PEG-SWCNT strongly induces platelet-granulocyte complex formation where the CD63 activation marker can be attributed solely to platelets - BIOCOMPATIBILITY OF PEG-SWCNT CAN BE QUESTIONED János Fent and Susan Lakatos Department of Pathophysiology, Research Institute, Medical Centre, Hungarian Defence Forces, H-1134 Róbert Károly krt. 44, Budapest, HUNGARY, latyakos24@gmail.com
INTRODUCTION
Experimental
• Single walled carbon nanotubes (SWCNT) gain more and more widespread utilization all over in our lives. • Biomedical applications demand biocompatible versions of these nanotubes. • Pegylated (PEG-SWCNT) version of single walled carbon nanotubes is claimed to be biocompatible. • Independently of the entry route of carbon nanotubes into the human body, sooner or later they get into the blood circulation and get into contact with blood cells.
Materials • citrated human blood • SWCNT and PEG-SWCNT --- Sigma Aldrich • ADP – physiological activator of platelets Dispersion of nanotubes into physiological medium • Sonication of nanotubes in distilled water • Stabilization of dispersion with human serum albumin • Sonication again • Adjusting physiological ionic strength and pH • Sonication again In vitro cell activation with nanotubes • Citrated human blood is incubated with 0.1 mg/ml nanotube dispersion at room temperature for 1 hour
Flow cytometry • identification of various blood cells with fluorescent monoclonal antibodies CD41+ – platelets CD15+ – granulocytes CD41+/15+ – platelet granulocyte complexes • Markers of cell activations CD63 – platelets and granulocytes as well • Staining protocol CD41/63/15 – multicolor staining of whole blood no lyse no wash protocol
AIM Aim of this study was to reveal and compare the effects of SWCNT and PEG-SWCNT exerted on human platelets and granulocytes Gating strategy
Measured data
single platelets (P)
single granulocyte (G)
platelet-granulocyte complex (C)
Mean fluorescence intensities (MFI) were measured Single platelets
Single granulocytes
Platelet-granulocyte complexes
CD41MFI
PCD41
GCD41
CCD41
CD63MFI
PCD63
GCD63
CCD63
Calculated data Number of platelets on one granulocyte in the complex N = CCD41 / PCD41 Fraction of CD63 within the complex attributable to platelets = N x PCD63 CD63 calculated in the complexes = (N x PCD63)+GCD63
data collection with CD15 trigger
data collection with CD41 trigger
RESULTS No CD63 expression on single granulocytes upon PEG-SWCNT treatment
PEG-SWCNT activates platelets
single granulocytes
platelets
platelet-granulocyte complexes
20
20 10
120
15 10
CD63 MFI
CD63 MFI
CD63 MFI
30
5 0
0 Control
ADP
SWCNT
complex % 80
*
*
Control
PEGSWCNT
PEG-SWCNT induces plateletgranulocyte complex formation
C%
CD63 activation marker on platelet-granulocyte complexes
ADP
SWCNT
PEGSWCNT
6
40
4
N
20
Control
ADP
SWCNT
PEGSWCNT
Control
ADP
SWCNT
PEGSWCNT
SWCNT
PEGSWCNT
I S U L C
N O
C • the cell surface expression of the CD63 activation marker,
• PEG-SWCNT induces degranulation of dense granules of platelets but not the azurophilic granules of granulocytes within the complex
ADP
S N O
• PEG-SWCNT promotes platelet granulocyte complex formation at higher extent than its pristine counterpart
the lysosomal membrane protein in the complex can be attributed solely to the bound platelets
30
NO EXTRA CD63 expression with GRANULOCYTE ORIGIN can be detected in the presence of SWCNT or PEG-SWCNT
2 0
0
60
Control
average number of platelets within the complexes
60
Calculated
0
PEG-SWCNT increases the number of platelets within the complexes *
90
Measured
BIOCOMPATIBILITY OF PEGSWCNT CAN BE QUESTIONED!