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Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells.

Zaske AM, Danila D, Queen MC, Golunski E, Conyers JL - J Pharm (Cairo) (2013)

Bottom Line: Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes.This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells.The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Internal Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX 77054, USA.

ABSTRACT
Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15-30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

No MeSH data available.


Related in: MedlinePlus

The internalization process of coupled liposomes was blocked when the cells were pretreated with 80 μM dynasore for 15 min before 60 min of incubation with “gold-liposomes.” The gold liposome clusters did not penetrate the cell membrane, remaining just attached to the external wall (arrow in (a)). The corresponding fluorescence image (b) shows positive signaling from the “gold liposomes” that were scanned. The cells were fixed in formalin 10% and imaged at 50 μm2 in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
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fig6: The internalization process of coupled liposomes was blocked when the cells were pretreated with 80 μM dynasore for 15 min before 60 min of incubation with “gold-liposomes.” The gold liposome clusters did not penetrate the cell membrane, remaining just attached to the external wall (arrow in (a)). The corresponding fluorescence image (b) shows positive signaling from the “gold liposomes” that were scanned. The cells were fixed in formalin 10% and imaged at 50 μm2 in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).

Mentions: Endocytosis is the process that cells use to take up nanovectors and other materials from the external environment, by encapsulating them in vesicles made from invaginations in the cell plasma membrane. It was of particular interest to investigate if the internalization of the gold-coupled liposomes could be hindered by a typical inhibitor of endocytosis. We used dynasore, a cell permeable, noncompetitive dynamin GTPase activity inhibitor, to block dynamin-dependent endocytosis of the liposomes (Figure 6). Our experiments indicated that the “gold-liposome” clusters barely attached to the external walls of cells treated 15 min with 80 μM dynasore and then incubated with the “gold-liposomes” for 60 min. In most of the cases, the “gold-liposomes” were easily removed from the cell membrane when probed with the AFM cantilever. The force loading exercised by the AFM probe, scanning to a velocity of 30 μm/s, removed ~90% of the liposomes. This indicated a poor binding efficiency with the plasma membrane and obstruction of the “gold-liposome” uptake after dynasore pretreatments.


Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells.

Zaske AM, Danila D, Queen MC, Golunski E, Conyers JL - J Pharm (Cairo) (2013)

The internalization process of coupled liposomes was blocked when the cells were pretreated with 80 μM dynasore for 15 min before 60 min of incubation with “gold-liposomes.” The gold liposome clusters did not penetrate the cell membrane, remaining just attached to the external wall (arrow in (a)). The corresponding fluorescence image (b) shows positive signaling from the “gold liposomes” that were scanned. The cells were fixed in formalin 10% and imaged at 50 μm2 in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4590807&req=5

fig6: The internalization process of coupled liposomes was blocked when the cells were pretreated with 80 μM dynasore for 15 min before 60 min of incubation with “gold-liposomes.” The gold liposome clusters did not penetrate the cell membrane, remaining just attached to the external wall (arrow in (a)). The corresponding fluorescence image (b) shows positive signaling from the “gold liposomes” that were scanned. The cells were fixed in formalin 10% and imaged at 50 μm2 in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
Mentions: Endocytosis is the process that cells use to take up nanovectors and other materials from the external environment, by encapsulating them in vesicles made from invaginations in the cell plasma membrane. It was of particular interest to investigate if the internalization of the gold-coupled liposomes could be hindered by a typical inhibitor of endocytosis. We used dynasore, a cell permeable, noncompetitive dynamin GTPase activity inhibitor, to block dynamin-dependent endocytosis of the liposomes (Figure 6). Our experiments indicated that the “gold-liposome” clusters barely attached to the external walls of cells treated 15 min with 80 μM dynasore and then incubated with the “gold-liposomes” for 60 min. In most of the cases, the “gold-liposomes” were easily removed from the cell membrane when probed with the AFM cantilever. The force loading exercised by the AFM probe, scanning to a velocity of 30 μm/s, removed ~90% of the liposomes. This indicated a poor binding efficiency with the plasma membrane and obstruction of the “gold-liposome” uptake after dynasore pretreatments.

Bottom Line: Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes.This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells.The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Internal Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, Houston, TX 77054, USA.

ABSTRACT
Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15-30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

No MeSH data available.


Related in: MedlinePlus