Accepted Manuscript

Research paper

Human skin penetration and local effects of topical nano zinc oxide after oc-

clusion and barrier impairment

V.R. Leite-Silva, W.Y. Sanchez, H. Studier, D.C. Liu, Y.H. Mohammed, A.M.

Holmes, E.M. Ryan, I.N. Haridass, N.C. Chandrasekaran, W. Becker, J.E. Grice,

H.A.E. Benson, M.S. Roberts

PII: S0939-6411(16)30146-1

DOI: http://dx.doi.org/10.1016/j.ejpb.2016.04.022

Reference: EJPB 12185

To appear in: European Journal of Pharmaceutics and Biophar-

maceutics

Received Date: 18 March 2016

Revised Date: 26 April 2016

Accepted Date: 26 April 2016

Please cite this article as: V.R. Leite-Silva, W.Y. Sanchez, H. Studier, D.C. Liu, Y.H. Mohammed, A.M. Holmes,

E.M. Ryan, I.N. Haridass, N.C. Chandrasekaran, W. Becker, J.E. Grice, H.A.E. Benson, M.S. Roberts, Human skin

penetration and local effects of topical nano zinc oxide after occlusion and barrier impairment, European Journal

of Pharmaceutics and Biopharmaceutics (2016), doi: http://dx.doi.org/10.1016/j.ejpb.2016.04.022

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

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1

Human skin penetration and local effects of topical nano zinc oxide after occlusion and barrier

impairment

Running head: Nano ZnO human skin penetration and effects.

Words, 3980: Tables, 0: Figures, 4

V. R. Leite-Silva1,2*

, W. Y. Sanchez2*

, H. Studier3, D. C. Liu

2, Y. H. Mohammed

2, A. M. Holmes

3, E. M. Ryan

2,

I. N. Haridass2, N. C. Chandrasekaran

2, W. Becker

4, J. E. Grice

2, H. A. E. Benson

5**, and M. S. Roberts

2,3**

*These authors contributed equally to this work. **Corresponding authors.

1Instituto de Ciências Ambientais Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema

SP, Brazil

2Therapeutics Research Centre, School of Medicine, The University of Queensland, Translational Research

Institute, QLD, Australia 4102

3School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia

4Becker & Hickl GmbH, Nahmitzer Damm 30, 12277, Berlin, Germany

5School of Pharmacy, CHIRI, Curtin University, GPO Box U1987, Perth, WA, Australia

Corresponding authors: Prof. Michael S. Roberts, Therapeutics Research Centre, School of Medicine, The

University of Queensland, Translational Research Institute, QLD, Australia 4102; +61 7 34438031;

m.roberts@uq.edu.au

Dr Heather A. E. Benson, School of Pharmacy, CHIRI, Curtin University, GPO Box U1987, Perth, WA,

Australia; +61 8 92662338; h.benson@curtin.edu.au

Funding source: This work was supported by the National Health & Medical Research Council of Australia.

Conflicts of interest: The authors declare no conflict of interest.

2

Abstract

Public health concerns continue to exist over the safety of zinc oxide nanoparticles that are commonly used

in sunscreen formulations. In this work, we assessed the effects of two conditions which may be

encountered in everyday sunscreen use, occlusion and a compromised skin barrier, on the penetration and

local toxicity of two topically applied zinc oxide nanoparticle products. Caprylic/capric triglyceride (CCT)

suspensions of commercially used zinc oxide nanoparticles, either uncoated or with a silane coating, were

applied to intact and barrier impaired skin of volunteers, without and with occlusion for a period of six hours.

The exposure time was chosen to simulate normal in-use conditions. Multiphoton tomography with

fluorescence lifetime imaging was used to noninvasively assess zinc oxide penetration and cellular metabolic

changes that could be indicative of toxicity. We found that zinc oxide nanoparticles did not penetrate into the

viable epidermis of intact or barrier impaired skin of volunteers, without or with occlusion. We also observed

no apparent toxicity in the viable epidermis below the application sites. These findings were validated by ex

vivo human skin studies in which zinc penetration was assessed by multiphoton tomography with

fluorescence lifetime imaging as well as Zinpyr-1 staining and toxicity was assessed by MTS assays in zinc

oxide treated skin cryosections. In conclusion, applications of zinc oxide nanoparticles under occlusive in-

use conditions to volunteers are not associated with any measurable zinc oxide penetration into, or local

toxicity in the viable epidermis below the application site.

Key words

zinc oxide nanoparticles; skin penetration; barrier impairment; occlusion; toxicity; sunscreens;

safety; in-use application

3

Introduction

There is a lack of consensus about the safety of zinc oxide nanoparticles (ZnO NP) applied topically

as a transparent and effective broad spectrum ultraviolet filter, with a range of views expressed by regulatory

authorities (1-3), consumer groups (4, 5) and in the scientific literature (6-9). These safety concerns should

be balanced against the potential benefits provided by ZnO NP-containing sunscreens in preventing adverse

outcomes resulting from excessive ultraviolet radiation exposure, which include sunburn and photo-allergy

due to accelerated skin ageing, immunosuppression and an increased risk of developing skin cancer (10).

One perception is that ZnO NP and other small inorganic NP currently used in commercially

available sunscreens may penetrate deep into the skin and cause a variety of adverse effects (11). Topically

applied NP occasionally reach the viable epidermis (12, 13) and zinc ions have even been found in human

blood and urine after topical ZnO NP application (14, 15), as well as in the SC and upper epidermis of

sunburned pig skin (16). Further, after short-term in vitro exposure above 15 μg.mL-1

, ZnO NP reduce

keratinocyte viability (17). An alternative view (18), supported by several in vivo human studies, is that ZnO

NP topically applied to un-occluded intact human skin penetrate only into the superficial layers of the stratum

corneum (SC), with no penetration to the viable epidermis (VE), both in vitro and in vivo (1-3).

