Light-triggered, self-immolative nucleic acid-drug nanostructures

Article abstract of DOI:10.1021/jacs.5b00795

The simultaneous intracellular delivery of multiple types of payloads, such as hydrophobic drugs and nucleic acids, typically requires complex carrier systems. Herein, we demonstrate a self-deliverable form of nucleic acid-drug nanostructure that is compo

Full text of DOI:10.1021/jacs.5b00795

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Communication
Light-triggered, Self-immolative Nucleic Acid-drug Nanostructures
Xuyu Tan, Ben B Li, Xueguang Lu, Fei Jia, Clarissa Santori,
Priyanka Menon, Hui Li, Bohan Zhang, Jean J Zhao, and Ke Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2015
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Xuyu Tan, Ben B. Li, Xueguang Lu, Fei Jia, Clarissa Santori, Priyanka Menon, Hui Li, Bohan
‡
§
†‡*
Zhang, Jean J. Zhao, Ke Zhang
†
Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
‡
Institute of Chemical Biology and Nanomedicine, Hunan University, Changsha 410081, China
§
Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02215, USA, and Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA
Supporting Information Placeholder
lent attachment to either the terminus of the oligonucleotides or
ABSTRACT: The simultaneous intracellular delivery of multi-
ple types of payloads, such as hydrophobic drugs and nucleic
acids, typically requires complex carrier systems. Herein, we
the gold nanoparticle surface. In these systems, however, the
cores of the nanoparticles do not play a significant role but con-
tribute to a non-bioresorbable metallic mass. Naturally, for appli-
cations where the gold core is not utilized, it is advantageous to
convert the core to a reservoir for payload molecules. Herein, we
construct and test a drug-cored nucleic acid nanostructure con-
taining hydrophobic chemotherapeutic agents. Such carrier-free,
single entity agent retains many of the unique properties of the
SNA while offering benefits including precise drug loading ra-
demonstrate
a self-deliverable form of nucleic acid-drug
nanostructure that is comprised almost entirely of payload mole-
cules. Upon light activation, the nanostructure sheds the nucleic
acid shell, while the core, which consists of prodrug molecules,
disintegrates via an irreversible self-immolative process, releas-
ing free drug molecules and small molecule fragments. We
demonstrate that the nanostructures exhibit enhanced stability
against DNase I compared with free DNA, and that the model
drug (camptothecin) released exhibits similar efficacy as free,
unmodified drugs towards cancer cells.
2
6
27,28
tio, improved drug solubility,
and spatiotemporally con-
29
trolled release.
In order to create a drug core, we designed and synthesized a
DNA-camptothecin (CPT) amphiphile (Scheme 1). It consists of
three CPT molecules connected to
a phenol-based self-
immolative linker. The phenol group is capped with a photolabile
2-nitrobenzyl ether moiety, to which DNA strands are attached.
Scheme 1. Schematics of the DNA-drug nanostructures assem-
bled from photolabile DNA-drug amphiphiles.
In recent years, it has been demonstrated that specific ar-
rangements of nucleic acids into unique three-dimensional archi-
tectures can give rise to unusual properties absent for linear or
1
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circular forms of the nucleic acid. For example, spherical nu-
cleic acids (SNAs), which are typically comprised of a dense
DNA shell surrounding a gold nanoparticle core, show high lev-
10
els of cellular uptake, enhanced binding with complementary
11
strands, and increased DNA stability against enzymatic degra-
12
dation. These properties have been shown to result from the
packing and orientation of the oligonucleotides on the surface of
the gold nanoparticles, and are not directly related to the metallic
13
core. Indeed, SNAs with different core compositions (poly-
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4
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18
mers, lipids, silica, metal oxides, and quantum dots ) or
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2
even those lacking a core all exhibit similar physiochemical and
biological properties. Thus, through arrangement of the DNA
strands, many problems associated with nucleic acid delivery19,
The amphiphilicity of the DNA-CPT conjugate allows it to as-
semble into micellar nanostructures (of different morphologies,
depending on DNA size and assembly condition, vide infra) in an
aqueous environment. The 2-nitrobenzyl group can be cleaved by
UV light (365 nm) or via a two-photon mechanism,
the DNA from the nanostructure and leaving a de-capped self-
immolative drug core, which can then rapidly undergo a sponta-
neous and irreversible degradation process, to generate free
CPT molecules. We expect this method to be relatively general
especially co-carrier-induced cytotoxicity and immunogenicity,
can be bypassed.
