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Functionalization of Carbon Nanotubes does not necessarily improve their Biocompatibility Biocompatibility János Fent, Susan Lakatos Department of pathophysiology, Research Institute, Military Hospital H-1134 Róbert K. krt. 44., Budapest, HUNGARY, latyakos24@gmail.com

CREDIT (k3-studio).

RESULTS

Methods

Introduction Whole Blood Aggregometry SINGLE-WALL CARBON NANOPARTICLES (SWCNT) SWCNT-PEG 1000

*

50

Impedance (AU)

60

p<0.001

40

*

30

*

*

C

A

800 600 400 200

0

500

2000

2500

10

SWCNT-OH SWCNT-PEG

SWCNT

SWCNTCONH2

SWCNTCOOH

SWCNT-OH

Dynamic Light Scattering Parameters

SWCNT-OH Control

C 1000 Impedance (AU)

ADP

No substantial aggregation of carbon nanoparticles occurs during the time period of our measurements (the polydispersity index is stable within the experimental).

A

800

0 0

500

800

1000

1500

2000

2500

400

SWCNT

The presence of SWCNTs inhibits collagen induced aggregation of the whole blood. The higher is the aggregation power of the SWCNT the more pronounced is the inhibition.

SWCNT-CONH2

SWCNT- SWCNT- SWCNT- SWCNTCOOH CONH2 OH PEG

HUMAN BLOOD SAMPLES Human blood was collected from the cubital vein of healthy volunteers into citrate anticoagulant containing Vacuette test tubes (Greiner, Austria, Kremsmünster). All samples were obtained with the approval of the local Ethical Committee after the donor had given informed consent.

1000 500

MEASUREMENT OF IN VITRO PLATELET ACTIVATION BY FLOW CYTOMETRY

0 0

500

1000

1500

2000

-500 Time (sec)

POLYDISPERSITY (PdI)

SWCNT-COOH

SWCNT-COOH

0.8

Control

The effect of nanoparticles on platelet activation in human whole blood was determined by measuring the P-selectin activation marker expression on platelets. 100 µl of citrated blood was incubated with 100 µl of vehicle or 100 µl of nanoparticle dispersion for 10 min at room temperature. As a positive control, samples were incubated with 1 µM ADP (final concentration) for 10 min.

A C

0.4 0.2 0.0 SWCNT

SWCNTCOOH

SWCNTCONH2

SWCNT-OH

SWCNTPEG

Impedance (AU)

0.6

EACH KIND OF CARBON NANOPARTICLE NEEDS TO BE TESTED INDIVIDUALLY

Nanoparticle dispersions were characterized by their Z-average equivalent hydrodanamic radius, by the polydispersity index as well as by Z-potential determined by Malvern Zetasiter using dynamic light scattering measurements.

Control

C

A Impedance (AU)

nm

600

0

Stock solutions of all the SWCNT samples were prepared at a concentration of 0.23 mg/ml in distilled water using sonication with 4.2 × 105 kJ/m3 specific energy for 2 minutes. Addition of human serum albumin at final concentration of 1.5 mg/ml was followed by sonication a sin the first step. Physiologic ionic strength was attained by adding 10× concentrated PBS solution, followed by a third sonication with the same energy. Control sample was prepared in the same way, but nanoparticle dispersion was replaced by distilled water.

200

SWCNT-CONH2

200

NanoAmor NanoShel

PHYSICO-CHEMICAL CHARACTERIZATION OF NANOPARTICLE DISPERSIONS

400

Z-AVERAGE DIAMETER

SWCNT and its forms functionalized with PEG, or COOH or OH -, but not with NH2 induce in vitro whole blood aggregation.

SES Research Sigma-Aldrich

600

Time (sec)

SWCNT and its forms functionalized with PEG, or COOH or CONH2-, but not with OH induce expression of CD62P, the platelet activation marker.

