A bioinspired, photostable UV-filter that protects mammalian cells against UV-induced cellular damage

Article abstract of DOI:10.1039/c9cc06323d

While commercially available suncare products are effective at absorbing ultraviolet (UV)-light, recent studies indicate systemic toxicities associated with many traditional chemical and physical UV-filters. We demonstrate the application of xanthommatin, a biochrome present in arthropods and cephalopods, as an alternative chemical UV-filter that is cytocompatible while maintaining its photostability and photoprotective properties.

Full text of DOI:10.1039/c9cc06323d

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A bioinspired, photostable UV-filter that protects
mammalian cells against UV-induced cellular
damage†
Cite this: Chem. Commun., 2019,
55, 12036
Received 15th August 2019,
Accepted 12th September 2019
a
Camille A. Martin,
Sean R. Dinneen,
Mahboobeh Rezaeeyazdi,b Thibault Colombani,b
a
bcd
a
Amrita Kumar,a Sidi A. Bencherif
and Leila F. Deravi
*
DOI: 10.1039/c9cc06323d
rsc.li/chemcomm
While commercially available suncare products are effective at
Typical commercial sunscreens contain a combination of
absorbing ultraviolet (UV)-light, recent studies indicate systemic chemical and/or physical UV-filters.24 Small molecules that
toxicities associated with many traditional chemical and physical absorb UV-radiation are known as chemical UV-filters; these
UV-filters. We demonstrate the application of xanthommatin, a compounds are generally conjugated systems that can readily
biochrome present in arthropods and cephalopods, as an alternative absorb incoming photons. More specifically, chemical UV-filters
chemical UV-filter that is cytocompatible while maintaining its photo- are composed of one or more benzene rings and/or carbonyls that
stability and photoprotective properties.
allow electron delocalization resulting in high molar absorptivity
in the UV-A and UV-B (290–320 nm) regions. Out of the 16 U.S.
The skin is a complex, multi-layered organ comprising a number Food and Drug Administration (FDA) approved UV-filters,
of biomolecules such as DNA, proteins, 7-dehydrocholesterol, avobenzone is the only chemical filter that provides long range
melanin, hemoglobin, and urocanic acid that have an ability to UV-A protection (lmax 360 nm).25 Unlike the chemical filters,
absorb a wide range of solar radiation (290–1440 nm).1–4 Solar physical UV-filters such as titanium dioxide and zinc oxide
ultraviolet (UV) (290–400 nm) alone can generate enough energy particles are designed to block, scatter, and reflect UV-light to
to transition many of these biomolecules from their stable states protect the skin.26 Regardless of the type, both components have
to reactive, excited states. As a result, these changes can trigger been reported to pose a threat to human health.27–29 For instance,
a cascade of biochemical reactions,2,4–9 including the over- many chemical UV-filters in commercially available sunscreens
production of reactive oxygen species (ROS),10,11 DNA mutations,12 can quickly absorb into the skin and interact with epidermal cells
and the activation of inflammatory signalling pathways.4,13–17 such as keratinocytes and Langerhan cells.30–32 Additionally, upon
UV-overexposure has also been linked to photoinduced aging exposure to solar radiation, some compounds degrade into
via the absorption of UV-A (321–400 nm) photons by DNA, reactive by-products that have been implicated as contributors
resulting in photosensitization and the generation of ROS and to reproductive and developmental toxicities in animals.27,33–35
nitrogen species (RNS).18 These events can initiate the activation of More recently, commonly used UV-filters such as avobenzone,
matrix metalloproteinases (MMPs),1,19 triggering the breakdown of oxybenzone, octocrylene, and ecamsule have been detected in the
collagen fibers, and ultimately causing the formation of coarse bloodstream at concentrations beyond FDA standards, prompting
wrinkles.20–22 In addition, these reactive species have been asso- their revaluation as human-safe materials by several regulatory
ciated with swelling in the epidermis, depletion of Langerhans agencies.36 Furthermore, early iterations of physical UV-filters
cells, and microvascular injury throughout the skin.3,23 Thus, there resulted in thick, white coatings that were aesthetically
has been a great effort to mitigate these negative effects through undesirable,37 leading manufacturers to develop micro- and
the application of topical interventions, such as sunscreens.
nano-sized particles that create more transparent films. How-
ever, reducing particle size increased skin penetration, contri-
buting to local and systemic toxicities.18,26 Therefore, there is a
need for new broad-spectrum filters that can minimize the
negative effects of solar radiation while being safe to humans.
