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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)
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
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
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
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. 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
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).
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.
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.
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
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).
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
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).
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).
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 ℃
Figure 3. Characterization of PDN
(A) RCA product was condensed with Mu peptide to produce DN.
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).
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).
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).
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)
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.
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.
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.
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.
Figure 8. Cell viability upon treatment with PAsoDN
Anticancer effect of ASOs were visualized by LIVE/DEAD Viability
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
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
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.
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.
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.
[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.
[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
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.
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
