Selenomelanin: An Abiotic Selenium Analogue of Pheomelanin

Selenomelanin: An Abiotic Selenium Analogue of Pheomelanin

Article

Wei Cao, Naneki C. McCallum, Qing Zhe Ni, Weiyao Li, Hannah Boyce, Haochuan Mao, Xuhao Zhou,

Hao Sun, Matthew P. Thompson, Claudia Battistella, Michael R. Wasielewski, Ali Dhinojwala,

Matthew D. Shawkey,* Michael D. Burkart,* Zheng Wang,* and Nathan C. Gianneschi*

Cite This: https://dx.doi.org/10.1021/jacs.0c05573

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sı Supporting Information

ABSTRACT: Melanins are a family of heterogeneous biopol-

ymers found ubiquitously across plant, animal, bacterial, and fungal

kingdoms where they act variously as pigments and as radiation

protection agents. There exist ﬁve multifunctional yet structurally

and biosynthetically incompletely understood varieties of melanin:

eumelanin, neuromelanin, pyomelanin, allomelanin, and pheome-

lanin. Although eumelanin and allomelanin have been the focus of

most radiation protection studies to date, some research suggests

that pheomelanin has a better absorption coeﬃcient for X-rays

than eumelanin. We reasoned that if a selenium enriched melanin

existed, it would be a better X-ray protector than the sulfur-

containing pheomelanin because the X-ray absorption coeﬃcient is

proportional to the fourth power of the atomic number (Z).

Notably, selenium is an essential micronutrient, with the amino acid selenocysteine being genetically encoded in 25 natural human

proteins. Therefore, we hypothesize that selenomelanin exists in nature, where it provides superior ionizing radiation protection to

organisms compared to known melanins. Here we introduce this novel selenium analogue of pheomelanin through chemical and

biosynthetic routes using selenocystine as a feedstock. The resulting selenomelanin is a structural mimic of pheomelanin. We found

selenomelanin eﬀectively prevented neonatal human epidermal keratinocytes (NHEK) from G2/M phase arrest under high-dose X-

ray irradiation. Provocatively, this beneﬁcial role of selenomelanin points to it as a sixth variety of yet to be discovered natural

melanin.

INTRODUCTION

optimization, and study of melanins for use in ionizing-

radiation protection for materials and living systems such as

the protection of healthy tissue during radiation-based cancer

■

Natural and synthetic eumelanins have been studied for use as

−5

6,7

pigments,

structural color, surface coatings, dry/wet

20,23

10−13

treatments has been a research focus.

Furthermore, given

adhesives, and free radical scavengers.

By contrast,

the increased interest in space travel (i.e., SpaceX, Virgin

pheomelanin is less studied, likely because it is structurally

more complex than eumelanin and because pure pheomelanin

is a rarity or is nonexistent, meaning it is never found without

Galactic) and the role of nuclear power in a noncarbon-based

electrical grid of the present and future, new lightweight

multifunctional and radioprotective materials are needed.

Selenium compounds have been reported to protect animals

against ionizing radiation, and inhibit apoptosis and inﬂam-

14,15

eumelanin in nature.

These features make its isolation

prohibitively diﬃcult. Pheomelanin is thought to be synthe-

sized biologically from tyrosine and cysteine and contains

benzothiazine subunits as evidenced by degradation studies

and analysis of intermediates.

pigmentation diﬀerences,

7,28

mation in model studies.

Selenium is an essential

6,17

In addition to structural and

some research suggests that

micronutrient. Indeed, the amino acid selenocysteine (Sec)

is encoded by a UGA codon when the SECIS (SElenoCysteine

4,18,19

pheomelanin absorbs X-rays more eﬃciently compared to

Insertion Sequence) is present in the mRNA and is hence

eumelanin.

30−32

incorporated in 25 natural human proteins.

