L. S. Eberlin, J. V. Mulcahy, A Tzabazis, J. Zhang, H. Liu, M. M.

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Article
pubs.acs.org/JACS
Visualizing Dermal Permeation of Sodium Channel Modulators by
Mass Spectrometric Imaging
Livia S. Eberlin,† John V. Mulcahy,† Alexander Tzabazis,‡ Jialing Zhang,†,§ Huwei Liu,§
Matthew M. Logan,† Heather J. Roberts,† Gordon K. Lee,∥ David C. Yeomans,‡ Justin Du Bois,*,†
and Richard N. Zare*,†
†
Department
Department
§
Department
∥
Department
‡
of
of
of
of
Chemistry, Stanford University, Stanford, California 94305-5080, United States
Anesthesia, Stanford University, Stanford, California 94305-5117, United States
Chemistry, Peking University, Beijing, China, 100871
Surgery, Stanford University, Stanford, California 94305-5641, United States
S Supporting Information
*
ABSTRACT: Determining permeability of a given compound
through human skin is a principal challenge owing to the
highly complex nature of dermal tissue. We describe the
application of an ambient mass spectrometry imaging method
for visualizing skin penetration of sodium channel modulators,
including novel synthetic analogs of natural neurotoxic
alkaloids, topically applied ex vivo to human skin. Our simple
and label-free approach enables successful mapping of the
transverse and lateral diffusion of small molecules having different physicochemical properties without the need for extensive
sample preparation.
■
INTRODUCTION
Transdermal delivery of therapeutic agents represents an
attractive alternative to oral or parenteral delivery and has
witnessed increased clinical application in the past two
decades.1 Transdermal delivery has many advantages in
comparison to other methods for drug administration.1,2
Although oral analgesics are commonly prescribed for the
treatment of acute and chronic pain, adverse, potentially fatal
effects such as respiratory depression, nausea, and addiction can
ensue from systemic drug exposure.3 Clinically effective drug
concentrations can be introduced at a peripherally located site
of injury or inflammation by topical administration without
resulting in high systemic concentrations that may increase the
likelihood of untoward side effects.3,4 Understanding drug
penetration and diffusivity through skin, however, poses
significant challenges given the structural complexity of this
organ and the many disparate mechanisms that transport small
molecules across the dermal layers.2 Methods for determining
transdermal drug absorption in skin include skin extraction
measurements,5 quantitative autoradiography,6 and spectroscopic methods (fluorescence7 and FTIR8). In this work, we
present a desorption ionization mass spectrometric imaging
technique for identifying small molecule analytes in skin and for
mapping compound distribution through the dermal layers. We
suggest that this technique represents a superior approach to
measuring the extent of skin permeation of topically applied
compounds.
Many clinically employed analgesics and anesthetics for
relieving pain modulate voltage-gated sodium channels (NaVs),
© 2014 American Chemical Society
which are a family of integral membrane proteins responsible
for the rising phase of action potentials in electrically
conducting cells. 9 One such example is lidocaine, a
prescription-based injectable and topical pharmaceutical
agent. In addition to clinically employed anesthetics, natural
neurotoxic alkaloids, including saxitoxin, neosaxitoxin,10
batrachotoxin,11 and aconitine,12,13 are sodium channel
modulators, some of which are currently being investigated
for potential usage as pain therapeutics owing to the high
affinity and specificity that such compounds demonstrate for
NaVs.10,14−16 Little is known about the ability of these
compounds to penetrate human skin to produce a topical
analgesic effect. Mass spectrometry imaging is a label-free
method with which the distribution of a multitude of
compounds can be mapped in biological tissues with high
specificity and offers an efficient, straightforward method for
this type of analysis.17−19
We have used high mass resolution/mass accuracy
desorption electrospray ionization mass spectrometric imaging
(DESI-MSI) to investigate the permeation of natural sodium
channel blockers and novel synthetic analogues following
topical application of these compounds to ex vivo human skin.
DESI-MSI enables two-dimensional mapping of a sample
analyte in the ambient environment, without the need for
extensive sample preparation. Samples are bombarded with
microdroplets of acetonitrile that dissolve hundreds of
Received: February 16, 2014
Published: April 7, 2014
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Journal of the American Chemical Society
Article
analogue of batrachotoxin, BTX-A,23 were observed as
protonated molecules at m/z 235.1805 (mass error of 0.09
ppm), m/z 646.3220 (mass error of 0.35 ppm), and m/z
504.2373 (mass error of 1.44 ppm), respectively. Errors in these
measurements were calculated from an average of a few mass
spectra. Figure 1 shows the DESI-MSI results obtained for
endogenous lipids and metabolites as well as exogenous
compounds present in tissue. The splash forms secondary
microdroplets that enter a mass spectrometer, providing a
detailed chemical map of the distribution of molecules within
the sample surface.20 After DESI-MSI, the same tissue section
can be stained, optically imaged, and compared to selected 2D
DESI-MS ion images.21 The compounds investigated in this
study include common analgesics such as lidocaine and
prilocaine as well as aconitine, an herbal neurotoxin used in
Chinese medicine, saxitoxin, a sodium channel inhibitor
synonymous with paralytic shellfish poisoning, and novel
synthetic analogues of both saxitoxin and batrachotoxin. The
effect of sunburn injury on the penetration of these sodium
channel blocking agents was also examined. Using DESI-MSI,
we demonstrate that the compound of interest can be detected
with unambiguous determination among the hundreds of other
endogenous skin compounds concurrently being analyzed. The
spatial distribution of these agents has been mapped and
directly compared to the morphology of the skin sections to
assess transdermal penetration.
