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Materials Technology
Advanced Performance Materials
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Carbon nanostructures grafted biopolymers for medical
applications
Nikola Slepickova Kasalkova, Pavlína Žáková, Ivan Stibor, Petr Slepička,
Zdeňka Kolská, Jana Karpíšková & Václav Švorčík
To cite this article: Nikola Slepickova Kasalkova, Pavlína Žáková, Ivan Stibor, Petr
Slepička, Zdeňka Kolská, Jana Karpíšková & Václav Švorčík (2019) Carbon nanostructures
grafted biopolymers for medical applications, Materials Technology, 34:7, 376-385, DOI:
10.1080/10667857.2019.1573943
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Carbon nanostructures grafted biopolymers for medical applications
Nikola Slepickova Kasalkovaa, Pavlína Žákováa, Ivan Stiborb, Petr Slepičkaa, Zdeňka Kolskác, Jana Karpíškováb
and Václav Švorčíka
aDepartment of Solid State Engineering, University of Chemistry and Technology Prague, Prague, Czech Republic; bInstitute for
Nanomaterials, Advanced Technologies and Innovation, Technical University of Liberec, Liberec, Czech Republic; cFaculty of Science, J. E.
Purkyně University, Ústí nad Labem, Czech Republic
ABSTRACT
Enhancing biopolymers with carbon nanostructures leads to the creation of attractive materials
suitable for diverse medical application. We studied surface properties and cytocompatibility of
amine functionalised carbon nanoparticles (CNPs) grafted on biopolymer film. Poly-L-lactic acid
and poly-3-hydroxybutyrate were treated in an inert argon plasma discharge and subsequently
grafted with three types of amine-functionalised CNPs. The surface properties of (i) CNPs and (ii)
grafted CNPs were studied using multiple methods. BET, electrokinetic and XPS analyses con-
firmed the successful grafting of amino-compounds on the surface and also into the pores of
CNPs. Goniometry approved the binding of themodified CNPs on the polymer surface. FromAFM
is evident that both the biopolymers show completely changed surface morphology after
grafting of CNPs. The cytocompatibility test with vascular smooth muscle cells showed that the
presence of the modified CNPs on polymer substrates has a more positive effect on cytocompat-
ibility of PLLA rather than PHB.
ARTICLE HISTORY
Received 15 August 2018
Accepted 21 January 2019
KEYWORDS
Biopolymers; grafting;
carbon nanoparticles; amine
functionalisation; surface
properties; cytocompatibility
Introduction
Tissue Engineering/Regenerative Medicine (TE/RM),
a multidisciplinary field, is a major revolution in the
field of medicine that focuses on the replacement and
regeneration of various tissues and organs of the
human body. Cell‒substrate interactions are crucial
features for the determination of materials applica-
tion in medical sciences. They play an important role
in determining cell growth, differentiation and orga-
nisation [1]. Biopolymer composites are of great sig-
nificance in tissue engineering because they provide a
favourable environment for the growth and differen-
tiation of cells, thus showing great promise in tissue
engineering research [2–4]. Carbon-based nanoma-
terials are currently very attractive nanomaterials
with their different forms, such as fullerenes, single-
and multiple-walled carbon nanotubes (CNTs), car-
bon nanoparticles (CNP), nanofibers, and so forth for
biological fields [5–7]. One of the issues related to the
use of carbon nanostructures, especially spherical
carbon nanoparticles (CNP), as therapeutic devices
is their cytotoxicity. Comparison of cytocompatibility
via measurement of cell viability the following trend
of cytocompatibility in various carbon nanostructures
(carbon nanodiamonds CND) was observed:
CND > CNP > CNTs [8]. Cytocompatibility of var-
ious carbon or other nanostructures strongly depends
on their surface chemistry, size, concentration or
pore size [8–10]. CNPs were proven to be
cytocompatible in lower concentration [11].
Fullerene molecules display a diverse range of biolo-
gical activity [12]. Their unique hollow cage-like
shape and structural analogy with clathrin-coated
vesicles in cells support the idea of the potential use
of fullerenes as drug or gene delivery agents.