There is a clear need to determine the in vivo penetration and local skin safety of ZnO or other NP in

humans after topical application under “in-use” conditions. Of particular importance are the ‘‘in-use’’

conditions of abrasion and increased skin hydration that are commonly encountered during work, sport and

beach activities. The current safety evaluation of ZnO NP applied to skin has focussed on a systemic margin

of safety, noting that the body has homeostatic mechanisms to deal with excessive zinc ion (Zn2+

) exposure

(19). We are not aware of any in vivo human study evaluating the local safety of ZnO NP applied to the skin

under occlusive conditions without or with impaired skin barrier function.

Here we explore the impact of occlusion on the topical penetration and local toxicity of ZnO NP into

the viable epidermis of intact and barrier-impaired human skin in vivo.

4

Materials and methods

ZnO nanoparticles

Uncoated and coated ZnO NP (Z-COTE® and Z-COTE

® HP1 respectively; BASF, Ludwigshafen,

Germany) were re-suspended in caprylic/capric triglyceride (CCT) for application to the skin. Transmission

electron microscopy (TEM) was performed on a JEOL 1400 microscope (JEOL Ltd, Tokyo, Japan) operating

at 120kV. Images were acquired using a TVIPS F416 CCD camera (TVIPS, Gauting, Germany) and

analysed by ImageJ to determine particle sizes.

Application of ZnO NP to the skin of human volunteers

The study was carried out on 10 healthy subjects between the ages 20 to 40 years with undamaged skin

and no history of cutaneous disease. Skin was considered undamaged if TEWL was less than 20 g.m-2

.hr-1

(20, 21). The volunteers gave informed consent and experiments were conducted with approval from the

University of Queensland Human Research Ethics Committee (No. 2008001342), in accordance with

Declaration of Helsinki protocols. Four treatment areas marked on each subjects’ volar forearm were either i)

untreated, ii) treated with vehicle (CCT), iii) barrier-impaired by tape stripping before treatment with 10% w/v

ZnO NP in CCT, or iv) treated with 10% w/v ZnO NP in CCT with the barrier intact, with the standard SPF-

testing dose (2 mg.cm-2

) (22). Areas on one arm were occluded by placing two adhesive dressings (Johnson

& Johnson Pacific Pty Ltd, Sydney, Australia) over the sites of application. Sites remained unoccluded on the

other arm. Coated ZnO NP were used in five subjects and uncoated ZnO NP in the other five. The barrier-

impaired areas (4 cm2) were tape-stripped 20 times with D-Squame tape (CuDerm, Dallas, US) immediately

prior to administration of the ZnO NP suspensions. All treatments were applied for 6 hours, after which the

excess ZnO NP were wiped off using a surgical swab moistened with water.

Multiphoton-FLIM imaging of autofluorescence and ZnO NP in the skin of human volunteers

A DermaInspect® system (JenLab GmbH, Jena, Germany) was used for multiphoton tomography

(MPT) as previously described (21). The skin was imaged at three depths from the skin surface,

corresponding to the SC (~5-10 µm), stratum granulosum (SG; ~15-20 µm) and stratum spinosum (SS; ~25-

30 µm). Fluorescence lifetime decay data from the MPT-FLIM images was analysed with SPCImage 5.0

software (23) after correction for the instrument response function.

ZnO NP were quantified by their quasi-continuous accumulated photoluminescence on laser pulsing

as illustrated in the sketch Figure 1A,B. Standard curves were created for uncoated and coated ZnO NP in

5

CCT (50, 100, 500 μg.mL-1

, and 1, 5 and 10 mg.mL-1

) on glass slides using bh SPCImage. GraphPad Prism

Ver 6.05 (GraphPad Software, Inc., La Jolla, CA) was used to conduct non-linear regression of standard

curves.

Data analysis

FLIM data were derived from the fluorescence lifetime decay curves registered in 256 time channels

for each pixel using bh SPCImage 5.0 software (23), as previously described (21).

For autofluorescence metabolic analysis, a distinct region of interest was defined to encapsulate the

viable cells within the SG and SS using SPCImage. To visualize, isolate, and measure the level of ZnO NP

distributed in the skin and within targeted regions of interest, the baseline value of the decay curve of each

pixel was used. These values (‘offset’ value in bh SPCImage) were pseudocolored in red to create images.

Quantification of ZnO NP

ZnO NP can be identified by their ability to generate a second-harmonic (SHG) signal. By using this

SHG signal, high sensitivity quantitation of ZnO can be achieved. However, melanin and keratin also exhibit

strong fluorescence signals with a very short fluorescence lifetime component that can be mistaken for ZnO

NP SHG emission. ZnO NP can also be identified by their characteristically slow photoluminescence (PL)

decay times. To eliminate interference from endogenous fluorophores, it is thus preferable to use the slow

PL signal that is at least one order of magnitude longer than any other endogenous skin component. The

compromise for achieving higher specificity is that the sensitivity is decreased. The long decay components

can be detected by conventional or advanced phosphorescence lifetime imaging (PLIM) techniques (24).

