Spherical Nucleic Acids for Topical Treatment of Hyperpigmentation

Article abstract of DOI:10.1021/jacs.0c12044

Oligonucleotide-based materials such as spherical nucleic acid (SNA) have been reported to exhibit improved penetration through the epidermis and the dermis of the skin upon topical application. Herein, we report a self-assembled, skin-depigmenting SNA structure, which is based upon a bifunctional oligonucleotide amphiphile containing an antisense oligonucleotide and a tyrosinase inhibitor prodrug. The two components work synergistically to increase oligonucleotide cellular uptake, enhance drug solubility, and promote skin penetration. The particles were shown to reduce melanin content in B16F10 melanoma cells and exhibited a potent antimelanogenic effect in an ultraviolet B-induced hyperpigmentation mouse model.

Full text of DOI:10.1021/jacs.0c12044

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Spherical Nucleic Acids for Topical Treatment of Hyperpigmentation
Yang Fang, Xueguang Lu, Dali Wang, Jiansong Cai, Yuyan Wang, Peiru Chen, Mengqi Ren, Hao Lu,
Jennifer Union, Lei Zhang, Yehui Sun, Fei Jia, Xi Kang, Xuyu Tan, and Ke Zhang*
Cite This: J. Am. Chem. Soc. 2021, 143, 1296−1300
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ABSTRACT: Oligonucleotide-based materials such as spherical nucleic acid (SNA) have been reported to exhibit improved
penetration through the epidermis and the dermis of the skin upon topical application. Herein, we report a self-assembled, skin-
depigmenting SNA structure, which is based upon a bifunctional oligonucleotide amphiphile containing an antisense oligonucleotide
and a tyrosinase inhibitor prodrug. The two components work synergistically to increase oligonucleotide cellular uptake, enhance
drug solubility, and promote skin penetration. The particles were shown to reduce melanin content in B16F10 melanoma cells and
exhibited a potent antimelanogenic effect in an ultraviolet B-induced hyperpigmentation mouse model.
yperpigmentation, a common condition associated with
(metallic or nonmetallic particles), have been shown to
Hexposure to ultraviolet (UV) light or skin inflammation, penetrate the skin barrier and abolish target gene expression
occurs when excess melanin forms deposits in the skin.1
in the epidermis and dermis with only 3 weeks of topical
treatment in the absence of a transfection agent.20,21
Melanogenesis takes place in melanocytes, which are located at
the basal layer of the epidermis.2 It is mainly regulated by
Here, we report an SNA-based skin depigmentation agent
incorporating an antisense oligonucleotide (ASO) targeting
MC1R (to downregulate new TYR synthesis) and a
polymerized PR prodrug, which releases active PR bio-
reductively upon cell internalization (to disable existing
TYR). The SNA takes advantage of the amphiphilicity of the
ASO−drug conjugate,22,23 allowing for the formation of a
spherical micelle with a dense DNA shell that is structurally
analogous to prototypical SNAs (Scheme 1). The ASO
component functions as both a carrier and a payload,
enhancing drug water solubility and transdermal delivery of
both components while being an active agent for gene
regulation. It has been shown that the antisense SNAs regulate
gene expression by RNase H-mediated degradation of target
mRNA without the ASO being released.24
The amphiphile is assembled by copper-free click chemistry
using an ASO modified with a 5′-dibenzocyclooctyne (DBCO)
and an azide-functionalized prodrug polymer (Scheme S1).
Because of the lack of an established MC1R ASO sequence, we
screened a number of sequences against the MC1R mRNA in
murine melanoma B16F10 cells using lipofectamine 2k-
formulated ASOs and identified a sequence with reasonable
antisense activity (sequence: 5′-TTCTCCACCA-
GACTCACCA-3′, Figure S1).25−27 The polymer is synthe-
sized via sequential ring-opening metathesis polymerization of
two monomers, an oxanorbornene bromide (N-Br)28 and a
norbornene disulfide PR (N-SS-PR),23 which gives a narrowly
tyrosinase (TYR) and other tyrosinase-related proteins
(TRPs).3,4 These enzymes are transcriptionally regulated by
the microphthalmia-associated transcription factor (MITF).5
Melanin biosynthesis can be stimulated with the increased
production of MITF, which is a result of melanocortin 1
receptor (MC1R) binding to the α-melanocyte-stimulating
hormones (α-MSHs).6−8 Current treatments for hyper-
pigmentation include physical means such as laser/chemical
skin resurfacing and microdermabrasion, which need to be
carefully managed to minimize skin damage,9 and transdermal
delivery of TYR inhibitors. The latter is a less invasive and
more convenient alternative that does not interfere with the
patient’s normal daily life.
