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약학석사 학위 논문
Aptamer-tethered and
polydopamine-shelled DNA
nanostructures for targeted delivery
앱타머로 수식되고 폴리-도파민으로 코팅된
DNA 나노구조체에 의한 표적 약물 전달
2019 년 2 월
서울대학교 대학원
약학과 물리약학 전공
양 건
Aptamer-tethered and
polydopamine-shelled DNA
nanostructures for targeted delivery
앱타머로 수식되고 폴리-도파민으로 코팅된
DNA 나노구조체에 의한 표적 약물 전달
지도교수 오 유 경
이 논문을 약학석사 학위논문으로 제출함
2018 년 12 월
서울대학교 대학원
약학과 물리약학 전공
양 건
양건의 석사 학위논문을 인준함
2018 년 12 월
위 원 장 이 봉 진 (인)
부 위 원 장 변 영 로 (인)
위 원 오 유 경 (인)
- i -
Abstract
Aptamer-tethered and
polydopamine-shelled DNA
nanostructures for targeted delivery
Geon Yang
Physical Pharmacy, Department of Pharmacy
The Graduate School
Seoul National University
Due to biocompatible, biodegradable, and non-immunogenic advantages,
DNA nanostructures have been investigated as delivery systems of
various cargoes. However, the plain DNA nanostructures loaded with
therapeutic agents have little specificity in target cell-directed delivery.
Here, we report various therapeutic cargo-loadable DNA nanostructure
shelled in polydopamine (PDA) and tethered with targeting aptamer
ligand. The DNA nanostructure was formed by rolling circle
amplification and condensation with adenovirus-derived cationic Mu
peptide. The DNA nanostructure was loaded with antisense
oligonucleotide. Therapeutic agent-loaded DNA nanostructure was then
shelled with PDA, and decorated with poly adenine tailed nucleic acid
aptamer (PTA) specific for PTK-7. Antisense oligonucleotide-loaded
- ii -
DNA nanostructures with PDA shell and PTA targeting ligand showed
enhanced cellular uptake and reduced the expression of target proteins.
These results suggest the potential of PTA-tethered and PDA-shelled
DNA nanostructures for targeted delivery of nucleic acids.
Keywords : DNA nanostructure, polydopamine shell, nucleic acid
aptamer, antisense oligonucleotide
Student Number : 2017-29915
- iii -
Contents
Abstract
Contents
List of Figures
1. Introduction
2. Materials and methods
3. Results
4. Discussion
5. Conclusion
6. References
국문 초록
ⅰ
ⅲ
ⅳ
1
5
10
21
24
25
29
- iv -
List of Figures
Figure 1. Components of ASO-loaded DNA nanostructure with
PDA shell and PTA decoration
Figure 2. Schematic illustration
Figure 3. Characterization of PDN
Figure 4. Tethering ability of poly adenine tailed aptamer on PDN
Figure 5. Cellular uptake of PDN
Figure 6. Intracellular delivery of ASO by PAsoDN
Figure 7. Target protein knockdown by PAsoDN
Figure 8. Cell viability upon treatment with PAsoDN
3
4
11
14
16
17
18
20
- 1 -
1. Introduction
DNA has emerged as a new source of biomaterial and nanostructures.
As a potential biomaterial, DNA has received attention due to its
natural biocompatibility, non-immunogenicity, and biodegradability [1].
DNA-based nanostructures have been studied exploiting the
sequence-specific complementary hybridization feature of DNA. Various
nanostructures such as DNA origami [2], DNA tetrahedron [3], and
DNA nanoribbon [4] were designed by controlling the sequences of
DNA template. Although these nanostructures suggested the potentiality
of DNA as a new source of nanomaterials, the application of various
shapes of nanostructures as delivery systems has not been fully
investigated.
Recently, rolling circle amplification (RCA) has been utilized for
polymerized DNA-based structures in nanoscale or microscale. Long
concatenated single strand DNA which was generated from circular
template of RCA can self-assemble into nano-, or micro- structures [5,
6]. These RCA-derived DNA nanostructures have been studied to
deliver various type of therapeutic agents such as antineoplastic [7],
protein [8], nucleic acid [9] and Cas9/sgRNA complex [10]. Although
RCA-derived polymeric DNA structures have advantages of generating
the amplified specific sequences in a few hours, they still suffer from
the difficulty of surface modifications with functional ligands for
targeted delivery. In this regard, the surface coating of DNA structures
with secondary polymeric layer may enable the sequential modification
with targeting ligands.