1000 800 600 400 200 0 0

500

1000

1500

2000

2500

3000

Time (sec)

ζ −POTENTIAL

ABSTRACT

SWCNT

SWCNT

Control

Whole blood was stained according to the manufacturer's instructions with PE-labeled anti-CD62P antibodies (Dako, Glostrup, Denmark) and FITC-labeled anti-CD41 antibodies (Immunotech, Marseilles, France). After staining, samples were diluted 50fold with PBS-BSA buffer (500-fold final dilution of blood). To minimize the spontaneous activation of platelets, no washing steps were used.The CD41 platelet marker was used as a trigger signal for data collection, the platelets were gated on the FS-SS dot plot and the mean CD62P fluorescence intensity (MFI) was measured for CD41 positive events. AGGREGOMETRY

A C

0 -2 -4 -6 -8 -10 -12 -14

Impedance (AU)

mV

Recently various engineered nanoparticles are popular candidates for implementing them into diagnostics, therapeutics or using them for drug delivery. In some extent all these procedures involve direct contact between the nanoparticles and the blood constituents. Most of the nanoparticles are more or less toxic or have prothrombotic effects. A great deal of efforts has been made to decrease these unfavorable effects, mainly by attaching various functional groups to the surface of the nanoparticles. The main goal of our study was to compare single-wall carbon nanotubes (SWCNT) with their functionalized counterparts bearing –CONH2, -OH, -COOH groups or polyethylene glycol (PEG) in terms of their effect exerting on platelets. All these SWCNTs, but SWCNT-CONH2 induce in vitro activation of human blood platelets and platelet aggregation, although at different extent, as judged by flow cytometry and aggregometry. Although attaching PEG to various nanoparticles seems to be widely accepted as a mean to increase biocompatibility, according to our experience SWCNT-PEG has the strongest platelet activation power. Our results indicate that there is no “universally good” functional group; each kind of functionalized nanoparticle needs to be tested individually.

1500

Time (sec)

CONCLUSIONS

THERE IS NO „UNIVERSALLY GOOD” FUNCTIONAL GROUP TO INCREASE BIOCOMPATIBILITY OF A SINGLE-WALL CARBON NANOTUBE

1000

20

DV

SWCNT SWCNT-PEG SWCNT- CONH2 SWCNT-COOH SWCNT-OH

PREPARATION OF NANOPARTICLE DISPERSIONS

0

0

Incipient instability characterizes our nanoparticle dispersions according to their Z-potential values.

SWCNT-PEG Control

CD62P MFI

In order to utilize various nanoparticles in biological systems either for drug delivery or for therapeutics or for diagnostics they need to meet certain requirements. First of all they must not to be toxic and they need to form a more or less stable dispersion in aqueous solutions. Many efforts have been implemented to satisfy these conditions for the engineered nanoparticles. Pegylation, which is considered to be a biocompatible modification, proved to be very effective in increasing the dispersibility of carbon nanotubes in aqueous media. Pharmaco-kinetic properties of single-wall carbon nanotubes (SWCNT) can be perturbed by attaching various chemical groups, bearing different charges – like CONH2, COOH, OH. Since in case of human applications nanoparticles - either modified or not - come into close contact with the blood cells, their effect exerted on the haemostasis needs to be controlled, and the ratio of benefits to the detrimental effects has to be considered prior to their human administration. The protrombotic effect of the single-wall carbon nanotubes together with their functionalized counterparts will be presented.

CD62P, the Platelet Activation Marker Expression in Whole Blood

SWCNT

SWCNTCOOH

SWCNT- SWCNT- SWCNTCONH2 OH PEG

1000 800 600 400 200 0 0

500

1000

1500

Time (sec)

A = addition of nanoparticle C = addition of collagen

2000

2500

The effect of nanoparticles on in vitro aggregation of human platelets was detected with a Chronolog Whole Blood Lumi-Aggregometer type 560 C (Chrono-Log, Havertown, USA) by measuring impedance change in plastic cuvettes at 37 °C with continuous stirring (1000 rpm). The reaction mixture contained 750 µl of citrated blood and 750 µl of 0.2 mg/ml nanoparticles or vehicle solution. After running samples for at least 20 min, 5 µM of collagen was added to check the aggregation ability of platelets. Data were collected both with a two-channel recorder and a computer.

P12-07  

nanopaprika poster 2012

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