비닐술폰 기능화 PEG-PLLA 미셀을 이용한 암표적 치료에 관한 연구
Vinyl sulfone-Functionalized PEG-PLLA Micelles for Cancer-Targeted Therapy
- 주제(키워드) Cancer nanotechnology , Micelle , Vinyl sulfone , Cancer targeting , Surface functionalization
- 발행기관 아주대학교
- 지도교수 박기동
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000009582
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
지난 수십 년 동안 cancer nanotechnology의 많은 진보와 더불어 기존의 암치료법을 대체하기 위한 다양한 치료 기법이 개발되고 있다. 특히, 나노전달체를 이용한 암치료는 암을 완치하기 위한 매우 혁신적인 진단 및 치료 방법으로서 광범위하게 개발응용되고 있다. 그 중, 소수성 및 친수성 블록을 갖는 양친매성 거대분자의 자가조립으로 형성된 고분자 미셀은 전달체로서의 적절한 크기와 함께 우수한 체내안정성, 항암제의 조절방출능력 등의 장점으로 인해 우수한 나노전달체들 중의 하나로 많은 각광을 받고 있다. 그럼에도 불구하고, 고분자 미셀 시스템은 합성 과정 상의 표면 기능화의 어려움으로 인해 임상적용에 있어 많은 제약이 보고되고 있다. 1장에서는 암의 발생과정과 현재 암표적 치료에 사용되고 있는 두 가지 표적 전략에 대한 간략한 소개와 더불어, 암표적 치료를 위한 고분자 미셀의 현재 연구동향 및 본 연구의 목적을 서술하였다. 2장에서는 암표적 치료를 위한 새로운 기능성 나노전달체로서 비닐술폰 기능화 PEG-PLLA 고분자 미셀 개발에 관한 연구내용을 기술하였다. 현재 단백질 PEGylation을 위한 가장 효과적인 반응으로 알려진 Michael-type addition을 이용하여, 미셀 형성을 위한 양친매성 공중합체는 미셀 표면에 위치하는 친수성 PEG 한쪽 말단에 비닐술폰기를 갖도록 설계하였다. 동시에 생체적합성 및 생분해성을 갖는 폴리락티드를 소수성 블록으로 이용하여 임상적용이 용이하도록 하였다. 비닐술폰 함유 PEG-PLLA 공중합체 합성을 위한 비닐술폰기 함유 PEG는 PEG의 선택적 monotosylation으로부터 여러 단계의 합성과정을 거쳐 제조하였으며, 비닐술폰 함유 PEG-PLLA 공중합체는 L-lactide의 개환중합으로부터 합성하였다. 1H-NMR을 통해 각 단계별 PEG 유도체들 및 공중합체의 구조를 확인하였고, 소수성 PLLA 블록 길이를 조절함에 따라 공중합체의 물에 대한 용해도를 쉽게 변화시킬 수 있었다. 공중합체의 임계미셀농도 및 미셀크기는 각각 59.8 mg/L 및 37 nm으로 측정되었고, 비닐술폰 기능화 고분자 미셀의 반응성을 평가하기 위해 4 종류의 cysteine 함유 펩타이드 (L-cysteine, CREKA, c(RGDfC), TAT peptide)를 사용하였다. 그 결과, 용액 pH, 펩타이드 크기 및 서열에 따라 반응속도에 차이가 있었으며, 대부분의 펩타이드가 수용액 내에서 1시간 이내의 빠른 반응성을 나타내었다. 3장에서는 표적 리간드가 결합된 PEG-PLLA 미셀의 특성을 평가하였다. c(RGDfC) 또는 galactosamine이 결합된 공중합체는 미셀을 형성할 수 있었으며, 그 크기는 대조군 (33.5 nm)에 비해 약간 증가한 크기 (38.6 및 44.8 nm)를 나타내었다. 뿐만 아니라, 표적 리간드가 결합된 미셀의 표면은 약한 음전하를 띄고 있었으며, 이는 암표적화 미셀의 우수한 체내안정성에도 기여할 것으로 판단된다. 4장에서는 항암제인 doxorubicin (DOX)의 표적전달을 위한 c(RGDfC)-PEG-PLLA 미셀의 효과를 평가하였다. 비닐술폰 기능화 PEG-PLLA 미셀의 DOX 함유효율은 6.64  0.32 %였으며, 약물방출거동 평가에서 DOX는 4일 동안 미셀로부터 방출되었으나 약 80 %의 DOX가 하룻동안 방출되었다. HeLa 세포를 이용한 세포독성평가에서 비닐술폰 함유 PEG-PLLA 미셀은 10 mg/ml의 높은 농도에서조차 독성을 나타내지 않았다. DOX-loaded micelles의 경우, 0.1 g/ml 이하에서 세포독성을 보인 free DOX에 비해 뚜렷한 세포독성을 보였으나, c(RGDfC) 효과는 나타나지 않았다. FACS 및 CLSM을 통해 DOX를 함유한 c(RGDfC)-PEG-PLLA 미셀의 세포질내 유입을 확인하였으며, 방출된 DOX의 대부분은 3시간 후 세포핵과 효과적 결합을 하는 것으로 관찰되었다. 따라서, 본 연구에서 개발된 비닐술폰 기능화 PEG-PLLA는 현재 많이 사용되고 있는 다양한 종류의 암표적 리간드를 쉽게 도입할 수 있을 뿐 아니라 항암제 및 진단제를 함유 및 전달할 수 있어 앞으로의 암 치료연구에 매우 유용할 것으로 판단된다.