Meanwhile, mussel-inspired polydopamine (PDA) has been known as a
nonspecific coating material. PDA is synthesized by auto-oxidation and
polymerization of dopamine in alkaline solution [11]. Upon
polymerization, PDA deposits on the surfaces of various materials
- 2 -
including glass, nobel metals (Au and Cu) [12], gold nanoparticles [13],
lipid nanoparticles [14] and poly(lactic-co-glycolic acid) nanoparticles
[15].
DNA aptamers have been studied as targeting ligands of cancer cells.
DNA aptamers such as AS1411 and sgc8 have been used to increase
the delivery of nanoparticles such as gold [16] and protein nanoparticles
[17]. In these studies, DNA aptamers were anchored by covalent
modification onto the surfaces of nanoparticles. Recently, graphene
oxide nanosheets were reported to bind to the single stranded DNA by
hydrophobic interaction with nucleobase [18]. The non-covalent
modification of PDA-coated nanoparticles with DNA aptamers have not
been reported.
In this study, we hypothesized that PDA may have ability to deposit
onto therapeutic agent-loaded DNA structures. Moreover, if so, we
hypothesized that nucleic acid aptamers may be non-covalently tethered
onto the PDA surfaces providing the specific cell targeting ability. To
test these hypotheses, we tested the surface coating of PDA onto
antisense oligonucleotide (ASO)-loaded DNA nanostructures, and further
modified the surfaces of PDA-wrapped DNA nanostructure with poly
adenine tailed aptamer(PTA) (Figure 1). Here, we report that the
enhanced cellular uptake and efficacy of antisense oligonucleotide
delivered in the DNA matrix coated with PDA layer and further
modified with PTK7-specific nucleic acid aptamer (Figure 2).
- 3 -
Figure 1. Components of ASO-loaded DNA nanostructure with PDA
shell and PTA decoration
RCA product was condensed with Mu peptide and resultant DN was
coated with PDA by self-polymerization of dopamine. Then, PTA was
tethered on the surface of PDN by π-π stacking interaction. In the core
of two ASOs-Dz13 and OGX-427-were encapsulated.
- 4 -
Figure 2. Schematic illustration
PTA/PDN can be taken up by the PTK-7 overexpressing cells by
receptor mediated endocytosis. After entry, released ASO which targeted
c-jun and hsp27 mRNA increased cancer cell apoptosis.
- 5 -
2. Materials and methods
2.1. Preparation of rolling circle amplification products
The polymerized DNA structures were prepared by rolling circle
amplification as previously reported [9]. In brief, linear RCA template
was circularized by annealing with primers (Macrogen Inc., Daejeon,
Republic of Korea) in hybridization buffer (10 mM Tris-HCl, 1 mM
EDTA, 100 mM NaCl, pH 8.0). The RCA template sequence was
5'-ATC TGA CTA GTA TAT ACG GGA GGA AAA GGC GTT GGA
AGT GTA GTG GGA CGC GGC ACT CGG TCA TAG TAA T-3'
where the Dz13 binding sequence was marked in bold and OGX-427
binding sequence was italicized. The primer sequence was 5'-TAT ATA
CTA GTC AGA TAT TAC T-3'. Next, the nick in the circularized
template was closed with T4 DNA ligase (125 units/mL) (Thermo
Scientific, Waltham, MA, USA). After heat-inactivating T4 DNA ligase
at 70 ℃, RCA template was amplified by phi29 DNA polymerase (100
units/mL, Thermo Scientific). For removal of unincorporated dNTPs, the
product was centrifuged at 11,000 × g for 20 min and re-suspended in
triple-distilled water. The DNA concentrations of RCA products were
measured by an UV/Vis spectrophotometer (Nanodrop spectrophotometer,
Thermo scientific).
2.2. PDA coating of ASO-loaded DNA nanoballs
For preparation of ASO-loaded DNA nanoballs (DN), RCA products
were loaded with ASO, condensed to make DN, and coated with PDA.
To load ASO to RCA products, two types of ASOs Dz-13 (125 μg)
and OGX-427 (125 μg) were hybridized with 1 mL of RCA products
(500 μg DNA/mL) by heating at 95 ℃ for 10 min and cooled down
gradually to room temperature. Unloaded ASO was removed by
- 6 -
centrifugation at 11,000 × g for 5 min. After loading therapeutic
agents, Mu peptide (NH2-MRRAHHRRRRASHRRMRGG-COOH,
Peptron, Daejeon, Republic of Korea) was complexed with RCA
products at an 1:1 weight ratio to condense ASO-loaded RCA products
to DN. ASO-loaded DN (AsoDN) were coated with PDA by placing in
freshly prepared dopamine solution (20 mg/mL in Tris buffer, pH 8.0)
for 10 min. The resulting PDA-coated Dz13/OGX-427 ASO-loaded DN
(PAsoDN) were washed and collected using an Amicon Ultra
centrifugal filter (MWCO 100 K, Merck Millipore, Burlington, MA,
USA). In some experiments, PDA-coated DN without ASO (PDN) was
used.