To identify ZnO NP, we used a novel approach to analyse FLIM data that contain a signature left by

long decay components (μs scale) in the recorded fluorescence decay curves (typically ns scale). When

excited by laser pulses with an 80 MHz repetition rate, the slow PL for hundreds of excitation pulses

accumulates and forms a quasi-continuous background in the recorded decay functions. We used the fact

that this background is significantly larger than the background caused by possible incomplete decay of

endogenous fluorescence. The amount of background contained in the decay functions can be considered

as a ‘by-product’ of the fitting procedure with SPCImage (25) and can be used as a measure of the amount

of ZnO NP present in the tissue. Under the chosen measurement conditions that were kept constant

throughout this study, the ZnO NP ‘offset’-signal (‘offset’ parameter within bh SPCImage determines the

baseline of the fluorescence decay curve) was adjusted for the cellular autofluorescence which was found to

lie between 0 and 5 counts.

6

A standard curve was created for uncoated and coated ZnO NP in CCT (50, 100, 500 μg.mL-1

, and 1, 5 and

10 mg.mL-1

) using the normalised offset values. FLIM data were analysed and exported using SPCImage to

calculate the normalized offset signal, which was used to plot the standard curve. GraphPad Prism was used

for non-linear regression of standard curves to generate a line of best fit which were interpolated for

unknown background and ZnO NP normalised offset signal measured in the skin.

Validation of ZnO NP penetration using ex vivo human skin

Human epidermal membranes were prepared from freshly excised abdominal skin by a heat

separation technique (26). After tape-stripping the epidermis 20 times to simulate damaged (barrier-

impaired) skin, CCT vehicle or uncoated ZnO NP were applied for 6 hours with occlusion and excess

material was swabbed off afterwards. Multiphoton imaging of the epidermis was conducted on a Zeiss LSM

710 microscope equipped with a bh SPC-152 TCSPC module, bh GaAsP hybrid detectors (HPM100-40) and

a Ti:Sa laser (Spectra-Physics Mai Tai®). Excitation was at 800 nm, with the detection band between 400

and 477 nm.

Labile Zn concentrations within the VE after topical application of uncoated ZnO NP were compared

with endogenous concentrations in untreated skin. Vehicle (CCT) was applied to intact VE, while ZnO NP in

CCT was applied at the standard in vivo dose of 2 mg.cm-2

to both intact and tape-stripped (x20) VE . After 6

hrs, excess material was wiped off and tissue was cryosectioned. The sections were stained with 10 µL of 10

µM Zinpyr-1 (ZP1, Cayman Chemical Co., Ann Arbor MI) for 10 minutes and excess ZP1 was removed with

ultrapure water. Images were taken after excitation at 488 nm using an argon laser and the ZP1 signal

intensity was detected at 520-560 nm.

MTS assay on biopsy of viable ex vivo skin

Female abdominal skin samples (1 x 1 cm2) were left intact or tape-stripped x 20. Skin was dosed at

2 mg.cm-2

with uncoated ZnO NP (10% in CCT), occluded and placed in a water bath at 35°C ± 0.1°C. After

6 hrs, excess ZnO was removed, 3 mm biopsies were taken (n=3) and the remaining skin was set aside for

MPT-FLIM analysis. The biopsies were placed in a 96 well plate and 100 µL MTS stock solution (Cell Titer

96® aqueous one solution assay, Promega) was added to each. After shaking at 150 rpm for 2 hours at

37°C, the biopsies were removed and washed with 1 mL of sterile PBS. Biopsies were minced with surgical

scissors and placed in an Eppendorf tube with 200 µL PBS for extraction, done by 60 min soaking followed

by 30 min in an ultrasonic bath and centrifugation at 10,000 rpm for 5 minutes (all at ambient temperature

and light protected). Absorbance of supernatants was measured at 490 nm on a plate reader.

7

Statistics

The average fluorescence lifetime was expressed as mean ± 95% confidence intervals. A p-value less than

0.05 was considered as statistically significant. A Kruskal-Wallis test was performed to calculate the

statistical differences between groups with a post-hoc Dunn’s multiple comparisons test (GraphPad Prism).

Results

Characterisation and quantitation of uncoated and coated ZnO NP applied to in vivo human skin

Representative TEM images and size distribution histograms for uncoated (65.5 ± 35.6 nm) and

coated (74.3 ± 32.3 nm) ZnO NP are shown in Supplementary Fig. S1. Our approach to quantification of

ZnO NP in human skin is illustrated in Fig. 1. Fig. 1A,B shows the offset representation of the long-time

component of the photoluminescence emission recorded with the time-correlated single photon counting

(TCSPC) technique during femtosecond laser pulsing used to provide a high specificity for ZnO NP in skin.

Fig. 1C shows the calibration curves derived from the pixel intensity found for different concentrations of

uncoated and coated ZnO NP. It is evident from the linear log-log plots that pixel intensity is related to ZnO

NP concentration over a wide concentration range. The limit of detection (LOD) for coated and uncoated

ZnO NP associated with these plots is ~ 50 μg.mL-1

and ~100 μg.mL-1

based on signal to noise ratios of 5

and 10 respectively after adjustment for background autofluorescence in the SG of in vivo skin. Control

images for topically applied coated and uncoated ZnO NP on the intact skin surface, in the SG and in the

skin furrows showed that ZnO NP are present only on the surface, in the SC and in the furrows.

