Magnetic Separation of Autophagosomes from Mammalian Cells Using Magnetic–Plasmonic Hybrid Nanobeads

Abstract

Developments in subcellular fractionation strategies have provided the means to analyze the protein and lipid composition of organelles by proteomics. Here, we developed ultrasmall magnetic–plasmonic hybrid nanobeads and applied them to the isolation of autophagosomes by applying a magnetic field. The beads were chemically synthesized and comprised an Ag/FeCo/Ag core/shell/shell structure with a mean diameter of 15 nm. The Ag core and the FeCo shell conferred imaging and magnetic separation capabilities, respectively. The nanobeads were transfected into mammalian cells by lipofection. Thirty minutes after lipofection, the nanobeads colocalized with Vps26 and subsequently with LC3. Cell lysates were prepared at the appropriate time points and were subjected to magnetic separation. The separated fraction contained LC3-II, transferrin receptor, and LAMP2, but not LC3-I, suggesting that autophagosomes engulfing endosomal origin had been isolated. The magnetic separation process was completed in less than 30 min, providing a rapid method for isolation of autophagosomes. The present organelle isolation technique using the hybrid nanobeads with imaging and magnetic separation capabilities is highly promising for isolation of other types of organelles such as endosomes and endosome-related organelles.

Basic Principles and Recent Advances in Magnetic Cell Separation

“…Magnetic cell separation is used for a wide range of applications and in various contexts, such as disease diagnostics, environment monitoring, fundamental biological research, etc. It is a crucial step in diverse biosensor assays and, along with the development of new cell separation systems, lots of efforts are also devoted to the synthesis of multifunctional magnetic nanoparticles displaying fluorescent [204][205][206][207], electrochemical [208,209] or plasmonic [210,211] properties, to enable both magnetic capture and downstream detection. In this section, we present examples of applications with a particular emphasis on CTCs and pathogenic bacteria isolation, representing considerable research efforts from the community.…”

Magnetically Controlled Hyaluronic Acid–Maghemite Nanocomposites with Embedded Doxorubicin

“…Biomedical applications require stabilized magnetic polymer conjugates with certain fundamental and operational characteristics, including a specific size which controls the magnetic properties of the conjugates [17], the total negative charge of particles responsible for their colloidal stability in a biological environment [18], the sufficient capacity of particles to encapsulate the drug [19], negligible toxicity [20] and the ability of the particles to biodegrade after completing their transport function [21]. Following these requirements, magnetic polymer formulations have been synthesized for magnetic resonance imaging [22], the separation of mammalian and bacterial cells [23,24], genetic engineering [25] and medical diagnostics and treatments [26][27][28][29].…”

Quick and Mild Isolation of Intact Lysosomes Using Magnetic–Plasmonic Hybrid Nanoparticles

“…In our previous works, we developed Ag/FeCo/Ag core/shell/ shell magnetic−plasmonic hybrid nanoparticles (MPNPs) 25 and demonstrated the feasibility of using them for plasmonic imaging and magnetic separation of autophagosomes. 26 To deliver the MPNPs to the lumen of autophagosomes, transfection of MPNPs was performed because it invoked xerophagy and the MPNPs were incorporated in autophagosomes automatically. To use the MPNPs for isolating intact lysosomes, surface functionalization of MPNPs is crucial for targeting them into the lumen of lysosomes.…”

“…Plasmonic nanoparticles such as Au and Ag nanoparticles have been considered as alternative bioimaging probes to fluorescent dyes because of their intense light scattering derived from localized surface plasmon resonance, and they are photobleaching free. , The combination of a superparamagnetic component with plasmonic nanoparticles will satisfy both intrinsic bioimaging and magnetic separation capabilities. In our previous works, we developed Ag/FeCo/Ag core/shell/shell magnetic–plasmonic hybrid nanoparticles (MPNPs) and demonstrated the feasibility of using them for plasmonic imaging and magnetic separation of autophagosomes . To deliver the MPNPs to the lumen of autophagosomes, transfection of MPNPs was performed because it invoked xerophagy and the MPNPs were incorporated in autophagosomes automatically.…”