2.3. Measurement of physicochemical properties
As physicochemical features, size, zeta potential, morphology, and
photothermal properties were measured. The sizes of various samples
were assessed by dynamic light scattering using an ELS-Z 1000
instrument (Photal, Osaka, Japan). The zeta potential was determined by
laser Doppler microelectrophoresis at an angle of 22 º (ELS-Z 1000,
Photal). The morphology of each sample was visualized by a scanning
electron microscope (SEM, Supra 55VP system, Carl Zeiss, Oberkochen,
Germany) and a transmission electron microscope (TEM, JEM1010,
JEOL, Japan). For evaluating photothermal property, the samples were
exposed to 808 nm near infrared (NIR) laser of 1.5 W/cm2 for 5 min
(Changchun New Industries Optoelectronics Technology, Changchun,
China). Temperature and thermal images were recorded on an IR
thermal imaging system (FLIR T420; FLIR Systems Inc., Danderyd,
Sweden).
- 7 -
2.4. Surface tethering of PDN with aptamers
PDN were tethered with plain or poly adenine tailed nucleic acid
aptamer (PTA). As a model aptamer, PTK-7 aptamer was used. To test
the binding affinity of plain or poly adenine tailed aptamers, PDN
(DNA 10 μg) were mixed with 0.2 nmole of PTK-7 aptamer in a plain
PTK-7 aptamer (Apt) or PTA and electrophoresed on a 1 % agarose
gel (GelDocXR, Bio-Rad, CA, USA). The sequences of Apt and PTA
were 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA
CGG TTA GA-3', and 5'-AAA AAA AAA ATC TAA CTG CTG CGC
CGC CGG GAA AAT ACT GTA CGG TTA GA-3', respectively. As a
control, poly adenine tailed scrambled aptamer (PTSA), 5'-AAA AAA
AAA GAG CGG ACT CAA GCT TCC GAT GAT CGG TTA TCG
AAC AAC GT-3', was used. The amount of PTA bound to PDN was
measured using fluorescein amidite (FAM)-labelled PTA (Bioneer,
Daejeon, Republic of Korea). FAM-labelled PTA was incubated with
PDN and unbound PTA was removed by centrifugation at 11,000 × g
for 10 min. The amount of unloaded FAM-labelled PTA were
calculated by measuring the fluorescence intensity from the supernatant
by Spectramax Gemini XS microplate fluorometer (Molecular Devices
Cooperation, Sunnyvale, CA, USA). The concentration of FAM-labelled
PTA was calculated from calibration curve for FAM-labelled PTA. The
amount of FAM-labeled PTA tethered on the surfaces of PDN was
calculated by subtraction of unloaded aptamers.
2.5. Cellular uptake test
The cellular uptake of aptamer-tethered PAsoDN was tested using FAM
labelled Dz13/Carboxy-X-rhodamine (ROX) labelled OGX-427. Leukemia
CCRF-CEM were maintained in RPMI-1640 medium supplemented with
- 8 -
10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml
streptomycin. For the cellular uptake of PAsoDN, 4 × 104 of each cells
were seeded onto 24-well plates. Next day, cells were treated with PTA
with AsoDN, PAsoDN, the mixture of Apt and PAsoDN,
PTSA/PAsoDN, or with PTA/PAsoDN (DNA 5 μg). One hr later, the
cells were fixed with 4 % paraformaldehyde and stained with 4',
6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St.
Louis, MO, USA). LSM 710 Confocal Laser Microscope (Carl Zeiss)
was used to observe the cellular fluorescence. For visualization of PDN,
cells were observed using a dark field microscope (Axioimager M1,
Carl Zeiss).
2.6. Western blot
The reduction of target protein expression by PAsoDN was tested by
western blot. CCRF-CEM cells were seeded in a 6-well plate (4 × 104
cells/well) and incubated with plain PAsoDN, PTSA/PAsoDN or
PTA/PAsoDN for an hour. Then cell medium was changed and
incubated for 23 hr. For SDS-PAGE, extracted proteins from whole-cell
lysates were quantified by a BCA protein assay kit (Thermo scientific).
