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Surface Modification of Layered Double Hydroxides (LDHs) for Cellular Tracking and Target Delivery

Surface Modification of Layered Double Hydroxides (LDHs) for Cellular Tracking and Target Delivery
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대학원 화학·나노과학과
이화여자대학교 대학원
약물 및 생기능성 분자의 효율적인 전달을 위한 벡터로 다양한 무기 나노 입자에 관한 연구가 그동안 진행되어 왔다. 그 중에서도 금속 이중층 수산화물 (LDH)은 음이온성 약물 및 분자를 담지 할 수 있으며 낮은 독성으로 세포 내 투과율이 높기 때문에 약물 전달체로써 많은 관심이 집중되어 있다. 그러나 금속 이중층 수산화물은 암세포에 대한 선택성이 부족함으로 효과적인 약물 전달체로써 한계성을 갖는다. 본 연구에서는 암세포에 대한 특정한 리간드인 folic acid를 금속 이중층 수산화물의 표면에 부착함으로써, folate receptor와 리간드간의 상호작용으로 타겟팅 효과를 높이고자 하였다. 잘 알려져 있듯이 folate receptor는 암세포 표면에 과 발현되어 있다. 또한 세포/생체 내 에서 추적이 불가능한 금속 이중충 수산화물의 한계점을 보완하고자, rhodamine-β-isothiocyanate (RITC)의 형광체를 표지 하는 실험도 수행하였다. 우선 수열합성 방법을 통해 100nm 크기의 금속 이중충 수산화물을 합성하였다. 그 다음 silane coupling 반응을 통해 amino propyl triethoxysilane (APS)를 금속 이중충 수산화물의 표면에 부착하였고, 다음단계로 수용체 특이적 리간드인 folic acid로 표면 개질을 하였다. 금속 이중층 수산화물이 효과적으로 표면 개질이 되었는지는 X-선 회절 분석 및 분광학적 분석을 통하여 확인하였다. 다음 단계로 FITC의 형광체를 표지해 세포/생체 내 효과적 추적을 가능하게 하였다. 금속 이중충 수산화물의 표면 개질을 통해 정상세포/종양세포를 구분할 수 있는 목표지향성을 부가함으로써 smart delivery system을 구현 할 수 있도록 기반을 마련하고자 하였으며 folic acid receptor가 과 발현된 KB cell을 사용하여 표적지향성 실험을 실시하였으며 그 결과, folic acid로 표면 개질된 금속 이중충 수산화물의 경우가 그렇지 않은 경우보다 표적지향성이 월등함을 확인할 수 있었다. 또한 RITC의 형광체를 표지한 금속 이중충 수산화물을 쥐에 주입한 실험결과, 형광체를 표지한 금속 이중충 수산화물이 뇌, 심장을 뺀 간, 허파, 비장, 콩팥에서 발견됨을 알 수 있었다. 이러한 결과에 따라 금속 이중충 수산화물이 여러 생물학적 응용에 사용될 수 있음을 알 수 있고, folic acid로 표면 개질 된 금속 이중충 수산화물이 암세포에 대한 표적지향성을 가진 약물 전달 체로써 널리 사용될 수 있음을 입증하였다.;In recent, layered double hydroxides (LDHs) nanoparticles, consisting of positively charged brucite-like layers and charge compensating interlayer anions, have attracted great attention as nano-reservoir or drug delivery carriers, since LDHs showed high cellular uptake behavior as well as low toxicity. However, the systemic application of LDH as drug delivery carrier has been quite limited because of its low selectivity for malignant cells. In this study, we have attempted to modify the surface of LDH nanoparticles with the cancer cell specific ligand like folic acid (FA), an immunogenically stable ligand, in order to enhance the targeting effect based on the ligand-folate receptor interaction. As well known, the folate receptors (FRs) are generally overexpressed in cancer cells. Also, no luminescent property of LDHs makes it infeasible to trace them both in vivo and in vitro. In this study, for the effective tracking of LDH in cellular and systemic level, we prepared LDH nanoparticles which are covalently labeled with rhodamine-B- isothiocyanate (RITC). The LDH (Mg₂ Al (OH)_(6)(CO₃)_(0.5) 0.1H₂O) nanoparticles with narrow size distribution (100?25nm) were prepared and then conjugated with folic acid. The covalent bond between folic acid and LDH was realized stepwisely with silane at first and then 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC). The surface hydroxyl groups of LDH were modified by 3-aminopropyl- triethoxysilane (APS), and then EDC coupling reaction was carried out to form a peptide bond between amine group of the grafted APS and carboxylic acid group of folic acid. According to the powder X-ray diffraction patterns and spectroscopy analysis, all LDH nanoparticles were well modified with folic acid and RITC, respectively. In order to evaluate the cancer cell targeting efficacy of LDH-APS-FA by immunofluorescence assay, both LDHs with and without folic acid moiety were labelled with fluorescein 5?-isothiocyanate (FITC). The uptake of the LDH and LDH-APS-FA into KB (human nasopharygeal carcinoma cell) and A549 (human lung adenocarcinoma epithelial cell) cells was visualized using fluorescence microscopy and the latter was determined to be more significant than the former, LDH only. This is evidence that the folic acid molecules are indeed conjugated to the LDH nanoparticles giving rise to an enhanced selectivity to the cancer cell. The cellular localization was observed by fluorescence microscopy and both free RITC and LDH-APS-RITC were found to be localized in cytoplasm, but in a different manner. Free RITC was diffused throughout the cytoplasm, while LDH-APS-RITC showed a punctuated form. Different tissue distribution of LDH-APS-RITC compared to free RITC in mice was also determined. This study on cellular tracking of LDH nanoparticles in vitro and in vivo will provide critical information about their practical biological applications. In summary, we attempted to modify the surface of LDH nanoparticles with cancer cell specific ligand, FA, through step-by-step coupling reactions with APS and EDC, and found that the FA conjugated LDH became more selective to cancer cells than LDH itself. So we came up with a conclusion that LDH could play a role as a delivery carrier with targeting function thanks to the conjugation with folate ligand.
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