This makes it

Unwanted exposure to ionizing radiation occurs during a

vast array of common human activities from air travel, to X-ray

diagnosis, to clinical radiation therapy, and in extreme cases

Received: May 22, 2020

such as nuclear reactor malfunction. Melanized organisms are

found in high radiation exposure environments such as

1,22

Chernobyl

and in orbiting spacecraft. Melanin may act

as a radioprotective shield by direct attenuation and/or by

quenching of radical species. Therefore, the synthesis,

XXXX American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 1. Selenomelanin chemical synthesis and characterization. (A) Synthetic route for selenomelanin NP via oxidative copolymerization of L-

DOPA and selenocysteine. (B) Representative TEM image showing spherical SeNPs. Scale bar 200 nm. (C) Images of selenomelanin suspensions,

−

−1

mg mL (left) and 0.02 mg mL (right). (D) Extinction coeﬃcient of SeNP and L-DOPA NP. (E) XPS Se 3d spectrum. (F) EPR spectrum of

SeNP. (G) C ssNMR spectra of SeNP overlaid with L-DOPA NP and monomers. Carbon assignments are listed adjacent to each main NMR

peak based on the monomer peak comparison and previous literature. Colored dots in G correspond with the proposed structure in A. Spinning

side bands of SeNP are labeled with asterisks (*).

a good candidate for examining selenium’s role as a substitute

in biomaterials that are known to naturally utilize cysteine.

This is despite the diﬃculty of working with small molecule

selenium compounds that have notorious odors, are redox

sensitive, and undergo constant metathesis triggered by visible

to synthetic pheomelanin or eumelanin. Furthermore, we show

that radioprotective selenomelanin may be biosynthesized by

bacteria fed selenocystine and L-3,4-dihydroxyphenylalanine

(L-DOPA).

light.

RESULTS AND DISCUSSION

■

We hypothesized that if a selenium enriched melanin existed

in nature or could be generated in the laboratory, it would

protect against X-rays better than the known sulfur-containing

pheomelanin. Selenium is a heavier chalcogen than sulfur, with

Chemical Synthesis of Selenomelanin. The synthesis of

selenomelanin was inspired by Lee and co-workers’ synthesis

of pheomelanin (Figure 1).

A typical synthesis of

selenomelanin involves the oxidation of L-DOPA in the

presence of selenocysteine formed in situ by selenocystine

reduction (Figure 1A). The reaction yields nanoparticles 160

nm in diameter (Figure 1B, Figure S1) with broadband

absorption across the UV−visible region, as is typical for

X-ray absorption proportional to Z . However, until

selenomelanin is discovered naturally, a synthetic route must

be devised to test its properties. The nucleophilic 1,6-addition

of the cysteine thiol group to enzymatically generated

dopaquinone is key for pheomelanin biosynthesis, so it

follows that incorporation of the more nucleophilic selenocys-

teine would yield a selenium version of pheomelanin.

melanin (Figure 1C,D). Note that KMnO has been previously

5,36

used for melanin synthesis.

conditions (1 M H SO ), 4 mM KMnO

decomposition of the polymer skeleton: a method used for

Under strongly acidic

Herein, we report the design, synthesis, and biosynthesis of

selenomelanin. We demonstrate that synthetic selenomelanin

contains benzoselenazine subunits, making it a direct analog to

the known, naturally occurring, sulfur-containing pheomelanin.

We prepare selenomelanin nanoparticles (SeNPs) which form

perinuclear caps, or microparasols, in neonatal human

epidermal keratinocyte (NHEK) cells, and signiﬁcantly

decrease cell cycle arrest following X-ray insult as compared

can lead to

7−39

HPLC-type analysis of these materials.

experiments, the pH of the reaction was kept at approximately

7.0 and the KMnO concentration was lower (1.4 mM). The

However, in our

extinction coeﬃcient (ε) in the UV−Vis region is lower than

that for nanoparticles made purely from L-DOPA (Figure 1D).

Fourier-transform infrared (FTIR) spectroscopy of SeNPs

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 2. Pheomelanin nanoparticle (PNP) synthesis and comparative ssNMR with natural pheomelanin. (A) Synthetic route and proposed

structure of PNP. (B) Representative TEM image of PNP. Scale bar 200 nm. (C) C ssNMR spectra indicate uncyclized cysteine in PNP. The

PNP NMR spectrum was processed with 60 Hz Gaussian line broadening for clarity. Carbon assignments of PNP are listed adjacent to each NMR

peak based on the monomer peak comparison. The aromatic peaks have low intensity, and assignment is impractical. Inset is the zoomed aromatic

region of cysteine, PNP, and pheomelanin from bird feathers. The PNP spinning sideband is labeled with an asterisk (*). Rooster pheomelanin

peaks at 172, 72, and 33 ppm were assigned to lipids. (D) Natural pheomelanin extraction from covert feathers of a Rhode Island red rooster shown

in photography.