After separated by SDS-PAGE on a 10% polyacrylamide gel, proteins
were transferred to polyvinylidene difluoride membrane (Hybond-ECL;
Amersham Bioscience, NJ, USA). Target proteins were detected by
specific antibodies to C-Jun (1:500, sc-1694, Lot B2013; Santa Cruz
Biotechnology, CA, USA), Hsp27 (1:500, sc-9012; Lot A0511; Santa
Cruz Biotechnology), and glyceral-dehyde-3-phosphate dehydrogenase
(GAPDH; 1:1000, sc25778; Lot D30115; Santa Cruz Biotechnology).
- 9 -
2.7. Cell viability test
The viability of cells treated with various samples was tested using a
Cell Counting kit 8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan)
or fluorescence microscopy. CCRF-CEM cells were seeded at a density
of 4 × 104 cells/well in a 48-well plate. Next day, the cells were
treated with various samples for an hour. And each sample was
incubated for 23 hr. The cells were then added with 20 μl of CCK-8
solution, and incubated for 30 min. The absorbance at 450 nm was
measured by an ELISA reader (Sunrise-Basic TECAN, Mannedorf,
Switzerland). In some experiments, viable cells were stained with
LIVE/DEAD Viability Assay kit (Thermo Scientific). Images were
obtained from fluorescence microscopy (Leica DM IL).
2.8. Statistics
Analysis of variance (ANOVA), with a post-hoc Student-Newman-Keuls
test, was used for statistical evaluation of experimental data. All
statistical analyses were two-tailed and were performed using SigmaStat
software (version 3.5; Systat Software, Richmond, CA, USA). P-values
< 0.05 were considered significant. (*P< .05; **P< .01; ***P< .005).
- 10 -
3. Results
3.1. Construction and characterization of PDN
The simplified structure of PDN is illustrated in Figure 3A. First, the
characterization of plain PDN was performed. TEM images (Figure 3B),
SEM images (Figure 3C) showed the morphology and sizes of RCA
products, positively charged Mu peptide-condensed DN, and PDN. Both
TEM (Figure 3B) and SEM (Figure 3C) images revealed that RCA
products were ball-like spherical shapes. The Mu peptide complexation
condensed the RCA products to DN. The mean sizes of RCA products
were microscale, larger than 1.4 μm (Figure 3D). Upon Mu peptide
complexation, the resulting DN showed the sizes of 256.9 ± 27.4 nm.
Surface coating with PDA increased the sizes, showing that the mean
size of PDN was 449.3 ± 19.3 nm. Zeta potential values were the
lowest in the RCA products, and increased in DN by positively charged
Mu peptide condensation (Figure 3D). The highest zeta potential value
was observed in PDN. To confirm the presence of PDA on PDN, the
NIR-responsive photothermal property was measured (Figure 3E). Both
the RCA products and DN did not show NIR-responsive temperature
increase. In contrast, upon NIR irradiation, PDN revealed the increase
of temperature up to 22.7 ± 0.2 ℃
- 11 -
Figure 3. Characterization of PDN
(A) RCA product was condensed with Mu peptide to produce DN.
- 12 -
Then polymerization of dopamine on the surfaces of DN resulted in
PDN. Morphological features of RP, DN and PDN were examined by
TEM (B) and SEM (C). Scale bar: 100 nm (D) Particle sizes of RP,
DN and PDN were determined by dynamic light scattering. Zeta
potentials of those were measured by laser Doppler microelectrophoresis.
(E) Photothermal activity of RP, DN and PDN. Temperature increases
of RP, DN and PDN in the course of NIR laser irradiation (1.5
W/cm2) were observed by IR thermal imaging system (***P<.005).
- 13 -
3.2. Tethering of poly adenine tailed aptamer on PDN
For anchoring aptamers on the surface of PDN, poly adenine tail was
used as a linker. To test whether the extension of aptamer sequences
with poly adenine tail could affect the folded structure of aptamers, the
folding structures were predicted by m-fold calculator
(http://unafold.rna.albany.edu/) (Figure 4A, 4B). The presence of poly
adenine tail did not change the structure of PTK-7 aptamer. The
tethering of various aptamers on PDN was tested by gel retardation
(Figure 4C). Free Apt, PTA migrated down on gel electrophoresis. The
mixture of Apt plus PDN showed the migration of unbound Apt in gel
electrophoresis. However, PTA/PDN group showed gel retardation,
without any migrated PTA. Consistent with the gel retardation data, the
amounts bound on the PDN was 3.0-fold higher in PTA-treated group
as compared to Apt (Figure 4D).