Commonly used TYR inhibitors include kojic acid, phenyl-
ethyl resorcinol, and hydroquinone, which perform well in vitro
but suffer from side-effects such as skin irritation and/or
limited absorption.10,11 Phenylethyl resorcinol (PR), a
particularly potent TYR inhibitor,12 which inhibits mushroom
tyrosinase ∼22 times more effectively than kojic acid,13 has
been adopted as the active ingredient in depigmenting
commercial products.14 However, the poor aqueous solubility
of PR reduces skin absorption and overall efficacy.14 Attempts
to target upstream proteins using anti-MITF or anti-MC1R
siRNA together with a transdermal peptide have been shown
to deplete target transcripts and produce a skin-lightening
effect on patients with hyperpigmented facial lesions after 12
weeks of topical treatment.15 However, efficient topical
delivery of oligonucleotide drugs across intact skin remains a
lasting challenge due to the stratum corneum,16 which is a
strong barrier of the epidermis that impedes a wide range of
agents intended for the skin.17−19 Recently, spherical nucleic
acids (SNAs), which are core−shell nanoparticles with a
densely functionalized oligonucleotide shell and a rigid core
Received: November 17, 2020
Published: January 12, 2021
https://dx.doi.org/10.1021/jacs.0c12044
J. Am. Chem. Soc. 2021, 143, 1296−1300
© 2021 American Chemical Society
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shows the presence of nanoparticles with a number-average
hydrodynamic diameter of 18.4 5.2 nm (Figure S3, Table
S2) and a zeta potential of −21.8 4.8 mV. The formation of
micelles is also confirmed by transmission electron microscopy
(TEM), which shows spherical particles with dry-state sizes
Scheme 1. Structure of ASO-PR10 Prodrug Conjugate and
Their Micellar SNA Assembly
a
averaging 15.2
1.4 nm (Figure 1B). To test if the PR
component can be released, dithiothreitol (DTT) is used to
simulate the reductive intracellular environment. When treated
with 10 mM DTT at 37 °C for 1−4 h, a faster-migrating band
emerged during gel electrophoresis, which is likely the
monomeric form of the PR-free DNA−polymer conjugate
(Figure S4). The released compound matches unmodified PR
in mass (measured by electrospray ionization mass spectrom-
etry) and in retention time during reverse-phase HPLC
analysis (Figure S5).
To examine the cellular uptake of the ASO-PR10 nano-
particles, B16F10 cells were treated with Cy5-labeled nano-
particles or free DNA in the concentration range of 0.2 to 2
μM (DNA basis). ASO-PR10-treated cells exhibit ∼100 times
higher cellular uptake than free ASO-treated cells, as evidenced
by flow cytometry (Figure 1C,D). The high level of uptake is
corroborated by confocal laser scanning microscopy, which
shows strong fluorescence signals in the cytoplasm of the cells
treated with ASO-PR10 nanoparticles but only background
signals for ASO- and vehicle (phosphate-buffered saline, PBS)-
treated cells (Figure 1E). The high cell uptake of SNA-type
nanoparticles has been shown to be the result of class A
scavenger receptors-mediated endocytosis via a lipid-raft-
dependent, caveolae-mediated pathway (Figure S6).30 Pre-
treatment of B16F10 cells with the pharmacological inhibitor
methyl-β-cyclodextrin (which depletes and removes cholester-
ol) significantly reduces intracellular accumulation of Cy5-
labeled ASO-PR10, which is consistent with prior mechanistic
studies. Despite high cellular uptake, the ASO-PR10 particles
exhibit negligible levels of cytotoxicity in the concentration
range tested, 1−20 μM (PR basis), by a 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay
(Figure S7). In addition, the cells are morphologically identical
to untreated cells by microscopic evaluation.
