Intracellular mRNA regulation with self-assembled locked nucleic acid polymer nanoparticles

Article abstract of DOI:10.1021/ja503598z

We present an untemplated, single-component antisense oligonucleotide delivery system capable of regulating mRNA abundance in live human cells. While most approaches to nucleic acid delivery rely on secondary carriers and complex multicomponent charge-neutralizing formulations, we demonstrate efficient delivery using a simple locked nucleic acid (LNA)-polymer conjugate that assembles into spherical micellar nanoparticles displaying a dense shell of nucleic acid at the surface. Cellular uptake of soft LNA nanoparticles occurs rapidly within minutes as evidenced by flow cytometry and fluorescence microscopy. Importantly, these LNA nanoparticles knockdown survivin mRNA, an established target for cancer therapy, in a sequence-specific fashion as analyzed by RT-PCR.

Full text of DOI:10.1021/ja503598z

Communication
pubs.acs.org/JACS
Intracellular mRNA Regulation with Self-Assembled Locked Nucleic
Acid Polymer Nanoparticles
Anthony M. Rush,† David A. Nelles,§,⊥ Angela P. Blum,† Sarah A. Barnhill,† Erick T. Tatro,∥
Gene W. Yeo,‡,§ and Nathan C. Gianneschi*,†,⊥
‡
§
†Department of Chemistry & Biochemistry, Stem Cell Program and Institute for Genomic Medicine, Department of Cellular and
∥
⊥
Molecular Medicine, Department of Psychiatry, Materials Science and Engineering, University of California, San Diego, La Jolla,
California 92093, United States
S
* Supporting Information
abundance in cellulo without the need for the incorporation of a
ABSTRACT: We present an untemplated, single-compo-
nent antisense oligonucleotide delivery system capable of
regulating mRNA abundance in live human cells. While
most approaches to nucleic acid delivery rely on secondary
carriers and complex multicomponent charge-neutralizing
formulations, we demonstrate efficient delivery using a
simple locked nucleic acid (LNA)-polymer conjugate that
assembles into spherical micellar nanoparticles displaying a
dense shell of nucleic acid at the surface. Cellular uptake of
soft LNA nanoparticles occurs rapidly within minutes as
evidenced by flow cytometry and fluorescence microscopy.
Importantly, these LNA nanoparticles knockdown survivin
mRNA, an established target for cancer therapy, in a
sequence-specific fashion as analyzed by RT-PCR.
cellular internalizing component have only been demonstrated
using metal-templated SNAs.16,17 Despite the success of gold-
core SNAs, the requirement for metal templation imposes
certain constraints and limitations on the resulting SNAs
including oligo attachment chemistry, chemical diversity of the
core itself, and maximum nucleic acid density achievable in the
nanoparticle shell.18 Furthermore, in order to avoid toxicity
associated with gold nanoparticle accumulation,19,20 the
template must be chemically dissolved once the material has
been synthesized.21 In the interest of developing multifunc-
tional and nontoxic oligonucleotide delivery agents with novel
properties, it is necessary to develop new strategies toward
accessing and expanding upon this unique class of materials. In
this work we demonstrate that efficient cellular uptake and
potent mRNA regulation can be achieved with a new class of
spherical nucleic acid, namely LNA-polymer amphiphile (LPA)
nanoparticles.
