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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

AFM images ((a), (c), and (e)) and corresponding fluorescence images ((b), (d), and (f)) of HCAECs incubated for 15 ((a) and (b)), 60 ((c) and (d)), and 120 min ((e) and (f)) with FITC-labeled liposomes conjugated with 90 nm gold particles. The cells with positive signaling in the bright field images (squares in the right-hand panels) were selected for AFM scanning. (a) Typical AFM image of an HCAEC incubated for 15 min, showing a gold liposome cluster attached to the cell membrane. (c) AFM image demonstrating the internalization process occurring in an HCAEC incubated for 60 min with “gold liposomes.” (e) The AFM micrograph of an HCAEC incubated for 120 min corroborated that the “gold liposomes” internalized almost completely at this time point. Cells fixed in formalin 10% and scanned in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
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fig4: AFM images ((a), (c), and (e)) and corresponding fluorescence images ((b), (d), and (f)) of HCAECs incubated for 15 ((a) and (b)), 60 ((c) and (d)), and 120 min ((e) and (f)) with FITC-labeled liposomes conjugated with 90 nm gold particles. The cells with positive signaling in the bright field images (squares in the right-hand panels) were selected for AFM scanning. (a) Typical AFM image of an HCAEC incubated for 15 min, showing a gold liposome cluster attached to the cell membrane. (c) AFM image demonstrating the internalization process occurring in an HCAEC incubated for 60 min with “gold liposomes.” (e) The AFM micrograph of an HCAEC incubated for 120 min corroborated that the “gold liposomes” internalized almost completely at this time point. Cells fixed in formalin 10% and scanned in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).

Mentions: The surface topology and characteristics of biological membranes can routinely be described using biological AFM instruments [23, 24]. Nevertheless, the application of this technique has rarely been approached to resolve kinetics for liposome cell uptake. To investigate how endocytosis takes place within endothelial cells, we used 90 nm gold particles to track FITC-labeled liposomes. Figure 4 shows sequential AFM images illustrating how the “gold-liposomes” internalize the plasma membrane.


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)

AFM images ((a), (c), and (e)) and corresponding fluorescence images ((b), (d), and (f)) of HCAECs incubated for 15 ((a) and (b)), 60 ((c) and (d)), and 120 min ((e) and (f)) with FITC-labeled liposomes conjugated with 90 nm gold particles. The cells with positive signaling in the bright field images (squares in the right-hand panels) were selected for AFM scanning. (a) Typical AFM image of an HCAEC incubated for 15 min, showing a gold liposome cluster attached to the cell membrane. (c) AFM image demonstrating the internalization process occurring in an HCAEC incubated for 60 min with “gold liposomes.” (e) The AFM micrograph of an HCAEC incubated for 120 min corroborated that the “gold liposomes” internalized almost completely at this time point. Cells fixed in formalin 10% and scanned in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: AFM images ((a), (c), and (e)) and corresponding fluorescence images ((b), (d), and (f)) of HCAECs incubated for 15 ((a) and (b)), 60 ((c) and (d)), and 120 min ((e) and (f)) with FITC-labeled liposomes conjugated with 90 nm gold particles. The cells with positive signaling in the bright field images (squares in the right-hand panels) were selected for AFM scanning. (a) Typical AFM image of an HCAEC incubated for 15 min, showing a gold liposome cluster attached to the cell membrane. (c) AFM image demonstrating the internalization process occurring in an HCAEC incubated for 60 min with “gold liposomes.” (e) The AFM micrograph of an HCAEC incubated for 120 min corroborated that the “gold liposomes” internalized almost completely at this time point. Cells fixed in formalin 10% and scanned in contact mode in liquid (DNP-S fo = 12–24 kHz, k = 0.06 N/m).
Mentions: The surface topology and characteristics of biological membranes can routinely be described using biological AFM instruments [23, 24]. Nevertheless, the application of this technique has rarely been approached to resolve kinetics for liposome cell uptake. To investigate how endocytosis takes place within endothelial cells, we used 90 nm gold particles to track FITC-labeled liposomes. Figure 4 shows sequential AFM images illustrating how the “gold-liposomes” internalize the plasma membrane.

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