Impact of barrier impairment on the penetration of uncoated and coated ZnO NP into human skin in

vivo

Fig. 2 shows representative multiphoton images recorded in the SG for ZnO NP penetration into

intact and barrier-impaired (tape stripped) skin of volunteers for coated and uncoated ZnO NP after topical

application. Fig. 2A (top images) shows that the ZnO NP signal is localised within the SC and skin furrows in

intact skin, with no detectable penetration into the VE. In barrier-impaired skin, the ZnO NP signal is

predominantly aggregated within the furrows, but signals are also observed on or in the exposed SG layer of

the VE in 2 out of 5 volunteers when the tape stripping is sufficient to expose the VE (Fig. 2A, bottom

images). ZnO NP levels within the furrow (Fig. 2A) were variable, but >10 mg.mL-1, within the regions of

interest (ROIs) analysed. In the exposed VE of barrier-impaired skin, the concentrations of uncoated and

coated ZnO NP within the indicated ROIs (Fig. 2A, white-dotted squares) were above 10 mg.mL-1

and

approximately 5 mg.mL-1

respectively. Fig. 2B shows the estimated levels of ZnO NP, both uncoated

8

(indicated by squares) and coated (indicated by triangles) within the VE based on representative ROIs

measured in 5 volunteers for each treatment group. Overall, the mean concentration of uncoated and coated

ZnO NP in barrier-impaired skin was 0.60 mg.mL-1

(Range, 0 – 1.6) and 1.2 mg.mL-1

(0 – 3.2), which were

not statistically significantly different (p>0.05) from other treatment groups or controls.

Effect of occlusion on ZnO NP penetration into in vivo intact and barrier-impaired human skin

The impact of occlusion is shown in Fig. 2C. Both uncoated and coated ZnO NP failed to penetrate

into the VE of occluded intact human skin, with no detectable levels above the non-specific background. In

contrast, uncoated and coated ZnO NP were found in the upper layer of the VE of barrier-impaired occluded

skin in 3 out of 5 volunteers. In aggregate, the viable epidermal levels of uncoated and coated ZnO NP in

barrier-impaired occluded skin were determined to be 9.6 mg.mL-1

(Range, 0 – 22.9) and 8.4 mg.mL-1

(Range, 0 – 8.5) respectively (Fig. 2D). While the mean concentrations of uncoated and coated ZnO-NP

were higher in barrier-impaired occluded skin, compared to intact skin, the differences were not statistically

significant (p>0.05).

There were no morphological changes apparent in the VE without and with occlusion and without

and with barrier impairment (Fig. 3A). The unbound/bound ratios (a1/a2) and the mean lifetimes (m) of

predominantly NAD(P)H (and to a minor degree, FAD) in SG of the viable epidermis are shown in Fig. 3B

and 3C respectively. As indicated in Figs. 3B and 3C, there were no significant differences in a1/a2 or m

between the controls and any treatment of ZnO NP suspensions, with or without occlusion, in intact or

barrier-impaired skin. The viable cells immediately below the superficial viable SG cells of those volunteers

with tape stripping sufficient for the VE to be exposed to ZnO NP had no significant changes in the

free/bound NAD(P)H ratio a1/a2 and the mean lifetime m compared with controls.

Validation of the lack of ZnO NP penetration and toxicity with ex vivo occluded barrier-impaired

human epidermis

To validate our findings from the in vivo study we used freshly excised human skin. Ex vivo z-stack

skin imaging confirmed that topically applied ZnO NP did not penetrate into the VE after application to

barrier-impaired epidermal membranes (Supplementary Fig. F2). After application of ZnO NP for 6 h to intact

and barrier impaired skin no significant increase of labile zinc was found when compared to the controls (Fig.

4). In addition, there was no significant change in the metabolic state of the skin when ZnO NP dosed skin

was compared to vehicle controls. An MTS assay from the biopsies showed no significant differences in

viability when the ZnO NP treated skin (median abs. 0.28) was compared to untreated skin (median abs.

0.29), with the assay validated by a decrease in cell viability (median abs. 0.20) when DMSO was used as a

9

positive control. FLIM images of NAD(P)H within the VE of freshly excised skin also showed that the

localized metabolic state of the keratinocytes within the SG did not change upon ZnO NP application,

whereas DMSO dosed skin showed an increase in NAD(P)H mean lifetime. The mechanical action of tape

stripping led to an increase in NAD(P)H mean lifetime for both ZnO NP treated and its respective vehicle

control.

Discussion

This work has focussed on assessing the penetration and effects of ZnO NP applied to in vivo

human skin under “in-use” conditions, as regulatory authorities have suggested this data is presently

inadequate (1-3). We also addressed the issue raised by the SCCS: “Any cosmetic products containing ZnO

particles (nano or non-nano) with coatings that can promote dermal penetration will also be of concern.” We

used an improved technique for quantifying ZnO NP in skin by MPT-FLIM imaging to provide enhanced

specificity for the ZnO NP signal in the skin. The limits of detection for coated and uncoated ZnO NP found

here (~ 50 μg.mL-1

and ~100 μg.mL-1

respectively) are similar in magnitude to those reported for SHG/HRS

of 0.08 fg.µm-3

(or 0.03 fg ZnO NP in the focal area of the MPM on one pixel, or ~80 μg.mL-1

) based on a

signal-to-noise ratio of ~10 (27). We also minimised the effects of out-of-focus emission signals, one of the

potential limitations of all imaging, by flattening the skin with a minimal continuous pressure between the

coverslip and the skin by taping and sealing the coverslip in place. If this is not done, and pressure is

released and reapplied a number of times, it may result in any ZnO NP remaining on the skin surface (and

there may be some, even after wiping off before imaging), tending to accumulate unevenly on the glass

under the coverslip. Secondly, a gap between the coverslip and the skin may result in out of focus ZnO

agglomerates “swimming” through the upper excitation cone of the laser, giving randomly aberrant signals. It

is therefore crucial to apply the soft continuous pressure at all times to minimize these disturbing effects.