Table 1. Synthetic Melanin Nanoparticles Used in This Work

Monomer

Oxidant

Size (R , nm)

ζ potential (mV)

Molar ratio of Sec/Cys to L-DOPA

CD Signal

SeNP

PNP

Selenocysteine, L-DOPA

Cysteine, L-DOPA

L-DOPA

KMnO₄

−20.3

−16.4

−52.2

0.44:1

0.65:1

−

L-DOPA NP

The molar ratio was calculated from ICP-OES results. CD spectra are shown in Figures S7 and S10 .

revealed absorption bands typical of melanins at 3600−2400

pheomelanin. On the basis of this data, we argue that the

adapted Raper-Mason model is applicable to the selenomela-

nin material (Figure S4). Attempts to obtain Se NMR were

−

−1

cm (−OH, −COOH, −NH), 1715 cm (C=O), and 1616

−

(−NH , aromatic ring C=C) (Figure S2). X-ray

photoelectron spectroscopy (XPS) conﬁrmed the presence of

the monoselenide (C−Se−C) functional group (Figure 1E,

Figure S1 ). Electron paramagnetic resonance (EPR) spectros-

unsuccessful, likely due to the low mobility of selenium in the

polymer and the intrinsic paramagnetic nature of selenomela-

nin. The integrated areas of both carbonyl peaks in the SeNP

spectrum suggest the molar ratio of selenocysteine and L-

DOPA to be 0.42:1(Figure S5 ). Inductively coupled plasma

optical emission spectrometry (ICP-OES) indicated a similar

molar ratio of 0.44:1, corresponding to a benzoselenazine

subunit content of ∼55% (w/w). In addition, circular

dichroism (CD) spectroscopy (Figure S6 ) showed that despite

using two chiral starting materials, intramolecular cyclization

leads to an achiral structure.

copy (Figure 1F) indicates the material contains stable

_u _n _p _a _i _r _e _d_e_l_e_c_t_r_o_n_s_w_i_t_h₁_.₁₂_×₁₀₁8 spins per gram (Figure

S3 ), consistent with typical synthetic melanins.

The characterization of melanin is generally challenging due

to its insolubility, hierarchical structure, and multiscale

disorder (from covalent bonds, to redox states, to noncovalent

interactions). Researchers have commonly relied on high

performance liquid chromatography (HPLC) to separate

17,20

Synthesis of Pheomelanin Nanoparticles (PNPs) and

Eumelanin Nanoparticles (L-DOPA NPs). PNPs were

synthesized using L-cysteine and L-DOPA (Figure 2, Table 1,

components arising from degraded melanin.

However,

solid-state nuclear magnetic resonance (ssNMR) oﬀers key

insights into the intact material. Therefore, we employed

comparative ssNMR analysis to study as prepared selenome-

lanin without disruptive sample preparation. A selenomelanin

Figure S7 ) according to literature procedures. PNPs have a

sulfur content of ∼7.5% as determined by ICP-OES, which is

similar to that of natural pheomelanin. XPS conﬁrmed the

C NMR spectrum was overlaid with that of synthetic

eumelanin derived from pure L-DOPA as the only monomer

Figure 1G). The peak at 163-ppm was ascribed to the

COOH of selenocysteine (ssNMR of selenocystine is

presence of sulfur (Figure S7 ), yet ssNMR (Figure 2C)

revealed the absence of benzothiazine groups in the ﬁ nal

product. CD spectroscopy of PNP shows a clear signal (Figure

S7 ) indicating a chiral structure, which is consistent with the

ssNMR results and the presence of noncyclized cysteine

adducts. The oxidation of L-DOPA and cysteine using

(

−

displayed for comparison, Figure 1G), while the decrease in

the peak at 143-ppm suggests a change in the catechol group

(

ssNMR of L-DOPA is overlaid for comparison, Figure 1G).