odulation of intracellular RNA abundance provides an
Mexceptional opportunity to study and influence gene
function and cellular behavior. In order to systematically exploit
this opportunity, the delivery of nucleic acids to relevant
biological compartments has been extensively investigated for
the past 50 years.1,2 Despite exhaustive efforts, nucleic acid−
based therapies have realized limited success. This shortcoming
is largely due to insufficient biostability of nucleic acids, off-
target effects of modified nucleic acids, and ultimately the
inability to deliver naked nucleic acids across phospholipid
membranes.3,4 The success of nucleic acid-based therapies relies
on the ability to rationally design well-defined and stable
materials capable of overcoming these barriers. Multicompo-
nent, vector-facilitated nucleic acid delivery has emerged as a
powerful tool in the past decade due to convenience,
effectiveness, and the ability to adapt materials for in vivo
experimentation.5−7 However, progress in multicomponent
nucleic acid delivery using viral vectors, lipoplex formulations,
or cationic transfection agents has been hindered by numerous
setbacks including toxicity, immunogenicity, DNA release, and
nucleic acid instability.8,9 More recently, single-component
nucleic acid based materials have been developed as well-
defined alternatives to multicomponent DNA delivery
systems.10−13 Spherical nucleic acids (SNAs)14 represent a
unique class of stable15 DNA delivery vehicles that display
nucleic acids at the surface of the nanomaterial, hence
eliminating the need to release nucleic acids from a condensed
or sequestered state. Materials capable of regulating mRNA
LPA nanoparticles are discrete assemblies of a well-defined
polymer-LNA conjugate prepared via solid-phase coupling of a
carboxylic acid terminated norbornyl polymer with an amine-
modified LNA oligonucleotide on controlled pore glass (CPG)
beads. After conjugation, LPAs are cleaved from the solid
support with an aqueous base to yield well-defined spherical
polymeric micellar nanoparticles (Figure 1). LPA nanoparticle
formation in aqueous solution is driven by the hydrophobic
effect,22 hence the micelles are composed of a hydrophobic
polynorbornyl core with each polymer covalently bound
through an amide linkage to one solvated hydrophilic
oligonucleotide in the shell. This chemistry is important in
that it drives the dense packing of negatively charged, self-
repulsive nucleic acids in the micelle corona via energetically
favorable solvent exclusion governed by the hydrophobic
polymer core. It is noteworthy to mention that nucleic acid
density achieved in the nanoparticle shell is exceptionally high
as evidenced in previous work from our laboratory demonstrat-
ing that analogous DNA-based nanoparticles can render DNA
resistant to degradation by both endo- and exonucleases.23 LPA
nanoparticles average 20 nm in diameter as evidenced by
transmission electron microscopy (TEM) and dynamic light
scattering (DLS; see Figure 1 and Supporting Information
Figures S1−S3). These materials are the first example of a
Received: April 10, 2014
© XXXX American Chemical Society
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Figure 1. LPA composition and charcterization by electron
microscopy. LPAs assemble into spherical micellar nanoparticles as
they are released from solid support and dispersed into aqueous
solution. The resulting nanoparticles are roughly 20 nm in diameter as
evidenced by negative stain TEM and DLS. LPA nanoparticles consist
of a hydrophobic polynorbornyl core and a fluorescently labeled
hydrophilic LNA shell designed to be complementary (antisense) or
noncomplementary (nonsense) to a 20-base region of mRNA
responsible for synthesizing the protein survivin. LNA bases are
indicated in orange, “Pol” indicates the norbornene polymer
conjugated to the 5′ end of the LNA sequence, F and FdT represent
fluorescein-modified thymidine, and Cy5 represents an incorporated
cyanine 5 phosphoramidite.
Figure 2. Uptake of dye-labeled LPA nanoparticles in HeLa cells. (A)
FACS distributions showing the intensity of fluorescence among HeLa
cells treated with the antisense fluorescein-labeled LNA-polymer
amphiphile (AS-FL-LPA) after a 2 h incubation in the presence of
nanoparticles. Data are gated on 2500 total events, λex = 488 nm, λem
=
533 15 nm; see Supporting Information Figure S5 for details. (B)
FACS data showing fluorescent cell population due to antisense Cy5-
labeled (AS-CY5-LPA) micelle uptake in HeLa cells after incubation
for 2 h. Data are gated on 2500 total events, λex = 640 nm, λem = 675
12.5 nm; see Supporting Information Figure S6 for details. (C−E)
Confocal fluorescence microscopy of AS-FL-LPA-treated HeLa cells
(C, λex= 488 nm), untreated HeLa cells (D, λex= 488 nm, 635 nm),
and antisense Cy5 LPA nanoparticle-treated HeLa cells (E, λex = 635
nm) showing material uptake and distribution in a single z-slice. Time
points are indicated on each panel; scale bars are 50 μm. See
Supporting Information Figures S7 and S8 for z-stack images.
nontemplated and purely organic single-component nano-
particle to demonstrate efficient cellular uptake and subsequent
mRNA regulation via antisense activity in live cells.