more초록/요약
With remarkable progress in cancer nanotechnology during the last decade, various nanocarrier systems have been extensively emerged for the purpose of early detection and cure of cancer. Polymeric micelle, composed of amphiphilic macromolecules that have distinct hydrophobic and hydrophilic block domains, is one of promising nanocarriers on account of their small size, controlled delivery of anticancer drugs or imaging agents, and prolonged blood circulation times. However, several kinds of limitations in micellar systems have been addressed and in particular, surface functionalization of the micelles is regarded as a critical issue for cancer targeted therapy. Chapter 1 provides general information for cancer treatment. A clear understanding of carcinogenesis is essential for effectively achieving cancer treatment. Although the targeting strategies for cancer-targeted therapy can be classified into passive and active targeting, current approaches are commonly based on their combinative targeting. Also, this chapter describes overall objectives of this study, with a brief introduction to the current status of cancer-targeted polymeric micelles. In Chapter 2, we report vinyl sulfone-functionalized PEG-PLLA polymeric micelles as a new functional nanocarrier system for cancer-targeted therapy. Based on Michael-type addition as one of the most effective reaction for protein PEGylation, this copolymer was designed to bear a vinyl sulfone (VS) group at one terminus of polyethylene glycol (PEG), which locates at the micellar surface. In addition, we choose a PLLA block as a hydrophobic segment of the copolymer because it was already approved by FDA for clinical use. Prior to the synthesis of VS-terminated PEG-PLLA copolymer, hetero-bifunctional PEG (Mw 3.4K) with a VS group was prepared by a multi–step synthetic procedure started from selective monotosylation of PEG diol. The 1H-NMR spectra of VS-terminated PEG indicated its successful preparation. Then, VS-terminated PEG-PLLA copolymer was prepared by ring opening polymerization of L-lactide with VS-terminated hetero-bifunctional PEG. The 1H-NMR spectra of VS-terminated PEG-PLLA exhibited the characteristic peaks of both PLLA and PEG which represent the degree of end group conversion over 90 %. For micellar properties of VS-terminated PEG-PLLA (3400/3000), the critical micelle concentration and the mean size were determined to be around 59.8 mg/L and 37 nm, respectively. The kinetic studies of VS-functionalized polymeric micelles via Michael-type addition showed that cysteine-contained peptides reacted rapidly within 1 h. After then, the reaction rates decreased constantly due to the difficulties against molecular diffusion and alignment of thiols to the reactive micellar surfaces. In addition, the reaction rates could be significantly affected by the solution pH, peptide sizes and sequences. In Chapter 3, VS-terminated PEG-PLLA copolymer was functionalized via Michael-type addition of two types of targeting ligands which contain thiol or amine. After chemical conjugation of c(RGDfC) and galactosamine, the obtained copolymers could form micelles and the micelles had spherical shapes. The size of c(RGDfC)- or galactosamine-conjugated micelles increased slightly (38.6 nm and 44.8 nm, respectively) but it was insignificant levels compared to the blank micelles (33.5 nm). The ligands-conjugated micelles had negatively-charged surfaces which decreased slightly, compared to the blank micelles. It is expected that the micellar characteristics after the conjugation of cancer targeting ligands are not only favorable enough for long circulation in the body but also effective for enhanced permeation and retention (EPR) effect. Finally, we tested the effect of c(RGDfC)-conjugated PEG-PLLA micelles for targeted delivery of doxorubicin (DOX). The loading