■
RESULTS AND DISCUSSION
Dermal Permeation in ex Vivo Human Skin. Human
skin samples were obtained from four patients undergoing
surgery at Stanford Medical School. All patients gave written
informed consent following an approved IRB protocol. The
skin graft was placed in a shallow dish partially filled with
synthetic interstitial fluid. Confined circular treatment areas
were created by delineating the application site with a felt-tip
pen and encircling this mark with petroleum jelly. The
compounds were directly applied to the treatment area with a
pipet, and after 1, 4, or 10 h of application, the skin was frozen
and cross sectional tissue samples carefully prepared to avoid
cross-contamination. Positive ion mode DESI-MSI was
performed using an Orbitrap for mass analysis at a mass
resolution of 60 000 (see the Supporting Information for
experimental details). Rich and distinctive molecular profiles
were observed from the different layers of skin (epidermis,
dermis, hypodermis) by high-resolution DESI-MS imaging.
Many of the ions were identified as sodium or potassium
adducts of complex glycerophospholipids: glycerophosphocholines (PC), lyso-PC, sphingomyelins, glycerophosphoethanolamines, and glycerophosphoglycerols; and glycerolipids:
triacylglycerols (TG) and diacylglycerols (Table S1). Ion
images showing the distribution of these compounds within
the different layers of the skin samples are shown in Figure S1,
alongside the optical image of the hematoxylin and eosin
(H&E) stained tissue sections. Pathological evaluation of the
H&E stained human skin tissue sections reveals the presence of
epidermis (30−60 μm thickness), superficial dermis (250−450
μm thickness), deep dermis (2200−3000 μm thickness) and
hypodermis (600−1200 μm thickness) layers within all the
human samples analyzed.
Topical application of local anesthetics and NaV modulators
was initially performed using ethanol as the vehicle, the alcohol
most commonly used as a permeation enhancer.22 Despite the
complexity of the DESI mass spectra obtained from the
different layers of the human skin, high mass resolution/high
mass accuracy measurements allowed for clear mass spectral
separation and identification of the compounds of interest.
Saxitoxin was detected at m/z 282.1303 (mass error of 2.14
ppm), corresponding to the protonated molecule with loss of
an equivalent of H2O, whereas lidocaine, aconitine, and an
Figure 1. Penetration of the sodium channel blocker compounds
tested in human skin of the same donor using ethanol as the vehicle.
Chemical structures are shown for (a) saxitoxin, (d) lidocaine, (g)
aconitine, and (j) BTX-A. Positive ion mode DESI-MS ion images of
the compounds are shown for human skin in which (b) saxitoxin, (e)
lidocaine, (h) aconitine, and (k) BTX-A were topically applied. Optical
images of cross sections of the same tissue sections imaged by DESI
after H&E stain are shown in (c) for saxitoxin, (f) for lidocaine, (i) for
aconitine, and (l) for BTX-A.
human skin tissue sections from the same patient in which
saxitoxin, lidocaine, aconitine, and BTX-A were topically
applied following the protocol outlined above. The optical
image of the H&E stained skin sample following DESI-MSI is
also depicted to assist visualization of the outline of the tissue
section. As observed in the DESI-MS ion image, saxitoxin did
not exhibit any penetration in the human skin tissue (Figure
1b). The molecule is completely localized to the application site
at the top of the tissue section. The low molecular weight of
this compound notwithstanding,1 the inability of saxitoxin to
penetrate the epidermis is unsurprising given its charged,
hydrophilic nature. By contrast, the DESI-MS ion image of
lidocaine (Figure 1e) shows that this molecule readily diffuses
throughout the tissue section, penetrating to the deep dermis
layer (as determined by comparison with the H&E stained
tissue section). Similar to lidocaine, DESI-MS images of
aconitine and BTX-A also reflect extensive tissue penetration.
