강력하고 선택적인 인공 결합 쌍을 이용한 생체 이미징

Abstract

Supramolecular chemistry has contributed significantly to the development of bioimaging tools for the visualization of locations and behavior of biomolecules. For example, protein-based noncovalent tool anchors a functionality to the other materials, providing new functional materials and tools for bioapplications, however; its large size (> 100 kDa), enzymatic degradation and difficulty in chemical modifications limit their uses for broad applications. Although synthetic binding pair systems can be alternatives for noncovalent anchoring because they are typically small (~ 1 kDa), chemically tractable and scalable, and resistant to enzymatic degradation, they have an intrinsic limitation for in vivo applications because of their low binding affinity (K < 104 M-1) in physiological environments. Cucurbit[n]urils (CB[n], n = 5-8, 10, 13-15), a family of synthetic receptors, exhibit remarkable guest recognition properties: they bind specific guest molecules with high binding affinity and specificity under physiological conditions and the resulting host-guest complexes are not easily dissociated. While the high-affinity synthetic binding pairs have been exploited for various biological applications, their applications in in vivo bioimaging have not been studied much. This thesis describes how the high–affinity synthetic binding pairs can be used for bioimaging in live animals. Chapter 2 describes the use of CB[6]-based polymer nanocapsules (CB[6]PNs) as an in vivo multimodal bioimaging platform. The surface of CB[6]PNs was easily functionalized with various spermidine (spmd)-conjugated functional tags (spmd-tags) for multimodalities in a non-covalent and modular manner by simple mixing of spmd-tags with CB[6]PNs. Dye-release assays using a dialysis method revealed the anchoring stability of spmd-tags to CB[6]PNs under serum condition, proving its suitability for bioimaging. Treatment of CB[6]PNs with Cyanine7 (Cy7)-spmd, 64Cu-labeled 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA)-spmd or Arginylglycylaspartic acid (RGD)-spmd for near infra-red (NIR), positron emission tomography (PET), or cancer targeting, respectively, produced materials for multimodal imaging. CB[6]PNs with three different modalities showed successful cancer-targeted multimodal imaging of tumors in cancer-bearing mice, demonstrating the potential use of strong and stable CB[6]-spmd binding pair as a platform for in vivo bioimaging. Chapter 3 describes the development of supramolecular tools for molecular recognition in in vitro & in vivo environments using CB[7] conjugated Erbitux (Erbitux-CB[7]) and Cyanine 5 conjugated adamantylammonium (Cy5-AdA). For active localization of CB[7] units on a cancer site, monocarboxylic acid-hexaethylene glycol-CB[7] was conjugated to Erbitux to afford Erbitux-CB[7]. Cy5-AdA was synthesized for tracking the occurrence of host-guest interaction through fluorescence signals. Confocal laser scanning microscopy (CLSM) and flow cytometry revealed that Erbitux-CB[7] retains its cancer-targeting ability after the conjugation. Supramolecular latching of Cy5-AdA to Erbitux-CB[7] on the cancer cells analyzed by CLSM and flow cytometry additionally supports not only conjugation of CB[7] units on Erbitux again, but also accessibility of the portal of CB[7] units on Erbitux to AdA using ultrastable and bioorthogonal host-guest chemistry under in vitro environment. In in vivo assessments with cancer-bearing mice, only the mouse pretreated with Erbitux-CB[7] showed selective accumulation of Cy5-AdA on the cancer site after intravenous injection of Cy5-AdA. It demonstrates that Cy5-AdA can be noncovalently anchored on Erbitux-CB[7] that is prelocalized in a cancer site in a live mouse. These findings may further provide insight into the development of new chemical, biological tools, and medical therapeutic systems for live animals. Chapter 4 describes our efforts to develop an efficient in vivo immuno-pretargeted PET imaging system via high binding affinity, specificity, and small size of CB[7]-AdA pairs. For efficient binding of 18F-labeled AdA to Erbitux-CB[7] prelocalized in the tumor site, three AdA-containing materials were synthesized. CB[7]-AdA pairs are anticipated to show great potential in fast and precise cancer-targeted PET imaging through both its rapid kinetics and bio-orthogonality in in vivo environments. These supramolecular tools could be a good candidate for the development of in vivo immuno-pretargeted PET imaging system that can reduce radiation exposure, and broaden the scope of supramolecular chemistry into practical applications.

생체 이미징은 비외과적인 방법으로 생체 과정들을 실시간으로 볼 수 있는 방법이다. 정확하고 효율적인 생체 이미징을 하기 위해서 여러 물질들을 이용하고 있다. 초분자는 두 개 이상의 분자를 비공유결합으로 결합하여 만든 또 다른 분자 (특정한 구조와 성질을 갖는 분자의 집합체)를 말한다. 최근, 초분자 화학의 중요한 핵심 개념인 분자 인식과 자기 조립 원리를 이용하여, 목적에 맞는 화학적 물리적 성질을 가지는 기능성 나노 재료를 구현하기 위해 많은 연구가 진행되고 있다. 그 중에서도 쿠커비투[n]릴 (cucurbit[n]uril, CB[n])은 반복단위체인 글리콜우릴로 형성된 거대 고리분자로, 소수성을 지닌 동공내부와 부분적으로 음전하를 띄는 입구를 가지고 있어, 소수성 상호작용과 쌍극자 이온상호작용을 통해 방향족 아민이나 알킬아민 화합물을 선택적으로 인지하고 동공 내부에 담지 할 수 있다. 이렇게 특정 손님 분자와 특이적 상호작용이 가능한 인공 수용체로 많은 관심을 받고 있으나, 유기 용매에서의 낮은 용해도와 추가적인 화학 작용기 도입의 어려움으로, 그 용도가 제한적이었다. 그러나 최근 CB[n]에 다양한 작용기 리간드를 도입하는 기술 개발로 용해도 문제를 극복했을 뿐만 아니라, CB[n] 유도체를 이용한 다양한 응용이 가능하게 되었다. 본 연구단은 12개의 알릴기가 도입된 CB[6]를 합성하였고, dithiol과 광축합 반응을 통해 고분자 나노캡슐을 성공적으로 합성하였다. 고분자 나노캡슐은 표면이 CB[6]로 되어있어, 손님 분자를 이용한 손쉬운 표면 개질로 다양한 생의료분야에 적용 가능성을 보였다. 또한, 동족체 중 하나인 CB[7]은 아민기가 도입된 아다만텐 유도체와 매우 강한 상호 작용을 보이며, 그 결합력이 자연계에서 가장 강한 비공유 결합으로 알려진 단백질 결합 쌍, 스트렙타아비딘-바이오틴 결합력 (K ~ 1015 M-1)에 상응하는 강력한 결합력을 보여준다. 강력하고 선택적인 주인-손님 결합의 장점을 이용하면 새로운 분자 인식 도구로 사용가능할 것이라 예상된다. 본 논문에서는 강력하고 선택적인 인공 결합 쌍을 이용한 생체 이미징에 대해 보고하고자 한다.