- 14 -
Figure 4. Tethering ability of poly adenine tailed aptamer on PDN
Conceptual diagram of various aptamers (A) and aptamer tethered on
PDN (B). (A) Structure and simplified figures of aptamers were
presented. (B) Interaction between aptamers and PDN was illustrated
(C) Binding of PTA to PDN was examined by electrophoretic mobility
shift assay. (D) The amount of Apt and PTA tethered on PDN was
evaluated using FAM labelled Apt and PTA (***P<.005).
- 15 -
3.3. Targeting ability of PTA bound on PDN
Cellular entry level of PDN was increased due to PTA tethering on
PDN. Dark field microscopy revealed significantly higher cellular uptake
of PDN in PTA/PDN treated group rather than other groups (Figure 5)
3.4. PTA/PAsoDN for intracellular delivery of ASO
The presence of PTA on the surface of PAsoDN increased the
intracellular delivery of ASO. As ASO, Dz13 and OGX-427 were
co-loaded in PAsoDN. The intracellular delivery of ASO was tested
using FAM-conjugated Dz13 and ROX-conjugated OGX-427. In PTK-7
overexpressing CCRF-CEM cells, PTA/PAsoDN-treated group showed
the highest uptake of both Dz13 and OGX-427 compared to other
groups (Figure 6)
3.5. Reduced expression of target protein by
PTA/PAsoDN
To test that the cellularly delivered ASO exerted the silencing of target
protein, the target protein levels were western blotted. In the groups
treated with plain PAsoDN or with PTSA/PAsoDN, the expression
levels of target proteins were not significantly different from the
untreated group. In contrast, the cells treated with PTA/PAsoDN
showed the highest silencing of C-Jun and HSP27 proteins, which are
the target proteins of Dz13, and OGX-427, respectively (Figure 7)
- 16 -
Figure 5. Cellular uptake of PDN
CCRF-CEM cells were treated with PTA+DN, PDN, Apt+PDN,
PTSA/PDN and PTA/PDN. PDN in cells were observed by dark field
microscopy. Scale bar: 10 μm.
- 17 -
Figure 6. Intracellular delivery of ASO by PAsoDN
Cellular upatake of two ASOs-Dz13/OGX-427-by CCRF-CEM cells.
CCRF-CEM cells were treated with PTA+AsoDN, PAsoDN,
Apt+PAsoDN, PTSA/PAsoDN and PTA/PAsoDN encapsulating FAM
conjugated Dz13 and ROX conjugated OGX-427. Uptake of these ASOs
was visualized by confocal microscopy. Scale bar: 20 μm.
- 18 -
Figure 7. Target protein knockdown by PAsoDN
The expression of ASO target protein, C-Jun and HSP27 was evaluated
by western blotting. After CCRF-CEM cells were treated with various
PAsoDNs, proteins extracted from whole-cell lysates were analyzed.
- 19 -
3.6. Antineoplastic effect of PAsoDN
Consistent with the enhanced delivery of ASO, PTA/PAsoDN-treated
group showed the highest anticancer effect against PTK-7
overexpressing CCRF-CEM cells. The population of live cells (Figure
8A) and the viability of CCRF-CEM cells (Figure 8B) were the lowest
in the group treated with PTA/PAsoDN.
- 20 -
Figure 8. Cell viability upon treatment with PAsoDN
Anticancer effect of ASOs were visualized by LIVE/DEAD Viability
Assay kit (A) and quantified by CCK-8 assay (B). (***P<.005).
Scale bar: 100 μm.
- 21 -
4. Discussion
In this study, we wrapped DN with PDA and tethered their surfaces
with PTA. The resulting PDN was demonstrated to be applicable for
delivery of antisense oligonucleotides.
We show that DN could serve as a matrix for delivery of nucleic
acids. Two ASOs were loaded to DN by hybridization with
complementary sequences in DN, which was introduced in the design
of RCA template [9]. After uptake in the cells, the loaded cargoes
were expected to be released from DN in endosomes, and diffuse to
the cytoplasm. Recently delivery of Crispr-Cas9 system by RCA
products has been reported [10]. If the aptamer was incorporated in
RCA product, there will be limitations to load these kinds of
therapeutics for maintaining of ligand functionality. Our aptamer-free
RCA product can be fully utilized for drug loading.