These results for in vivo human skin using our newer, sensitive detection method for ZnO NP are

consistent with our earlier work (21), suggesting that topically applied coated and uncoated ZnO NP are

found only on the skin surface, in the SC and in the furrows of the skin (Fig. 2A top images). Similar findings

were found with occlusion of both uncoated and coated ZnO NP (Fig. 2B). These findings for intact skin are

in agreement with previous human in vivo ZnO NP penetration studies showing that topically applied NP

remain on the skin surface and accumulate within the SC furrows and hair follicles (27, 28), with little to no

observable penetration into the VE (21, 29) after repeated washing with water (30).

Although skin occlusion can greatly increase the penetration of topically applied chemicals (31),

there are very few studies which have explored the impact of occlusion on the human in vivo skin penetration

10

of nanoparticles. Here, we showed that occlusion did not enhance the penetration of uncoated or coated

ZnO NP into the VE of intact skin in volunteers (Fig. 2). This is also generally consistent with biopsy results

showing no significant nanoparticle penetration from a combined TiO2 NP/ZnO NP sunscreen applied to the

skin of volunteers for 48 hours (32).

In contrast, after barrier impairment, whilst the ZnO NP signal was predominantly aggregated within

the furrows, ZnO NP signals were also observed within the SG layer of the VE in two of the five volunteers

after non-occluded application (Fig. 2A, bottom images) and three of the five volunteers after occluded

application (Fig. 2C). This may be related to the known inter-subject variability in the amount of stratum

corneum removed by 20 tape strips, so that the SG may be exposed to varying degrees (33). Twenty tape

strips have been shown to remove approximately 50% of stratum corneum thickness from human volunteers

in our laboratory (unpublished data) and by the Lademann group (34). However, it is also clear from

histological studies (35) and from the images shown here in Fig. 2 that the stratum corneum may not be

removed evenly over the entire treated surface and that tape stripping can leave “islands” of intact skin

surrounded by areas in which the VE is exposed. This could occur regardless of the care taken in applying

tapes to the skin consistently. In view of this variability, we suggest that the tape stripping pressure used in

our earlier work (29) was not sufficient to significantly expose the VE. In any case, however, our in vivo

results here are consistent with our ex vivo results showing that ZnO NP penetration was limited to the upper

region of the SG, while not actually penetrating the viable cells (Fig. 4).

The barrier-impaired studies were undertaken in response to general concern amongst authorities

and consumer advocacy groups about the lack of investigations of barrier-impaired/sunburned skin. We

previously showed that the levels of coated ZnO NP within the VE of skin lesions in patients with

psoriasis/atopic dermatitis were similar to normal skin regions after topical application (29). This present

work shows that the specific concerns raised by Faunce that ZnO NP may penetrate into the VE of barrier-

compromised skin (36) may be justified. However, our in vivo (Fig. 2) and ex vivo (Fig. 3) results both

indicate that penetration is limited to the outermost viable epidermal cells, without and with occlusion. The

current results are also consistent with the superficial epidermal penetration reported for the same ZnO NP

(Z-COTE® HP1) in sunburned pig skin (16).

Currently, there are limited data on the local toxicity of ZnO NP applied to the skin. The most recent

summary on the scientific opinions of the safety of ZnO NP, referred to as “Zinc Oxide (Nano Form)”,

focuses on the margin of safety of zinc absorbed into the systemic circulation and notes that the body has

efficient homeostatic mechanisms to handle the balance of systemically absorbed zinc (19). We found in this

work that topical application of uncoated and coated ZnO NP, with and without occlusion and to intact and

11

barrier-impaired skin, did not cause local toxicity to the viable epidermal cells, as evidenced by no detectable

changes in cellular morphology or fluorescence lifetime properties of the VE (Fig. 3). Cellular toxicity, via

apoptosis, can be clearly measured by MPT-FLIM as it is associated with an increase in the mean

fluorescence lifetime of NAD(P)H and the unbound/bound ratio of NAD(P)H (37-42). As ZnO NP associated

toxicity is reported to occur via the induction of apoptosis (43-45), the absence of autofluorescence FLIM

changes associated with apoptosis suggest that “in-use” exposure (in barrier-impaired skin) does not lead to

toxicity within a 6 hour time frame.

In their in vivo human studies of ZnO NP sunscreen application under in-use conditions, Gulson et

al. reported very small elevations of zinc concentrations in blood and urine (1/1000th of total zinc) using a

68Zn enriched ZnO NP formulation, although this was detected over 5 days with repeated applications (14,

15). One mechanism by which this could occur is penetration of zinc ions, following hydrolysis of ZnO on the

skin surface (46) and indeed there is in vivo evidence in rats (47) and humans (48) that zinc ions, applied in

both aqueous (48) and oil-based (47, 48) formulations, are able to penetrate the stratum corneum. In the

case of the rats, this led to an apparent increase in plasma zinc levels after 8 hours (47), whereas in the

humans, increased zinc was detected in tape strips from the upper stratum corneum after 3 hours (48). To

our knowledge, there are no published human studies of zinc levels measured in the viable epidermis after

zinc ion application in vivo, nor have the effects of Zn2+

applied under occlusion been reported. Although

these mechanisms of hydrolysis and Zn2+

penetration are both possible in the case of topical ZnO

application, our current findings are consistent with our previous observations that a 6h application time does

not result in a significant increase of labile zinc within the VE (46). This is important, given that Zn2+

ions are

implicated in keratinocyte cytotoxicity, albeit in cultured cells in vitro (17). Our findings may be partly

explained by a minimal dissolution of zinc in the organic vehicle, CCT, particularly after the short contact

time. No significant localised toxicity was observed after application of ZnO NP to intact and barrier-impaired

skin, either in vivo or in freshly excised human skin. A conventional MTS assay that is sensitive to changes

in NAD(P)H confirmed that ZnO NP produced no significant changes in tissue viability. The mechanical

action of removing 20 tape strips from the skin resulted in an observed mean NAD(P)H lifetime change on ex

vivo viable skin although this was not observed in vivo. This finding warrants further investigation to

characterise the differences between the use of in vivo and freshly excised skin.

