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Magnesium Research 2016; 29 (2): 35-42 ORIGINAL ARTICLE

Permeation of topically appliedMagnesium ions through humanskin is facilitated by hair folliclesNavin Chandrakanth Chandrasekaran1,2, Washington Y. Sanchez2,Yousuf H. Mohammed2, Jeffrey E. Grice2, Michael S. Roberts2,3,Ross T. Barnard1

1 School of Chemistry and Molecular Biosciences, Australian Infectious Diseases ResearchCentre, The University of Queensland, Brisbane, Queensland, 4072 Australia; 2 TherapeuticsResearch Centre, School of Medicine, Translational Research Institute, The University ofQueensland, 37 Kent St. Woolloongabba, Queensland, 76131, Australia; 3 School of Phar-macy and Medical Sciences, University of South Australia, City East Campus, North Terrace,Adelaide, South Australia, 5001, AustraliaCorrespondence: Prof. Ross T. Barnard, School of Chemistry and Molecular Biosciences, Australian Infectious
Diseases Research Centre, The University of Queensland, Brisbane, Queensland, 4072 Australia.<[email protected]>
Abstract. Magnesium is an important micronutrient essential for various bio-logical processes and its deficiency has been linked to several inflammatorydisorders in humans. Topical magnesium delivery is one of the oldest forms oftherapy for skin diseases, for example Dead Sea therapy and Epsom salt baths.

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Some anecdotal evidence and a few puration of inflammatory skin conditionsOn the other hand, transport of magnof skin, the stratum corneum, is contwas to estimate the extent of magnesiand the role of hair follicles in facilitacation of magnesium solution, we fouhuman stratum corneum and it dependWe also found that hair follicles makepenetration.

Key words: magnesium, hair follicle, multi

Mineral based therapies such as Dead Seatherapy have been used for several centuriesand have been associated anecdotally with arange of health benefits. These are mostly skinconditions where the stratum corneum (SC) iscompromised, such as psoriasis and dermatitis [1].In all these therapies, magnesium is believed tobe the key component involved in ameliorating orsubduing inflammatory response. Much evidenceexists demonstrating that increased levels of

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To cite this article: Chandrasekaran NC, Sanchez WY, Mohamof topically applied Magnesium ions through human skin is fdoi:10.1684/mrh.2016.0402

ished reports have attributed amelio-the topical application of magnesium.um ions across the protective barrierious. Our primary aim in this studyion permeation through human skin

g the permeation. Upon topical appli-that magnesium penetrates throughn concentration and time of exposure.ignificant contribution to magnesium

photon microscopy, mag-fura-2

agnesium through oral supplementation, orietary intake can ameliorate inflammatory disor-ers [2-4]. However, in order for topically appliedagnesium to be effective in treating inflamma-

ory skin conditions, transport of its ions acrosshe SC is a critical precondition.

Transdermal delivery is a commonly usedoute of administration for treatments that arentended to have both local and systemic effects.ermeability of the skin to magnesium ions

35med YH, Grice JE, Roberts MS, Barnard RT. Permeationacilitated by hair follicles. Magnes Res 2016; 29(2): 35-42

mailto:[email protected]

10.1684/mrh.2016.0402

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could be dependent on pathways associated withappendages, glands, hair follicles, hydration stateor integrity of the SC [5]. The barrier prop-erty of SC has been widely discussed, with thegeneral view promulgated in the literature thatthe layer acts as a selective barrier to trans-port of ions [6, 7]. It has also been conjecturedthat the negatively charged SC would not allowpermeation of any charged molecules such asmagnesium ions. The radius of the hydrated mag-nesium ion has been reported to be 400 timeshigher than its dehydrated form [8], leading tothe assertion that it is almost impossible formagnesium ions to pass through biological mem-branes [8, 9]. However, when we re-examinedthis calculation, it was found that the volumeof the magnesium ion is 451.76 Å3 [ 4

3 �(4.76)3]in the hydrated state, but based on the ionicradii of dehydrated and hydrated magnesium ions,i.e., 0.87 Å and 4.76 Å respectively [10-13], wecalculated that the hydrated radius of the ionis only 5.47 fold ( 4.76 Å

0.87 Å) greater than its dehy-

drated radius. Based on our recalculation, and onpublished data showing systemic effects of top-ical magnesium [1, 5], we postulated that thehydrated magnesium ion could potentially pen-etrate by bulk diffusion through the 10 Å poresformed by protein subunits in the lipid mem-brane [5, 14], or by other means, such as hairfollicles. To test this postulate, we conductedexperiments to test and visualize magnesium pen-etration, localization and concentration in humanepidermis.

