항유전자성 단일클론항체 3D8 scFv 를 담지한 PLGA 나노입자에 의한 지속적인 항바이러스성 효과에 관한 연구
Sustained anti-virus effect of anti-DNA monoclonal antibody 3D8 scFv loaded PLGA nanoparticle
- 발행기관 아주대학교 대학원
- 지도교수 박기동
- 발행년도 2008
- 학위수여년월 2008. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Recently, therapeutic and diagnostic antibodies are an emerging drug for biopharmaceutical applications. 3D8 scFv, monoclonal antibody (mAb), is anti-DNA abs that has shown DNA hydrolyzing catalytic activities. Therefore, 3D8 scFv has potential to be used as biopharmaceutical drugs for antivirus therapy. However, the therapeutic efficacies of 3D8 scFv which require prolonged drug action largely depend on dose and duration of their availability and maintenance of stability at the intracellular region. Biodegradable Poly (glycolic-co-lactic acid) (PLGA) nanoparticles (NPs) have been widely studied as protein carriers. The advantage of this system for antiviral effect is sustained cytoplasmic release that could potentiate the therapeutic efficacy of the 3D8 scFv whose the site of action is cytoplasm. Nevertheless, protein stability still remains one of the most important hurdles for their successful incorporation in various carriers, such as biodegradable drug delivery system. In this study, various modifications were tried to improve the conformational and biological stability of a therapeutic agent of antivirus therapy, 3D8 scFv, with several stabilizers during the primary emulsification process of PLGA NP preparation. For that purpose, five stabilizers [i.e. heparin, PEG, mannitol, trehalose and HP- -CD] were incorporated together with 3D8 scFv into water phase. CD and fluorescence spectroscopy profiles of intact and 3D8 scFv incorporated stabilizers revealed that primary emulsification process was not a major cause of destabilizing structural stability even in the absence of the stabilizers. In contrast, all stabilizers except mannitol failed to maintain biological activity of 3D8 scFv when enzyme-linked immunosorbent assay (ELISA) and agarose gel electrophoresis were performed to evaluate DNA binding and hydrolyzing ability. And we showed the potential for the therapeutic use of 3D8 scFv through the systemic evaluations such as intracellular 3D8 scFv level, cytotoxicity, anti-virus effect of cytosolic released 3D8 scFv. The aims of this study are to develop the suitable formulation technique for 3D8 scFv - a new therapeutic agent of antiviral therapy - loaded PLGA NP and to investigate the sustained therapeutic efficacy (antiviral activity) of the 3D8 scFv encapsulated in PLGA NPs and its intracellular localization.
more초록/요약