tyrosinase gave similar results to the permanganate oxidation

The catechol carbon shifted downﬁeld could be attributed to

cyclization generating the benzoselenazine functional group

analogous to the benzothiazine proposed for natural

as determined by C ssNMR spectroscopy (Figure S8 ). We

reasoned that while the addition of cysteine occurred, the

subsequent intramolecular cyclization step was hindered by

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 3. SeNPs form perinuclear caps and protect NHEK cells against X-ray. (A) Schematic showing SeNP perinuclear cap formation in NHEK

cells. Cells treated with SeNP for 24 h, and imaged via CLSM (B, Scale bar 20 μm), and STEM (C, scale bar 2 μm, D, scale bar 1 μm). For B, cells

were stained with wheat germ agglutinin (WGA) (red) and Hoechst 33342 (blue). (E) SeNPs protect cells against cell cycle changes induced by X-

rays. G2/M phase populations for each NP were plotted against X-ray dosage. Error bars represent standard deviation from at least three

−

−4

−6

experiments. NS means no statistical diﬀerence (P > 0.01), ***P < 10 , ****P < 10 , ******P < 10 , with reference to no NP control at same

X-ray dose, determined by Student’s two-tailed t test. (F) Attenuation of NP suspension measured by EBT3 Radiochromic ﬁlm. Error bars

represent standard deviation from three experiments. (G) CDKN1a gene expression level decreased by SeNP treatment. Error bars represent

standard deviation from three experiments. (H) Live-cell imaging of ROS scavenging by CLSM showing DCFDA ROS-positive signal (green), and

nuclei stained with Hoechst 33342 (blue). Scale bar 20 μm.

electron-donation of the sulﬁde group to the quinone, whereas

for selenocysteine, the weaker electron-donating capability of

selenide groups results in a more electrophilic ortho quinone

group due to orbital mismatch between selenium and quinone.

It should be noted that, to the best of our knowledge, papers

reporting the synthesis of pheomelanins have omitted ssNMR

data. Here, we ﬁnd that the published method in our hands

results in an absence of benzothiazine rings and the presence of

dangling cysteines, bringing into question how analogous this

synthetic pheomelanin is to that from natural sources.

Regardless, for comparison, we designate this material as a

synthetic pheomelanin and utilize it in functional studies

ssNMR spectrum (Figure 2C inset), corresponding to the

proposed benzothiazine structure (Figure S9).

Eumelanin control particles (L-DOPA NP) were prepared

via oxidative polymerization of L-DOPA (Figure S10). The

three types of melanin NPs (SeNP, PNP and L-DOPA NP) are

all spherical, with similar radii and negative ζ potentials (Table

1). Their stability was investigated under various conditions,

including dilution, ionic strength, and varying pH (Figures S11,

S12 ). SeNPs showed good stability under ambient conditions,

low refractive index (Figure S13 ), and solubility in alkaline

conditions.

Cytotoxicity and Perinuclear Cap Formation. With

melanin nanoparticles in hand, we explored their toxicity and

subcellular distribution in NHEK cells. Our prior work has

shown that polydopamine-based synthetic melanin can mimic

natural eumelanin and form artiﬁcial perinuclear caps in

against the SeNP analogue (vide infra). We note that in

addition to the synthetic route, and for structure veriﬁcation,

natural pheomelanin was extracted from the covert feathers of

a Rhode Island red rooster (Figure 2D) which have high

pheomelanin content. This natural pheomelanin has an

aromatic region analogous to what we observe in the SeNP

keratinocytes which protect them against UV damage.

Cytotoxicity experiments using NHEK cells (Figure S14)

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 4. Radioprotecting selenomelanin synthesized by living cells. (A) SEM of selenomelanin ghosts made by V. natriegens fed with selenocystine

and L-DOPA. Scale bar 1 μm. (B) C ssNMR of selenomelanin nanoparticles made by V. natriegens in culture medium. (C) XPS Se 3d spectrum of

selenomelanin ghosts. (D) Representative CLSM image showing perinuclear caps in NHEK cells formed by selenomelanin ghosts. Cell membranes

were stained with WGA (red), and nuclei with Hoechst 33342 (blue). Scale bar 20 μm. (E) Representative STEM micrograph showing the

perinuclear caps formed in NHEK cells. Scale bar 2 μm. (F) Zoomed STEM image showing semihollow selenomelanin ghosts in the perinuclear

region. Scale bar 1 μm. (G) Selenomelanin ghosts protect against cell cycle arrest induced by X-rays. Error bars represent standard deviation from

