Magnetic Control of Cells by Chemical Fabrication of Melanin

概要

pubs.acs.org/JACS

Communication

Magnetic Control of Cells by Chemical Fabrication of Melanin
Kosuke Nishio, Kohei Toh, Amelie Perron, Masato Goto, Masahiro Abo, Yuichi Shimakawa,*
and Motonari Uesugi*
Cite This: J. Am. Chem. Soc. 2022, 144, 16720−16725

Downloaded via KYOTO UNIV on October 7, 2022 at 05:44:29 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ACCESS

Metrics & More

Read Online

Article Recommendations

sı Supporting Information
*

ABSTRACT: Melanin is an organic material biosynthesized from tyrosine in pigment-producing cells. The present study reports a
simple method to generate tailored functional materials in mammalian cells by chemically fabricating intracellular melanin. Our
approach exploits synthetic tyrosine derivatives to hijack the melanin biosynthesis pathway in pigment-producing cells. Its
application was exemplified by synthesizing and using a paramagnetic tyrosine derivative, m-YR, which endowed melanoma cells
with responsiveness to external magnetic fields. The mechanical force generated by the magnet-responsive melanin forced the cells to
elongate and align parallel to the magnetic power lines. Critically, even non-pigment cells were similarly remote-controlled by
external magnetic fields once engineered to express tyrosinase and treated with m-YR, suggesting the versatility of the approach. The
present methodology may potentially provide a new avenue for mechanobiology and magnetogenetic studies and a framework for
magnetic control of specific cells.

M

elanin is a biologically produced organic material found
across all families of living organisms including plants,
microorganisms, and animals.1 Its major functions in animal
hair, skin, and neurosensorial tissues include UV protection,
coloration, and radical scavenging.2,3 Animal melanin is
synthesized in melanosome, a cellular organelle specialized
for melanin synthesis in pigment-producing cells. The major
precursor of melanin is tyrosine, which is oxidized by the
enzyme tyrosinase triggering a polymerization reaction.
Tyrosinase, amyloid fibrils, and substrates are usually confined
in melanosomes for regulated cellular melanin synthesis.1,4
The unique properties of this biopolymer have attracted
attention in the field of materials science. As a result, a number
of melanin-mimicking materials have been developed by
implementing tyrosinase as a catalyst for oxidative polymerization.5−7 The cellular tyrosinase in melanoma cells has
recently been exploited for intracellular synthesis of bioactive
peptide polymers for potential therapy.8,9 These studies have
demonstrated that tyrosinase accepts a wide range of tyrosinecontaining substrates in vitro and in cellulo for oxidative
polymerization. In the present study, we envisioned designing
synthetic tyrosine derivatives that hijack the melanin biosynthesis pathway and copolymerize with tyrosine to generate
tailored functional materials in mammalian cells (Figure 1).
To test the concept, we generated a tyrosine probe linked to
an azide handle that selectively reacts with a click-reaction
reagent. The resulting probe, Y-N3 (Figure 2A), was mixed
with tyrosinase in vitro and allowed for the reaction for 24 h.
The polymerization reaction readily proceeded, yielding a
water-insoluble dark precipitate (Figure S1). In contrast, a
phenylalanine version of the probe, F-N3, failed to form
precipitates, underscoring the importance of the phenolic
group for polymerization. When a 1:1 mixture of Y-N3 and
tyrosine was treated with tyrosinase, we were able to detect the
formation of an intermediate whose molecular mass is
© 2022 American Chemical Society

Figure 1. Chemical fabrication of intracellular melanin with synthetic
tyrosine derivatives.

consistent with that of an oxidized covalent heterotrimer
(Figure S2), suggesting the copolymerization of Y-N3 and
tyrosine. The copolymerization was further supported by the
optical properties of the tyrosinase-treated products of the 1:1
mixture (Figure S3). Interestingly, the Y-N3 homopolymer was
found to exhibit autofluorescence in 525 nm confocal images
due to unknown reasons. When mixed with the nonfluorescent tyrosine polymer, mosaic-like fluorescent materials
were observed under a confocal microscope. No such
fluorescence was detected in the tyrosinase-treated products
of the 1:1 mixture, indicating that this product is not a simple
mixture of two homopolymers but most likely a copolymer.
Received: June 23, 2022
Published: September 12, 2022