In this article, we have demonstrated a methodto visualise magnesium ions in human skin sec-tions using mag-fura-2, tetrapotassium salt, afluorescent dye that specifically binds to magne-
sium ions. This enabled us to identify the influxor localisation of magnesium ions in human skin.Subsequently, we varied the duration of treatmentand concentrations of magnesium chloride solu-tions applied to the skin. We used 5 mM MgCl2
solution, as it was closer to the physiological lev-els found in human tissues. The concentrations52 mM and 1.9 M, even though far from physi-ological levels, are equivalents of MgCl2 in oceanwater and Dead Sea water, respectively, were usedto understand the relevance of Dead Sea ther-apy. Moreover, we developed a novel method toplug hair follicles in an attempt to investigatepossible magnesium permeation through hairfollicles.

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ethods

opical magnesium solution application

e obtained excised human skin from patientsndergoing abdominoplasty. We then topicallypplied deionised water (milliQ) (vehicle control)nd 5 mM MgCl2 solution (pH 7.2) for 30 minutes,t 23-25 ◦C using the “donor” (upper) chamberf a Franz cell. We also applied 5 mM MgCl2 tokin that was tape stripped 30 times, in order toacilitate the permeation of magnesium throughpidermis and represent a positive control. Wereated another set of excised skin in the same ways above, with 52 mM and 1.9 M MgCl2 solutionspH 7.4 and 7.8 respectively) for 5, 15, and 60 min-tes to study the effect of time and concentration

n magnesium permeation.

air follicle plugging

e developed a novel method to plug the hairollicles on excised skin to evaluate their contri-ution in magnesium permeability across the skinfigure 1). According to this method we closelydentified all the hair follicles in a marked regionsing a Zeiss Primostar microscope and imageaptured using Zeiss AxioCam Erc 5s connected toxiocam software (Zeiss, Oberkochen, Germany).he plugging procedure involved applying 0.1 �Lcriflavine solution (dissolved in milliQ water)o form a globule around the follicle and sub-equently adding 0.1 �L of ethylcyanoacrylateLoctite Super glue) (Henkel, Ohio, USA) on topf it. The polar monomers of ethylcyanoacrylateolymerize when they come in contact with theater molecules in the presence of acriflavine dye.
his dye-sensitised polymerisation forms long andtrong chains, thus plugging the skin and hair fol-icle. We then treated the plugged and unpluggedkin with 1.9 M MgCl2 solution for 15 minutesnder the conditions described above.
alidation for hair folliclelugging method

he efficacy of this new method was tested byonducting an in vitro skin absorption study inFranz diffusion cell with an effective diffusion

rea of 1.33 cm2 and a 3.4 mL receptor chamberapacity. The skin obtained from abdominoplasty

Journal Identification = MRH Article Identification = 0402 Date: August 24, 2016 Time: 6:39 pm

elHaou

acdthe system at 1 mL/min flow rate. The column

A B

Figure 1. Novel hair follicle plugging method. Novcontribution towards magnesium permeation. A)water (1 mg/mL) around the hair. C) Add equal am

