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Intracellular Delivery of Plasmid DNA and Antiviral Peptides Using Nanoparticles and Cell-Penetrating Peptides : 나노파티클과 세포 투과성 펩타이드를 이용한 플라스미드 유전자와 항바이러스성 펩타이드의 세포내 전달

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dc.contributor.advisor이 연-
dc.contributor.author장상목-
dc.date.accessioned2017-07-14T05:53:24Z-
dc.date.available2017-07-14T05:53:24Z-
dc.date.issued2014-08-
dc.identifier.other000000020946-
dc.identifier.urihttps://hdl.handle.net/10371/125258-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2014. 8. 이연.-
dc.description.abstract세포막을 통과하여 핵산, 펩타이드, 단백질들과 같은 생체고분자들을 전달하는 어려움은 새로운, 유력한 전달 전략들의 개발을 위한 필요성을 정당화한다. 특히, 지질과 고분자를 이용하여 세포안으로 핵산을 전달하는 현재의 비바이러스성 전략들은 제약 산업의 성공에 중대한 장벽을 나타낸다. 그러므로, 안전하고 효과적인 핵산 전달체의 개발이 빠르게 진행중이다. 칼슘인 기반 시스템이 고안할 수 있는 수많은 전달 전략들 중 한 신뢰할 수 있는 전달 시스템으로 간주한다. 그러나, 칼슘인 침전체들의 생리적 불안정성과 계속적인 성장이 생체시스템 내에서 효율적인 핵산 전달체로써의 적용을 제한한다. 이 장벽들을 극복하기 위하여, 나는 인산 기반 블록 공중합체 (PEG-b-PMOEP)를 합성하였고, 효율적이고 안전한 한 세포내의 유전자 전달체로써 블록 공중합체로 코팅된 칼슘인 나노파티클들을 구성하였다.PEG 블록이 칼슘인 침전체의 조절할 수 없는 성장을 막기 위하여 한 껍질을 형성하는 동안, PMOEP 블록은 칼슘인 속으로 삽입되어질 수 있다. 칼슘인 나노파티클들은 생리적 pH 7.4에서 봉입된 pDNA를 방출하지 않았으나, 엔도좀 pH 5.0에서는 인산 형태의 pH 의존적 양성자화를 통하여 pDNA를 방출하였다. PEG-b-PMOEP/CaP/pDNA 나노파티클들은 미래의 유전자 치료제 적용을 위한 유전자 전달체로서 큰 잠재성을 나타낸다.
한 비바이러스성 전달체로써 세포투과성 펩타이드 (CPPs)의 사용은 CPPs가 높은 세포 투과력을 성취하기 위해 필수적인 마이크로몰내의 CPPs의 농도를 필요로 한다. 그러나, 현재의 기술들은 단 시간내에 펩타이드의 불완전한 투과와 세포 내에서 마이크로몰 농도의 펩타이드의 장시간 노출 때문에 세포막 손상과 같은 다양한 부작용과 연관되어 있다. 그러므로, 나는 나노몰 농도에서 CPPs의 적용을 위해 류신과 라이신으로 이루어진 단량체 LK-1과 LK-2 뿐만 아니라 펩타이드 스테이플링 기술을 사용하여 LK-3와 LK-4로 이름지어진 이량체 알파나선형 세포투과 펩타이드를 구성하였다. 환원성의 이황화 결합 혹은 비환원성 결합을 사용한 이량화 전략은 수용액에서도 높은 알파나선형 성질을 초래하였다. 특히, 펩타이드의 알파나선형과 세포투과성 사이의 강한 관련성을 본 연구에서 관찰하였다. 소위, 류신과 라이신으로 이루어진 LK 펩타이드들은 이전 논문에서 HIV-1의 TAR RNA에 대하여 강한 결합 친화성을 보였다. HIV-1의 TAR RNA는 바이러스성 Tat과 결합함에 의해 전사 활성화에 참여한다. LK 이량체들은 포유 세포들에서 효율적인 세포 투과와 Tat-TAR 상호작용의 저해를 위해 낮은 나노몰 수준에서 독성을 가지지 않은 채 세포막을 투과할 수 있다. T-림프아구성 세포들에서 HIV-1 복제의 효과적인 저해는 LK 이량체들, 특히 LK-3가 항바이러스성 펩타이드로서의 강한 잠재성을 지님을 제안한다.		-
dc.description.abstractThe difficulty in delivering biomacromolecules such as nucleic acids, peptides, and proteins into cells through the cellular membranes warrants the need for the development of novel, potent delivery strategies. In particular, current non-viral strategies for delivering nucleic acids into cells using lipids and polymers present a significant hurdle to the success in pharmaceutical industry. Therefore, the development of safe and efficient nucleic acid delivery carriers has been proceeding rapidly. The CaP-based system is regarded as a reliable delivery system among the numerous delivery strategies being