Fullerene C60 deposited in the form of thin film
gave good support to the adhesion, spreading, growth
and viability of human osteoblast-like MG 63 cells
[13]. The discovery of the possible intercalation of
fullerenes into biological membranes has encouraged
many research groups to study the potential antimi-
crobial effects of C60 [14]. An amorphous form of
carbon – DLC – can be readily tuned by doping or
alloying with different elements to enhance its bio-
compatibility and control its conductivity [15]. Thin
carbon layer or carbon nanoparticles (CNTs, carbon
nanohorns, graphene) can be used to improve the
properties of the bioelectronic materials such as
neural electrodes [16]. The nanoparticles (graphene
oxide, carbon nanotubes, carbon nanohorns) may not
be used alone, but may be added to other material to
create hybrid materials with improved properties
[17,18] such as hybrid silicone systems with CNTs
(high strength-at-break (increase of ≥500% over
stand-alone silicone), undiminished intrinsic ductility
of silicone high cytocompatibility, and stimulated
osteoblasts functions and cellular interactions [19].
CNTs have appeared as a promising scaffold material
CONTACT Nikola Slepickova Kasalkova nikola.kasalkova@vscht.cz Department of Solid State Engineering, University of Chemistry and
Technology Prague, Prague 166 28, Czech Republic
MATERIALS TECHNOLOGY
2019, VOL. 34, NO. 7, 376–385
https://doi.org/10.1080/10667857.2019.1573943
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for in vitro culture of cells for their possible use in
tissue engineering and regenerative medicine [20].
CNTs can be heterogeneously surface-functionalised
and stained cytochemically with nonquenching and
non-photobleaching. Accordingly, CNTs may be sui-
table for bioapplications in biorecognition and drug
delivery systems [5]. Carbon nanoparticles (CNPs)
functionalised with amine groups and subsequently
chemically grafted onto the surface of polyethylene-
terephthalate (PET) and high-density polyethylene
(HDPE) had a positive effect on adhesion and pro-
liferation of VSMC on the polymers’ surface [21].
The CNPs can be used in the form nanodots. Their
advantage is that carbon nanodots can be prepared by
green synthesis, their low cost, photostability, low
toxicity, excellent water solubility, high sensitivity to
target analytes, tunable fluorescence emission and
excitation,and high quantum yield. Moreover, they
can be functionalised and rendered biocompatible.
Based on the excellent properties, carbon dots can
be made viable for sensors such as biosensors for
DNA, protein or metal ions [22,23]. Although few
applications of carbon dots as nanosensors for che-
mical analysis have been reported, the results indicate
that they can be easily functionalised and immobi-
lised in the polymer matrix for chemical or biochem-
ical analysis [23]. The CNPs or carbon layer cannot
always be on top of the substrates or scaffold; thus,
the possibility of the incorporation of CNM into
polymeric matrices can be considered. This material
not only improves the physical properties of the
scaffolds but also leads to the enhancement of cell
behaviour and physiological properties of the cardiac
tissue construct [24]. Carbon nanowalls can thus be
used to mediate locational cell growth and migration
[25]. Because of their tendency to aggregate carbon
nanoparticles have been modified using different
approaches to increase their stability in suspended
states. Commonly used approach is by introducing
polar groups onto the carbon surface [26,27].
However, for this purpose, the TE/RM cells need to
be ‘anchored’ to a substrate. Between adequate mate-
rials for such application belong polymers. In the area
of polymers, specific natural and synthetic materials
may be used to prepare the cellular carriers that allow
the body's own cells to grow and shape in the new
tissue, while the carrier is gradually absorbed. The
very commonly used materials are biopolymers, for
example, chitosan, polyglycolic acid (PGA) or poly-
lactic acid (PLLA) [28,29]. An innovative approach to
significantly enhance and tune the antimicrobial
activity of silver is to anchor or attach AgNPs along
the long axis of carbon nanotubes (CNTs) [30,31].
Biopolymers that are naturally derived are suitable for
medical applications because of their biocompatibility,
biodegradability, non-toxicity [32,33]. Biopolymers can
be divided into three groups according to their chemical
structure and source: (i) polysaccharides, (ii) proteins,
and (iii) microbial polymers. Microbial production of the
biopolymer is very versatile and its significance increases
due to the fact that they belong to renewable resources
[34,35]. Genetic manipulation of microorganisms allows
the biotechnological production of biopolymers with
tailored properties that make them suitable for high-
value medical applications such as tissue engineering
[2]. Microbial biopolymers can be of various chemical
structures such as polysaccharides, polyesters or polya-
mides [2,36]. The interaction of a graft with its surround-
ing tissue is the key factor for successful application of
tissue replacements. This interaction involves cell adhe-
sion, adsorption of extracellular molecules [21] and sup-
port of cell proliferation [37,38].