efficiency of DOX in VS-functionalized PEG-PLLA micelles was determined to be 6.64  0.32 %. In DOX release test, the encapsulated DOX was continuously released from the micelles until 4 days but the DOX release of about 80 % was shown within 1 day. The cytotoxicity test using HeLa cells explained that free DOX showed considerable killing effect when DOX concentration was below 0.1 g/ml. In addition, cell viability decreased dramatically when two types of micelles containing DOX were treated. However, the result did not show the effect of c(RGDfC) conjugation on the cells. VS-terminated PEG-PLLA micelles had no cytotoxicity against HeLa cells even above 10 mg/ml. Intracellular uptake studies using flow cytometry and confocal laser scanning microscopy demonstrated that c(RGDfC)-conjugated micelles containing DOX could be effectively internalized within the cytoplasm and then the released DOX was accumulated into the cell nuclei. Therefore, VSterminated-PEG-PLLA can be very useful as a functional micellar system for the effective conjugation of thiol- and amine-contained targeting ligands and it can provide a promising alternative for conventional cancer chemotherapy.
more목차
Acknowledgement
Abstract
Table of Contents
List of Figures
List of Tables
Chapter 1. General Introduction
1. Nanotechnology-based cancer diagnosis and therapy
2. Cancer targeting strategies based on carcinogenesis
2.1. Understanding of carcinogenesis
2.2. Passive versus active targeting
2.3. Cancer targeting ligands
2.4. Rationale for targeted cancer therapy
3. Polymeric micelles for cancer-specific drug delivery
3.1. General features of polymeric micelles
3.2. Biological significance of polymeric micelles
3.3. Surface functionalization of polymeric micelles for cancer targeting
4. Design of targeting ligands tethered polymeric micelles
4.1. Aliphatic polyester-based micelles
4.2. Previous studies for functionalized micelles
4.3. Michael-type addition for bioconjugates
4.4. Anticancer drugs in micellar systems
5. Overall Objectives
6. References
Chapter 2. Vinyl sulfone-terminated PEG-PLLA block copolymer for thiol-reactive micelles and the micellar reactivity toward thiols
1. Introduction
2. Materials and Methods
2.1. Synthesis of monotosyl-PEG
2.2. Synthesis of monothiol-PEG and VS conjugation
2.3. Synthesis of VS-terminated PEG-PLLA block copolymer
2.4. Polymer characterizations
2.5. Preparation and characterizations of VS-functionalized PEG-PLLA micelles
2.6. Kinetic studies of VS-functionalized micelles toward thiols
3. Results and Discussion
3.1. Synthesis of VS-terminated PEG and PEG-PLLA copolymer
3.2. Critical micelle concentration and size distribution of micelles
3.3. Kinetic studies of VS-functionalized micelles toward thiols
4. Conclusions
5. References
Chapter 3. Conjugation of cancer targeted ligands to VS-terminated PEG-PLLA and their micellar properties
1. Introduction
2. Materials and Methods
2.1. Preparation of c(RGDfC)-conjugated PEG-PLLA micelles
2.2. Preparation of galactosamine-conjugated PEG-PLLA micelles
2.3. Micellar characterization
3. Results and Discussion
3.1. Characterizations of cRGDfC- and galactosamine-conjugated PEG-PLLA
3.2. Micellar characterizations
4. Conclusions
5. References
Chapter 4. c(RGDfC)-conjugated PEG-PLLA micelles for targeted doxorubicin delivery
1. Introduction
2. Materials and Methods
2.1. Preparation of c(RGDfC)-conjugated PEG-PLLA micelles
2.2. Preparation of DOX-loaded micelles and loading efficiency
2.3. Micellar characterizations
2.4. DOX loading and in vitro DOX release test
2.5. In vitro cytotoxicity against HeLa cells
2.6. Intracellular uptake studies
3. Results and Discussion
3.1. DOX loading and in vitro release
3.2. In vitro cytotoxicity
3.3. Analysis of intracellular uptake
4. Conclusions
5. References
General Conclusion
Abstract in Korean
List of Publications