To examine the efficiency of LPA nanoparticle cellular
uptake and subsequent interaction with intracellular mRNA,
three different LPA nanoparticles were designed and
synthesized (Figure 1 and Supporting Information Figure
S4). The first micelle, termed antisense fluorescein-labeled LPA
(AS-FL-LPA) nanoparticle, contains a fluorescently labeled
LNA sequence complementary to a 20-base region located in
the second exon of survivin mRNA in HeLa cells. As a control,
a second micelle was synthesized, termed nonsense fluorescein-
labeled LPA (NS-FL-LPA) nanoparticle, in which the
nucleotide sequence was scrambled. We anticipated that
comparison of the activity of these two distinct materials
would facilitate determination of the sequence-specific nature
of LPA nanoparticle mediated survivin mRNA regulation. A
third micelle, termed antisense cyanine 5-labeled LPA (AS-
CY5-LPA) nanoparticle, was designed to interrogate the
influence of the incorporated dye on LPA nanoparticle uptake
in HeLa cells.24−26
As an initial experiment, AS-FL-LPA nanoparticles and the
corresponding naked single-stranded fluorescein-labeled LNA
sequence were incubated with HeLa cells to investigate the
extent of uptake of each species measured by fluorescence-
activated cell sorting (FACS, Figure 2). After incubation with 5
nM AS-FL-LPA nanoparticle or 1 μM (the equivalent
concentration with respect to LNA and fluorescein dye)
naked fluorescein-labeled ssLNA analogue for 2 h, FACS
analysis reveals an approximately 10-fold increase in pop-
ulation-wide fluorescence at 533 nm for those cells treated with
LPA nanoparticles as compared to those treated with the
ssLNA analogue (Figure 2A and Supporting Information Figure
S5). Uptake for Cy5-labeled LPA nanoparticles shows a similar
trend (Figure 2B and Supporting Information Figure S6).
However, in contrast to fluorescein-labeled ssLNA, there is
observable association of the naked Cy5-labeled ssLNA
analogue with HeLa cells. As the LNA nucleobase sequence
is identical to that of the fluorescein-labeled ssLNA, this effect
may be a result of cyanine 5 incorporation, as certain dye
molecules are known to associate with cell membranes.24−26
Nevertheless, HeLa cells incubated with 5 nM AS-CY5-LPA
nanoparticles demonstrate a ca. 10-fold increase in fluorescence
per cell at 675 nm as compared to those cells treated with the
Cy5-labeled ssLNA analogue. These results underscore the
importance of the three-dimensional arrangement of oligonu-
cleotides in facilitating cellular association.14 Indeed, it has been
recently demonstrated that other varieties of nanostructured
DNA-based materials undergo cellular uptake more efficiently
than single-stranded analogues.27,28
Having established that LPA nanoparticles associate with
HeLa cells by FACS, we performed live-cell z-stack confocal
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fluorescence microscopy to determine the extent of nano-
particle internalization as well as intracellular distribution
(Figure 2 C−E and Supporting Information Figures S7 and
S8). Live HeLa cells were imaged after LPA nanoparticle
incubation in order to discern the distribution of LPA
nanoparticles relative to cellular boundaries without the
introduction of artifacts associated with cell fixation. Based on
confocal fluorescence images it is apparent that LPA nano-
particles are distributed diffusely throughout the cell body in
some cells (ca. 70% of cells, as determined by visual inspection
of the imaged area) after 10 min of incubation and in almost all
imaged cells (ca. 98% of cells, as determined by visual
inspection of the imaged area) after 2 h of incubation with
LPA nanoparticles. This, along with FACS data, suggests that
micelles are rapidly taken up into HeLa cells and effectively
distributed in the cytosol.
RNA plays a central role in regulating and propagating
genetic information; hence numerous efforts have demon-
strated the utility of manipulating the expression of disease-
causing genes via interference with various RNAs.29 To
determine if LPA nanoparticles are able to modulate intra-
cellular mRNA levels, we designed nanoparticles capable of
base-pairing in an antisense fashion specifically with a 20-base
region of survivin mRNA, an RNA associated with proliferation
of HeLa and other cancerous cells.30 After treatment with
antisense (AS-FL-LPA) or nonsense (NS-FL-LPA) micelles,
total HeLa RNA was harvested and analyzed for relative
abundance of survivin mRNA (Figure 3B). Treatment with
the kinetics, cytotoxicity, and potential mechanism for LPA
nanoparticle uptake (Figure 4). Previous reports concerning
Figure 4. AS-FL-LPA Nanoparticle uptake kinetics, dependence on
cholesterol, and cytotoxicity. (A) Compiled FACS data showing LPA
nanoparticle uptake over time in various cell lines. Data are gated on a
minumum of 2500 events, λex = 488 nm, λem = 533 15 nm; see
Supporting Information Figures S10 and S11 for more information.
(B) Compiled FACS data showing a decrease in LPA nanoparticle
uptake after HeLa cells were treated with methyl-β-cyclodextrin λex=
488 nm, λem = 533 15 nm; see Supporting Information Figures S13,
S14, and Tables S4 and S5 for more information. (C) Compiled FACS
data showing relative cytotoxicity for 5 nM and 10 nM LPA
nanoparticle treatments versus treatment with 0.25% Triton-X 100.
