A magnetic switch for the control of cell death signalling i

A magnetic switch for the control of cell death signalling in in vitro and in vivo systems

Mi Hyeon Cho 1?,Eun Jung Lee 1,2?,Mina Son 2?,Jae-Hyun Lee 1,Dongwon Yoo 1,Ji-wook Kim 1,Seung Woo Park 3,Jeon-Soo Shin 2,4*and Jinwoo Cheon 1,2*

The regulation of cellular activities in a controlled manner is one of the most challenging issues in ?elds ranging from cell biology to biomedicine 1,2.Nanoparticles have the potential of becoming useful tools for controlling cell signalling pathways in a space and time selective fashion 3,4.Here,we have developed magnetic nanoparticles that turn on apoptosis cell signalling by using a magnetic ?eld in a remote and non-invasive manner.The magnetic switch consists of zinc-doped iron oxide magnetic nanoparticles 5(Zn 0.4Fe 2.6O 4),conjugated with a targeting antibody for death receptor 4(DR4)of DLD-1colon cancer cells.The magnetic switch,in its On mode when a magnetic ?eld is applied to aggregate magnetic nanoparticle-bound DR4s,promotes apoptosis signalling pathways.We have also demonstrated that the magnetic switch is operable at the micrometre scale and that it can be applied in an in vivo system where apoptotic morphological changes of zebra?sh are successfully induced.

Cell signalling is an important process in biological systems for exchanging information through networks of various signal molecules to control cellular activities,such as differentiation,growth,metabolism and death 6.Owing to their newly developed high precision and accuracy,physical stimuli using optical,electrical and magnetic methods have been devised to regulate cell signalling 1–4.Among these,magnetic techniques are uniquely advantageous because magnetic fields can penetrate deeply with negligible attenuation into biological tissues 7,8.Consequently,it has distinctive benefits for in vivo applications.Moreover,when coupled with magnetic nanoparticles,magnetic fields can be transformed into other forms of energy,such as heat and mechanical force 9–16.The magnetic heat induction has been used for gating of the thermosensitive ion channel 9as well as for hyperthermia therapy 10.Although relatively large mechanical force (in the piconewton range)has been used in in vitro and in vivo systems for direct stretching of ion channels and cytoskeletal stimulation 11–13,two recent in vitro studies have revealed that the induction of calcium influx 15and tubulogenesis 16using receptor clustering is also possible by using nanoparticles.Magnetic nanoparticles can exert a gentle force (in the femtonewton range)on membrane receptors to induce their clustering without disturbing the rheological and cytoskeletal properties.Furthermore,the nanoscale dimensions of nanoparticles conjugated with targeting molecules make them beneficial for probing cellular sensory structures and functions at the molecular level and for inducing specific cellular activation processes 7,8.Nonetheless,the nanoscale magnetic switching technique for receptor clustering is still at too

1Department

of Chemistry,Yonsei University,Seoul 120-749,Korea,2Graduate Program for Nanomedical Science,Yonsei University,Seoul 120-749,Korea,

3Department of Internal Medicine,Institute of Gastroenterology,College of Medicine,Yonsei University,Seoul 120-752,Korea,4Department of

Microbiology,Severance Biomedical Science Institute,Institute for Immunology and Immunological Diseases,College of Medicine,Yonsei University,Seoul 120-752,Korea.?These authors contributed equally to this work.*e-mail:jsshin6203@yuhs.ac;jcheon@yonsei.ac.kr.

early a stage of development to guarantee that it will be generally applicable to the control of cell signalling in other biologically important systems.In addition,it is not known whether it will be effective in in vivo systems.

Apoptosis,programmed cell death,is known to be a major factor in maintaining homeostasis and removing undesired cells 17–19.Recently,an extrinsic apoptosis signalling pathway that is initiated by death receptors has emerged as one of the main targets for cancer therapy 20–22.Extrinsic apoptosis signalling is usually activated by clustering of death receptors through docking of biochemical ligands,such as the TNF-related apoptosis inducing ligand (TRAIL)that is a potent inducer of apoptosis 23,24.However,direct use of such ligands in clinical applications is limited by the short plasma half-life and the ease of degradation 25,26.

In the study described below,we have developed a magnetic switch for apoptosis signalling,and demonstrate its in vivo feasi-bility through the receptor clustering process (Fig.1).The mag-netic switch for cell signalling consists of DR4monoclonal anti-body conjugated to magnetic nanoparticles (Ab–MNPs).DR4s are highly expressed on tumour cells 20–27and magnetic nanoparticles are designed to bind DR4s through a specific monoclonal anti-body interaction.Zinc-doped iron oxide magnetic nanoparticles (Zn 0.4Fe 2.6O 4MNPs,15nm)are chosen (Fig.2a)owing to their high saturation magnetization value (161e .m .u .g ?1),which is essential for effective utilization of magnetic force 5(Supplementary Section S1).For preparation of the Ab–MNPs,protein A is conjugated to thiolated MNPs through a sulpho-SMCC (sulphosuccinimidyl-4-[N -maleimidomethyl]cyclohexane-1-carboxylate)crosslinker.The DR4antibody is then conjugated to protein A on MNPs in a DR4antibody/MNP stoichiometric ratio of 1:1(Fig.2b and Supple-mentary Section S1).

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