For wrapping DN, PDA was chosen. PDA coating was used for
convenient noncovalent tethering with PTA. PTA was tethered onto
PDN surfaces by physical adsorption due to the inherent binding
affinity of ssDNA to PDA [13]. Due to the rich quinone group, PDA
coating has been used for covalent modification of ligands [15]. In
covalent modification, the quinone group of PDA was known to be
involved in Michael addition or Schiff base formation between amine
group or thiol group in alkaline condition [15]. Even though
conjugation between PDA and ligand is quite simple, our method is
incredibly easier; just simple mixing of poly adenine tailed aptamer and
PDA coated material.
The surface coating of DN with PDA enabled various modification of
PDN with targeting molecules. In this study, we used poly adenine
tailed PTK-7 aptamers for anchoring onto PDN. However, other nucleic
acid aptamers with poly adenine tail can be anchored on the surfaces
- 22 -
of PDN.
In PTA, poly adenine tail was incorporated to the aptamer for tethering
moiety on PDN. Unlike the covalent modification, the physical
adsorption of poly adenine tail and PDA may be attributed to the
hydrophobic interactions between the nucleobase groups of adenine and
PDA. A previous study reported the interaction energy of nucleobases
with hydrophobic surfaces of graphene [19, 20]. Among the
nucleobases, guanine has the highest energy and adenine, thymine,
cytidine, uracil followed. Because the poly G tail can form
G-quadruplex itself, we selected poly adenine tail for hydrophobic
interaction with PDA surface.
PDA coating of DN is also expected to protect the DN in the
bloodstream. The previously studied RCA product alone may have
limitation in the surface modification. Moreover, DNA-based RCA
product may suffer from the limited stability due to degradation by
nucleases in the circulation after intravenous injection.
We observed the highest uptake of Dz13 and OGX-427 delivered by
PTA/PAsoDN. Dz13 is known to be a DNAzyme cutting c-jun mRNA
and inhibit cell proliferation [21, 22]. OGX-427 is reported to reduce
the expression of Hsp27, which plays a major role in drug resistance
and survival of cancer cells [23, 24]. Both Dz13 and OGX-427 are in
the clinical phase of drug development [21, 25]. Previously, synergistic
anticancer activity has been reported by reducing the expression of two
tumorigenic proteins working in different pathways [9]. Since the targets
and working pathways of Dz13 and OGX-27 are different, the dual
delivery of Dz13 and OGX-427 was done aiming for synergistic
activity.
In addition to nucleic acids, the scope of therapy can be expanded
depending on the nature of loaded cargo molecules in PDN. For
- 23 -
example, small molecules such as methylene blue or doxorubicin can be
loaded to DN by DNA intercalation [26, 27]. Other than DNA
intercalator, positively charged anticancer drugs may be loaded to DN
by charge-charge interaction. Moreover, the use of PDA shell wrapping
the DNA nanostructure enable the combination of each therapeutic
module with photothermal therapy.
- 24 -
5. Conclusion
We provided evidences that the wrapping of DN with PDA and facile
decoration with PTA could substantially increase the delivery of
entrapped nucleic acid to target cells. The wrapping of DN with PDA
enabled the noncovalent tethering of PTA. The concept of poly adenine
tailing for anchoring to PDA can also be applied to the design of other
nucleic acid aptamers. Although ASO was loaded to the PTA/PDN,
these are the examples of therapeutic agents. Other nucleic acid-based
products, DNA interacting molecules, and positively charged chemical
drugs might be loaded to DN. Our results support the wide feasibility
of PTA/PDN as a platform system for targeted delivery of versatile
therapeutic agents.
- 25 -
6. References
[1] Jin Y., Li Z., Liu H., Chen S., Wang F., Wang L., Li N., Ge K.,
Yang X., Liang X.-J., Zhang J. Biodegradable, multifunctional
DNAzyme nanoflowers for enhanced cancer therapy, NPG Asia Mater
2017;9:e365.
[2] Zhuang X., Ma X., Xue X., Jiang Q., Song L., Dai L., Zhang C.,
Jin S., Yang K., Ding B., Wang P.C., Liang X.J. A
photosensitizer-loaded DNA origami nanosystem for photodynamic
therapy, ACS Nano 2016;10(3):3486-3495.
[3] Lee H., Lytton-Jean A.K., Chen Y., Love K.T., Park A.I.,
Karagiannis E.D., Sehgal A., Querbes W., Zurenko C.S., Jayaraman M.,
Peng C.G., Charisse K., Borodovsky A., Manoharan M., Donahoe J.S.,
Truelove J., Nahrendorf M., Langer R., Anderson D.G. Molecularly
self-assembled nucleic acid nanoparticles for targeted in vivo siRNA
delivery, Nat Nanotechnol 2012;7(6):389-393.