−

−5

three experiments. NS P > 0.01, ***P < 10 , *****P < 10 , determined by Student’s two-tailed t test.

indicated that SeNP has good biocompatibility below 0.02 mg

eliminate possible quenching of the propidium iodide

−

mL , similar to L-DOPA NP. Next, the intracellular

distribution was studied after incubating NHEK cells with

NPs for 24 h (Figure 3). Artiﬁcial perinuclear caps were

observed by confocal laser scanning microscopy (CLSM)

ﬂuorescence) and then irradiated with either 0, 2, 4, or 6 Gy

of X-ray radiation. It is worth noting that a typical clinical dose

used for cancer radiotherapy patients is 1.8−2 Gy per day,

while 5 Gy of radiation is lethal to humans. For SeNPs, even

after 6 Gy X-ray irradiation, the G2/M phase population

remained at 17%, which is statistically identical to non-X-ray

treated cells (P = 0.22). Another eumelanin control, namely,

polydopamine nanoparticles (PDA NPs), was synthesized and

utilized. Cell cycle experiments showed that after 6 Gy

radiation, the G2/M phase population increased to 33%

(Figure S21). This result further conﬁrms the superior

protection eﬀect of selenomelanin. The FDA approved drug

amifostine for cytoprotection against radiotherapy was also

tested, yet no diﬀerence was observed compared to the control

where particles were omitted (Figure S22). Moreover, the

viability of cells protected by SeNPs was maintained after 6 Gy

irradiation (Figures S23, S24 and Table S2 ). These results

indicate that SeNPs can protect human skin cells from X-ray-

induced cell cycle changes. While using selenomelanin for X-

ray protection, direct exposure to sunlight should be avoided

since pheomelanin has pro-oxidant properties upon photo

(

Figure 3A,B).

Scanning transmission electron microscopy (STEM) studies

Figure 3C,D) detected SeNPs inside vesicles in the

(

perinuclear area of keratinocytes and dispersed in keratin

ﬁlaments, suggesting the same distribution as natural

melanosomes. Similar behaviors were observed for PNP and

L-DOPA NPs (Figure S15, S16). Note that in this scenario, the

comparison to pheomelanin is speciﬁc to the synthetic

pheomelanin made by the commonly employed literature

methods and more work is needed to develop a high quality

synthetic mimic for further testing.

Superior Protection Observed for SeNP against X-ray

Irradiation As Compared to PNP or L-DOPA NP. We then

studied the protection eﬀects of NPs against X-ray irradiation

by assaying for G2/M phase cell cycle arrest (Figure 3E). In

initial studies, NHEK cell cycle distributions were determined

by ﬂow cytometry at diﬀerent time points following 6-Gy X-ray

irradiation. Untreated NHEK cells showed an increase from

excitation.

5% to 46% in the population of cells in the G2/M phase 24 h

To explore the mechanism of protection by SeNPs, we

assessed the antioxidant properties of the diﬀerent particles.

Exposure of a living system to high energy radiation leads to

the ionization of water and the subsequent production of toxic

reactive oxygen species (ROS), which accounts for approx-

imately two-thirds of the biological damage produced by X-

rays. Live-cell imaging shows strong green ﬂuorescence from

the ﬂuorogenic ROS probe (2′,7′-dichloroﬂuorescein diac-

etate, DCFDA) after 6 Gy X-ray irradiation in the non-NP

treated control (Figure 3H). Conversely, a very low ROS signal

postirradiation (Figures S17, S18 ); therefore, 24 h time points

were used for further cell cycle studies. NHEK cells were

otherwise morphologically intact 24 h following X-ray

treatment (Figure S19 ).

Cell cycle experiments on treated cells revealed that of the

three particles, only SeNP prevented G2/M arrest induced by

X-rays (Figure 3E, Figure S20 , Table S1 ). In these studies,

NHEK cells were treated with one of the synthetic melanin

−

NPs (NP concentrations were kept at 0.004 mg mL to

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

https://pubs.acs.org/doi/10.1021/jacs.0c05573.