devised. However, the physiological instability and uncontrolled growth of CaP precipitates limit their application as efficient nucleic acid delivery carriers in biosystems. To overcome these barriers, I have synthesized a phosphate-based block copolymer, PEG-b-PMOEP (poly(ethylene glycol)-b-poly(2-methacryloyloxyethyl phosphate)), and have constructed calcium phosphate nanoparticles (CaPNs) coated with the block copolymer as an efficient and safe intracellular gene delivery carrier. While PEG block forms a shell to prevent uncontrolled growth of CaP precipitates, PMOEP block could be inserted into the calcium phosphate (CaP) core to entrap pDNA. The CaPNs showed no release of entrapped pDNA at physiological pH 7.4, but released pDNA at an endosomal pH 5.0 through a pH-dependent protonation of phosphate moieties. The PEG-b-PMOEP/CaP/pDNA nanoparticles exhibited great potential as gene delivery vehicles for future gene therapy applications.
The use of cell-penetrating peptides (CPPs) as a non-viral delivery vehicle requires the concentration of CPPs in the micromolar range, which is essential for CPPs to achieve a high cell-penetration ability. However, current technologies are associated with various disadvantages such as incomplete penetration of the peptides within a short time period and cell membrane damage due to the long-term exposure to the micromolar peptides. Therefore, I constructed monomeric LK-1 and LK-2 peptides, as well as dimeric alpha-helical cell-penetrating peptides (DHCPPs), named as LK-3 and LK-4, consisting of leucines (L) and lysines (K), by using a peptide stapling technique to enable the use of CPPs in the nanomolar range. The dimerization strategy based on a reducible disulfide bond or a non-reducible bond resulted in the high alpha-helical propensities of the peptides even in aqueous media. In particular, a strong correlation between alpha-helicity and cell-penetrating ability of the peptides was identified in the study. The LK peptides exhibited a strong binding affinity for the transactivation response element (TAR) RNA of human immunodeficiency virus-1 (HIV-1), as reported in the previous study. The TAR RNA of HIV-1 participates in the transcriptional elongation by interacting with the viral Tat protein. The LK dimers are able to efficiently penetrate the cell membrane for inhibiting the Tat-TAR interaction in mammalian cells without showing toxicity in the low concentration (i.e., nanomolar) range. The effective inhibition of HIV-1 replication in T-lymphoblastoid cells strongly suggests that the LK dimers have exhibited great potentials for use as antiviral peptides.		-
dc.description.tableofcontentsContents