[4] Chen G., Liu D., He C., Gannett T.R., Lin W., Weizmann Y.
Enzymatic synthesis of periodic DNA nanoribbons for intracellular pH
sensing and gene silencing, J Am Chem Soc 2015;137(11):3844-3851.
[5] Sun W., Lu Y., Gu Z. Rolling circle replication for engineering
drug delivery carriers, Ther Deliv 2015;6(7):765-768.
[6] Baker Y.R., Chen J., Brown J., El-Sagheer A.H., Wiseman P.,
Johnson E., Goddard P., Brown T. Preparation and characterization of
manganese, cobalt and zinc DNA nanoflowers with tuneable
morphology, DNA content and size, Nucleic Acids Res
2018;46(15):7495-7505.
[7] Hu R., Zhang X., Zhao Z., Zhu G., Chen T., Fu T., Tan W. DNA
nanoflowers for multiplexed cellular imaging and traceable targeted drug
delivery, Angew Chem Int Ed Engl 2014;53(23):5821-5826.
- 26 -
[8] Kim E., Zwi-Dantsis L., Reznikov N., Hansel C.S., Agarwal S.,
Stevens M.M. One-pot synthesis of multiple protein-encapsulated DNA
flowers and their application in intracellular protein delivery, Adv Mater
2017;29(26):1701086.
[9] Kim M.G., Park J.Y., Shim G., Choi H.G., Oh Y.K. Biomimetic
DNA nanoballs for oligonucleotide delivery, Biomaterials
2015;62:155-163.
[10] Sun W., Ji W., Hall J.M., Hu Q., Wang C., Beisel C.L., Gu Z.
Self-assembled DNA nanoclews for the efficient delivery of
CRISPR-Cas9 for genome editing, Angew Chem Int Ed Engl
2015;54(41):12029-12033.
[11] Liu Y., Ai K., Lu L. Polydopamine and its derivative materials:
synthesis and promising applications in energy, environmental, and
biomedical fields, Chem Rev 2014;114(9):5057-5115.
[12] Lee H., Dellatore S.M., Miller W.M., Messersmith P.B.
Mussel-inspired surface chemistry for multifunctional coatings, Science
2007;318(5849):426-430.
[13] Choi C.K., Li J., Wei K., Xu Y.J., Ho L.W., Zhu M., To K.K.,
Choi C.H., Bian L. A gold@polydopamine core-shell nanoprobe for
long-term intracellular detection of microRNAs in differentiating stem
cells, J Am Chem Soc 2015;137(23):7337-7346.
[14] Zhang R., Su S., Hu K., Shao L., Deng X., Sheng W., Wu Y.
Smart micelle@polydopamine core-shell nanoparticles for highly
effective chemo-photothermal combination therapy, Nanoscale
2015;7(46):19722-19731.
[15] Park J., Brust T.F., Lee H.J., Lee S.C., Watts V.J., Yeo Y.
Polydopamine-based simple and versatile surface modification of
polymeric nano drug carriers, ACS Nano 2014;8(4):3347-3356.
- 27 -
[16] Li C.H., Kuo T.R., Su H.J., Lai W.Y., Yang P.C., Chen J.S.,
Wang D.Y., Wu Y.C., Chen C.C. Fluorescence-Guided Probes of
Aptamer-Targeted Gold Nanoparticles with Computed Tomography
Imaging Accesses for in Vivo Tumor Resection, Sci Rep 2015;5:15675.
[17] Wu J., Song C., Jiang C., Shen X., Qiao Q., Hu Y. Nucleolin
targeting AS1411 modified protein nanoparticle for antitumor drugs
delivery, Mol Pharm 2013;10(10):3555-3563.
[18] Kim M.G., Park J.Y., Miao W., Lee J., Oh Y.K. Polyaptamer
DNA nanothread-anchored, reduced graphene oxide nanosheets for
targeted delivery, Biomaterials 2015;48:129-136.
[19] Antony J., Grimme S. Structures and interaction energies of
stacked graphene-nucleobase complexes, Phys Chem Chem Phys
2008;10(19):2722-2729.
[20] Lee J.H., Choi Y.K., Kim H.J., Scheicher R.H., Cho J.H.
Physisorption of DNA Nucleobases on h-BN and Graphene:
vdW-Corrected DFT Calculations, J Phys Chem C
2013;117(26):13435-13441.