was cut into disc pieces and mounted in the Franzdiffusion cell between donor and receptor com-partments, with the stratum corneum side facingthe donor chamber and the dermal side facing
the receptor chamber. We used 1 mL of 3% (w/w)caffeine totally solubilized in aqueous solution asdonor solution, as it is known to permeate throughhair follicles and PBS buffer as receptor solutionat pH 7.4 and 35 ◦C (physiological skin tempera-ture). The donor chamber was then wrapped withParafilm after addition of the solution to pre-vent evaporation. At different point times over12 hours, 200 �L of the receptor solution waswithdrawn and replaced with the same amount offresh PBS buffer. The sample thus withdrawn wasanalysed for caffeine levels by sensitive and rapidhigh performance liquid chromatography (HPLC),consisting of Shimadzu SIL-20 a HT, CBM-20Asystem controller, a SPD-20A detector, LC-20AD
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Figure 2. Validation for hair follicle plugging methodmethod by topical application of 3% caffeine solution ohair follicle over 8 hours using a Franz cell setup. Twas sampled every hour from the receptor chamber o

Permeation of Magnesium ions through human skin

C D

hair follicle plugging method to evaluate follicularir follicle. B) Add 0.2 �L acriflavine dissolved innt of cyanoacrylate. D) All hair follicles plugged.

pump and an auto injector. The mobile phase ofaffeine (95% water, 2% acetonitrile, 2% tetrahy-rofuran and 0.5% acetic acid) was pumped across

sed was Phenomenx Luna 5 �m, c18 (150 mm4.6 mm); and caffeine was detected at 273 nm

figure 2).

istochemistry staining

fter treating the skin with the magnesium solu-ions, the pieces were embedded in OCT and 60 �mryosections section were obtained. The sectionsere immediately stained with 30 �L of Mag-fura-, tetrapotassium salt (Biotium, Fremont, CA,SA) (10 �g/mL) and incubated for 5 minutes in a

ight-proof chamber. The dye was washed off withilliQ water for 10 seconds. The section was thenounted on the slide.

4

e (h)5 6 7 8

. Test of the effectiveness of hair follicle pluggingn both normal skin (open hair follicle) and pluggedhe resulting transdermal permeation of caffeinef the Franz cell and evaluated using HPLC.

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%)

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75

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300 400 500

Wavelength (nm)

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Figure 3. Imaging Mag-fura-2 fluorescence usingemission spectra (520 nm) of mag-fura-2, tetra pomultiphoton system with a tunable titanium Sappwavelength was 740 nm and emission was detecte485 to 550 nm, and 593 to 600 nm.

Multiphoton Microscopy

We used a LaVision Biotec Nikon multiphoton sys-tem with a tunable titanium Sapphire laser tovisualize the magnesium skin sections (figure 3).The slides were imaged with excitation wave-length was 740 nm and emission was detectedusing three non-descanned filters: 447 to 460 nm,485 to 550 nm, and 593 to 600 nm, and resultingimages were analysed using ImageJ to measureCTCF (corrected total cell fluorescence) of Mag-fura fluorescence in the epidermis. A 20 × 0.95 NAwater immersion objective (Olympus) was used.CTCF was calculated using the formula:

CTCF = Integrated densityViable epidermis

− (AreaViable epidermis×Mean fluorescenceBackground

)

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Channelsmerged

ltiphoton microscopy. A) Excitation (320 nm) andssium salt. B) We used a LaVision Biotec Nikonre laser to visualize the skin sections. Excitationsing three non-descanned filters: 447 to 460 nm,

esults

opically applied magnesium permeateshrough human stratum corneum

ryosections of human skin pretreated with 5 mMgCl2 solution for 30minutes showed increased

uorescence intensity relative to sections thatere not pretreated, when stained with Mag-

ura-2 dye. This increase was observed in bothtratum corneum intact, and tape-stripped skinndicating the permeability of stratum corneumo magnesium ions. We also found a greaterncrease in fluorescence intensity in tape-strippedkin that was subsequently treated with 5 mMgCl2. In control skin sections not treated withgCl2, we observed that the Mag-fura-2 dye emit-

ed some background fluorescence upon binding



2×107

No Mag-fura-2

No MgCl2 No MgCl2

Mag-fura-2 stained

5 mM MgCl2

nor

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1.5×107

1×107

5×106

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2×107

1.5×107

1×107

tens

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epid

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Figure 4. Magnesium ions penetrate through skiness. A) Images from three donors showed incintact skin treated with 5 mM MgCl2 solution foB) Histograms showing normalized fluorescence i= 50 �m.