Article

was observed for NP treated groups, suggesting the eﬃcient

elimination of ROS (Figures S25, S26). A subsequent DPPH

assay (2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl) (Figure

S27 ) revealed that L-DOPA NPs are faster at quenching

radicals than SeNPs and PNPs. This rapid scavenging

detoxiﬁes cells with respect to ROS caused by X-ray irradiation

and maintains a robust redox balance in cells.

by X-rays (Figure 4G, Table S3). These promising results show

that selenomelanin is a feasible natural melanin and that it

could be beneﬁcial to some organisms where high selenium

and high radiation exposure are found in natural ecosystems.

This work points to the possibility that melanin acts as a

repository for selenium and that it may be an important part of

selenium metabolism.

The radiation-related gene expression level of cyclin-

dependent kinase inhibitor, a regulator of cell cycle

progression, was also assessed. Reverse transcription polymer-

ase chain reaction (RT-PCR) studies (Figure 3G) revealed a 3-

fold upregulation of the cyclin-dependent kinase inhibitor 1a

CONCLUSIONS

■

In summary, selenomelanin, a new, abiotic form of melanin,

was prepared. Its synthesis was achieved via copolymerization

of the naturally occurring amino acid selenocysteine and the

tyrosine oxidation product, L-DOPA. The nanoparticle form of

selenomelanin, SeNPs, form perinuclear caps within human

keratinocyte cells, where they scavenge ROS and protect

against G2/M phase arrest caused by impinging ionizing

radiation. We demonstrated that selenomelanin could be

biosynthesized in live cells and that the material showed similar

properties to the synthetic version. We anticipate that this line

of research may guide the discovery of selenomelanin in nature

where an abundant source of selenium and high radiation are

both present.

(

CDKN1a) gene 3 h after 6 Gy X-ray radiation (P < 0.0001).

SeNP treatment suppressed CDKN1a expression in NHEK

cells signiﬁcantly (P = 0.01). Additionally, we compared the X-

ray attenuation capability of the diﬀerent NPs. Nanoparticle

suspensions were placed over radiochromic ﬁlm and exposed

to X-ray radiation. The ﬁlm underneath the SeNPs had the

lowest absorbance at 635 nm (Figure 3F, Figure S28), while

the L-DOPA NP and PNP suspensions had no statistical

diﬀerence in the ﬁlm absorption, suggesting that SeNPs can

better attenuate X-ray radiation. Taken together, ROS

scavenging, gene expression regulation, and physical attenu-

ation all contribute to the superior protective eﬀects of SeNPs.

Biosynthesis of selenomelanin. It is known that fungi

and bacteria metabolize selenium species contributing to

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

■

44,45

selenium cycling in nature.

In addition, eukaryotic species

such as mammals, ancient fungi, nematodes, and Drosophila

30,46,47

produce one or more selenoproteins.

Furthermore, it

Detailed experimental protocols, materials and methods,

additional characterization of SeNP, PNP, NHEK cells,

and selenomelanin made by Vibrio natriegens (PDF)

was recently reported that when zebraﬁsh larvae were treated

with selenium compounds, selenium preferentially accumu-

lated in melanized cells. However, the authors did not argue

that the melanin itself acted as a repository for the selenium

but rather that proteins in those cells had incorporated the

element. These facts lead to our reasoning that selenomelanin

should be a part of the melanin family. Therefore, we set out to

show whether bacteria genetically modiﬁed with a tyrosinase

gene can produce selenomelanin in vitro if given selenocystine.

To test this, we fed selenocystine and L-DOPA to engineered

AUTHOR INFORMATION

■

Corresponding Authors

Matthew D. Shawkey − Evolution and Optics of Nanostructures

Group, Department of Biology, The University of Ghent, 9000

Ghent, Belgium; Email: matthew.shawkey@ugent.be

Michael D. Burkart − Department of Chemistry &

Biochemistry, University of California, San Diego, La Jolla,

California 92093, United States of America; orcid.org/

tyrosinase-producing Vibrio natriegens (Figure 4, Figure S29).