a
Upon skin penetration, bioactive PR and ASO are released.
dispersed diblock copolymer (PDI = 1.2, Mw = 7.0 kDa, Figure
S2) with high yield (∼98%). Azide substitution of the bromide
is carried out postpolymerization. A bioreductive, self-
immolative disulfide linker is incorporated in N-SS-PR,29
which can be cleaved under intracellular reductive conditions
and release the active form of the drug. The ratio between PR
and DNA is designed to be 10:1 (mol:mol) by controlling the
stoichiometries during polymerization and final coupling. This
ratio is balanced to provide sufficient driving force for
micellization while also maintaining solubility during polymer-
ization and coupling. The coupling reaction is performed in a
dimethylformamide (DMF)/water (5:1, v/v) mixture, giving a
yield of ∼68% based on gel electrophoresis band densitometry
analysis (Figure 1A).
The ASO-PR10 amphiphile readily forms spherical micellar
nanoparticles in an aqueous buffer. Dynamic light scattering
Next, we compare the antimelanogenic efficacy of ASO-PR10
nanoparticles, free PR, DNA-PR10 nanoparticle with a
scrambled sequence (Scr-PR10), and PR-devoid ASO micelles
in vitro by measuring tyrosinase inhibition and melanin
content. B16F10 cells were treated with α-MSH (200 nM)
to stimulate melanin formation. When ASO-PR10 nanoparticles
and equivalent amounts of free PR (2 to 20 μM PR) were
added, the effects of α-MSH were reversed in a dose-
dependent manner. Remarkably, the ASO-PR10 particles
show comparable reduction in tyrosinase activity to molecular
PR despite that the latter is able to freely diffuse into the cell
(inferred from an octanol/water partition coefficient value of
3.35),31 while the particles require bioreductive activation
(Figure 2A). Furthermore, a potent antimelanogenic effect is
observed for both ASO-PR10 and PR as determined by
measuring the intracellular melanin content using light
absorbance at 405 nm, corroborating the efficient intracellular
release of PR in the active form by the micelles (Figures 2B,
S8). To measure the antisense activity of the ASO-PR10 SNAs,
MC1R levels from B16F10 cell lysates were measured by
Western blot. SNAs reduced MC1R levels in a dosage-
dependent manner, with a maximum reduction level (83%)
achieved at 2 μM ASO (20 μM PR). The effect is not a
feedback loop of released molecular PR, as PR itself does not
Figure 1. (A) Agarose gel (2%) electrophoresis of the conjugation
reaction mixture (lane 1) between N3-PR10 and DBCO-ASO, purified
ASO-PR10 conjugate (lane 2), and free ASO (lane 3). (B) TEM image
of the ASO-PR10 nanoparticles. Samples were stained with uranyl
acetate (2%). (C, D) Flow cytometry measurement of B16F10 cells
treated with Cy5-labeled ASO-PR10 SNAs or Cy5-labeled free ASO
(0.2−2 μM ASO, 4 h), showing significantly (>100×) higher uptake
for the SNAs. Error bar: mean SD; n = 3. (E) Confocal microscopy
imaging of cells treated with 2 μM of Cy5-labeled ASO-PR10
nanoparticles or free ASO. Images taken with identical settings.
Scale bar: 20 μm.
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Figure 2. Tyrosinase activity (A) and melanin content (B) upon
treatment with ASO-PR10, DNA-PR10 with a scrambled sequence
(Scr-PR10), PR-devoid micelles (in PBS), or free PR (in DMSO). α-
MSH is used to stimulate melanogenesis. Tyrosinase activity and
melanin content are recorded as percent changes compared with α-
MSH-treated cells (2 to 20 μM PR, 48 h treatment). PR-devoid
micelles: 1 μM ASO. (C, D) MC1R levels in B16F10 cells after
treatment with PR, ASO-PR10, and controls (2 to 20 μM PR, 72 h
treatment) as determined by Western blot analysis. DNA−lipofect-
amine 2k complexes: 10 μM ASO. *p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001 (two-tailed test).