The rapid endocytosis of SNAs also makes them suitable for
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delivery applications.
It is hypothesized that the co-delivery
releasing
of nucleic acids and small molecule drugs can allow one to inde-
pendently access gene and protein targets, thereby circumventing
drug resistance, which one-dimensional therapeutic strategies
32
23
cannot address. Payload molecules such as platinum (IV) pro-
2
4
25
drugs and doxorubicin have been delivered to cells via cova-
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with regard to the kind of drug molecules because many chemo-
therapeutic drugs are hydrophobic.
The photocleavage and self-immolation reactions are visual-
ized by gel electrophoresis. The DNA -CPT conjugate is first
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electrophoresed for 30 min (free Cy3-DNA used as a control),
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and thereafter UV light (365 nm, 10 mJ•s cm ) is applied for 5
min, followed by another 30 min of electrophoresis. After the
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Figure 2. TEM images (stained by 2% uranyl acetate) of (A) the
DNA -CPT conjugate prepared from PBS buffer, and (B) the
5
DNA -CPT conjugate prepared from PBS buffer with 5 mM
20
Figure 1. (A) Multiplex gel images of the DNA -CPT conjugate
20
MgCl2.
and free Cy3-DNA20 (1% agarose, 0.5x TBE) before (left) and
after (right) UV treatment. The top of the gel lanes is marked
initial run, the conjugate shows nearly the same migration dis-
tance as the free Cy3-DNA20 strand (Figure 1A). However, UV
treatment causes the conjugate to split into two bands: a green
band, which traveled the same overall distance as the free Cy3-
with a dashed line. (B) Release kinetics of the DNA -CPT con-
20
-
jugate as a function of light exposure times (365 nm, 10 mJ•s
1
-2
cm ). (C) Reverse-phase HPLC chromatograms of the DNA -
20
DNA , and a broad, slower-migrating red band, which correlates
CPT conjugate before (top) and after (bottom) light exposure,
showing the conjugate peak splitting into two, which match the
elution times of free DNA and CPT. (D) Multiplex gel images
20
with free CPT.
In order to determine the kinetics of the payload release, we
used reverse-phase HPLC to monitor the extent of drug and DNA
released as a function of UV exposure times. Without UV expo-
sure, a single peak was observed at both Cy3 (548 nm) and CPT
(350 nm) channels. With only 1 min of UV irradiation (365 nm,
(5% agarose, 0.05x PBS) of DNA-drug conjugates (red) or free
DNA (green) in the absence (left) or presence (right) of 2 mM
2+
Mg . The top of the gel lanes is marked with a dashed line. From
left to right: DNA -CPT, DNA , DNA -CPT, DNA12F, DNA9-
17
16F
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CPT, DNA , DNA -CPT, and DNA
10 mJ•s cm ), ca. 73% of the conjugate is cleaved, but to
reach >90% cleavage, 8 min of light irradiation is required (Fig-
ure 1B). Two new peaks are observed following UV treatment,
and their elution times match that of free CPT and DNA (Figures
8F
5
4F.
The successful synthesis of the linker is confirmed by Matrix-
Assisted Laser Desorption Ionization-Time of Flight Mass Spec-
+
trometry (MALDI-ToF MS, 395.0 Da [M+Na] , calc. 396.1 Da),
1
C, S5). MALDI-ToF MS also confirms that the DNA20 released
NMR, and IR (Figures S1-2). CPT molecules are attached to the
linker by forming carbonate linkages between the CPT 4-
hydroxyl group and the linker’s three benzyl alcohol groups.
Although singly and doubly modified linkers are formed, the
gains an extra ca. 190 Da of mass compared with the free azide-
Cy3-DNA , which is mass of the phenyl aldehyde residue of the
20
cleaved linker. Of note, we did not observe a two-stage release
profile, i.e. DNA released first followed by CPT, despite the fact
that the release of free CPT requires an additional step (the self-
immolation process, Scheme S3). It is likely that the time scale of
the HPLC analysis is much longer than that of the self-
immolation reaction.
