課題番号 ：S-16-NM-0006
利用形態 ：機器利用
利用課題名（日本語） ：
Program Title (English) ：Lipid membrane interaction with varying sizes and types of silver nanoparticle
利用者名（日本語） ：佐村祐弥
Username (English) ：Y. Samura
所属名（日本語） ：フリンダース大学大学院ナノテクノロジー研究科　
Affiliation (English) ：Centre for NanoScale Sciences and Technologies, Faculuty of Chemistry and Physical Sciences Flinders University

１．概要（Summary）
Main theme for the research is observation of nanoparticle-membrane interaction. Silver nanoparticles (AgNPs), which are currently used in varieties of medical products due to anti-bacterial property, were observed for their nature of interactions. Living cells and artificial membrane were used to confirm nanoparticle location upon exposure.

２．実験（Experimental）
【利用した主な装置】
 Transmission electron microscope
 Fluorescence emission spectrophotometer
 Dynamic light scattering
 Zeta potential particle analyzer
 Microplate reader
 Confocal microscope
 Cell culture incubator
 Clean bench
 UV-visible spectroscopy

【実験方法】
Silver nanoparticle interaction was measured using various techniques listed as above. AgNPs were synthesized at laboratory resulting citrate capped AgNPs size less than 100nm (approximately 20nm in TEM). Also, AgNPs were purchased from Sigma-Aldrich for comparison, their difference in size and surface morphology were observed using TEM.

Figure1: TEM image of citrate capped silver nanoparticles that synthesized at NIMS

DPhyPC (1, 2-diphytanoyl-sn-glycero-3-phosphocholine). This DPhyPC lipid was used to prepare human membrane like vesicles, as phosphocholine lipids makes up major part of human cell membrane. A549 human cell line and DPhyPC vesicles were exposed with the nanoparticle. Interaction were confirmed by confocal microscope, successfully obtaining images suggesting the nanoparticle uptake through membrane interaction.

Also the degree of the interaction are currently investigated at Flinders University through fluorescence quenching study and isothermal calorimetric measurements.

３．結果と考察 （Results and Discussion）
Nature of their interaction was confirmed through equipment at NIMS including UV-visible spectroscopy, phase contrast microscope, TEM, fluoresce　emission spectroscopy and confocal microscopy. We confirmed the following aspects.
Most importantly, interaction with lipid membrane was confirmed. AgNPs exposed artificial vesicles prepared using DPhyPC sugggested AgNPs localization. Clear confocal images were taken supporting the nanoparticle-membrane interaction.

Cells used for the interaction study was A549 cell line provided from NIMS, and A549 cells were also exposed with AgNPs. Their interaction are shown in images below.

Figure 2: Confocal image of A549 human cell line upon exposure of AgNPs.

Figure 2 shows A549 cell line observed using confocal microscope. Arrow marks on A2 and blue color on B2 shows the localization of AgNPs upon exposure, while the red color on C2 shows the cell membrane stained with fluorescence dye.

Furthermore, nature of interaction was observed using UV-Visible spectroscopy, fluoresce emission spectroscopy through determining the degree of interaction through quenching study. These techniques exhibit the alternative evidence to suggest their nature of interactions, which include binding constant and Stern-Volmer rate constant that suggests the mechanisms of interactions.

４．その他・特記事項（Others）
今回はこのような素晴らしい環境で働かせていただいたこと、また至らないわたしに温かい支援の手を差し伸べてくださった分子・物質合成プラットフォームの皆さまへ感謝を申し上げます。研究の方針へのアドバイスや意見を頂いた担当者の花方信孝様。様々な支援をしてい頂いたShanmugavel Chinnathambi様。書類の管理やNIMS内での行動で便宜をはかってくださった星川朱美様、前野由華様。物質・合成プラットフォームにて様々な実験器具の扱い方に関して支援してくださった服部普也様、森田浩美様、箕輪貴司様、竹村太郎様。特に服部様と森田様からはConfocal　Microscopeに関して支援を、竹村様からは細胞培養とDarkfield　Microscopeに関して支援を受け、感謝しております。今後も、なにかとお世話になる機会があるかと思います、どうぞよろしくお願いいたします。

５．論文・学会発表（Publication/Presentation）
なし。

６．関連特許（Patent）
なし。