Bio-derived materials are great alternatives to commercially
available UV-filters due to their innate cytocompatibility, structural
complexity, photostability, and high molar absorptivity.38–41 One
example is the mycosporine-like amino acids (MAAs) isolated from
marine cyanobacteria and algae, which have been explored as
a Department of Chemistry and Chemical Biology, Northeastern University, Boston,
MA 02115, USA. E-mail: l.deravi@northeastern.edu
b Department of Chemical Engineering, Northeastern University, Boston, MA 02115,
USA
c Department of Bioengineering, Northeastern University, Boston, MA 02115, USA
d John A. Paulson School of Engineering and Applied Sciences, Harvard University,
Cambridge, MA 02128, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc06323d
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UV-filters due to their broad spectrum absorbance and photo- (Fig. 1A and ESI,† Fig. S1). Next, we integrated the spectral
stability.42–44 While these unconventional materials, and others absorbance curve from 290 to 400 nm to identify the effective
alike, show great promise as sunscreens, their commercialization critical wavelength. According to the FDA, UV-filters can be
has seen limited success due to the continued dependence on their considered ‘‘broad-spectrum’’ if they have a critical wavelength
biological source coupled with low extraction yields.45
of 370 nm.25 We observed that Xa has a critical wavelength of
In this report, we explore the application and utility of an 385 nm, indicating it can be considered as a broad-spectrum
unexpected material, xanthommatin (Xa), as an alternative UV-filter. From the absorption profiles in Fig. 1A, we calculated
UV-filter. Both natural and synthetic forms of Xa provide a the sun protection factor (SPF) of Xa at varying concentrations
broad absorption and scattering profile that spans UV through (see ESI† for details). We measured a concentration dependent
short-wave infrared regions (lmax = 360 and 480 nm).46,47 Given SPF that ranged from 1 Æ 0 to 18 Æ 1 (Fig. 1B), suggesting that Xa
these features, we hypothesize that Xa could be repurposed and is a tunable UV-filter. The SPF values generated by Xa were also
packaged as an alternative UV-filter. The rationale is that the comparable to other commercially available UV-filters within this
photostability innate to Xa can mitigate UV-induced cellular specified range (ESI,† Fig. S1).
damage by absorbing and dissipating a broad spectrum of solar
Given its UV-filtering capability, we next characterized the
radiation. In this study, we investigated the cytocompatibility photostability of Xa both with and without application of solar
and bioactivity of soluble Xa and Xa-based coatings in vitro as a irradiation. To test this, thin films were first constructed by
broad-spectrum UV-filter. An added, unexpected feature is that varying Xa concentration from 0.30 to 5.00 mg cmÀ2 on
Xa also behaves as a free radical scavenger, suggesting its utility UV-transmitting PDMS substrates (Fig. 2A). Next, these films
in both suncare protection and as a potential therapeutic agent. were analysed using a standard plate reader, where the PDMS
As a phenoxazone-based biochrome abundant in arthropods films were used as the plate cover over a standard 96-well
and cephalopods, Xa contains a variety of functional groups plate. From the measured absorption profiles, SPF values of
such as carbonyls, amines, hydroxyl groups and unsaturated Xa films were calculated, exhibiting a range from 3 Æ 0 to
regions (Fig. 1). Given these features, we tested whether Xa 20 Æ 2 (ESI,† Fig. S2). Like the solution-based experiments,
could be used as a new UV-filter, where we began by evaluating Xa films exhibited SPF values that increased based on the
the photoprotective properties of soluble Xa. We measured the concentration used during fabrication.
UV-absorption of Xa (0.03–1.00 mM) and observed a broad profile
Because Xa contains several auxochrome functional groups
that increased in intensity as a function of concentration (e.g., hydroxyl, amino, aldehyde, carboxylic acid) that are known
to enhance the photostability of UV-absorbing molecules,48 we
postulated that Xa would remain stable when subjected to solar
radiation. To test this hypothesis, we monitored the photo-
stability of Xa-coated PDMS films under solar simulated light.
Fig. 2 Xa as photostable coatings. (A) An illustration of Xa-based coatings
and controls prepared on imprinted PDMS substrates. Photostability was
Fig. 1 Tunable optical properties of Xa. (A) Absorption spectra of Xa
(0.03–1.00 mM) in the UV-region. Solutions tested in PBS, pH 7.4.
(B) The concentration dependent SPF of Xa as calculated from the Sayre
derived Mansur equation. Values represent mean Æ SD (n = 3). Inset is the
non-ionized chemical structure of Xa.
tested both with and without application of solar radiation. (B) Solar
simulated light (820 W mÀ2) was applied to the coatings for 240 min,
and the AUCI was calculated at various time points. All irradiated samples
were compared to non-irradiated samples. Values represent mean Æ
SD (n = 3).
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In this study, films comprising of 5.00 mg cmÀ2 Xa were confluency in both treated and untreated conditions were com-
exposed to 820 W mÀ2 total solar radiation. This is equivalent parable. These results suggest that Xa is cytocompatible at UV-
to a 4 hour global irradiance at solar noon on a clear day in protective concentrations.
