Nucleus-targeted Gene and Anticancer Drug Delivery Using α-Synuclein-Gold Nanoparticle Conjugates = 알파-시뉴클레인-금나노입자 콘쥬게이트를 이용한 세포핵 대상 유전자 및 항암약물 전달

국문 초록 (Abstract)

유전자 전달 기술은 유전자 치료 및 유전자 편집부터 암 치료와 줄기세포 연구에 이르기까지 다양한 응용 분야를 포괄하는 생명과학의 핵심 기술로 활용되고 있으며, 동시에 핵 표적 약물 전달은 핵심적인 세포 과정의 교란을 통해 항암 효능을 증대시키는 유망한 전략으로 주목받고 있다. 본 연구에서는 세포 분열에 의존하지 않는 유전자 형질 도입 및 핵 표적화 약물 전달을 동시에 수행할 수 있는 알파-시뉴클레인-금나노입자(αS-AuNP) 기반 다기능 전달 시스템을 개발하였다.
알파-시뉴클레인(αS)은 비정형 단백질(intrinsically disordered protein, IDP)로서 지질막과 상호작용 시 무작위 코일 구조에서 알파-나선 구조로의 전이가 일어나는 특성을 갖는다. 이를 기반으로, αS의 C-말단 타이로신(Y136)을 시스테인(C)으로 치환한 αS(Y136C) 변이체를 활용하여, 특정한 방향으로 금나노입자(AuNP) 표면에 공유 결합시킨 αS(Y136C)-AuNPs를 제작하였다. 이 방향성은 나선 형성에 기여하는 염기성 N-말단을 노출시켜 세포 내 유입을 촉진하였다. 본 복합체는 αS(S9C)-AuNP와 달리, 세포 독성 없이 유전자를 세포 내로 전달할 뿐만 아니라, 신속한 유전자 발현을 유도함을 확인하였다. 또한, 엔도좀 의존 및 비엔도좀 경로를 통한 세포 내 유입이 모두 가능하며, 핵으로의 DNA 전달 과정이 세포 분열과 무관하게 진행됨을 입증하였다. 본 시스템은 그랜자임 A 유전자의 전달에 효과적으로 활용되었으며, 파이롭토시스(pyroptosis) 세포 사멸을 성공적으로 유도하였다.
나아가, αS-AuNP 기반 전달 시스템을 항암 치료로 확장하기 위해 독소루비신을 열 민감성 가교제를 이용하여 αS(Y136C)-AuNP에 결합하였으며, 이를 근적외선(NIR) 조사에 의해 제어된 약물 방출 시스템으로 개발하였다. 금나노입자의 광열 효과 의해 국소적인 고온 상태가 발생하면서 가교제의 아조 결합(azo bond)이 절단되었다. 이 과정은 독소루비신의 핵 내 방출을 효과적으로 촉진하여 항암 효능을 극대화함을 확인하였다. 이러한 핵 표적 전략은 기존 항암제의 세포 내 유입률 저하 및 핵 내 축적의 어려움과 같은 암 치료의 주요 한계를 극복할 수 있는 강력한 전략을 제시한다.
본 연구에서 개발한 αS(Y136C)-AuNP 기반 전달 시스템은 유전자 형질 도입과 핵 표적화 약물 전달을 동시에 수행할 수 있는 다기능 플랫폼으로, 정밀 의학, 유전자 치료 및 표적 항암 치료 등의 다양한 생명공학 및 의생명과학 분야에서 활용될 수 있을 것으로 기대된다. 추가적으로 항체 고정화, RNA·단백질·융합 생체재료의 DNA 및 약물 대체 적용 등을 통해 본 전달 시스템의 적용 범위를 더욱 확장할 수 있을 것이다.

