First, a ketoprofen solution in deuterated PG was applied ex vivo to an excised piece of mouse ear, which was subsequently sealed between two coverslips and placed on the SRS microscope stage. Three-dimensional volumes of SRS data were then acquired over an area of approximately 250 × 250 μm2
and to a depth of ~100 μm. The images obtained extended from within the drug-in-solvent layer above the skin down to the subcutaneous fat. The lipid architecture was first mapped from a series of images based on the CH2
stretching absorbance at 2845 cm−1
. Attention was focused on the major barrier to skin penetration of chemicals, the SC (the outermost ~15 μm of the skin) which comprises tightly packed layers of terminally differentiated, keratin-filled corneocytes (roughly hexagonal in shape, ~20 μm across, yet only ~0.5 μm in thickness), the spaces between which are filled with a mixture of lipids organized into lamellar bilayers. SRS delineated individual cells clearly via the CH2
signal emanating from the intercellular lipids () in the images recorded from different depths (Z), indicated down the left side of the figure, at the beginning (t = 11 minutes post-application of the ketoprofen in PG solution) and end (t = 158 minutes) of the penetration experiments. Registration of images to the same tissue region is possible, an attribute thereby permitting useful and novel mechanistic information to be deduced. An off-resonance image taken after the final CH2
-stretching dataset shows no signal, demonstrating the label-free contrast and chemical selectivity of SRS. When the pump laser is tuned off-resonance (i.e., away from the CH2
stretching absorbance frequency), the signal level drops to the background which is <1% of the peak CH2
signal (and <5% of the peak signal from PG-d8
discussed below), making the image effectively black and featureless. This outcome highlights a major advantage of SRS over the related coherent anti-Stokes Raman scattering (CARS) technique for which strong background signals from tissue complicate data analysis4
Figure 2 Architecture of mouse skin based on SRS imaging. Images at t = 11 and 158 minutes are acquired with CH2-stretching contrast and show the lipid architecture of the skin at various depths. The off resonance images demonstrate the chemical contrast of SRS. (more ...)
Then, on the same tissue sample, the laser was tuned either to the aromatic CH stretching absorbance frequency, or to the CD2 (carbon-deuterium) stretching band to visualize ketoprofen and PG, respectively. Because it was possible to recognize individual cells in the skin, the 3-dimensional stacks could be registered to minimize the impact of drift and swelling of the tissue. This meant that changes in signal from the topically applied chemicals, as a function of depth within the SC, could be visualized throughout the experiment. Such time lapse images for deuterated PG and ketoprofen are in . The image series at the skin surface (Z = 0 μm) shows minimal changes in intensity because the chemical concentrations above the tissue do not change significantly during the experiment. In contrast, images in the stratum corneum (Z = 12 and 15 μm) show increasing signal over time. The data acquisition time in these experiments was about 20 seconds per frame (256 × 256 pixels). Each stack of images from the skin surface to the deeper layers of the tissue therefore required about 10 minutes to acquire. Alternating stacks of images from the drug and from the cosolvent were obtained by re-tuning the laser between each stack. It is apparent that both compounds penetrate via the intercellular lipids of the stratum corneum and, as well, through the hair shafts. Only by 3-dimensional resolution of the penetration maps is this type of direct mechanistic insight achievable, and other analytical techniques, including those based on spontaneous Raman scattering, lack sufficient spatiotemporal resolution for these measurements.
Figure 3 Imaging the penetration of deuterated PG (upper panel) and ketoprofen (lower panel) across the stratum corneum. Images acquired at the depths indicated down the left-hand side of the figure and times indicated along the top show the penetration of cosolvent (more ...)
Next, integration of the total signal as a function of depth from the registered 3-dimensional maps provided an effective concentration profile of the penetrating species. A representative result for deuterated PG as a function of time post-application of the formulation is in . Experimental data points were obtained by integrating comparable regions of interest in . The relative amount of PG, as a function of position within the skin, at each time, is determined by the ratio of the integrated total signal at each depth to that at the surface (located at approximately Z = 0 μm). Thus, each normalized profile decreases from a value of 1 at the surface with increasing distance of penetration into the skin. Normalization of the profiles to 1 at the top layer assumes that the chemical's concentration above the sample does not change significantly during the experiment (a reasonable approximation given the effectively infinite `dose' of solution applied at t = 0). Monotonically-increasing penetration profiles over time are observed; that is, the area under the normalized signal versus distance profiles increases as more of the cosolvent is absorbed into the SC.
Figure 4 Integrated depth-profiling analysis of percutaneous penetration. (A) Depth profiles of deuterated PG as a function of time. (B) Comparison of the areas under the normalised SRS signal versus skin depth profiles (AUCs) of PG and ketoprofen at different (more ...)
Data obtained nearly simultaneously from ketoprofen showed similar behavior, although the slower rate of change of the normalized signal versus distance profile with increasing time demonstrated that PG penetrates the stratum corneum more efficiently than the drug (). This finding is consistent with tape-stripping and attenuated total reflectance infrared spectroscopy experiments that tracked the uptake of ibuprofen and PG into human SC9–12
, and emphasises the importance of tracking not only the drug when attempting to understand the performance of a topical formulation.13
The temporal evolution of the effective concentration profiles in and their monotonic decrease from the skin surface into the barrier are both qualitatively and quantitatively comparable with data from the far more invasive and labor-intensive tape-stripping results observed previously9,11,12
, and suggest a diffusion lag-time on the order of 2 hours. More refined interpretation of these results will be possible with the acquisition of SRS images in finer steps (e.g., 1 μm, rather than every 3 μm), and an improved use of the CH2
signal to define SC thickness.