16720

https://doi.org/10.1021/jacs.2c06555
J. Am. Chem. Soc. 2022, 144, 16720−16725

Journal of the American Chemical Society

pubs.acs.org/JACS

Communication

DBCO than those from the tyrosine or F-N3-treated cells
(Figure 2D). To gain insights into the subcellular localization
of the fabricated melanin, the Y-N3-treated cells were fixed and
subsequently reacted with TAMRA-DBCO. Confocal microscopic imaging of the cells revealed that the fabricated melanin
co-localized with gp100, a melanosome marker (Figure S4).
These results demonstrate that the cellular machinery of
melanin biosynthesis recognized Y-N3 as a building block and
integrated it into cellular melanin. Intriguingly, they further
indicate that cellular melanin can be chemically fabricated by
simply treating pigment cells with tyrosine derivatives.
We then set out to confer mammalian cells with a nonnatural capability by chemically fabricating intracellular
melanin. We envisioned that a proof of concept of this
capability would be the responsiveness of the ensuing cells to
magnetic fields. Magnetic actuation on cells is appealing
because magnetic fields provide easy, rapid, and non-invasive
control.10,11 Paramagnetic molecules with unpaired electrons
often become highly responsive to magnetic fields when
accumulated at high densities. The covalent condensation of
paramagnetic tyrosine derivatives in intracellular melanin may
impart melanin and its host cells with magnetic responsiveness
(Figure 3A). Toward this goal, we synthesized a water-soluble
paramagnetic tyrosine derivative, m-YR (Figure 3B), by
conjugating a tyrosine-arginine dipeptide with N,N′-bis(salicylidene)ethylenediamine-iron(III) complex, an organoiron compound often used in organocatalysis12 and magnetic
hyperthermia anti-cancer therapy.13−17 When mixed with
tyrosinase in vitro, m-YR formed a water-insoluble dark
precipitate (poly(m-YR)), which was attracted by a 315-mT
neodymium magnet (Figure 3C). Without tyrosinase, in
contrast, m-YR displayed no detectable levels of magnetically
attracted materials. The phenylalanine counterpart of m-YR
(m-FR) failed to generate any melanin-like polymer even after
tyrosinase treatment (Figure S5). These results indicate that
tyrosinase is capable of accepting m-YR as a substrate to
produce magnet-responsive melanin-like polymers in vitro.
Under a cellular environment, m-YR is supposed to form
copolymers with endogenous tyrosine. Tyrosinase treatment of
equal amounts of m-YR and tyrosine in vitro yielded a black
precipitate that was attracted by a 315-mT magnet, whereas
co-treatment of tyrosine and m-FR formed a precipitate that
exhibited no detectable response to the magnet (Figure S6).
The magnetic properties of the m-YR-tyrosine copolymer were
quantified by a superconducting quantum interference device
(SQUID) (Figure 3D). The m-YR-tyrosine copolymer
displayed a positive and linear magnetization-magnetic field
relationship with a calculated magnetic susceptibility (Xm) of
1.54 × 10−5 emu g−1 Oe−1, confirming its paramagnetic nature.
On the other hand, the melanin polymer consisting of tyrosine
alone displayed little magnetic susceptibility as low as −4.42 ×
10−7 emu g−1 Oe−1, indicating that natural melanin polymer is
magnetically inactive.
Encouraged by the in vitro results, we examined the effects of
m-YR on α-MSH-stimulated B16F10 melanoma cells. Cell
viability assays showed that 96-h incubation with high
concentrations of m-YR decreased cell proliferation with the
maximum tolerated dose of 100 μM (Figure S7). RT-PCR
analysis of a panel of 84 cell death-linked genes indicated that
the treatment with 100 μM m-YR had little effect on the
expression of the cell-death genes, except for only a subset of
the genes related to DNA-damaging responses (Figure S8). We
therefore used 100 μM m-YR to examine its ability to confer

Figure 2. Chemical fabrication of intracellular melanin with a tyrosine
click chemistry probe. (A) Chemical structures of monomers and a
TAMARA-conjugated click reagent. (B) Experimental workflow of
the melanin fabrication in B16F10 melanoma cells. (C) Macroscopic
images of isolated melanin after the TAMRA-DBCO treatment. Scale
bar: 50 μm. (D) Consumption rates of TAMRA-DBCO. Experiments
were replicated at least in duplicate. n = 3. Data represent mean ± SD.
Significance was determined using an unpaired two-tailed Student’s t
test. *p < 0.005.

These results collectively support our notion that tyrosinase
copolymerizes Y-N3 and tyrosine.
To examine whether Y-N3 can be incorporated into
intracellular melanin, we added Y-N3 to the culture medium
of B16F10 melanoma cells and induced melanin biosynthesis
by adding α-MSH, a peptide hormone that stimulates
melanocytes. After 96 h, the cells were collected, lysed, and
treated with TAMRA-DBCO, a red dye-conjugated clickreaction reagent (Figure 2B). Melanin polymers were purified
from the lysates by centrifugation in RIPA buffer. As expected,
the insoluble melanin particles displayed red color, while the
addition of tyrosine or F-N3 led to the formation of the blacker
pigments (Figure 2C). Spectrophotometric quantification of
TAMRA-DBCO remaining in the supernatant showed that
melanin from the Y-N3-treated cells consumed more TAMRA16721

https://doi.org/10.1021/jacs.2c06555
J. Am. Chem. Soc. ...

この論文で使われている画像