Contents ....................................................................................................... i
List of figures .............................................................................................. v
List of tables ................................................................................................ x

Part I. Delivery of plasmid DNA using hybrid
nanoparticles consisting of calcium phosphate and block
copolymer with phosphomonoester moieties

1. Abstract ................................................................................................... 1
2. Introduction ............................................................................................ 2
3. Materials and Methods ........................................................................... 5
3.1. Materials ........................................................................................... 5
3.2. Synthesis of PEG macroinitiator ...................................................... 6
3.3. Synthesis of MPDME
(2-methacryloyloxyethyl phosphoryldimethylester) monomer .............. 6
3.4. Synthesis of PEG-b-PMPDME
(PEG-block-poly(2-methacryloyloxyethyl phosphoryldimethylester))... 6
3.5. Synthesis of PEG-b-PMOEP
(PEG-block-poly(2-methacryloyloxyethyl phosphate)) .......................... 7
3.6. PEG-b-PMOEP/CaP/pDNA nanoparticle preparation ................... 8
3.7. Measurement of pCN-Luci DNA (pDNA) entrapment efficiency ... 8
3.8. pH-sensitive release of pDNA ........................................................... 9
3.9. Cytotoxicity assay ............................................................................. 9
3.10. Statistical analysis ......................................................................... 10
3.11. Transfection and luciferase assay ................................................. 10
4. Results and Discussion .......................................................................... 12
4.1. Synthesis of PEG-b-PMOEP .......................................................... 12
4.2. Preparation of PEG-b-PMOEP/CaP/pDNA nanoparticles ........... 13
4.3. Measurement of pDNA entrapment efficiency and zeta potential
from PEG-b-PMOEP/CaP/pDNA nanoparticles .................................. 14
4.4. pH-sensitive release of pDNA from PEG-b-PMOEP/CaP/pDNA
nanoparticles ......................................................................................... 15
4.5. Cytotoxicity assay of HeLa cells by PEG-b-PMOEP and PEG-b-
PMOEP/CaP/pDNA nanoparticles ....................................................... 16
4.6. Confocal Laser Scanning Microscopy (CLSM) observation of
HeLa cells by cy5-labeled pDNA .......................................................... 16
4.7. Transfection of HeLa cells and luciferase assay by PEG-b-
PMOEP/CaP/pDNA nanoparticles ....................................................... 17
5. Conclusions ........................................................................................... 19
6. References ............................................................................................. 20

Part II. Delivery of antiviral peptide drugs using dimeric
alpha-helical cell-penetrating peptides with cell penetration
activity

1. Abstract ................................................................................................. 43
2. Introduction .......................................................................................... 45
3. Materials and Methods ......................................................................... 48
3.1. Materials ......................................................................................... 48
3.2. Syntheses of peptides ...................................................................... 49
3.3. Fluorescence Anisotropy (FA) analysis .......................................... 54
3.4. Circular Dichroism (CD) analysis ........................................................55
3.5. Statistical analysis ..................................................................................55
3.6. Fluorescence Activated Cell Sorting (FACS) analysis ......................56
3.7. Confocal Laser Scanning Microscopy (CLSM) observation ......... 56
3.8. mRNA inhibition assay using quantitative RT-PCR ...................... 57
3.9. Transfection and luciferase inhibition assay .................................. 58
3.10. Cytotoxicity assays ........................................................................ 59
3.11. HIV-1 viral antigen assay.............................................................. 60
4. Results and Discussion .......................................................................... 61
4.1. Binding affinities and alpha-helicities of peptides ................................ 61
4.2. Comparison between cell penetration activity and membrane
destabilization activity by FITC-labeled peptides at a high concentration
in HeLa cells ................................................................... 62
4.3. Cell penetration activity of FITC-labeled peptides in HeLa and RAW
264.7 cells ............................................................................................... 63
4.4. Cell penetration mechanism of FITC-labeled LK peptides in HeLa
cells......................................................................................................... 64
4.5. mRNA inhibition assay by LK peptides in HeLa cells ................... 65
4.6. Luciferase inhibition assay by LK peptides after transfection of
HeLa and RAW 264.7 cells by pLTR and pTat ..................................... 67
4.7. Cytotoxicity assays of HeLa and RAW 264.7 cells by LK peptides 67
4.8. Anti-HIV-1 activity by LK peptides after HIV-1 infection in Tlymphoblastoid
cells .............................................................................. 68
5. Conclusion ............................................................................................. 69
6. References ............................................................................................. 70

List of figures

Part I. Delivery of plasmid DNA using hybrid
nanoparticles consisting of calcium phosphate and block
copolymer with phosphomonoester moieties