spring in Phoenix, AZ. Absorption spectra were then measured
Next, the photoprotective activity of Xa-based coatings was
every 30 minutes, and the area under the curve index (AUCI) evaluated at a fixed concentration representing SPF = 19 Æ 4
was calculated for each condition. As shown in Fig. 2B, the (ESI,† Fig. S4). In this study, cells were irradiated for 30 minutes
AUCI for Xa-coated PDMS films remained 40.90 over the with 73 W mÀ2 UVR, representing 11 MED for skin type III (see
course of the 4-hour experiment which was similar to our solar ESI† for details) in the presence of Xa-containing and Xa-free
radiation-free, control films. This set of data suggests that Xa is PDMS films. Upon irradiation, we measured the concentration
photostable within the window of maximum activity of topical of pyrimidine (6-4) pyrimidone photoproducts (6-4PP), a bio-
sunscreens.49
marker of UV-induced DNA damage by ELISA.6,7 As expected, the
As a first step in assessing its cytocompatibility, we per- Xa-based coatings substantially reduced UV-induced cell damage,
formed a proof-of-concept study where Xa was incubated in a where we observed a 46 Æ 20% reduction of 6-4PP when
2D cell culture model with murine fibroblasts (NIH 3T3). As compared to unprotected cells (Fig. 4A). Altogether, these results
fibroblasts are the primary cell type in the dermis, looking into indicated that Xa provided broad-spectrum protection against the
their interactions with Xa in vitro will provide valuable insight harmful effects of UV-light in our experimental setup.
into Xa as a safe material. To test Xa’s cytocompatibility, cells
Due to its unique structural complexity, photostability,
were exposed to the concentrations used to define the SPF and photoprotective properties, we explored whether Xa had
range (0.03–1.00 mM) for a 24 hour incubation period. As additional features to protect mammalian cells. To that end,
shown in Fig. 3 and ESI,† Fig. S3, cell viability was found to we investigated Xa’s antioxidant activity in vitro using the
be 94% Æ 6% and 96% Æ 5% as quantified by fixable dead and 2,2-diphenyl-1-picrylhydrazyl (DPPHꢀ) assay, where we compared
AlamarBlue assays, respectively. These data indicate that Xa its antioxidant activity with vitamin C (ascorbic acid), a commonly
treatment does not alter mammalian cell behavior, with viability used antioxidant.11,50–52 Based on our findings, the half maximal
similar to the control group (Xa-free incubated cells) (Fig. 3A). To effective concentration (EC50) of Xa was found to be 1.00 mM. In
further assess the impact of Xa treatment on fibroblasts, we comparison, ascorbic acid’s EC50 was found to be 0.13 mM as
looked at cell morphology and proliferation using confocal micro- shown in Fig. 4B. Although the antioxidant capacity of Xa is not as
scopy. Following a 24 hour incubation, Xa treatment at 1.00 mM strong as ascorbic acid, it is significantly higher than avobenzone
did not alter cell morphology, as the elongated and spindle- and oxybenzone, the two main commercially available sun
shaped cell characteristics were comparable to untreated cells blockers (ESI,† Fig. S5).
(Fig. 3B). Additionally, cell proliferation as well as cell number and
In this study, we found that Xa is a potent UV-absorbing
compound. As a material, Xa is photostable and provides
broad-spectrum UV-R protection over multiple hours. When
tested in vitro with fibroblasts, Xa is safe and cytocompatible.
Fig. 3 Cytotoxicity of Xa monitored in vitro. (A) The cytotoxicity of Xa was
monitored by the AlamarBlue and fixable dead cell staining assays. In both
assays, the cell viability was measured after 24 hours incubation of 0.03 to
1.00 mM Xa with fibroblasts. The cell viability was determined to be
statistically similar between the two assays except for the 0.03 mM
condition (p o 0.05). Data represents mean Æ SD from both assays
(N = 9 per concentration). (B) Confocal microscopy images of cells
cultured for 24 hours in 0 mM (untreated) and 1.00 mM Xa. Here, cell
nuclei were stained with DAPI (blue), dead cells with far-red staining (red),
and actin with AlexaFluor 488-phalloidin (green). Confocal images are
representative of n = 9 samples per condition. Scale bar = 50 mm.
Fig. 4 Bio-activity of Xa monitored in vitro. (A) The bio-activity of Xa as
an UV-protectant was evaluated by ELISA. The percent of normalized
[6-4PPs] corresponds to the relative concentration of 6-4PPs in samples
covered with PDMS substrates (uncoated) compared to Xa-coated PDMS
substrates and control (not irradiated) samples. Data was analysed by
Student’s two tail t-test where + denotes statistical similarity, p = 0.453,
and ++ denotes statistically significant difference, p = 0.003. Values
represent mean Æ SD (n = 6). (B) The inhibitory effect of DPPH by a
commercial antioxidant ascorbic acid was compared to Xa at concentra-
tions ranging from 0.03–1.00 mM. Values represent mean and SD (n = 3).
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This work was supported in part by the Army Research Office
(W911NF-16-1-0455), the US Army Environmental Quality
Basic Research Program, the National Science Foundation
(DMR- 1700720), and Northeastern University’s Department of
Chemistry and Chemical Biology (C. A. M., S. R. D., A. K., L. F. D.).
S. A. B. acknowledges the support from Northeastern University
Seed Grant/Proof of Concept Tier 1 Research Grant, Burroughs
Wellcome Fund (1018798) award, Thomas Jefferson/Face founda-
tions award, DFCI/NU Joint Program Grant, and NSF CAREER
award (1847843).
Conflicts of interest
There are no conflicts to declare.
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