A. Chirianc, Giorgiana Ion, Z. Faiyad, I. Poeata


Romanian Neurosurgery (2019) XXXIII (4): pp. 404-410 
DOI: 10.33962/roneuro-2019-085 
www.journals.lapub.co.uk/index.php/roneurosurgery 

 
 

 

Preliminary study of thrombogenicity 
induced by the nanoparticle surface 
coating of intracranial stents  
 

 
A. Chiriac1, Georgiana Ion1, G. Stan2, T. Popescu2, 

Mihaela Sofronie2, I. Poeata1 

  
1 “Gr. T. Popa” University of Medicine and Pharmacy, Iasi, ROMANIA 
2 National Institute of Materials Physics, Bucharest-Magurele, ROMANIA 

 

 
 

ABSTRACT 
Endovascular treatment of intracranial aneurysms with intracranial stents was 

proven to be clinically safe and effective, but is still associated with a risk of 

thromboembolic complications. Stent thrombosis could be a sever complication 

associated with specific stent surface coatings and designs. Standardized in vitro tests 

for investigation of thrombogenicity induced by different nanomaterials were used 

as the basic method in carrying out the present study. Therefore, the aim of this study 

was to evaluate the thrombogenicity of three different nanomaterials (ZnO, TiO2 si 

Fe3O4) possible used as surface coating for intracranial stents. This study is based on 

a procedure for in vitro analyses of plasma coagulation time. To measure the plasma 

coagulation time, platelet-poor plasma from human whole blood was in vitro 

exposed to nanoparticles and analysed in prothrombin (PT) and activated partial 

thromboplastin (APTT). 

 
INTRODUCTION 

A variety of intracranial stents are used in permanent blood contact. 

Thrombogenicity is the property of a material to induce a thrombus 

formation, which may result in partial or complete blood vessel 

occlusion. Thus, thrombogenicity of intracranial stent may have 

important implication in the long-term outcome of neurosurgical 

patients that may lead to a life-threatening condition such as stroke. 

Stent / blood contact surface activity has been a widely subject 

discussed and reported and, notwithstanding the clinical significance of 

this phenomenon is still controversial.  

Moreover, the surface coatings of these implants that are in 

permanent contact with blood are often subjected to tests on 

coagulation in order to use the least thrombogenic materials. Activated 

partial thromboplastin time (aPTT), prothrombin time (PT) and the 

international normalized ratio (INR) are indicated as the techniques of 

choice used to help detect and diagnose a bleeding disorder or 

excessive clotting disorder. In ou r study, an in vitro model was 

established, which allows investigation of neurovascular stents with 

Keywords 
intracranial stents, 
thrombogenicity, 

nanomaterials, 
surface coating   

 
 

 
 

Corresponding author: 
A. Chiriac 

 
“Gr. T. Popa” University of Medicine 

and Pharmacy, Iasi, Romania 
 

chiriac_a@hotmail.com 
 
 

 
 

Copyright and usage. This is an Open Access 
article, distributed under the terms of the Creative 
Commons Attribution Non–Commercial No 
Derivatives License (https://creativecommons 
.org/licenses/by-nc-nd/4.0/) which permits non-
commercial re-use, distribution, and reproduction 
in any medium, provided the original work is 
unaltered and is properly cited. 
The written permission of the Romanian Society of 
Neurosurgery must be obtained for commercial 
re-use or in order to create a derivative work. 
 

 
ISSN online 2344-4959 
© Romanian Society of 

Neurosurgery 
 

 
 

First published 
December 2019 by 

London Academic Publishing 
www.lapub.co.uk 

 

http://www.lapub.co.uk/


 405 
Preliminary study of thrombogenicity induced by the nanoparticle surface coating of intracranial stents 

regard to thrombogenicity and coagulation 

activation. The preliminary study was designed to 

compare these 3 methods to address the 

coagulation property of three biomaterials (ZnO, 

TiO2 si Fe3O4) with emphasis on the sensitivity and 

reproducibility of the test. These three biomaterials 

prepared in the form of nanoparticles were 

evaluated for their coagulation profile during 

incubation in human plasma. The study presents a 

revised version of these methods to include updated 

details on sample preparation of nanoparticles, 

selection of nanoparticle concentration for in vitro 

study, and updated details on test controls [4,5,6]. 

 

MATERIALS AND METHODS 

Platelet rich plasma (PRP) is obtained from fresh 

human blood derivative incubated with 

nanoparticles for 15 minutes at 37 ° C. This probe is 

subsequently analysed by evaluating the plasma 

coagulation time and how the nanoparticles affect 

this part of the haemostasis control system.  

 

The materials listed below are required for 

coagulation tests for the study of nanoparticles (Fig. 

1): 

1. Vacuum test tubes for coagulation testing; 

2. 12% sodium citrate. 

