聚乙二醇包覆之四氧化三鐵奈米粒子應用於外泌體純化之研究

Exosomes purification based on PEG coated Fe3O4

巢沛禹(Pei-Yu Chao)

指導教授 ： 章明

中原大學/工學院/機械工程研究所/碩士(2016年)

https://doi.org/10.6840/cycu201600710

摘要

多年來，癌症的死亡率居高不下，若能在早期發現並提早治療，
則能大大的提升存活機率。當癌細胞開始大量增生，或即將要轉移時，
會分泌出大量的外泌體，因此若能檢測體內的外泌體數量及種類，當
某些特定外泌體數量不正常增加時，便有可能是癌症轉移的前兆。
外泌體會出現於各項生物流體中，其中以血液為大宗，但血液內
還有大量其他物質，即便處理成血清仍含有許多蛋白雜質，如球蛋白、
白蛋白等，會影響外泌體的檢測。故本研究擬結合磁控奈米粒子技術
與聚乙二醇蛋白質過濾法，以聚乙二醇包覆四氧化三鐵奈米粒子用於
過濾血清中的蛋白質，達到純化血清中外泌體的目標，提供醫學方面
的檢測應用，避免進行生物流體檢測時其餘雜質所造成的誤差。
本研究以化學共沉法製備聚乙二醇包覆四氧化三鐵奈米粒子，單
顆粒子的粒徑約為20nm，團聚物則約從30 至400 奈米，經FTIR、
XRD 及FE‐SEM 的檢測證實聚乙二醇確實有包覆在四氧化三鐵奈米粒
子的表面。在抓取蛋白質的實驗中，將粒子與血清充分混合後，用強
力磁鐵將粒子吸附於底部，取其上清液進行檢測，結果顯示能將血清
中的蛋白質濃度降低至原先的39.9%，與傳統的聚乙二醇離心過濾方
式的71.9%相比大幅提升，將過濾完畢後的溶液經粒徑分析以及膠體電泳的檢測確認只有將血液中的雜蛋白給濾除，並沒有誤將外泌體也
過濾掉，此初步結果證實了以聚乙二醇包覆的四氧化三鐵奈米粒子有
蛋白質的過濾能力，且能保留下純淨的外泌體，能做為純化外泌體的
一種方法。

關鍵字

四氧化三鐵 ； 奈米粒子 ； 聚乙二醇 ； 蛋白質過濾 ； 外泌體 ； 生物流體檢測 ； 癌症 ； 純化

並列摘要

Cancer also known as malignant tumors, is a group of diseases involving
abnormal cell growth with the potential to invade or spread to other parts
of the body, for many years, cancer is a disease of the highest mortality,
over 100 cancers affect humans. When cancer metastasis, cancer cells
secretes a kind of microvesicles called “Exosome” to create the
microenvironment at the destination. So, if we can detect the quantity of
exosomes in human body, perhaps have the opportunity to check out the
cancer at an early stage.
Exosomes appears in biological fluids like blood, but it is impurity that
contain lots of protein like globulin and albumin, will reduce the accuracy
of liquid biopsy. So this study focuses on exosome isolation with magnetic
nanoparticles. The use of chemical co-precipitation method to synthesize
Polyethylene glycol(PEG)-coated Fe3O4 nanoparticles, the surface of the
Fe3O4 has the reticular structure of PEG, can produce space crowing effect
to make protein precipitated from the original location. The particles are
uniformly mixed with serum, let particles catch protein, then use magnet
to separate the particles and the supernatant, the supernatant contain pure
exosomes. In order to use the protein precipitation, the purification of
exosomes can be achieved.
The diameter of PEG-coated Fe3O4 nanoparticles is about 20 nm, and
the agglomerates size is about several hundred nanometers. The use of FESEM,
XRD and FTIR to check the PEG is indeed coated on the surface of
Fe3O4 nanoparticles. In the experiment of protein isolation, through ELISA to detect, the particles can decline the protein concentration to 39.9% of
stock solution. To confirm exosomes are still in the supernatant, the result
of Nanosight shows the supernatant content 1.6x1010 particles/ml the size
of 30-200nm inclusions, same as the stock solution, SDS-PAGE shows the
CD63 exosomes band (~53kD) both are equally show in supernatant and
stock solution clearly, the immunoglobulin and albumin band (160~188kD
and ~66kD) are shown in stock solution but disappear in supernatant, the
result shows the exosome still in the supernatant but the other impurity
protein are filtered.
As a result, the PEG-coated Fe3O4 nanoparticles indeed can catch the
protein and retain the exosome, this is a practical method in exosome
isolation.

並列關鍵字

Fe3O4 ； nanoparticles ； exosomes ； Polyethylene glycol (PEG) ； Cancer ； liquid biopsy ； isolation

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