Both compounds were detected deep within the tissue section,
the former reaching the hypodermis of the skin. Ion images for
samples treated with either aconitine or BTX-A reveal a higher
relative signal intensity in the top layers of the skin than in the
hypodermis (Figure 1h,k, respectively).1 DESI-MS ion images
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Figure 2. Penetration of novel synthetic analogue tested in human skin of the same donor. The chemical structure of STX-ge is shown in (a).
Positive ion mode DESI-MS ion images of the compound are shown for human skin in which STX-ge was topically applied using (b) ethanol and
(d) DMSO as vehicles. Optical images of the same tissue sections imaged by DESI after H&E stain are shown in (c) and (f), respectively. DESI mass
spectra of selected skin regions outlined with a small black box in the optical images in (c) and (f) are shown for STX-ge (d) in ethanol and (g) in
DMSO.
and prilocaine in a ratio of 1:1 by weight. We have investigated
the skin penetration of these compounds in EMLA cream
applications to human skin in addition to samples in which
aconitine, BTX-A, saxitoxin, and STX-ge were each added.
DESI-MS ion images of lidocaine and prilocaine (m/z
221.1651, mass error of 0.3 ppm) show complete transverse
skin penetration to the hypodermis as well as diffuse lateral
distribution within the skin section in all 14 samples analyzed
(Figure 3). When aconitine is included to the EMLA cream
for saxitoxin, lidocaine, aconitine, and BTX-A using ethanol as
vehicle were reproducible in skin sections obtained from the
three different donors. Overall, the average penetration depth
of lidocaine was 1.24 mm (5 samples), of aconitine was 3.05
mm (6 samples), of BTX-A was 2.00 mm (4 samples), and no
penetration was observed for saxitoxin (7 samples).
A synthetic derivative of saxitoxin bearing a long chain,
glycerol ether group (STX-ge) was also examined in human
skin DESI-MS imaging experiments.24,25 The attachment of a
lipophilic chain to saxitoxin was expected to facilitate
transdermal penetration of this compound. Detection of
STX-ge was observed in the positive ion mode with
protonation and loss of H2O at m/z 681.4652 (mass error of
0.56 ppm) and in its protonated form m/z 699.4760 (mass
error of 0.55 ppm). An endogenous lipid, ubiquitously
distributed throughout the skin tissue, was detected at the
same nominal m/z 699.4 of STX-ge. This lipid (measured at m/
z 699.4082) could be completely resolved from toxin derivative
using high mass resolution DESI-MSI, thereby enabling
accurate mapping of the exogenous compound in tissue (Figure
S2). The DESI-MS ion image for STX-ge is shown in Figure
2b. As is noted in Figure 2b, despite the hydrocarbon
appendage, STX-ge does not penetrate human skin, similar to
the results with saxitoxin (ethanol as vehicle, 6 samples). This
compound was only detected outside of the tissue section, and
only lipids were identified in the mass spectra of the epidermis
and superficial dermis of the skin (Figure 2d).
In an attempt to improve the transdermal penetrability of
STX-ge, ethanol was replaced with dimethyl sulfoxide (DMSO)
as the vehicle. DMSO is a well-known skin penetration
enhancer that interacts with lipids in the stratum cornea and
epidermis and induces conformational changes in keratin.26
Our DESI-MSI results show that some degree of skin
penetration by STX-ge is achieved when DMSO is employed
(Figure 2e). Figure 2g shows the mass spectra of the superficial
layer of the skin dermis in which STX-ge can be clearly
detected together with the complex phospholipids that
characterize this layer of tissue. An average penetration depth
of 0.62 mm was measured for STX-ge using DMSO (3
samples).
In the clinic, eutectic mixture of local anesthetics (EMLA)
cream is prescribed as a dermal anesthetic. EMLA cream is an
oil phase emulsion containing a eutectic mixture of lidocaine
Figure 3. DESI-MS ion images showing the penetration of (a)
aconitine, (b) BTX-A and (c) STX-ge tested in human skin of the
same donor using EMLA cream. Ion images showing the penetration
of lidocaine and prilocaine, compounds with are both present in the
EMLA cream formulation, are also presented for the three sections
analyzed.
formulation, similar penetration behavior was observed
(average depth 2.49 mm, 4 samples); however, lateral spread
of the compound was far less pronounced (Figure 3a). BTX-A
also shows complete skin penetration (average depth 1.10 mm,
4 samples), albeit not to the same depth as lidocaine/prilocaine
(Figure 3b); no penetration was observed for saxitoxin (4
samples). STX-ge showed partial skin penetration, with
permeation observed through the skin epidermis reaching the
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Journal of the American Chemical Society
Article
superficial dermis, similar to results obtained using DMSO as
the vehicle (Figure 3c). In a few skin sections in which less
compound was detected, complete penetration of the STX-ge
compound up to the superficial dermis was seen (average
penetration depth of 0.45 mm for 4 samples) (Figure S3).