Figure 3. (A) Fluorescence micrographs of mouse ear sections
(paraffin-embedded) after treatment with Cy5-labeled ASO-PR10
(red). Cell nuclei are stained with Hoechst 33342 (blue). ASO-
PR10 fluorescence is present throughout the stratum corneum,
epidermis, and dermis. Scale bar: 20 μm. (B) Brightness levels (L*
value) in mouse left (blue/red) and right (green/black) ears upon
treatment with sample/vehicle groups for 32 days. ****p < 0.0001
(two-tailed test).
CIELAB color space. The darkest black is represented by L* =
0, and the brightest white at L* = 100. The L* values of both
ears of mice were recorded daily (Figure 3B). During days 8 to
22, UVB irradiation induced significant melanogenesis in both
vehicle-treated groups as reflected in a decrease of L* values,
with an average ΔL* of −2.9 0.5 (water) and −2.8 0.6
(1,2-propanediol/ethanol). Strikingly, the ASO-PR10- and PR-
treated groups both exhibited insignificant changes in L*,
suggesting successful blockage of melanogenesis. Upon
termination of UVB irradiation, the vehicle-treated groups
cease to further darken, while the ASO-PR10- and PR-treated
groups lighten slightly, with ΔL* being ∼+0.5 0.7 and +0.4
0.3, respectively. Overall, comparing day 32 to day 1, both
PR and SNA produced a net skin lightening effect despite UVB
irradiation for 2 weeks, while both control groups exhibited a
visible darkening effect (Figure S11). Collectively, these results
support that the PR-loaded SNA can deliver the active
ingredients through the stratum corneum to induce a
protective, antimelanogenic effect and avoid hyperpigmenta-
tion upon topical application.
To further study the effect of ASO-PR10 on a tissue level, the
distribution of melanin at the epidermis layer was visualized by
Fontana−Masson staining. There is clearly a higher level of
melanin in the epidermis of the vehicle-treated left ear, as
demonstrated by deposits of melanin (indicated by the
arrows), whereas treatment by PR or ASO-PR10 decreased
melanin content in the epidermis to levels comparable to
nontreated control (Figure 4A). To test the antisense efficacy
of the SNA in suppressing MC1R expression, MC1R protein
was detected by immunohistochemistry (IHC) staining
(Figure S12). While both vehicle- and SNA-treated tissues
exhibit fluorescence associated with MC1R, the intensity for
the ASO-PR10 is ∼50% that of the vehicle-treated control
(Figure 4B). However, the reduction may not have contributed
to the overall phenotypic response, as the SNA does not show
an advantage in activity despite having the ability to reduce
MC1R levels. This observation may be due to insufficient
treatment time, incomplete depletion of target protein,15 or the
cause changes in MC1R levels at the same PR concentration
(Figure 2C,D). Interestingly, ASO-PR10 particles show slightly
better reduction in tyrosinase activity and melanin content
than both Scr-PR10 and PR-devoid particles, suggesting some
level of synergy between the ASO and PR. Collectively, these
results show that the drug and ASO components in the ASO-
PR10 serve as an effective delivery vehicle for each other
through the formation of SNAs but retain their bioactivity
upon cellular uptake.
In order to examine the degrees of skin penetration and
depigmentation, C57BL/6 mice are used as a model. Because
the epidermis of the mice ear skin contains epidermal
melanocytes, unlike the trunk/fur-bearing skin, the ear skin
region is selected for testing.32,33 Cy5-labeled ASO-PR10 SNAs,
Cy5-labeled free ASO, and free Cy5 dye (equivalent to 10 μM
DNA, dispersed in Nanopure water) were topically applied to
hairless ear skin every 8 h for a total of four times.