Very interesting group of materials suitable for bio-
medical applications are stimuli-responsive polymers
and injectable hydrogels based on biocompatible and
biodegradable precursors that disintegrate into non-
toxic degradation products on exposure with available
external conditions. Most of the natural biopolymers
such as polysaccharides are biocompatible and biode-
gradable. However, they do not have the stimuli-
responsive groups in their structures which render the
usage of resulting hydrogels. Stimuli-responsive func-
tional groups on these polymers can be introduced by
modification reactions [39]. These polymers find poten-
tial applications in drug delivery, emulsification, sen-
sing or detection, catalysis, self-healing, and so forth.
The response of these smart polymers to stimuli such as
pH, temperature, redox, magnetic field, light (ultravio-
let or visible), and so forth in macromolecular architec-
tures such as block, graft, star, cyclic, and crosslinked/
hyperbranched polymers has been extensively studied
[40,41]. Polymeric systems which respond to the com-
bination of two or multiple stimuli would be more
attractive for the release of the guest materials.
Hydrogels are more often used for the delivery of
active cells for soft and hard tissue regeneration
where the patient lacks organ formation [41]. The
advantage of this material is that in the injectable
hydrogel scaffolds the incorporation of cells, neces-
sary growth factors and other bioactive molecules are
suspended in the gel precursors prior to injection and
the suspension of gel precursors is injected at the
desired site of the body as a sol which results into
the in-situ gelation. Furthermore, injectables can
occupy the irregular defected site easily and homo-
geneously which avoid the requirement of any selec-
tive shape of the scaffold implant [42]
This work is focused on the study of plasma mod-
ified and subsequently amine-functionalised carbon
nanoparticles grafted biopolymers (poly-3-hydroxy-
butyrate and poly-L-lactic acid). Such modified sub-
strates were studied by analytical methods such as
goniometry, atomic force microscopy or XPS.
Cytocompatibility of selected samples was tested in
MATERIALS TECHNOLOGY 377
vitro using vascular smooth muscle cells from the
aorta of a rat (our work was focused on adhesion,
proliferation and viability of seeded cells).
Experimental
Material, plasma treatment, carbon nanoparticles
activation and their grafting
The experiments were carried out on poly-3-hydroxy-
butyrate with 8% polyhydroxyvalerate (PHBV) and
poly-L-lactic acid (PLLA) in the form of foils: (i)
PHBV (thickness 50 μm, 1.25 g cm−3, Goodfellow
Ltd., UK) and (ii) PLLA (50 µm, density 1.25 g cm−3,
Goodfellow Ltd., UK).
The polymers were treated by Ar+ plasma in Balzers
SCD 050 under the following conditions: gas purity
99.997%, pressure 10 Pa, electrode distance 50 mm,
with power 3 and 8 W and treatment time 120 s.
The carbon nanoparticles (Activated charcoal-
DARCO® KB-G, Sigma Aldrich, D, size 20–40 nm)
were used in this study. The carbon nanoparticles
(CNPs) were modified with ethylenediamine (ED),
triethylenetetramine (TET) and tris[2-(methylamino)
ethyl]amine (TMAE) in a three-step synthesis [43].
Such modified CNPs (molecular structure – see
Figure 1) were activated in 1 mol l−1 water solution
of HCl (1 h, room temperature (RT)).
The plasma treated polymers’ surfaces were grafted
from activated CNPs suspension (24 hours, RT, con-
stant stirring). In this work, we studied several types
of polymeric substrates samples: (i) pristine, (ii)
plasma treated, (iii) plasma treated and etched in
water solution of hydrochloric acid (environment
from which the CNPs are grafted) and (iv) plasma
treated, etched and CNPs grafted polymers. Scheme
of the experiment can be seen in Figure 2.
Used analytical methods
The properties of pristine, plasma treated and CNPs
grafted samples were studied using multiple analytical
methods.
