J Vet Sci.  2006 Dec;7(4):321-326. 10.4142/jvs.2006.7.4.321.

Cellular uptake of magnetic nanoparticle is mediated through energydependent endocytosis in A549 cells

• Kim JS
• Yoon TJ
• Yu KN
• Noh MS
• Woo M
• Kim BG
• Lee KH
• Sohn BH
• Park SB
• Lee JK
• Cho MH

• KMID: 1089477
• DOI: http://doi.org/10.4142/jvs.2006.7.4.321

Abstract

Biocompatible silica-overcoated magnetic nanoparticles containing an organic fluorescence dye, rhodamine B isothiocyanate (RITC), within a silica shell [50 nm size, MNP@SiO2(RITC)s] were synthesized. For future application of the MNP@SiO2(RITC)s into diverse areas of research such as drug or gene delivery, bioimaging, and biosensors, detailed information of the cellular uptake process of the nanoparticles is essential. Thus, this study was performed to elucidate the precise mechanism by which the lung cancer cells uptake the magnetic nanoparticles. Lung cells were chosen for this study because inhalation is the most likely route of exposure and lung cancer cells were also found to uptake magnetic nanoparticles rapidly in preliminary experiments. The lung cells were pretreated with different metabolic inhibitors. Our results revealed that low temperature disturbed the uptake of magnetic nanoparticles into the cells. Metabolic inhibitors also prevented the delivery of the materials into cells. Use of TEM clearly demonstrated that uptake of the nanoparticles was mediated through endosomes. Taken together, our results demonstrate that magnetic nanoparticles can be internalized into the cells through an energy-dependent endosomal-lysosomal mechanism.

Keyword

A549 cells; cellular uptake; endocytosis; magnetic nanoparticle

MeSH Terms

Biocompatible Materials/*pharmacokinetics
Cell Line, Tumor
Drug Delivery Systems/methods
Endocytosis/*physiology
Endosomes/physiology
Humans
Lung Neoplasms/drug therapy/*metabolism
Macrolides/pharmacology
Microscopy, Confocal
Microscopy, Electron, Transmission
Nanoparticles/*administration & dosage
Sodium Azide/pharmacology
Sucrose/pharmacology
Temperature

Figure

• 

  Fig. 1 Representative transmission electron micrograph (TEM) of cobalt ferrite magnetic-silica (core-shell) nanoparticles, MNP@SiO2(RITC)s. The average size of the particle is approximately 50 nm. In the low magnitude image, the size distribution is revealed to be narrow (scale bar = 200 nm). In the high magnitude image (left insert image), core-shell structure is clearly shown by different image contrast, which shows the core of MNPs being darker than the silica shell due to electron density. The right insert image represents the detailed structure of MNP.

• 

  Fig. 2 Confocal laser scanning microscope (CLSM) images of MNP@SiO2(RITC) uptake under low temperature conditions. In order to confirm whether nanoparticle uptake was possible at low temperature, A549 cells were incubated at 37℃ and 4℃ (after preincubation at 4℃ for 10 min) for 30 min. Concentration of magnetic nanoparticles is 0.4 mg/ml, and the uptake pattern was observed by CLSM. A549 cells were incubated at 37℃ (A) and 4℃ (B) for 30 min. The left panel shows the fluorescence image (emission spectrum is 488 nm.), the middle panel shows the optical microscopic image, and the right panel shows the images merged together. bars = 20 µm.

• 

  Fig. 3 Confocal laser scanning microscope (CLSM) images of MNP@SiO2(RITC) uptake in the presence of several metabolic inhibitors. To confirm whether MNP uptake was possible under metabolic inhibition, A549 cells were co-incubated with MNP@SiO2(RITC)s and metabolic inhibitors. (A) A549 cells were incubated with the magnetic nanoparticles only, (B) A549 cells pretreated with 0.1% sodium azide, (C) 0.45 M sucrose, or (D) 0.05 mM bafilomycin A were incubated with the nanoparticles at 37℃ for 30 min. The left panel shows the fluorescence image (emission spectrum is 488 nm), and the right panel shows the optical microscopic image. bars = 20 µm.

• 

  Fig. 4 Representative transmission electron micrographs (TEM) of A549 cells treated with MNP@SiO2(RITC). To elucidate the detailed information of MNP uptake through the endosome-lysosomal mechanism, TEM was performed. The treated cells were fixed with glutaraldehyde, paraformaldehyde, and osmium tetroxide, and were subsequently embedded with epoxy resin. Thin sections were cut using an ultramicrotome; sections were not stained with any reagent for detecting of uptake of nanoparticles into the cells. Images were collected using a transmission electron microscope and a digital camera. (A) Uptake of MNPs was initiated upon the invagination of the plasma membrane. (B) Some nanoparticles had already been internalized into the cells (solid line box 2), while some cells still in the process of uptake at the plasma membrane (solid line box 1). (C) Uptaken MNP@SiO2(RITC)s were trapped inside the lysosome.

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