yield for the triply modified linker (linker-CPT ) is satisfactory
3
(ca. 30%), and impurities can be easily removed by HPLC. The
mass of the product is determined by MALDI-ToF MS
+
(
m/z=1518.9 Da [M+Na] , calc. 1518.4 Da, Figure S3). Next,
azide-modified DNA strands of various lengths (5, 9, 13, 17 and
0 bases, Table S1) are attached to linker-CPT3 by Cu(I)-
2
Next, we investigated how the DNA-CPT conjugates can self-
assemble to form micellar nanostructures in solution. Agarose gel
catalyzed click chemistry, followed by HPLC purification. The
DNA strand with 20 bases (DNA ) is labeled with a Cy3 dye at
electrophoresis (5%, 0.05x PBS buffer) of the DNA
gate shows a sharp band corresponding to a much higher MW
compared with free DNA , which indicates the formation of self-
-CPT conju-
2
0
5
the 5’ end to enable tracking by fluorescence and UV-Vis spec-
troscopy. The UV-Vis spectrum of the DNA -CPT conjugate
2
0
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shows three absorption maxima at 260, 370, and 551 nm, which
are indicative of the presence of DNA, CPT, and Cy3, respective-
ly (Figure S4). The molar ratio of CPT: Cy3 is calculated based
assembled structures (Figure 1D). The number-average hydrody-
namic diameter (Dh(n)) of the nanostructure is 32±13 nm as de-
termined by Dynamic Light Scattering (DLS, Figure S8). Trans-
-
1
-1
on free CPT and Cy3 extinction coefficients (19.9 L mmol cm
mission Electron Microscopy (TEM) shows that the DNA -CPT
5
-
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-1
and 136 L mmol cm ) to be 3.4:1, which is close to 3:1. The
conjugate is also characterized by agarose gel electrophoresis
conjugate assembled into a spherical morphology with a dry-state
diameter of 28±5 nm (Figure 2A). The MW of the aggregates
decreases with increasing DNA length, presumably due to re-
duced aggregation numbers resulting from increased amphiphile
head group volume. When the DNA strand is longer than 9 bases,
the migration rate of the conjugate becomes identical to that of
free DNA, which indicates that micelle formation is no longer
thermodynamically favorable. However, in the presence of 2-5
(Figure 1A). Multiplex gel imaging (Cy3 and Cy2 excita-
tion/emission filters applied) shows a single band emitting both
Cy3 (green) and CPT (red) fluorescence. These results indicate
that the DNA-CPT conjugate is successfully synthesized and
purified. The molecular purity of the conjugate ensures a constant
DNA:drug molar ratio, which is important for consistency in drug
3
3
2+
formulation and efficacy. In addition, the conjugate can mask
the toxicity of the chemotherapeutic agent before it is released at
targeted site by light triggering.
mM divalent Mg ion, which is within the physiological range
2+
34
for Mg concentration, self-assembly takes place even for
DNA20-CPT (longest DNA tested), as indicated by agarose gel
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electrophoresis (Figure S9). This is likely because divalent Mg
Under these conditions, photocytotoxicity is undetectable (cell
viability near 100%). We next investigated whether a non-cell
killing dose of UV light can activate the cytotoxicity of the DNA-
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ions are able to bind with the charged DNA phosphate groups and
bridge different strands, and thereby attenuate the entropy penalty
to form higher order, densely packed DNA nanostructures. Inter-
estingly, the mode of packing for the DNA -CPT conjugate is
drug nanostructure. SK-BR-3 cells were first treated with DNA20-
CPT or free CPT, and after 6 h, UV light was applied. Non-UV-
2
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different from that of smaller conjugates. The hydrodynamic
diameter (Dh(n)) of the assembled structure is 22±8 nm as deter-
mined by DLS (Figure S8). TEM reveals that rod-like nanostruc-
tures have formed. The rods appear to be relatively rigid, with
length on the order of ca. 53±14 nm and cross-section diameter
approximately 8±1 nm (Figure 2B). It is possible that the rods are
formed by stacking circularly arranged DNA -CPT conjugates in
a vertical fashion, which results a cylindrical morphology. The
fact that longer hydrophilic segments give rise to rod-like struc-
tures appears to be against the common rules of amphiphilic
block copolymer assembly; however, tunable attractive interac-
tions mediated by ions between the hydrophilic segments of 3d6i,f3-7
ferent chains are oftentimes ignored in canonical theories.
Nonetheless, these morphological results are preliminary, and
additional characterization is needed to fully rule out artifacts
resulting from TEM sample preparation and imaging. According-
ly, a more detailed self-assembly study is planned.
treated cells were used as controls. Because some extent of cell
uptake has occurred within the initial 6 h of drug treatment, the
CPT is expected to be released both intracellularly and extracellu-
larly. The DNA -CPT exhibited much reduced toxicity com-
pared with free CPT in the absence of UV light (IC50 13 µM vs.