Melanin was produced in cell-free supernatant as nanoparticles,

or in association with the cell walls, to form micron-size

melanin ghosts (Figure 4A, Figure S30). ssNMR of the

nanoparticles (Figure 4B) showed similar features to those of

synthetic selenomelanin (Figure 1G). FTIR spectra (Figure

S31) suggested the presence of amide bonds from

peptidoglycans. ICP-OES indicated the presence of selenium,

while XPS (Figure 4C, Figure S32) showed increased counts of

the Se 3d band with longer beam etching times, suggesting that

selenomelanin is buried inside the bacterial cell wall, further

aﬃrming that selenomelanin could be naturally incorporated in

live cells through biosynthetic pathways. Indeed, the successful

incorporation of selenium in biosynthesized melanin provides a

possible answer to the question of selenium accumulation in

000-0002-4472-2254 ; Email: mburkart@ucsd.edu

Zheng Wang − Center for Bio/Molecular Science and

Engineering, US Naval Research Laboratory, Washington, D.C.

0375, United States of America; Email: zheng.wang@

nrl.navy.mil

Nathan C. Gianneschi − Department of Chemistry and

Department of Materials Science and Engineering, Department

of Biomedical Engineering, Department of Pharmacology,

International Institute for Nanotechnology, Simpson-Querrey

Institute, Chemistry of Life Processes Institute, Lurie Cancer

Center, Northwestern University, Evanston, Illinois 60208,

United States of America; orcid.org/0000-0001-9945-

475 ; Email: nathan.gianneschi@northwestern.edu

melanized tissues of zebraﬁsh in previous studies.

We ﬁnally explored whether the biosynthesized selenome-

Authors

lanin could provide any X-ray protection. Light microscopy

Wei Cao − Department of Chemistry and Department of

(

Figure 4D) showed that selenomelanin ghosts were

Materials Science and Engineering, Department of Biomedical

Engineering, Department of Pharmacology, International

Institute for Nanotechnology, Simpson-Querrey Institute,

Chemistry of Life Processes Institute, Lurie Cancer Center,

Northwestern University, Evanston, Illinois 60208, United

States of America; orcid.org/0000-0002-5048-7991

internalized and formed perinuclear caps in NHEK cells.

STEM microscopy also supported the melanosome-mimicking

distribution (Figure 4E) and the semihollow structure of the

ghosts (Figure 4F). Cell cycle distribution studies showed that

biosynthetic selenomelanin also decreased G2/M arrest caused

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

orcid.org/0000-0002-8512-295X

Article

Naneki C. McCallum − Department of Chemistry,

Northwestern University, Evanston, Illinois 60208, United

States of America; orcid.org/0000-0001-9210-3493

Qing Zhe Ni − Department of Chemistry & Biochemistry,

University of California, San Diego, La Jolla, California 92093,

United States of America

Weiyao Li − Department of Polymer Science, The University of

Akron, Akron, Ohio 44325, United States of America;

Hannah Boyce − Department of Chemistry, Northwestern

University, Evanston, Illinois 60208, United States of America

Haochuan Mao − Department of Chemistry and Institute for

Sustainability and Energy, Northwestern University, Evanston,

Illinois 60208, United States of America; orcid.org/0000-

natriegens. W.C., M.P.T., and N.C.G. cowrote the manuscript

with contributions from all authors. All authors discussed and

interpreted the results and revised the manuscript.

Funding

The work was supported by the Air Force Oﬃce of Scientiﬁc

Research through a MURI Grant (FA9550-18-1-0142), and

supplemental grant (AFOSR FA9550-18-1-0477) and FWO

grant G007117N by the Assistant Secretary of Defense for

Research and Engineering (ASD (R&E)) through the Applied

Research for Advancement of S&T Priorities Synthetic Biology

for Military Environments program which supports Z. W. EPR

spectroscopy was supported by the National Science

Foundation under Award NO. CHE-1900422 (M.R.W.). H.

B. was supported primarily by the National Science

Foundation under NSF Award Number EEC-1757618.

001-8742-089X

Xuhao Zhou − Department of Chemistry, Northwestern

University, Evanston, Illinois 60208, United States of America;

orcid.org/0000-0002-7181-607X

Notes

The authors declare the following competing ﬁnancial

interest(s): N. C. G. and W. C. have ﬁled a provisional patent

US 62/928,129 and are listed as inventors for selenium-

containing analogues of pheomelanin, related materials, and

synthetic methods.