Figure 1. Synthetic scheme of PEG-b-PMOEP (6) by ATRP........................ 25
Figure 2. Maldi-TOF mass spectra of PEG (1) and PEG macroinitiator (3). 26
Figure 3. 1H NMR spectra of MOEP monomer (4) in CDCl3....................... 27
Figure 4. 1H NMR spectra of PEG-b-PMPDME (5) in CD3OD. .................. 28
Figure 5. 1H NMR spectra of PEG-b-PMOEP (6) in CD3OD ...................... 29
Figure 6. Polymer concentration dependence of the size of the PEG-b-PMOEP
coated CaP nanoparticles. ............................................................................ 30
Figure 7. Polymer concentration dependence of the PDI of the PEG-b-PMOEP
coated CaP nanoparticles. ............................................................................ 31
Figure 8. The sized distribution of the CaP nanoparticles (PEG-b-PMOEP
concentration = 500 μg/mL) ........................................................................ 32
Figure 9. Entrapment efficiency of pDNA loaded PEG-b-PMOEP/CaP
nanoparticles (PEG-b-PMOEP concentration of CaPN 1, CaPN 2, CaPN 3 = 0,
440, 500 μg/mL, respectively) ..................................................................... 33
Figure 10. pH-sensitive release of pDNA from PEG-b-PMOEP/CaP
nanoparticles according to the incubation time on pH 7.4 and pH 5.0,
respectively. ................................................................................................ 34
Figure 11. Relative amount of pDNA from PEG-b-PMOEP/CaP nanoparticles
according to the incubation time on (a) pH 7.4 and (b) pH 5.0, respectively . 35
Figure 12. Cytotoxicity of PEG-b-PMOEP (○) and ExGen500 (●) at various
polymer concentration in HeLa cells. .......................................................... 36
Figure 13. Cytotoxicity of CaPN 2 (○) and ExGen 500/DNA polyplex (N/P=6)
(●) at various pDNA concentration in HeLa cells. ....................................... 37
Figure 14. CLSM images of Hela cells transfected with CaP normal (a, b, c,
and d). ........................................................................................................ 38
Figure 15. CLSM images of Hela cells transfected with CaPN2 (a, b, c, and d).
................................................................................................................... 39
Figure 16. Transfection efficiency of PEG-b-PMOEP/CaP nanoparticles in
HeLa cells at a pDNA concentration of 1.39 μg/mL..................................... 40
Figure 17. Transfection efficiency of PEG-b-PMOEP/CaP nanoparticles
(CaPN 2) (○) and ExGen 500 (N/P=6) (●) at various pDNA concentration in
HeLa cells. ................................................................... 41

Part II. Delivery of antiviral peptide drugs using dimeric
alpha-helical cell-penetrating peptides with cell penetration
activity