진단 및 치료를 목적으로 하는 항체는 최근 들어 생물학적 제재의 응용분야에서 그 중요성이 증대되고 있다. 단일클론항체중의 하나인 3D8 scFv 는 DNA 를 가수분해하는 활성을 가진 항유전자성 (anti-DNA) 항체의 일종이며, 이러한 성질을 이용하여 항 바이러스성 치료에 응용 될 수 있다. 그러나 3D8 scFv 를 이러한 치료의 목적으로 사용하기 위해서는 그것의 작용부위인 세포질 내에서의 장기간에 걸친 활성을 가질 수 있어야 하는데, 이를 위해 세포 내부로 전달되는 적정량의 3D8 scFv 양이 세밀히 조절되어야 하고, 장기간 동안 안정성 유지가 보장 되어야 한다. 단백질 약물 전달을 위한 생분해성 PLGA 나노입자는 매우 광범위하고 활발하게 연구 되고 있는 분야이다. 이러한 PLGA 나노입자를 이용한 항바이러스성 치료의 가장 큰 강점은 3D8 scFv 의 작용부위인 세포질 내부에서 약물의 서방형 방출이 가능하며 이를 통한 약효의 지속화를 보장할 수 있다는 것이다. 하지만, 이러한 큰 강점에도 불구하고 약물을 담지 하는 과정상에 유발되는 단백질 약물의 불안정성과 비효율적인 세포내부로의 전달 효율은 PLGA 나노입자의 이용에 많은 제약이 되어 왔다. 이에, 본 연구에서는 여러 가지 안정제를 항체가 함유된 수용액상에 첨가 함으로써 PLGA 나노입자의 제조 과정상에서 유발되는 3D8 scFv 의 구조적, 생물학적 불안정성을 극복하기 위한 시도를 하였고, CD 와 fluorescence spectroscopy 를 통해 구조적 안정성 여부를, ELISA 와 전기영동을 실시 함으로써 약물의 생물학적 활성의 유지여부를 체계적으로 분석 함으로써 3D8 scFv 의 활성 유지에 가장 최적화된 조건을 확립하였다. 이렇게 제조된 나노입자를 이용한 생체 외 실험인 3D8 scFv 의 세포 내 반감기와 방출거동, 세포 독성 평가 등을 실시하여 여러 실험 조건들을 확립함으로써 3D8 scFv 의 항바이러스성 제재로서의 가치를 극대화하여 3D8 scFv 의 약물로써 응용의 가능성을 제시하였다.
more목차
I. INTRODUCTION = 1
A. Theoretical background = 1
1. Drug delivery system (DDS) = 1
1.1. Nanomedicine = 1
1.2. Nanoparticulate systems for drug delivery = 1
2. Anti-DNA monoclonal antibody 3D8 scFv = 3
2.1. Monoclonal antibody = 3
2.2. Anti-DNA monoclonal antibody 3D8 scFv = 4
3. Biodegradable nanoparticles as mab delivery system = 5
3.1. PLGA based drug delivery system = 6
3.2. PLGA based formulations for Protein/peptide delivery = 8
4.Mab instability = 9
4.1. Mab instability in formulation process of PLGA NPs = 9
4.2. Effect of formulation excipients in mab instability = 9
5. Intracellular delivery of therapeutics = 10
5.1. PLGA NP for intracellular uptake = 11
5.2. Cellular internalization of NPs: mechanism and factors = 11
II. EXPERIMENTS = 14
A. Materials = 14
B. Preparation of 3D8 scFv loaded PLGA nanoparticle = 15
C. Characterizations of 3D8 scFv loaded PLGA NP = 17
D. Stabilization of 3D8 scFv in PLGA NPs = 18
1. Recovery of 3D8 scFv after primary emulsification with or without PLGA = 18
2. Circular dichroism (CD) = 18
3. Fluorescence spectroscopy = 18
4. DNA binding assay by enzyme-linked immunosorbent assay (ELISA) = 19
5. DNA hydrolyzing assay by agarose gel electrophoresis = 19
E. Intracellular uptake of 3D8 scFv loaded PLGA NP = 20
1. Cell culture = 20
2. Confocal microscopy = 20
3. Fluorescent activated cell sorter (FACS) = 20
F. Sustained intracellular 3D8 scFv levels with NPs = 21
G. Cytotoxicity test = 21
H. Anti-virus activity test = 21
III. RESULTS AND DISCUSSION = 22
A. Characterizations of 3D8 scFv loaded PLGA NP = 22
1. The effect of formulation parameters on physical properties of PLGA NPs = 22
2. The effect of formulation parameters on the loading efficiency of 3D8 scFv = 28
B. Recovery of 3D8 scFv after primary emulsification with or without PLGA = 30
C. Effects of additives on structural stability of 3D8 scFv = 33
D. Effect of additives on biological activity of 3D8 scFv = 36
E. Intracellular uptake of 3D8 scFv loaded PLGA NP = 41
F. Sustained intracellular 3D8 scFv levels with NPs = 44
G. Cytotoxicity test = 47
H. Anti-virus activity = 49
IV. CONCLUSIONS = 52
REFERENCES = 53