다국어 초록 (Multilingual Abstract)

Gene delivery is a fundamental technology in biomedical science, spanning applications from gene therapy and gene editing to cancer treatment and stem cell research. Simultaneously, nucleus-targeted drug delivery has emerged as a powerful strategy for enhancing anticancer efficacy by disrupting key nuclear processes. In this study, we introduce a multifunctional delivery system based on α-synuclein-gold nanoparticle conjugates (αS-AuNPs), designed for both cell division-independent gene transfection and nucleus-directed drug delivery.
α-Synuclein (αS), an intrinsically disordered protein (IDP) known for its membrane interactions, undergoes a structural transition from a random coil to an α-helix upon binding to lipid membranes. Leveraging this property, we engineered αS(Y136C)-AuNPs, where a cysteine-substituted mutant αS(Y136C) was covalently anchored to the AuNP surface in a specific orientation. This orientation exposed the helix-forming basic N-termini to enhance cellular uptake. Unlike αS(S9C)-AuNPs, the αS(Y136C)-AuNP conjugates facilitated rapid and non-disruptive DNA transfection, enabling cell division-independent gene delivery into nucleus via both endosomal and non-endosomal pathways. The system was effectively utilized for the delivery of the granzyme A gene. This mechanism subsequently induced pyroptotic cell death.
To extend the application of αS-AuNPs to cancer therapy, doxorubicin (Dox) was conjugated to αS(Y136C)-AuNPs via a heat-sensitive crosslinker, enabling controlled drug release upon near-infrared (NIR) irradiation. The photothermal effect of AuNPs induced localized hyperthermia, which cleaved the azo bond in the linker. This process facilitated the nuclear release of Dox, significantly enhancing its anticancer activity. This nucleus-targeted strategy addresses key challenges in cancer therapy, including limited cellular uptake and poor nuclear localization of conventional anticancer agents.
The multifunctionality of αS(Y136C)-AuNPs, enabling both gene transfection and nucleus-directed drug delivery, presents a promising platform for future biomedical applications. Modifications such as antibody immobilization and the replacement of DNA and drug with RNA, proteins, or hybrid biomaterials further expand its potential for precision medicine, gene therapy, and targeted cancer treatment.

목차 (Table of Contents)

• Abstract i
• Contents iv
• Section I. Cell Division-independent Nuclear-targeting DNA Delivery Using α-Synuclein-Gold Nanoparticle Conjugates
• I-1. Introduction
• (1) Gene delivery 1
• (2) α-helix 2
• (3) α-synuclein 3
• I-2. Results and Discussion 5
• (1) Preparation of αS-AuNPs with αS(S9C) or αS(Y136C) and their distinctive cellular translocation properties
• (2) Preparation of αS(Y136C)-AuNP/DNA complex
• (3) αS(Y136C)-AuNP-mediated intracellular delivery of DNA and its rapid expression
• (4) Intracellular translocation of the αS(Y136C)-AuNP/DNA complex and its cell-division independent gene expression
• (5) Delivery of granzyme A gene with αS(Y136C)-AuNPs to develop anti-cancer therapeutics
• I-3. Conclusion 17
• Section II. Intranuclear Delivery of Anticancer Drug Doxorubicin Using α-Synuclein-Gold Nanoparticle Conjugates and a Thermosensitive Cross-linker
• II-1. Introduction
• (1) Nucleus-targeted cancer treatment 52
• (2) Heat-sensitive crosslinker 53
• (3) Photothermal effect 54
• II-2. Results and Discussion 55