12

Acknowledgments

This work was supported by the National Health & Medical Research Council of Australia and FAPESP

(Fundação de Amparo à Pesquisa do Estado de São Paulo: Prof Leite-Silva). We acknowledge Dr

Washington H. Sanchez, Clinton Hupple and Dr Jamie Riches for their valuable contributions.

13

References

1. SCCS. Opinion on Zinc oxide (nano form)

http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_103.pdf (Accessed on

14/09/2015)

2. SCCS. Addendum to the opinion SCCS/1489/12 on Zinc oxide (nano form)

http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_137.pdf (Accessed on

14/09/2015)

3. TGA. Literature review on the safety of titanium dioxide and zinc oxide nanoparticles in sunscreens.

https://www.tga.gov.au/literature-review-safety-titanium-dioxide-and-zinc-oxide-nanoparticles-

sunscreens (Accessed on 14/09/2015)

4. Miller G. Nanomaterials, Sunscreens and Cosmetics: Small Ingredients, Big Risks.

http://libcloud.s3.amazonaws.com/93/ce/0/633/Nanomaterials_sunscreens_and_cosmetics.pdf

(Accessed on 14/09/2015)

5. Sales L. What's the story with nanoparticles in sunscreen? http://www.foe.org.au/whats-story-

nanoparticles-sunscreen (Accessed on 14/09/2015)

6. Burnett M E, Wang S Q. Current sunscreen controversies: a critical review. Photodermatol

Photoimmunol Photomed 2011: 27: 58-67. doi: 10.1111/j.1600-0781.2011.00557.x

7. Newman M D, Stotland M, Ellis J I. The safety of nanosized particles in titanium dioxide- and zinc

oxide-based sunscreens. J Am Acad Dermatol 2009: 61: 685-692. doi: S0190-9622(09)00539-8 [pii]

10.1016/j.jaad.2009.02.051

8. Nohynek G J, Lademann J, Ribaud C, et al. Grey goo on the skin? Nanotechnology, cosmetic and

sunscreen safety. Crit Rev Toxicol 2007: 37: 251-277. doi: 10.1080/10408440601177780

9. Smijs T G, Pavel S. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety

and effectiveness. Nanotechnol Sci Appl 2011: 4: 95-112. doi: 10.2147/NSA.S19419

10. Nohynek G J, Dufour E K, Roberts M S. Nanotechnology, cosmetics and the skin: is there a health

risk? Skin Pharmacol Physiol 2008: 21: 136-149. doi: 000131078 [pii] 10.1159/000131078

11. Deng Y, Ediriwickrema A, Yang F, et al. A sunblock based on bioadhesive nanoparticles. Nature

materials 2015: 14: 1278-1285. doi: 10.1038/nmat4422

http://www.nature.com/nmat/journal/vaop/ncurrent/abs/nmat4422.html#supplementary-information

12. Baroli B, Ennas M G, Loffredo F, et al. Penetration of metallic nanoparticles in human full-thickness

skin. J Invest Dermatol 2007: 127: 1701-1712. doi: 10.1038/sj.jid.5700733

14

13. Labouta H I, el-Khordagui L K, Kraus T, et al. Mechanism and determinants of nanoparticle

penetration through human skin. Nanoscale 2011: 3: 4989-4999. doi: 10.1039/c1nr11109d

14. Gulson B, McCall M, Korsch M, et al. Small amounts of zinc from zinc oxide particles in sunscreens

applied outdoors are absorbed through human skin. Toxicol Sci 2010: 118: 140-149. doi:

10.1093/toxsci/kfq243

15. Gulson B, Wong H, Korsch M, et al. Comparison of dermal absorption of zinc from different sunscreen

formulations and differing UV exposure based on stable isotope tracing. The Science of the total

environment 2012: 420: 313-318. doi: 10.1016/j.scitotenv.2011.12.046

16. Monteiro-Riviere N A, Wiench K, Landsiedel R, et al. Safety evaluation of sunscreen formulations

containing titanium dioxide and zinc oxide nanoparticles in UVB sunburned skin: an in vitro and in vivo

study. Toxicol Sci 2011: 123: 264-280. doi: 10.1093/toxsci/kfr148

17. Kocbek P, Teskac K, Kreft M E, et al. Toxicological aspects of long-term treatment of keratinocytes

with ZnO and TiO2 nanoparticles. Small 2010: 6: 1908-1917. doi: 10.1002/smll.201000032

18. Loden M, Beitner H, Gonzalez H, et al. Sunscreen use: controversies, challenges and regulatory

aspects. Br J Dermatol 2011: 165: 255-262. doi: 10.1111/j.1365-2133.2011.10298.x

19. SCCS. Zinc oxide (nano form). http://ec.europa.eu/health/scientific_committees/opinions_layman/zinc-

oxide/en/l-3/6.htm (Accessed on 16/09/2015)