Hao Sun − Department of Chemistry and Department of

Materials Science and Engineering, Department of Biomedical

Engineering, Department of Pharmacology, International

Institute for Nanotechnology, Simpson-Querrey Institute,

Chemistry of Life Processes Institute, Lurie Cancer Center,

Northwestern University, Evanston, Illinois 60208, United

States of America; orcid.org/0000-0001-9153-4021

Matthew P. Thompson − Department of Chemistry and

Department of Materials Science and Engineering, Department

of Biomedical Engineering, Department of Pharmacology,

International Institute for Nanotechnology, Simpson-Querrey

Institute, Chemistry of Life Processes Institute, Lurie Cancer

Center, Northwestern University, Evanston, Illinois 60208,

United States of America

Claudia Battistella − Department of Chemistry and Department

of Materials Science and Engineering, Department of Biomedical

Engineering, Department of Pharmacology, International

Institute for Nanotechnology, Simpson-Querrey Institute,

Chemistry of Life Processes Institute, Lurie Cancer Center,

Northwestern University, Evanston, Illinois 60208, United

States of America

ACKNOWLEDGMENTS

■

The authors thank Paul Mehl in the Flow Cytometry Core

facility at Northwestern University. We thank Matthew Clutter

at the Center for Molecular Innovation and Drug Discovery at

Northwestern University for helpful discussions in the design

of X-ray radiation experiments. The authors acknowledge the

aid of Prof. Bethany Perez-White in the department of

dermatology at Northwestern Feinberg School of Medicine

for providing NHEK cells and for lending her expertise in

keratinocyte cell culture. We thank Prof. Dimitre H. Hristov in

the Department of Radiation Oncology, Stanford University

for help in the design of radiation experiments and Prof

Vythialingam Sathiaseelan at Northwestern Feinberg School of

Medicine for providing us with X-ray ﬁlm. Finally, we thank

Sara Fernandez Dunne at Northwestern’s High Throughput

Analysis Core for her assistance with conducting RT-PCR

experiments. We would also like to thank Northwestern

NUANCE BioCryo staﬀ scientists Eric W. Roth and Charlene

Wilke for their assistance in TEM cell embedding and

microtomy.

Michael R. Wasielewski − Department of Chemistry and

Institute for Sustainability and Energy, Northwestern University,

Evanston, Illinois 60208, United States of America;

orcid.org/0000-0003-2920-5440

Ali Dhinojwala − Department of Polymer Science, The

University of Akron, Akron, Ohio 44325, United States of

America; orcid.org/0000-0002-3935-7467

ABBREVIATIONS

■

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.0c05573

SeNP, selenomelanin nanoparticle; NP, nanoparticle; Sec,

selenocysteine; SECIS, SElenoCysteine Insertion Sequence; L-

DOPA, L-3,4-dihydroxyphenylalanine; EPR, electron para-

magnetic resonance; XPS, X-ray photoelectron spectroscopy;

NHEK, neonatal human epidermal keratinocytes; PDA NP,

polydopamine nanoparticle; TEM, transmission electron

microscopy; FTIR, Fourier-transform infrared spectroscopy;

ROS, reactive oxygen species; HPLC, high performance liquid

chromatography; ICP-OES, inductively coupled plasma optical

emission spectrometry; ssNMR, solid-state nuclear magnetic

resonance; PNPs, Pheomelanin nanoparticles; CLSM, confocal

laser scanning microscopy; STEM, Scanning transmission

electron microscopy; WGA, wheat germ agglutinin; DCFDA,

Author Contributions

W.C. and N.C.G. conceived the project and designed the

experiments. W. C. conducted the majority of experiments.

N.C.M. assisted with NHEK cell culture and obtained/

analyzed STEM and SEM images. Q.Z.N. and M.D.B.

designed, performed, and interpreted solid-state C NMR.

W.L. and A.D. measured the refractive index of selenomela-

nins. H.B. performed NP stability tests for aggregation by DLS.

C.B. assisted with cell culture. H.M. and M.R.W. performed

EPR. H.S. obtained CD spectra. X.Z. performed and analyzed

radical scavenging experiments. M.D.S. obtained samples and

developed methodologies for extraction of the natural

pheomelanin samples. Z.W. performed biosynthesis with V.

′,7’−dichloroﬂuorescein diacetate; DPPH, 2,2-diphenyl-1-

(2,4,6-trinitrophenyl) hydrazyl; RT-PCR, reverse transcription

polymerase chain reaction; CDKN1a, cyclin-dependent kinase

inhibitor 1a

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

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