Fluorescence microscopy of sectioned ear skin revealed
significant fluorescence in the matrix of the stratum corneum,
the cytoplasm of epidermal cells, and the dermis for the ASO-
PR10-treated sample but not for control-treated samples
(Figures 3A, S9). To test the efficacy of the SNA in
suppressing melanogenesis, the SNA, PR, or vehicle was
topically applied daily for 32 days to the dorsal ear skin of the
mouse. To minimize differences among individuals, the right
ear of each mouse receives SNA or PR while the left ear is
treated with an equal volume of vehicle. Due to the poor
solubility of PR in water, a mixed solvent of 1,2-propanediol/
ethanol (v/v 3:7) was used as vehicle, while the SNAs remain
dispersed in water. On days 8 through 22, mice were exposed
to 200 mJ/cm2 of UVB irradiation every other day to induce
hyperpigmentation (Figure S10). The degree of depigmenta-
tion is quantitatively evaluated by comparing the skin color
between the two ears of mice. The skin lightness was measured
in reflectance by colorimetry using a tristimulus colorimeter
and shown as L*, which is the lightness component in the
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Dali Wang − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Jiansong Cai − Department of Chemistry and Chemical
Biology, Northeastern University, Boston, Massachusetts
02115, United States
Yuyan Wang − Department of Chemistry and Chemical
Biology, Northeastern University, Boston, Massachusetts
02115, United States
Peiru Chen − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Mengqi Ren − Department of Chemistry and Chemical
Biology, Northeastern University, Boston, Massachusetts
02115, United States
Hao Lu − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Figure 4. (A) Histological analysis of sectioned mouse ear after
treatment with PR/ASO-PR10/vehicle groups (paraffin-embedded).
Melanin (indicated by black arrows) is stained by Fontana−Masson
staining. Scale bar: 50 μm. (B) Relative MC1R levels in ASO-PR10- vs
vehicle-treated mouse ears as determined by immunohistostaining.
*** p < 0.001 (two-tailed test).
Jennifer Union − Department of Chemistry and Chemical
Biology, Northeastern University, Boston, Massachusetts
02115, United States
Lei Zhang − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Yehui Sun − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Fei Jia − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Xi Kang − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Xuyu Tan − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States; Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139,
United States
fact that PR by itself can effectively suppress TYR activity once
delivered to the cell. However, the poor water solubility of PR
requires a harsh solvent condition to be used, which may cause
skin drying and irritations. For better synergy, future designs
may target alternative pathways such as MC1R-free tyrosinase
biosynthesis initiated by p53.34
In summary, we have synthesized an SNA-like micellar
nanoparticle with a tyrosinase inhibitor prodrug core and an
ASO shell that can inhibit the expression of a key receptor
involved in melanogenesis. This drug-cored SNA can penetrate
the ear skin of mice upon direct topical application. Once
inside the melanocytes, the drug component is released via
cleavage of a bioreductive, self-immolative linker, while the
ASO inhibits target gene expression. This approach takes
advantage of otherwise weaknesses associated with highly
hydrophobic drug molecules and non-cell-penetrating ASOs
and transforms them into essential features that enable their
co-delivery across the skin and into skin cells. The principles
demonstrated here should be broadly applicable for drug/
oligonucleotide combination therapies that target a variety of
skin-related disorders via topical application.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c12044
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c12044.
■
Author Contributions
sı
K.Z., Y.F., and X.T. devised the experiments and wrote the
manuscript. Y.F. conducted the synthesis of materials,
purification, and material/biological characterization. All
other authors contributed to material synthesis, purification,
and/or discussion of the results. All authors edited the
manuscript.
Materials and methods, experimental procedures,
instrumentation, supplemental figures, and references
(PDF)
AUTHOR INFORMATION
Corresponding Author
Ke Zhang − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States; orcid.org/0000-0002-8142-6702;
Email: k.zhang@northeastern.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This publication was made possible by the National Science
Foundation (DMR award number 2004947) and the National
Institute of General Medical Sciences (award number
1R01GM121612). The authors thank Dr. Heather Clark for
assistance with confocal microscopy, Dr. William Fowle for
electron microscopy, Dr. Jiahe Li for flow cytometry, and Dr.
Roman Manetsch for mass spectrometry.
Authors
Yang Fang − Department of Chemistry and Chemical Biology,
Northeastern University, Boston, Massachusetts 02115,
United States
Xueguang Lu − Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139,
United States
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conjugates for gene regulation. Proc. Natl. Acad. Sci. U. S. A. 2012,
109 (30), 11975.
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