3. Thermostat bath; 

4. Special centrifuge with tubes test stand; 

5. Fresh human blood completely anti-coagulated 

with sodium citrate and obtained from at least 

three healthy donors known to be out of any anti-

inflammatory and antihistamine drug, blood 

thinning agents and birth control pills; 

6. APTT and PT / INR test cards for Abrazo Cascade 

equipment; 

7. Coagulation tester analyzer Abrazo Cascade 

System from Helena Laboratories; 

8. Nanoparticle sample for analysis. 

 

 

 

 

 

 

 

 

 

A 

B 

C 

D 

E 



 406 
A. Chiriac, Georgiana Ion, G. Stan et al. 

 

 
Figure 1: A- aPTT tester card; B- PT / INR tester card; C- Abrazo 

Cascade Coagulation Test Equipment; D- Centrifuge; E- 

Vacuum test tubes with citrate; F- Thermostatic bath. 

 
PREPARATION OF THE STUDY SAMPLES 

The concentrations used in these assays are based 

on an estimated plasma concentration in a medium-

sized human patient at the desired therapeutic dose. 

In our study, we refer to this concentration as the 

theoretical concentration of plasma. The hypotheses 

and considerations for estimating theoretical plasma 

concentration have been reviewed in the literature 

[4] and are summarized below. 

For the calculation of nanoparticle concentration, 

we used in vitro test reports in mice. 

If the mouse dose used is approximately 123 mg / Kg 

in this example the human dose can be calculated as 

follows: 
 

 

 

It is known that blood volume accounts for about 8% 

of body weight (for example, an adult of 70 kg has 

about 5.6 L (8% of 70) blood. This allows an extremely 

crude estimate of maximum blood concentration. 

 

 

 

Each test evaluates a nanoparticle formulation of 

three different materials at preset concentrations. 

When the predicted therapeutic concentration is not 

known, the highest final concentration is 1 mg per 

ml.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2: A- Blood collection; B- Centrifuge with multiple cartridges; C- Vacutainer tubes with centrifuged plasma.

 

PLASMA PREPARATION  

When the blood collection procedure is started the 

first 10 ml of blood must be discarded [fig.2] due to 

necessity to prevent the activation of cells and 

plas ma bioch em ic al c as cades by th e v eno us 

puncture procedure. Exposure and storage of blood 

or plasma at extreme temperatures (<20 C or> 37 C) 

can affect the quality of test results and should be  

 

avoided. Two types of plasma can be obtained to 

perform this experiment: platelet rich plasma (PRP), 

platelet poor plasma (PPP). Plasma from individual 

donors can be analyzed separately or collected 

together. The combined plasma is prepared by 

mixing plasma from at least two individual donors. 

 

F 



 407 
Preliminary study of thrombogenicity induced by the nanoparticle surface coating of intracranial stents 

NORMAL TEST PLASMA PREPARATION  

Fresh whole blood must be used within 1 hour after 

collection. The blood is then centrifuged for 10 min 

at 3000 g at 20–22 C and plasma is collected from at 

least two donors. The combined plasma is stable for 

8 hours at room temperature. The assay can also be 

performed in plasma from individual donors, when 

needed for mechanical tracking experiments. Two 

duplicates (four total samples) of plasma test are 

analyzed in each of the coagulation tests. A duplicate 

is performed prior to analyzing the nanoparticle 

samples and the second duplicate at the end of each 

run to verify that plasma functionality is not affected 

throughout the experiment. 

 

NANOPARTICLE-TREATED TEST PLASMA PREPARATION  

In a micro-tube, 0.125 mg of nanoparticles and 1 ml 

of plasma are combined. Mix well on a shaker and 

incubate for 30 min at 37 ° C. Three test tubes are 

prepared for each test sample (ie, when each 

nanoparticle is tested at four concentrations, one 

needs three test tubes for each concentration for a 

total of 12 test tubes per nanoparticle test). 

 

PLASMA COAGULATION TESTS (Fig. 3) 

1. Prepare the Cascade Abrazo equipment as 

described in the manufacturer's manual. 

2. Preheat all tubes to 37 C. 

3. Configure the instrument test parameters for 

each of the two tests (aPTT and PT / INR). 

4. Add to the micro-tubes with pre-loaded 

nanoparticles 1 ml of plasma. 

5. Stir micro-tubes for 5 minutes. 

6. Scan each specific tester card (aPTT and INR / 

PT) before testing; 

7. Insert the test card into the Cascade Abrazo 

device; 

8. Allow the instrument to warm up before use. 

9. Extract the test solution with the micropipette. 

10. When the test timer starts, transfer the solution 

to the test card's geode by pressing the PIP 

button to activate the pipette. 

11.  When the time is up, the device will beep and 

the recording of coagulation time will remain 

displayed on the screen until further testing 

begins. 

12. The average of the three values must be 

calculated for each control or test. 

13. The average for each control and test sample 

should be between 25%. 