Cytotoxicity was assessed via cell-associated propidium iodide
fluorescence, λex = 488 nm, λem filter = 670 nm LP. See Supporting
Information Figure S15 and Table S6 for more information.
spherical nucleic acids indicate rapid cellular internalization that
is dependent on Type A scavenger receptors and cholesterol-
dependent caveolae-mediated endocytosis.32 LPA nanoparticles
exhibit rapid uptake within 10 min of introduction to adherent
cells across five different cell lines including human embryonic
kidney cells (Figure 4A and Supporting Information Figures
S10−12, Table S3). LPA nanoparticle association with the cell
appears to reach a maximum between 30 and 60 min after
incubation for each of the four cancerous cell lines studied.
Furthermore, LPA nanoparticle uptake in HeLa cells appears to
be dependent on cholesterol, as treatment with methyl-β-
cyclodextrin significantly decreases association of LPA nano-
particles (Figure 4B). In our hands, treatment with other
pharmacological inhibitors or disruptors of the aforementioned
endocytotic pathways did not have a significant effect on
cellular association in HeLa cells (Supporting Information
Figures S13, S14 and Tables S4 and S5). Futhermore, LPA
nanoparticles show no appreciable cytotoxicity in HeLa cells
when analyzing membrane integrity after 1 h of incubation
using propidium iodide as a probe (Figure 4C and Supporting
Information Figure S15 and Table S6).
The development of hybrid nucleic acid-based materials
capable of facilitating potent and specific interactions in
complex biological milieu hinges upon the ability to create
well-defined elements with predictable attributes and diverse
composition. The straightforward synthesis, high-density dis-
play of covalently bound nucleic acids, and the potential for
chemical diversification make LPA nanoparticles ideal candi-
dates in forming the basis of next generation smart biomaterials
capable of recognizing and responding to particular gene
expression features.
Figure 3. LPA nanoparticle uptake and survivin mRNA depletion. (A)
FACS data showing similar uptake for both antisense and nonsense
LPA nanoparticles after incubation with HeLa cells for 2 h. λex= 488
nm, λem = 533 15 nm; see Suporting Information Figure S9 for more
details. (B) RT-PCR results showing sequence-selective survivin
mRNA knockdown due to treatment with antisense LPA nanoparticles
(AS-FL-LPA). See Supporting Information Tables S1 and S2 for more
information.
antisense LPA nanoparticles significantly depleted survivin
mRNA levels relative to endogenous GAPDH mRNA tran-
scripts, suggesting efficient, sequence-specific regulation of
mRNA levels (Figure 3B). Treatment with nonsense LPA
nanoparticles showed no significant effect on survivin mRNA
levels when compared to levels in untreated cells. To our
knowledge, other than gold-templated SNAs, there has been
only one other example in the literature of a DNA-based
nanomaterial demonstrating gene-specific interactions without
the need for an auxiliary uptake-enhancing component.31
Given that cellular uptake and internalization of LPA
nanoparticles appears to be rapid and efficient, we investigated
C
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(17) Prigodich, A. E.; Seferos, D. S.; Massich, M. D.; Giljohann, D.
A.; Lane, B. C.; Mirkin, C. A. ACS Nano 2009, 3, 2147−2152.
(18) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem.
2006, 78, 8313−8318.
(19) Alkilany, A. M.; Murphy, C. J. J. Nanopart. Res. 2010, 12, 2313−
2333.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed materials and sample preparation are included
together with additional data and images. This material is
available free of charge via the Internet at http://pubs.acs.org.
(20) Chompoosor, A.; Saha, K.; Ghosh, P. S.; Macarthy, D. J.;
Miranda, O. R.; Zhu, Z. J.; Arcaro, K. F.; Rotello, V. M. Small 2010, 6,
2246−2249.
AUTHOR INFORMATION
■
Corresponding Author
ngianneschi@ucsd.edu.
(21) Young, K. L.; Scott, A. W.; Hao, L. L.; Mirkin, S. E.; Liu, G. L.;
Mirkin, C. A. Nano Lett. 2012, 12, 3867−3871.
(22) Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4175−4176.
(23) Rush, A. M.; Thompson, M. P.; Tatro, E. T.; Gianneschi, N. C.
ACS Nano 2013, 7, 1379−1387.
Notes
The authors declare no competing financial interest.
(24) Rubalcav, B.; Demunoz, D. M.; Gitler, C. Biochemistry 1969, 8,
2742−2747.
ACKNOWLEDGMENTS
■
(25) Sims, P. J.; Waggoner, A. S.; Wang, C. H.; Hoffman, J. F.
Biochemistry 1974, 13, 3315−3330.