20. Farahmand S, Tien L, Hui X, et al. Measuring transepidermal water loss: a comparative in vivo study

of condenser-chamber, unventilated-chamber and open-chamber systems. Skin Res Technol 2009:

15: 392-398. doi: 10.1111/j.1600-0846.2009.00376.x

21. Leite-Silva V R, Lamer M L, Sanchez W Y, et al. The effect of formulation on the penetration of coated

and uncoated zinc oxide nanoparticles into the viable epidermis of human skin in vivo. Eur J Pharm

Biopharm 2013: 84: 297-308. doi: 10.1016/j.ejpb.2013.01.020

22. ISO. Determination of sunscreen UVA photoprotection in vitro: [ISO 24443:2012(en)].

http://www.iso.org/iso/catalogue_detail?csnumber=46522 (Accessed on 14/09/15)

23. Becker W. Fluorescence lifetime imaging--techniques and applications. Journal of microscopy 2012:

247: 119-136. doi: 10.1111/j.1365-2818.2012.03618.x

24. Becker W, Su B, Bergmann A, et al. Simultaneous Fluorescence and Phosphorescence Lifetime

Imaging. Proc Spie 2011: 7903. doi: Artn 790320

10.1117/12.875204

25. Becker W. The bh TCSPC Handbook. http://www.becker-hickl.com/handbookphp.htm (Accessed on

14/09/2015)

15

26. Kligman A M, Christophers E. Preparation of Isolated Sheets of Human Stratum Corneum. Arch

Dermatol 1963: 88: 702-705. doi:

27. Darvin M E, Konig K, Kellner-Hoefer M, et al. Safety assessment by multiphoton fluorescence/second

harmonic generation/hyper-Rayleigh scattering tomography of ZnO nanoparticles used in cosmetic

products. Skin Pharmacol Physiol 2012: 25: 219-226. doi: 10.1159/000338976

28. Roberts M S, Roberts M J, Robertson T A, et al. In vitro and in vivo imaging of xenobiotic transport in

human skin and in the rat liver. J Biophotonics 2008: 1: 478-493. doi: 10.1002/jbio.200810058

29. Lin L L, Grice J E, Butler M K, et al. Time-correlated single photon counting for simultaneous

monitoring of zinc oxide nanoparticles and NAD(P)H in intact and barrier-disrupted volunteer skin.

Pharmaceutical research 2011: 28: 2920-2930. doi: 10.1007/s11095-011-0515-5

30. Raphael A P, Sundh D, Grice J E, et al. Zinc oxide nanoparticle removal from wounded human skin.

Nanomedicine 2013: 8: 1751-1761. doi: 10.2217/nnm.12.196

31. Roberts M S, Bouwstra J A, Pirot F, et al. Skin Hydration—A Key Determinant in Topical Absorption.

In: Walters K A, Roberts M S, eds. Dermatologic, Cosmeceutic, and Cosmetic Development. New

York: Informa Healthcare USA, Inc., 2008: 115-128.

32. Filipe P, Silva J N, Silva R, et al. Stratum corneum is an effective barrier to TiO2 and ZnO nanoparticle

percutaneous absorption. Skin Pharmacol Physiol 2009: 22: 266-275. doi: 10.1159/000235554

33. Kalia Y N, Alberti I, Naik A, et al. Assessment of topical bioavailability in vivo: the importance of

stratum corneum thickness. Skin Pharmacol Appl Skin Physiol 2001: 14 Suppl 1: 82-86. doi: 56394

34. Lademann J, Ilgevicius A, Zurbau O, et al. Penetration studies of topically applied substances: Optical

determination of the amount of stratum corneum removed by tape stripping. Journal of biomedical

optics 2006: 11: 054026. doi: 10.1117/1.2359466

35. van 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: 514-518. doi:

36. Faunce T A. Toxicological and public good considerations for the regulation of nanomaterial-

containing medical products. Expert Opin Drug Saf 2008: 7: 103-106. doi: 10.1517/14740338.7.2.103

37. Ghukasyan V V, Kao F J. Monitoring Cellular Metabolism with Fluorescence Lifetime of Reduced

Nicotinamide Adenine Dinucleotide. J Phys Chem C 2009: 113: 11532-11540. doi: 10.1021/jp810931u

38. Heikal A A. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial

anomalies. Biomark Med 2010: 4: 241-263. doi: 10.2217/bmm.10.1

16

39. Sanchez W Y, Prow T W, Sanchez W H, et al. Analysis of the metabolic deterioration of ex vivo skin

from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and

fluorescence lifetime imaging microscopy. J Biomed Opt 2010: 15: 046008. doi: 10.1117/1.3466580

40. Su G C, Wei Y H, Wang H W. NADH fluorescence as a photobiological metric in 5-aminolevlinic acid

(ALA)-photodynamic therapy. Opt Express 2011: 19: 21145-21154. doi: 10.1364/OE.19.021145

41. Wang H W, Gukassyan V, Chen C T, et al. Differentiation of apoptosis from necrosis by dynamic

changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells. J Biomed Opt

2008: 13: 054011. doi: 10.1117/1.2975831

42. Yu J S, Guo H W, Wang C H, et al. Increase of reduced nicotinamide adenine dinucleotide

fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa

cells. J Biomed Opt 2011: 16: 036008. doi: 10.1117/1.3560513

43. Akhtar M J, Ahamed M, Kumar S, et al. Zinc oxide nanoparticles selectively induce apoptosis in

human cancer cells through reactive oxygen species. Int J Nanomedicine 2012: 7: 845-857. doi:

10.2147/IJN.S29129

44. Deng X, Luan Q, Chen W, et al. Nanosized zinc oxide particles induce neural stem cell apoptosis.

Nanotechnology 2009: 20: 115101. doi: 10.1088/0957-4484/20/11/115101

45. Sharma V, Anderson D, Dhawan A. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-

triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis 2012: 17: 852-

870. doi: 10.1007/s10495-012-0705-6

46. Holmes A M, Song Z, Moghimi H R, et al. Relative Penetration of Zinc Oxide and Zinc Ions into

Human Skin after Application of Different Zinc Oxide Formulations. ACS Nano 2016: 10: 1810-1819.

doi: 10.1021/acsnano.5b04148

47. Keen C L, Hurley L S. Zinc absorption through skin: correction of zinc deficiency in the rat. Am J Clin

Nutr 1977: 30: 528-530. doi:

48. Sun Q, Tran M, Smith B, et al. In-situ evaluation of barrier-cream performance on human skin using

laser-induced breakdown spectroscopy. Contact Dermatitis 2000: 43: 259-263. doi:

17

Figure legends

Figure 1. (A): ZnO NP fast and slow decaying photoluminescence, the latter accumulating on repeated

laser pulsing to create enhanced background signal related to ZnO NP concentration, defined as ‘offset5-25’.

(B): Fluorescence decay fit curves of representative pixels from the viable epidermis (grey), uncoated (red

solid line) and coated (red dashed line) ZnO NP within in vivo human skin after topical application. (C)

Standard curves for uncoated (solid line) and coated ZnO NP (dashed line), showing normalised signal

intensities plotted against ZnO NP concentrations in µg.mL-1

on a log-log scale.

Figure 2. Impact of occlusion and barrier impairment on the penetration of topically applied uncoated and

coated ZnO NP into in vivo human skin.

Representative multiphoton images of intact and barrier-impaired in vivo human skin recorded by TCSPC

FLIM.

A; without occlusion and C; with occlusion. The images were pseudocoloured according to the pixel ‘offset’

value (0-10 counts, blue-green-red, 10-25, red saturated). Images were measured 6 hours after the topical

application of the vehicle control (CCT), uncoated or coated ZnO NP (100 mg.mL-1

; 2 mg.cm-2

) on intact (In)

and barrier-impaired (BI) skin (tape-stripped 20 times). The white dotted box corresponds to ZnO NP signals

detected within the BI viable epidermis (VE). The white scale bar represents a length of 40 μm.

Also shown are mean ZnO NP levels.

B; Effect of barrier impairment. Mean ZnO NP levels (n=5, ± 95% CI) measured in unoccluded in vivo skin:

uncoated ZnO NP (blue squares) and coated ZnO NP (red triangles) applied to intact (closed symbols) and

barrier-impaired (open symbols) skin. Also shown are values for untreated controls [C (Untr), black closed

circles] and CCT vehicle controls [C (Veh), black open circles].

D; Effect of occlusion. Mean levels (n=5, ± 95% CI) of uncoated and coated ZnO-NP measured in intact and

barrier-impaired in vivo skin, with and without occlusion. Uncoated without occlusion – blue squares;

uncoated with occlusion – purple diamonds; Coated without occlusion – red triangles; Coated with occlusion

– orange inverted triangles. Open and closed symbols indicate intact and barrier-impaired skin respectively.

Figure 3. Effect of topical application of ZnO NP on the metabolic state of the viable epidermis (VE) in vivo.

(A) Representative multiphoton FLIM images pseudocoloured according to the ratio of the amplitudes a1 and

a2 of the two lifetime components 1, 2 of free and bound NADH (a1/a2). (B) a1/a2 ratios and (C) mean

lifetimes m for all volunteers (n=10, mean ± 95%CI) from the stratum granulosum.

18

Uncoated without occlusion – blue squares; uncoated with occlusion – purple diamonds; Coated without

occlusion – red triangles; Coated with occlusion – orange inverted triangles. Open and closed symbols

indicate intact and barrier-impaired skin respectively. Untreated controls – closed black circles; Vehicle

(CCT) controls – open black circles.

Figure 4. Ex vivo validation on the metabolic state of the viable epidermis after topical application of ZnO

NP; Effect on the labile Zn concentrations of ex vivo human viable epidermis after barrier impairment and

occlusion. Multiphoton images of cryosections of human skin; Top: vehicle control; Middle: intact skin;

Bottom: barrier-impaired (tape-stripped x20) skin. Treatment: topical application of uncoated ZnO NP (100

mg.mL-1

; 2 mg.cm-2

) for 6 hours, excess ZnO NP were wiped off using a surgical swab, cryosectioning,

staining with 10 µL of 10 µM Zinpyr-1 for 10 minutes. Zinpyr-1 signal intensities of the stratum corneum (SC)

and the viable epidermis after excitation with 488 nm Argon laser, representing labile Zn are shown in green

colour, ZnO SHG signal intensities shown in red colour. The white scale bar represents a length of 40 μm.

http://ees.elsevier.com/ejpb/download.aspx?id=288900&guid=9df4e034-171b-4feb-b856-776e50f289aa&scheme=1

http://ees.elsevier.com/ejpb/download.aspx?id=288909&guid=21159883-1831-4d93-ba4a-6300a590f050&scheme=1

http://ees.elsevier.com/ejpb/download.aspx?id=288901&guid=791bfd5a-edda-4014-922a-51984fca2d44&scheme=1

http://ees.elsevier.com/ejpb/download.aspx?id=288902&guid=1a223fe2-27ff-405a-a1e6-51de12a2511d&scheme=1

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Graphical abstract