14. If two duplicates of the same study sample 

showed different results more than 5%, this 

sample was reanalysed. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure 3: Schematic  

diagram of coagulation 

time measurement. 

 

Donors 
Fresh 

blood 

Plas

ma 

Control samples with 

nanoparticles 

Test 

settings 

Data recording and 

analysis 

Card scan Card insertion 

Add test 

solution 



 408 
A. Chiriac, Georgiana Ion, G. Stan et al. 

RESULTS 

The results shown in this study indicate that ZnO 

nanoparticles administered in the blood vessels can 

adsorb the coagulation factors in a specific form to 

the surface functional group, without activating 

these factors and that the adsorbed coagulation 

factors are not functional. Also, ZnO nanoparticles 

delayed the coagulation time with the size or surface 

functional group specificity, because all types of ZnO 

nanoparticles adsorbed the coagulation factors of 

the common pathway, some types of nanoparticles 

adsorbing more factors than others. The 

thrombogenicity study of TiO2 and Fe3O4 

nanoparticles suggests that nanoparticles should be 

an effective agent in prolonging the blood clotting 

time, thus exerting an anti-coagulant effect, thus 

improving the hemocompatibility of the coating. The 

ability of TiO2 and Fe3O4 nanoparticles to regulate 

platelet adhesion and plasma coagulation was thus 

demonstrated in vitro. 

 

 

 

 

 

DISCUSSIONS 

Treatment of neurosurgical patients with 

functionalized coating surfaces stents continues to 

evolve with the current emergence of nanoparticle 

functionalization technology that offers a 

combination of pharmacological and mechanical 

approaches to prevent thrombosis and arterial 

restenosis. Despite the promising short term and 

mid-term outcomes of surface coated stents, there 

are serious concerns about adverse clinical effects of 

late stent thrombosis. Certain nanoparticles 

intended for drug delivery application are designed 

PT COAGULATION TEST FOR NANOPARTICLES

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 409 
Preliminary study of thrombogenicity induced by the nanoparticle surface coating of intracranial stents 

to reduce the effect of thrombogenicity of an 

intracranial stent in the bloodstream for a prolonged 

coagulation time in the circulatory system. Thus, a 

larger therapeutic window is offered until systemic 

anti-aggregation is administered. Studies evaluating 

nanoparticles effects on plasma coagulation time 

and their tendency to initiate vascular thrombosis 

are useful to assess nanoparticle thrombogenicity 

[1,2,3].  

A number of nanotechnology approaches have 

been borrowed and applied in stent technology. 

Stents with biofunctionalized surfaces with 

nanoparticles with high stability and carrier capacity, 

able to reduce the interaction with blood 

components and to incorporate substances as 

carriers for bioactive factors and drugs have been 

manufactured. There are already implants in the 

form of drug-eluting or biodegradable stents that 

induce healing reactions by triggering the body's 

natural processes [2,3]. 

At present, there is not much data on the 

thrombogenicity of stent materials dictated by nano-

scale observations. Because thrombogenicity is a 

multiparametric process, our study tests the 

hypothesis of the influence of surface 

nanotopography on platelet activation, in order to 

produce nano-coatings with less thrombogenic stent 

by adapting their surface properties. It is in fact 

intended to implement a real-time study of platelet 

response to biomaterials to improve 

hemocompatibility. 

The primary phenomenon of blood-material 

interaction was the rapid and selective adsorption of 

proteins through a three-step process represented 

by the transport to the interface, the adsorption 

reaction and the conformational rearrangement. 

Thus, the engineering of the surface properties 

(physical and chemical characteristics) of the 

implants aims to reduce the adsorption of proteins 

and cellular interactions resulting in the 

improvement of implant biocompatibility. 

Platelets are the main cells present in the blood 

that play the most important role in blood-material 

interactions. The adhesion of platelets to a surface is 

conditioned by two independent mechanisms. These 

are the transport of platelets to the surface, which 

depends on the flow conditions and the reaction of 

platelets with the surface, which depends on the 

nature of the surface and the adsorbed proteins. 

In this paper, we want to verify the influence of 

nanotopography and surface characteristics on the 

thrombogenicity of different possible types of nano-

coatings (ZnO, TiO2 and Fe3O4). Thus, the results 

obtained by us are in line with previous 

hemocompatibility studies in the literature regarding 

the influence of these nanoparticles on coagulation. 

These lead us to the conclusion that 

nanotopography and surface roughness of 

biomaterials influence their biological behaviour. 

The studies within our project will be continued by 

thrombogenicity analyses on nanoparticle-coated 

metal bands. 

 

This study about thrombogenicity induced by the 

nanoparticle is the subject of the grant: "New 

diagnostic and treatment methodologies: current 

challenges and technological solutions based on 

nanoparticles and biomaterials", that won the 2017 

complex projects completed in consortia CDI, grant 

number: PN-III-P1-1.2-PCCDI-2017-0062, funded by 

CNCS – UEFISCDI Romania. 

 

 

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