Figure 1. HPLC chromatogram of (a) LK-1 and (b) LK-2 peptides ............. 75
Figure 2. Maldi-TOF mass spectra of LK-1 peptide .................................... 76
Figure 3. Maldi-TOF mass spectra of LK-2 peptide .................................... 77
Figure 4. HPLC chromatogram of R9 peptide ............................................. 78
Figure 5. Maldi-TOF mass spectra of R9 peptide ........................................ 79
Figure 6. HPLC chromatogram of (a) LK-3 and (b) LK-4 peptides ............. 80
Figure 7. Maldi-TOF mass spectra of LK-3 peptide .................................... 81
Figure 8. Maldi-TOF mass spectra of LK-4 peptide .................................... 82
Figure 9. FA experiments for the measurement of binding affinity of LK-3 and
LK-4 against TAR RNA .............................................................................. 83
Figure 10. CD spectra of peptides in PBS and 50% TFE in PBS. ............................ 84
Figure 11. CD spectra of dimeric peptides in presence of glutathione .......... 85
Figure 12. Comparison between (a) cell penetration activitity and (b)
membrane destabilization activity by peptides. ............................................ 86
Figure 13. FACS results for LK-1 (▲), LK-2 (■), LK-3 (●), LK-4 (♦), and R9
(▼) at various peptide concentrations in HeLa cells. ................................... 87
Figure 14. FACS results for LK-1 (▲), LK-2 (■), LK-3 (●), LK-4 (♦), and R9
(▼) at various peptide concentrations in RAW 264.7 cells........................... 88
Figure 15. CLSM images of HeLa cells after 12 h treatment of FITC-labeled
(a) LK-1, (b) LK-2, (c) LK-3, (d) LK-4, and (e) R9 peptide at 10 nM. ......... 89
Figure 16. CLSM images of HeLa cells after 12 h treatment of FITC-labeled
(a) LK-1, (b) LK-2, (c) LK-3, (d) LK-4, and (e) R9 peptide at 100 nM. ....... 90
Figure 17. The relative cell penetration activity of LK peptides at endocytosis inhibiting
conditions at (a) 10 nM and (b) 500 nM in HeLa cells after 12 h
incubation. .................................................................................................. 91
Figure 18. Inhibition of the Tat-mediated transcriptional elongation by LK
peptides in HeLa cells. Relative mRNA expression of (a) TAR-luc/beta-actin, (b)
TAR-luc/18S rRNA at 10 nM and 100 nM of each LK peptide. ................... 92
Figure 19. Inhibition of the Tat-mediated transcriptional elongation by LK
peptides in HeLa cells. Relative mRNA expression of TAR-luc/TAR at 10 nM
and 100 nM of each LK peptide. ................................................................. 93
Figure 20. Inhibition of luciferase expression by (a) LK-1 and (b) LK-2
peptides in HeLa cells. ................................................................................ 94
Figure 21. Inhibition of luciferase expression by (a) LK-3 and (b) LK-4
peptides in HeLa cells. ................................................................................ 95
Figure 22. Inhibition of luciferase expression by (a) LK-1 and (b) LK-2
peptides in RAW 264.7 cells. ...................................................................... 96
Figure 23. Inhibition of luciferase expression by (a) LK-3 and (b) LK-4
peptides in RAW 264.7 cells. ...................................................................... 97
Figure 24. Inhibition of luciferase expression by LK-2 and LK-3 peptides
according to pTat dose in HeLa cells. .......................................................... 98
Figure 25. Relative membrane destabilization of LK-1 (▲), LK-2 (■), LK-3 (●),
LK-4 (♦) in (a) HeLa cells and (b) RAW 264.7 cells. ............................ 99
Figure 26. Relative viability of (a) HeLa cells and (b) RAW 264.7 cells after
treatment with LK-1 (▲), LK-2 (■), LK-3 (●), and LK-4 (♦). ................... 100
Figure 27. Inhibition of HIV-1 replication by (a) LK-3 and (b) LK-4 peptides
in T-lymphoblastoid cells (MOLT-4/CCR5). .............................................. 101

List of TABLES

Part I. Delivery of plasmid DNA using hybrid nanoparticles
consisting of calcium phosphate and block copolymer with
phosphomonoester moieties

Table 1. Measurement of molecular weights of polymers ............................ 42

Part II. Delivery of antiviral peptide drugs using dimeric alpha-helical
cell penetrating peptides with cell penetration activity as well as
antiviral activity

Table 1. Binding affinities on TAR RNA and alpha-helicities of LK peptides .. 102
Table 2. Change of alpha-helicity of LK dimers by glutathione ....................... 103

List of Publications ............................................................................... 104
Abstract in Korean (국문 초록) ................................................... 107		-
dc.formatapplication/pdf-
dc.format.extent3492663 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoko-
dc.publisher서울대학교 대학원-
dc.subjectGene delivery-
dc.subjectblock copolymer-
dc.subjectCell-Penenetrating Peptides (CPPs)-
dc.subjectpeptide drugs-
dc.subject유전자 전달-
dc.subject블록 공중합체-
dc.subjectpH-민감성-
dc.subject세포 투과성 펩타이드 (CPPs)-
dc.subjectHIV-1-
dc.subject펩타이드 약물-
dc.subjectTat-TAR 상호작용-
dc.subject전사적 저해-
dc.subject.ddc540-
dc.titleIntracellular Delivery of Plasmid DNA and Antiviral Peptides Using Nanoparticles and Cell-Penetrating Peptides-
dc.title.alternative나노파티클과 세포 투과성 펩타이드를 이용한 플라스미드 유전자와 항바이러스성 펩타이드의 세포내 전달-
dc.typeThesis-
dc.contributor.AlternativeAuthorSangmok Jang-
dc.description.degreeDoctor-
dc.citation.pagesx, 124-
dc.contributor.affiliation자연과학대학 화학부-
dc.date.awarded2014-08-
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