled transfersomes in excised skin. The results were very similar to that observed for POPC LUVs (see Figure 3 and Supplementary Figure S3a, b online), with no observable cross-correlation at a depth of 4–6μm from the tissue surface. In addition, as shown in Figure 3, the diffusion process was independent of the lipid concentration used (labeled transfersomes vs. labeled plus unlabeled transfersomes, see Materials and Methods). As a control experiment, the ATTO-647N-PE/TMR-DEX-labeled transfersomes were injected ∼30μm under the skin SC. These transfersomes were observed as bright diffusing particles, and a clear cross-correlation was measured between the two fluorescent probes, D=0.35±0.10μm2s−1 (see Figure 2c and Supplementary Figure S3d–f online). To investigate whether or not the particular way to treat the excised skin (see Materials and Methods) may influence the measured diffusion coefficients, we decided to test our approach in experiments involving Franz cells. Again, the results (see Figures 2d, 3, and 4) were similar to those of the experiments reported above, i.e., no cross-correlation was observed within the SC. Figure 4 shows representative diffusion maps for ATTO-647N-PE and TMR-DEX obtained with ATTO-647N-PE/TMR-DEX-labeled transfersomes at 4μm under the SC surface, including a fluorescence intensity image of the measured area. The data show no correlation between the diffusion of ATTO-647N-PE and TMR-DEX probes. As a control, experiments were carried out by exposing skin mounted in Franz cells for 1, 5, and 24hours to ATTO-647N-PE/TMR-DEX-labeled transfersomes. No measurable differences in the measured diffusion coefficients were found between the different time points. The results obtained from the experiments with the free dyes clearly show that RICS is a suitable technique to measure local diffusion in skin tissue. RICS provides spatially resolved diffusion information of fluorescence compounds in a noninvasive manner, which is different from other approaches such as those based on the use of Franz cells or tape stripping. In addition, the inherent optical sectioning effect of the MPEM (up to 1mm; Helmchen and Denk, 2005Helmchen F. Denk W. Deep tissue two-photon microscopy.Nat Methods. 2005; 2: 932-940Crossref PubMed Scopus (3146) Google Scholar)) combined with RICS offers a rather broad axial depth range for exploring the tissue structure and associated dynamics. The major limitation of our approach, however, is that fluorescent substances are required in order to perform the experiments, and labeling of the compound of interest must be performed. This situation is problematic, particularly if the compounds of interest are on the size range of the fluorophores (i.e., small molecules), as strong modifications on the molecular structure may affect the properties of the compound of interest. This situation reflects a drawback of our approach compared with diffusion experiments obtained by other techniques in which no" @default.
• W2032926656 created "2016-06-24" @default.
• W2032926656 creator A5002764364 @default.
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• W2032926656 date "2013-05-01" @default.
• W2032926656 modified "2023-10-12" @default.
• W2032926656 title "Spatially Resolved Two-Color Diffusion Measurements in Human Skin Applied to Transdermal Liposome Penetration" @default.
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