• (1) Fabrication of Dox-αS-AuNP conjugte using αS(Y136C) and a heat-labile crosslinker
• (2) Intracellular translocation and nuclear localization of Dox-αS(Y136C)-AuNP
• (3) Anticancer effect of Dox-αS(Y136C)-AuNP triggered by near-infrared (NIR) light irradiation
• II-3. Conclusion 59
• Experimental Section 77
• (1) Expression and Purification of αS
• (2) Preparation of Plasmid DNA
• (3) Preparation of αS-AuNP/DNA Complex
• (4) Cell Culture
• (5) Confocal Laser Scanning Microscopy (CLSM)
• (6) Liposome Preparation
• (7) Fourier Transform Infrared Spectroscopy (FT-IR)
• (8) Gel Retardation Assay
• (9) Transmission Electron Microscopy (TEM)
• (10) Dynamic Light Scattering (DLS)
• (11) DNA Intercalation Assay
• (12) Luciferase Assay
• (13) Cell Counting Kit-8 (CCK-8) Assay
• (14) Trypan Blue Assay
• (15) Intracellular Translocation of αS-AuNP/DNA Complex
• (16) Field-Emission Scanning Electron Microscopy (FESEM)
• (17) Western Blot
• (18) Preparation of Doxorubicin-αS-AuNP Conjugate (Dox-αS-AuNP)
• (19) Photothermal Effect Experiment
• (20) Dox Release Assay
• References 90
• List of Figures
• Scheme 1. Schematic illustration 18
• Figure 1. Preparation of αS-AuNP conjugates 19
• Figure 2. Orientation-dependency of αS bound onto AuNPs for the intracellular translocation of αS-AuNP conjugates 20
• Figure 3. The property of αS-AuNP conjugates about nuclear localization 21
• Figure 4. The property of αS-AuNP conjugates about liposome binding 22
• Figure 5. FT-IR spectra of αS(Y136C)-AuNPs and αS(S9C)-AuNPs incubated with or without liposomes are monitored for the amide I band region (1600-1700 cm-1) 23
• Figure 6. Orientation-dependency of αS bound onto AuNPs for the complex formation with DNA 24
• Figure 7. Formation of αS(Y136C)-AuNP/DNA complex under various conditions 25
• Figure 8. Possible pH effect on the charged state of αS(Y136C)-AuNP/DNA complex 26
• Figure 9. pH effect on the charged state of αS(Y136C)-AuNP/DNA complex 27
• Figure 10. Breakdown of αS(Y136C)-AuNP/DNA complex under various conditions 28
• Figure 11. Separation of αS(Y136C)-AuNP/DNA complexes from dissociated DNA above pH 4.5 29
• Figure 12. FT-IR spectra of αS(Y136C)-AuNPs and αS(S9C)-AuNPs incubated with or without liposomes in the presence of DNA are monitored for the amide I band region (1600-1700 cm-1) 30
• Figure 13. Intracellular protein expression of HeLa cells transfected with αS(Y136C)-AuNP/DNA complex after 3 hrs of incubation 31
• Figure 14. Intracellular EGFP fluorescence intensity of HeLa cells transfected with αS(Y136C)-AuNP/DNA complex after 3 hrs of incubation 32
• Figure 15. Cell viability of HeLa cells transfected with αS(Y136C)-AuNP/DNA complex after 3 hrs of incubation 33
• Figure 16. Intracellular protein expression of HeLa cells transfected with DNA of either EGFP or firefly luciferase in absence and presence of lipofectamine or αS(Y136C)-AuNPs and incubated for 24 hrs 34
• Figure 17. Cell viability of HeLa cells transfected with DNA in absence and presence of lipofectamine or αS(Y136C)-AuNPs and incubated for 24 hrs 35
• Figure 18. Intracellular EGFP expression in HeLa cells transfected with αS(Y136C)-AuNP/DNA complex in the presence of chlorpromazine 36
• Figure 19. Intracellular EGFP expression in HeLa cells transfected with αS(Y136C)-AuNP/DNA complex under the conditions suppressing several discrete endocytic processes 37
• Figure 20. Intracellular pathway of αS(Y136C)-AuNP/DNA complex in HeLa cells 38