A major challenge in analyzing the 3-dimensional datasets presented here is to extract truly quantitative information from the images. Spatial drift and swelling of tissue over the time course of the experiment mean that the three-dimensional datasets must be registered with care in order to separate tissue movement from diffusion of the compounds of interest within the skin. In this work, this is achieved by visual inspection which permits linear 3-dimensional movements to be taken into account but cannot remove distortions or swelling of the tissue. In addition, the penetration of compounds, such as PG, can affect the linear optical properties of the tissue, affecting the depth profiles by changing signal levels because of alterations in laser power at the focus rather than changes in chemical concentration14
. Recent reports of optical clearing measurements in microscopy suggest that these linear optical property changes are relatively rapid (they occur in ~30 minutes),15
so that the measurements reported here, which began at 26 minutes post-initiation of the experiment, should not be significantly affected. Nevertheless, developing methods to compensate for the changing linear optical properties of tissue will improve quantitative analysis. Additionally, improving the imaging speed7
will ultimately allow experiments to be conducted in vivo
, where high speed imaging is a requirement because living samples inevitably move on the microscopic length scale, and result in blurred images if the acquisition speed is too low.
A further illustration of the ability of SRS microscopy to track chemical uptake and transport kinetics across the SC is illustrated in , which plots the normalized, integrated signal from PG at 12 and 15 μm from the skin surface (note that, in this case, the data have been normalized such that the final value is equal to 1 so as to better visualize the overall trends with time). The significant increase in cosolvent level in deeper tissue layers with increasing time is clearly shown; in contrast, the relative signal at the skin surface (Z = 0 μm) did not change, reflecting the essentially infinite dose of formulation applied.
Figure 5 (A) Temporal profiles of the signal intensity from PG at the skin surface (Z = 0 μm) and at two representative depths (12 and 15 μm) into the skin. (B) Temporal profiles of PG at a depth of Z = 6 μm at two specific sub-regions (more ...)
In addition, the three-dimensional imaging capability provided novel mechanistic insight into the topical drug delivery process (an achievement impossible with Raman depth profiling16,17
or tape stripping2,9,11,12
which are essentially one-dimensional techniques). Specifically, compares the penetration profile of deuterated PG via a hair shaft to that via the SC intercellular lipids (outlined respectively in the inset of by the green and red boxes). These results were generated by integrating the signal from two regions of interest, one a clearly visible follicle, the other an area encompassing the basket-weave structure typical of SC, at the same depth, as a function of time. While the signal from SC steadily increased with time, that from the hair shaft remained essentially unchanged and, importantly, significantly above background. Thus, PG penetration via the hair shaft was rapid and attained steady-state before the first measurement was recorded at 26 minutes post-application of the formulation. On the other hand, transport through the SC intercellular lipids was slower and the increasing signal over time during the ~2-hour experiment represented the non-steady state approach to a constant flux. These data provide direct experimental proof of behavior deduced over 40 years ago by Scheuplein18,19
who inferred that the initial drug molecules crossing the skin post-treatment came through low-resistance, “shunt” pathways (such as hair shafts) of limited capacity; the parallel, but slower, transport across the bulk of the SC eventually overwhelms the transient pathways, however, and completely dominates at steady-state. Despite additional, indirect support for the importance of the follicular pathway,20,21
the SRS microscopy data in are unique in their separation and real-time observation of molecular transport at two distinct rates occurring through two discrete and identifiable pathways across the skin.
In a final series of measurements, the apparently rapid penetration of the cosolvent, relative to that of drug (), was probed following topical application of deuterated ibuprofen in `normal' (i.e., undeuterated) PG. The drug concentration in the cosolvent represented about 90% of its saturation solubility9
. Less than 30 minutes post-application, the formation of solid ibuprofen crystals (with characteristic sizes of tens of microns - see ), which produced a strong Raman signal at the CD stretching frequency, was observed at the skin surface () on the SC (). Some drug penetration via the follicular “shunt” route was again seen (inset in ). The logical explanation for the precipitation of ibuprofen observed is that the faster penetration of PG into the skin (coupled, perhaps, with some evaporation from the surface) caused the drug concentration to increase above its saturation solubility. Again, while this phenomenon has been anticipated, and for which indirect evidence has been obtained,10
the 3-dimensional SRS imaging illustrated in represents new and direct proof of the so-called “metamorphosis”22–24
of a topical drug formulation post-application to the skin.
Figure 6 Crystal formation on the skin surface 25 minutes post-topical application of a solution of ibuprofen in PG. (A) Maximum intensity depth projection (of the upper ~30 μm) with SRS contrast at 2120 cm−1. (B) Depth projection down the yellow (more ...)
In summary, this SRS microscopy study of dermato-pharamacokinetics has revealed previously invisible features of the percutaneous penetration of skin-active compounds. As a label-free optical imaging technique, SRS permits non-destructive visualization of the drugs and excipients delivered from topically-applied formulations with high spatial and temporal resolution. The application of SRS to the delivery of ketoprofen and ibuprofen has unambiguously revealed distinct penetration pathways, across which the molecules transport at different rates, and has directly visualized the formation of drug crystals on the tissue surface in situ during the initial stages of the penetration process. Improved image acquisition speed, signal processing and analysis – especially in terms of defining more precisely the location of the skin surface - will allow further quantitative information to be extracted, and the technology may ultimately be applied in vivo (in models more relevant than the mouse skin used in this work) to understand and optimize formulations and delivery vehicles for topical and transdermal drug delivery.