with endogenous ions. We have also estimated themagnesium levels present in our sections by com-paring the CTCF to the CTCF of skin equilibratedwith known concentrations of MgCl2 solution. Thegreater fluorescence intensity and its progres-sive distribution through the viable epidermis inthe tape-stripped skin, compared to unstrippedskin, indicates that the stratum corneum offers
some resistance to the permeation of magnesiumions, as expected. However, where the stratumcorneum is intact, permeability to magnesium ionsstill takes place, with the level of penetration afunction of the thickness of the stratum corneum(which varies between donors) (figure 4).
Magnesium permeability varies basedon concentration and time of exposure

Higher fluorescence intensities relative to sectionsuntreated with MgCl2, were observed at 15 and60 minutes after the skin was exposed to 1.9 MMgCl2 concentration (p<0.05, one way ANOVA,Tukey’s test) and after 60 minutes of exposure

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0

ut the extent depends on stratum corneum thick-sed fluorescence in tape stripped (TS) and SC0 min compared to skin untreated with MgCl2.nsity in viable epidermis in each donor. Scale bar

o 52 mM MgCl2 (p<0.01). This indicates thatagnesium penetration increases with time, andigher concentration enhances permeation. How-ver, although there is a trend towards an increasen magnesium penetration 5 minutes after expo-ure to either 52 mM or 1.9 M MgCl2 it was notignificant (p>0.05). After 15 minutes treatmentith 1.9 M MgCl2 there was a significant increase

n measured magnesium penetration (p<0.05)nd a further increase at 60 minutes (figure 5B).fter treatment with 52mM MgCl2, a trend to

ncrease was apparent after 15 minutes of expo-ure, however, a significant increase was onlybserved at 60 minutes at this lower concentrationp<0.05) (figure 5).

air follicles have significantontribution towards magnesiumenetration through skin

e developed a novel method to plug the regionurrounding the hair follicle in order to test theole of hair follicles in ion transport. To validate

39



BA

2×107

3×107

4×107

Cl 2 . C

TC

F (

a.u)

Con

trol

-No

MgC

l 2

60 min 15 min 5 min

**

**

52 M

Mg

1.9

M M

gCl 2

Figure 5. Magnesium ion permeation at varying timindicating penetration of magnesium ions at varying twith 52 mM and 1.9 M MgCl2 solutions for 5, 15 and

the plugging method, we tested caffeine pene-tration through plugged and unplugged skin. Wefound reduced caffeine penetration through skinwhen hair follicles were plugged, compared tounplugged skin. The plugging method was effec-

ttaut

A B

Plugged follicles Unplugged follicles

1.9 M MgCl2 for 15 min

Figure 6. Hair follicles significantly contribute to mregion shown in blue). B) Unplugged skin sections alpenetration of magnesium ions in unplugged skin, (*

40

0

1×107

Nom

52 mM MgCl2 1.9 M MgCl2Control

Control-No MgCl2 treatment

60 min 15 min 5 min

es and concentrations. A) Representative imagesimes and concentrations. B) The skin was treated60 minutes. (* p<0.05, ** p<0.01).

ive, as there was no permeation in the firstwo hours, and very limited permeation evenfter four hours (figure 2). We then proceeded tose this method to test magnesium penetrationhrough hair follicles. Magnesium penetration

2.5x107

1.5x107

2x107

1x107

5x106

0

Plugged

C

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Unplugged

Nor

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agnesium permeation. A) Plugged skin (pluggedlow penetration of magnesium ions. C) Increasedp<0.05).

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was significantly decreased in the sections ofskin that were plugged, compared to penetrationthrough unplugged skin (figure 6). This indicatesthat hair follicles act as a major route of pen-etration of magnesium ions through the skin.We also observed fluorescence emission from theacriflavine-cyanoacrylate complex used to plugthe hair follicle. This aided with acquisition ofimages at the plugged region, which is difficult todo without this local labelling.