• Figure 21. Proportion of internalized αS(Y136C)-AuNP conjugates located in the nucleus and cytosol during 0.5 to 1.5 hrs of incubation following the transfection 39
• Figure 22. Intracellular EGFP expression in HeLa cells transfected with αS(Y136C)-AuNP/DNA complex in the presence of paclitaxel 40
• Figure 23. Cell-division independent DNA delivery of αS(Y136C)-AuNPs in non-dividing CCD-986sk cells 41
• Figure 24. Cell-division dependent DNA delivery of lipofectamine in non-dividing CCD-986sk cells 42
• Figure 25. Colocalization coefficients between αS(Y136C)-AuNPs and DNA and between the nucleus αS(Y136C)-AuNPs 43
• Figure 26. Nuclear localization property of N-terminus-truncated αS-AuNP conjugates 44
• Figure 27. Cellular excretion of αS(Y136C)-AuNPs 48 hrs after treatment 45
• Figure 28. Transfection of granzyme A gene with αS(Y136C)-AuNPs 46
• Figure 29. Transfection of granzyme A gene with αS(Y136C)-AuNPs and lipofectamine 47
• Figure 30. Morphological change of HeLa cells after transfection of granzyme A gene with αS(Y136C)-AuNPs 48
• Figure 31. Western blot analysis showing the expression of gasdermin B in the HeLa cells treated with αS(Y136C)-AuNP/granzyme A DNA complex or lipofectamine-DNA complex for 6 hrs 49
• Scheme 2. Schematic illustration 61
• Figure 32. Preparation of Dox-αS(Y136C)-AuNP conjugate 62
• Figure 33. DLS histogram of AuNPs, αS(Y136C)-AuNPs, and Dox-αS(Y136C)-AuNP 63
• Figure 34. Localized surface plasmon resonance (LSPR) spectra of AuNPs and Dox-αS(Y136C)-AuNP 64
• Figure 35. Crosslinking efficiency of Dox to αS(Y136C)-AuNPs was evaluated based on the concentration of free Dox remaining in the supernatant following the separation of Dox-αS(Y136C)-AuNP as the precipitates 65
• Figure 36. Orientation dependence of αS anchored onto AuNPs for the cell membrane translocation and nuclear localization 66
• Figure 37. FT-IR spectra of Dox-αS(Y136C)-AuNP before (left) and after (right) the interaction with phosphatidylcholine (PC) liposomes 67
• Figure 38. FT-IR spectra of Dox-αS(S9C)-AuNP before (left) and after (right) the interaction with phosphatidylcholine (PC) liposomes 68
• Figure 39. Temperature-dependent Dox release profile from Dox-αS(Y136C)-AuNP 69
• Figure 40. The photothermal effect of Dox-αS(Y136C)-AuNP during 5 min of NIR irradiation at 808 nm 70
• Figure 41. NIR thermographic images of the solutions containing Dox-αS(Y136C)-AuNP during 5 min of NIR irradiation at 808 nm 71
• Figure 42. Release of Dox from Dox-αS(Y136C)-AuNP upon NIR irradiation and its anti-cancer effect 72
• Figure 43. Cell viability of HeLa cells treated with αS(Y136C)-AuNPs in the presence and absence of Dox attached with and without NIR irradiation 73
• Figure 44. CLSM images of HeLa cells treated with free Dox and Dox-αS(Y136C)-AuNP in the presence and absence of NIR irradiation 74
• Figure 45. Colocalization coefficients between Dox and αS(Y136C)-AuNP and between the nucleus and αS(Y136C)-AuNP based on the CLSM image of HeLa cells treated with Dox-αS(Y136C)-AuNP in the absence of NIR irradiation 75
• List of Tables
• Table 1. Protein secondary structure of αS(Y136C) and αS(S9C) bound to AuNP in the absence or presence of liposomes 50
• Table 2. Protein secondary structure analysis of αS(Y136C) and αS(S9C) bound to AuNP with or without liposomes in the presence of DNA 51
• Table 3. Protein secondary structure analysis of αS(Y136C) and αS(S9C) in the Dox-αS-AuNP complex in the absence or presence of PC liposomes 76
• Abstract in Korean 88