We acknowledge primary support from the NIH via a
Director’s New Innovator Award (1DP2OD008724). A.P.B.
was supported by the American Cancer Society − North
Texans Creating Tomorrow’s Miracles Postdoctoral Fellow-
ship. D.A.N. is supported by an NSF-GRF (DGE-1144086).
This work was supported by grants from the NIH to G.W.Y.
(R01 HG004659 and R01 NS075449) and the California
Institute for Regenerative Medicine to G.W.Y. (RB3-05009).
This work was also supported by the Director, Office of
Science, and Office of Biological & Environmental Research of
the US DOE under Contract No. DE-AC02-05CH1123. E.T.T.
acknowledges support from the NIH (R21DA036423). G.W.Y.
and N.C.G. both gratefully acknowledge the Alfred P. Sloan
Foundation for research fellowships. We acknowledge the
UCSD Cryo-Electron Microscopy Facility (NIH, Agouron to
T. Baker) and the UCSD Light Microscopy Facility (P30 Grant
NS047101). We thank N. Olson and J. Santini for their
generous and insightful technical assistance.
(26) Johnson, L. V.; Walsh, M. L.; Chen, L. B. Proc. Natl. Acad. Sci.
U.S.A. 1980, 77, 990−994.
(27) Alemdaroglu, F. E.; Alemdaroglu, N. C.; Langguth, P.;
Herrmann, A. Macromol. Rapid Commun. 2008, 29, 326−329.
(28) Hamblin, G. D.; Carneiro, K. M. M.; Fakhoury, J. F.; Bujold, K.
E.; Sleiman, H. F. J. Am. Chem. Soc. 2012, 134, 2888−2891.
(29) Hannon, G. J. Nature 2002, 418, 244−251.
(30) Ambrosini, G.; Adida, C.; Altieri, D. C. Nat. Med. 1997, 3, 917−
921.
(31) Chen, T.; Wu, C. S.; Jimenez, E.; Zhu, Z.; Dajac, J. G.; You, M.
X.; Han, D.; Zhang, X. B.; Tan, W. H. Angew. Chem., Int. Ed. 2013, 52,
2012−2016.
(32) Choi, C. H. J.; Hao, L. L.; Narayan, S. P.; Auyeung, E.; Mirkin,
C. A. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 7625−7630.
REFERENCES
■
(1) Crawford, L.; Dulbecco, R.; Fried, M.; Montagnier, L.; Stoker, M.
Proc. Natl. Acad. Sci. U.S.A. 1964, 52, 148−152.
(2) Jackson, D. A.; Berg, P.; Symons, R. H. Proc. Natl. Acad. Sci.
U.S.A. 1972, 69, 2904−2909.
(3) Ledley, F. D. Hum. Gene Ther. 1995, 6, 1129−1144.
(4) Davis, M. E. Curr. Opin. Biotechnol. 2002, 13, 128−131.
(5) Wu, Z. J.; Asokan, A.; Samulski, R. J. Mol. Ther. 2006, 14, 316−
327.
(6) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33−37.
(7) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Nat. Mater.
2013, 12, 967−977.
(8) Crystal, R. G. Science 1995, 270, 404−410.
(9) Tripathy, S. K.; Black, H. B.; Goldwasser, E.; Leiden, J. M. Nat.
Med. 1996, 2, 545−550.
(10) Meade, B. R.; Dowdy, S. F. Discovery Med. 2009, 8, 253−256.
(11) Zheng, D.; Giljohann, D. A.; Chen, D. L.; Massich, M. D.;
Wang, X. Q.; Iordanov, H.; Mirkin, C. A.; Paller, A. S. Proc. Natl. Acad.
Sci. U.S.A. 2012, 109, 11975−11980.
(12) Jensen, S. A.; Day, E. S.; Ko, C. H.; Hurley, L. A.; Luciano, J. P.;
Kouri, F. M.; Merkel, T. J.; Luthi, A. J.; Patel, P. C.; Cutler, J. I.; Daniel,
W. L.; Scott, A. W.; Rotz, M. W.; Meade, T. J.; Giljohann, D. A.;
Mirkin, C. A.; Stegh, A. H. Science Trans. Med. 2013, 5, 209ra152.
(13) Averick, S. E.; Paredes, E.; Dey, S. K.; Snyder, K. M.; Tapinos,
N.; Matyjaszewski, K.; Das, S. R. J. Am. Chem. Soc. 2013, 135, 12508−
12511.
(14) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. J. Am. Chem. Soc. 2012,
134, 1376−1391.
(15) Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.;
Mirkin, C. A. Nano Lett. 2009, 9, 308−311.
(16) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.
R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027−1030.
D
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