Evaluation of Dermal Permeation in a UV Burn Injury
Model. Topical application of analgesics has been used for the
treatment of dermal pain caused by skin injuries such as
sunburn. To test if the penetrability and diffusivity of small
molecules in human skin is altered due to UV burn injuries, we
have performed DESI-MS experiments on a burn injury model
by applying UV radiation (300−450 nm) to ex vivo human
skin.27 Following exposure to UV light, compounds of interest
were applied as ethanol solutions. UV radiation of human skin
sufficient to induce DNA damage, as measured by increase in
growth arrest and DNA-damage-inducible Gadd45a protein,
did not significantly alter skin penetration of the five
compounds tested (Figure S4) when compared to our earlier
findings (see Figures 1 and 2).
Dermal Permeation in ex Vivo Human Skin at
Different Application Times. We have also evaluated the
penetration behavior of aconitine, lidocaine, and BTX-A at
different application times (1, 4, and 10 h) in ex vivo human
skin. Interestingly, aconitine was the only compound that
showed a pronounced change in penetration depth with
application time, with decreased depth penetration occurring at
1 h of application in comparison to 4 and 10 h (Figure S5). For
all the other compounds, no significant temporal difference was
observed.
Dermal Permeation in in Vivo Rat Skin. Transdermal
permeation experiments using live murine subjects were
performed for comparative purposes against ex Vivo data.
Solutions of aconitine and STX-ge in either ethanol or DMSO
were applied to the shaved back of male Sprague−Dawley rats
under general anesthesia (see Supporting Information for
experimental details).28 The animals were sacrificed, their skin
removed, sectioned, and analyzed following the same
experimental procedures used for ex vivo experiments.
Pathologic evaluation of the tissue sections revealed that in
all samples analyzed, besides skin epidermis, dermis, and
hypodermis layers, muscle tissue was also present. Complete
skin penetration of aconitine was noted using both ethanol and
DMSO as vehicles, and in both cases the compound reached
the muscle layer of the tissue section. Interestingly, many
samples showed a different spatial distribution of analyte within
the sectioned tissue, which varied depending on the choice of
vehicle (Figure 4). Ion images of aconitine applied in ethanol to
rat skin suggest that transverse diffusion of this compound is
facilitated through hair follicles, an important pathway for the
penetration of topically administered substances.29 The same
experiment performed with DMSO reveals a very different ion
map that is much more reminiscent of the ex vivo data.
Pathologic evaluation of the H&E stained tissue sections in
comparison with the DESI-MS ion images confirms that
aconitine is colocalized with the regions of hair follicles within
the tissue section. In the case of STX-ge, complete penetration
of this compound occurs using both ethanol and DMSO,
reaching the deep dermis layer of the skin (Figure S6). The
results from ethanol experiments stand in contrast to those
obtained with human skin. Overall, deeper tissue penetration of
both aconitine and STX-ge was achieved in vivo using DMSO
as vehicle (1.90 mm in ethanol for 7 samples versus 2.60 mm
for 3 samples in DMSO for aconitine; 0.90 mm in ethanol for 5
Figure 4. DESI-MS ion images showing the penetration of aconitine
tested in in vivo rat skin using (a) ethanol and (b) DMSO as vehicles.
Optical images of the same tissue sections imaged by DESI after H&E
stain are also shown.
samples versus 1.12 mm for 7 samples in DMSO for STX-ge)
(Table S2).
■
CONCLUSIONS
Our findings demonstrate that DESI-MSI is an operationally
simple yet sensitive and powerful tool for investigating
transdermal permeation of small molecules. We have
successfully employed high mass resolution DESI-MSI to
visualize the permeation of topically applied sodium channel
modulators on ex vivo human skin samples and on live animal
skin. These results lead the way for subsequent animal
behavioral studies to assess the local antinociceptive effect of
these agents when applied in different vehicles. We believe
DESI-MSI methodology has the potential to play a guiding role
in topical drug development and formulations research.
■
ASSOCIATED CONTENT
■
AUTHOR INFORMATION
* Supporting Information
S
Experimental details, supporting figures and tables are provided.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Corresponding Authors
[email protected]
[email protected]
Notes
The authors declare the following competing financial
interest(s): J.V.M., D.C.Y., and J.D.B. have an equity interest
in SiteOne Therapeutics, Inc., a start-up company focused on
pain research. The other authors declare no competing financial
interests.
■
ACKNOWLEDGMENTS
L.S.E. is grateful to the Center of Molecular Analysis and
Design (CMAD) for her postdoctoral fellowship. We thank
Richard Luong (Department of Comparative Medicine,
Stanford University) for the pathology services and assistance
provided. J.Z. thanks the China Scholarship Council affiliated
with the Ministry of Education of China (grant no.
201206010110). J.D.B. thanks the NIH for partial support of
this work (R01 NS045684).
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■
Article
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