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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

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- 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

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[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

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[3] Lee H., Lytton-Jean A.K., Chen Y., Love K.T., Park A.I.,

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Peng C.G., Charisse K., Borodovsky A., Manoharan M., Donahoe J.S.,

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[4] Chen G., Liu D., He C., Gannett T.R., Lin W., Weizmann Y.

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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