When 1.9 M MgCl2 solution was topicallyapplied over plugged and unplugged skin for 15minutes, we found that Mag-fura-2 fluorescenceintensity was higher in unplugged skin sections,demonstrating that hair follicles contribute signif-icantly to magnesium permeation (figure 6).

Discussion

The treatment solution used in our experimentswas magnesium chloride (MgCl26H2O) which hasbeen used for several years for therapeutic pur-poses. It has been reported to have greatereffectiveness and reduced toxicity, compared tosalts with other anions [15]. Once dissolved it dis-sociates into magnesium and chloride ions. Thequestion of fundamental importance is whetherions are able to traverse, by any mechanism, thestratum corneum layer of skin, which is negativelycharged [16-18].

Figure 4 illustrates the ability of magnesiumions to penetrate through both intact skin andtape-stripped skin, although the penetration ismore rapid in the latter. This confirms our hypoth-esis that magnesium ions could permeate through
skin. Utilization of a method to indirectly stainmagnesium ions in the various layers of skinsections further allowed us to investigate the pen-etration profile under prolonged exposure andincreased concentrations of magnesium chloridesolution. We observed that with the applicationof the 1.9 M MgCl2 solution Mg2+ ions penetratedrapidly, that is, within 15 minutes of treatment.In the case of the 52 mM MgCl2 solution, per-meated Mg2+ was visible only after 60 minutesapplication to the skin. According to Fick’s lawof diffusion, ion flux moves from regions of highconcentration to regions of low concentration, witha magnitude that is proportional to the concentra-tion gradient [19, 20]. Thus the observed increasein fluorescence intensity results from the move-
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Date: August 24, 2016 Time: 6:39 pm


ent of magnesium ions from a region of highoncentration to low concentration, with moreapid movement at higher external concentra-ions. However, mechanisms other than simpleiffusion could be operating.To further investigate the route of penetration ofagnesium ions, we topically applied MgCl2 solu-

ion onto equally marked out areas in excised skin,ith and without plugging of the hair follicles. Webserved a significantly higher fluorescence inten-ity indicating magnesium penetration when theair follicle orifices were open. The artificial block-

ng of the hair follicle orifices ensured they werexcluded as ion penetration routes. Consequently,g2+ should only pass through the interfollicu-

ar epidermis and its lipid domains, and possiblyhrough the sweat glands. Unlike the previousethods of hair follicle plugging with a micro-

rop of special varnish-wax-mixture [21], we usedcombination of acriflavine dye and cyanoacry-

ate. The resulting polymerization reaction ofyanoacrylate molecules when they contactedcriflavine solution formed a rigid bond aroundhe hair follicles blocking the orifice [22]. We alsoarnessed the fluorescence of acriflavine to visu-lize the plugged region of the follicle, along withag-fura-2 fluorescence when observed under theultiphoton microscope. In conclusion, we demon-

trate that magnesium ions are able to penetratehrough barrier-compromised SC, and, at a slowerate, through SC intact skin. We also found thatair follicles contribute significantly to the perme-tion of magnesium ions (an approximately 40%ncrease in Mag-fura-2 CTCF over 15 minutesfigure 6C). Our work establishes that magnesiumons are able to traverse the stratum corneum in aime dependent manner, and that this process is
acilitated by hair follicles.
cknowledgement

The authors acknowledge the technical assis-ance of Dr. Eman Abd with running of HPLC toalidate follicle plugging experiments.

isclosure

inancial support: This work was funded by theniversity of Queensland and in the preliminaryhase by Poolrite, Australia. Conflict of interest:one.

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Human Research Ethics Clearance: Thisresearch was approved by the Universityof Queensland and QPAH Ethics Commit-tees: HREC/16/QPAH/064, UQ HREC number2008001342.

Statement of Author Contributions. Allauthors participated in the design, interpretationof the studies, analysis of the data and review ofthe manuscript; NCC, WYS and YHM conductedthe experiments. NCC, RTB, JEG and MSR wrotethe manuscript.

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