[21] Cho E.A., Moloney F.J., Cai H., Au-Yeung A., China C., Scolyer
R.A., Yosufi B., Raftery M.J., Deng J.Z., Morton S.W., Hammond P.T.,
Arkenau H.T., Damian D.L., Francis D.J., Chesterman C.N., Barnetson
R.S., Halliday G.M., Khachigian L.M. Safety and tolerability of an
intratumorally injected DNAzyme, Dz13, in patients with nodular
basal-cell carcinoma: a phase 1 first-in-human trial (DISCOVER),
Lancet 2013;381(9880):1835-1843.
[22] Cai H., Santiago F.S., Prado-Lourenco L., Wang B., Patrikakis M.,
Davenport M.P., Maghzal G.J., Stocker R., Parish C.R., Chong B.H.,
Lieschke G.J., Wong T.W., Chesterman C.N., Francis D.J., Moloney
F.J., Barnetson R.S., Halliday G.M., Khachigian L.M. DNAzyme
targeting c-jun suppresses skin cancer growth, Sci Transl Med
- 28 -
2012;4(139):139ra182.
[23] Lamoureux F., Thomas C., Yin M.J., Fazli L., Zoubeidi A.,
Gleave M.E. Suppression of heat shock protein 27 using OGX-427
induces endoplasmic reticulum stress and potentiates heat shock protein
90 inhibitors to delay castrate-resistant prostate cancer, Eur Urol
2014;66(1):145-155.
[24] Chi K.N., Yu E.Y., Jacobs C., Bazov J., Kollmannsberger C.,
Higano C.S., Mukherjee S.D., Gleave M.E., Stewart P.S., Hotte S.J. A
phase I dose-escalation study of apatorsen (OGX-427), an antisense
inhibitor targeting heat shock protein 27 (Hsp27), in patients with
castration-resistant prostate cancer and other advanced cancers, Ann
Oncol 2016;27(6):1116-1122.
[25] Rosenberg J.E., Hahn N.M., Regan M.M., Werner L., Alva A.,
George S., Picus J., Alter R., Balar A., Hoffman-Censits J., Grivas P.,
Lauer R., Guancial E.A., Hoimes C., Sonpavde G., Albany C., Stein
M.N., Breen T., Jacobs C., Anderson K., Bellmunt J., Lalani A.A., Pal
S., Choueiri T.K. Apatorsen plus docetaxel versus docetaxel alone in
platinum-resistant metastatic urothelial carcinoma (Borealis-2), Br J
Cancer 2018;118(11):1434-1441.
[26] Kim J.H., Jang M., Kim Y.J., Ahn H.J. Design and application of
rolling circle amplification for a tumor-specific drug carrier, J Med
Chem 2015;58(19):7863-7873.
[27] Nogueira J.J., Oppel M., González L. Enhancing intersystem
crossing in phenotiazinium dyes by intercalation into DNA, Angew
Chem Int Ed Engl 2015;54(14):4375-4378.
- 29 -
국문초록
생체 적합성, 생분해성 그리고 비 면역원성이라는 장점 덕분에
DNA 나노 구조체는 다양한 약물에 대한 전달 시스템으로 제시되고
있다. 하지만 치료제를 탑재한 순수한 DNA 나노 구조체로는 표적
세포 특이적으로 약물을 전달하기 어렵다. 여기서 우리는 다양한
치료제를 탑재할 수 있는, 폴리-도파민(PDA)으로 감싸이고 표적
앱타머 리간드가 달린 DNA 나노 구조체를 보고하고자 한다. DNA
나노 구조체는 롤링-써클 증폭과 아데노 바이러스 유래 양 이온성
Mu 펩타이드 응축으로 형성되었다. DNA 나노 구조체에는 안티센스
올리고 뉴클레오타이드가 탑재되었다. 치료제가 탑재된 DNA 나노
구조체를 PDA로 코팅하고, 폴리-아데닌 꼬리가 달린 PTK-7 특이적
핵산 앱타머 (PTA)로 수식하였다. 안티센스 올리고
뉴클레오타이드가 탑재된, PDA 셸과 PTA 표적 리간드가 있는
DNA 나노 구조체는 세포 내 흡수를 향상하였고 표적 단백질의
발현을 줄였으며 항암 효과를 증진하였다. 이러한 결과들은 핵산
치료제의 전달을 위해 PDA 셸과 PTA 표적 리간드가 있는 DNA
나노 구조체를 사용할 수 있음을 의미한다.
주요어 : DNA nanostructure, polydopamine shell, nucleic acid aptamer,
antisense oligonucleotide
학 번 : 2017-29915