330 nM), as expected from the chemical modification and pack-
ing of CPT (Figure 4B). Furthermore, the addition of UV irradia-
tion did not increase the cytotoxicity of free CPT. On the other
hand, the cell-killing efficacy of the DNA20-CPT conjugate was
significantly amplified by UV treatment, which resulted in simi-
lar levels of toxicity as that of free CPT control (IC50 360 nM).
These results suggest that the DNA-CPT conjugates can achieve
controlled cytotoxicity by light triggering.
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A hallmark feature for SNAs is their resistance to nuclease
degradation. This property stems from the high local sodium ion
38
concentration and steric inaccessibility to the enzyme. It is of
interest to study whether the packing of the DNA-CPT conjugate
can also lower the rate of DNA degradation by nucleases. Be-
cause nucleic acid sequences with a length of 18-25 bases are
39
particularly useful for gene regulation applications, we focused
on the DNA -CPT conjugate. We synthesized a dabcyl-modified
20
complementary strand for DNA -CPT, and hybridized it to pre-
Figure 4. (A) Confocal microscopy image of SK-BR-3 cells
following incubation with DNA20-CPT nanostructures (1 µM) for
6 h (CPT channel). (B) Cytotoxicity measurement of CPT and
DNA20-CPT nanoparticles in the absence and presence of UV.
Error bars denote standard error of mean from three independent
experiments.
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formed, Cy3-modified DNA -CPT nanostructure. The dabcyl
2
0
quencher quenches the fluorescence of Cy3 when the duplex is
formed. Upon introduction of DNase I (a nuclease with prefer-
ence for phosphodiester linkages adjacent to pyrimidine nucleo-
In summary, we have demonstrated that nucleic acid/drug
nanostructures can be achieved by taking advantage of the am-
phiphilicity of nucleic acid-drug conjugates. The nanostructures
resulting from the solution assembly exhibit unique properties
often found with densely arranged nucleic acid nanostructures,
such as rapid cell uptake and enhanced nuclease stability, which
makes these structures suitable as carrier-free delivery platforms.
Furthermore, the photolabile DNA-CPT nanostructures show
localized, light-controlled cytotoxicity, providing a favorable
therapeutic window for potential clinical applications.
Figure 3. (A) Schematic of the DNA stability assay against
DNase I. (B) Kinetics of the enzymatic degradation of DNA -
20
ASSOCIATED CONTENT
Supporting Information
CPT and free Cy3-DNA20 duplexes (100 nM), expressed as Cy3
fluorescence as a function of time upon DNase I introduction.
tides), the Cy3 fluorophores are released and an increase in fluo-
rescence can be observed (Figure 3A). We found that the initial
rate of duplex degradation for the DNA -CPT nanostructures is
Materials and methods, additional characterization data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
2
0
approximately 60% of that for the free duplexes, and the time
needed for half degradation is ca. 4.5 times longer (Figure 3B).
After establishing that the DNA-drug nanostructures exhibit
SNA-like properties, we next characterized these nanostructures
in vitro for light-activated cytotoxicity, using the breast cancer
cell line SK-BR-3 as a model system. Confocal microscopy con-
firmed that DNA -CPT nanostructures can be taken up by SK-
AUTHOR INFORMATION
Corresponding Author
k.zhang@neu.edu
2
0
BR-3 cells following 6 h of incubation (1 µM) (Figure 4A). In
order to assess the efficacy of the DNA-drug nanostructure, we
first determined the proper dosage of UV light to minimize UV-
induced cell death. SK-BR-3 cells were irradiated with 0.5-4.5
Notes
The authors declare no competing financial interests.
-
1
-2
min of UV light (365 nm, 10 mJ•s cm ) in the absence of drug,
and cell viability assays were carried out after 24 h (Figure S10).
ACKNOWLEDGMENT
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The authors thank William Fowle at the NEU Biology Depart-
(34) Swaminathan, R. Clin. Biochem. Rev. 2003, 24, 47.
(35) Lin, Y. A.; Cheetham, A. G.; Zhang, P.; Ou, Y. C.; Li, Y.; Liu, G.;
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ment for help with the TEM. Financial support from Northeastern
University start-up, NEU-DFCI seed grant, and NSF CAREER
award (1453255) is gratefully acknowledged.
Hermida-Merino, D.; Hamley, I. W.; Cui, H. ACS Nano 2014, 8,
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