Surface contact angle (CA, wettability) wasmeasured
using the static (sessile) water drop contact angle
method. The measurements and evaluation were per-
formed using the See System (Advex Instruments, CZ).
The CA was measured at RT on 10 different positions
on the sample surface using water (8 µl). The contact
angles of all modified samples were measured 30 days
after their complete modification. It is known that after
such ageing time the CA achieves saturation and
remains constant [21].
Surface morphology and roughness of the pristine
and modified samples were determined using a
VEECO CP II AFM device (tapping mode) equipped
with Si probe, RTESPA-CP with a spring constant
20–80 N m−1. By repeated measurements in the same
region (2 × 2 or 3 × 3 µm2), it was shown that the
surface morphology did not change after three con-
secutive scans. The roughness value (Ra) represents
Figure 1. Scheme of CNPs functionalised with different amines: (i) ethylenediamine (ED), (ii) triethylenetetramine (TET) and (iii)
tris[2-(methylamino)ethyl]amine (TMAE). The dark balls represent CNPs.
Figure 2. Scheme of the experiment step by step.
378 N. SLEPIČKOVÁKASÁLKOVÁ ET AL.
the arithmetic average of the deviations from the
central plane of the sample.
X-ray photoelectron spectroscopy (XPS, Omicron
Nanotechnology ESCAProbeP spectrometer,
Omicron Nanotechnology Ltd.), was used for mea-
surement of the atomic concentration of O(1s), C(1s),
N(1s), on the surface of CNPs [43].
Surface area and the total pore volumes of CNPs
were determined from adsorption and desorption
isotherms (Quantachrome Instruments, NOVA3200)
using NovaWin software and 5 points of Brunauer-
Emmett-Teller (BET) analysis for the total surface
area determination and 40 points Barrett‒Joyner‒
Halenda (BJH) model for the pore volume. Samples
were degassing at temperature of liquid nitrogen for
15 h, then adsorption and desorption isotherms were
measured with the nitrogen (N2, Linde, 99.999% pur-
ity). Each sample was measured five times.
CNPs (0.01 g) dispersed in KCl water solution
(4 ml of 0.01 mol L−1) were tested by dynamic light
scatterinng (Zetasizer ZS90, Malvern software Ver.
6.32) method for determination of electrokinetic
potential. Each sample was measured three times.
Cell culture, adhesion and proliferation and
viability
The adhesion and proliferation of vascular smooth
muscle cells (VSMC) on pristine and modified poly-
mers were studied in vitro. The samples were steri-
lized for 1 h in 70% ethanol in a Petri dish. After that,
they were inserted to 12-well plates (TPP, CH, dia-
meter 2.14 cm) and fixed to the well bottom with
plastic rings. VSMC (fifth passage) were seeded on
the samples with the density of 50,000 cells/well (i.e.
17,000 cells cm−2) into 3 ml of Dulbecco´s modified
Eagle Minimum Essential Medium (DMEM, Sigma,
USA, Cat. No. D5648) containing 10% fetal bovine
serum (FS, Sebak GmbH, Aidenbach, Germany) and
40 µm/ml of gentamicin (LEK, Ljubljana, Slovenia).
The cells were cultivated on the samples for 24, 72
and 144 h at temperature 37°C, humidity 85% and air
atmosphere containing 5% CO2.
After the cultivation times, the samples were
rinsed in phosphate-buffered saline (PBS) and fixed
in a deep-frozen 70% ethanol (−20°C) for 45 min.
The samples were then stained for 1 h at room
temperature with the following combinations of two
fluorescent dyes: (i) Texas Red C2-maleimide
(Molecular Probes, Invitrogen, No. T6008; concentra-
tion 20 ng ml−1 in PBS) which stains the membrane
and cytoplasmic proteins (red color) and (ii) Hoechst
# 33342 (Sigma, USA, 5 µg ml−1 PBS) which stains
the cell nuclei (blue color). Before taking photo-
graphs, the samples were rinsed in PBS. The number
and morphology of cells on the sample surface were
then evaluated in photographs taken under an
Olympus IX 51 microscope (objective 20×, 20 photos
for each sample, visualised area of 0.136 mm2),
equipped with an Olympus DP 70 digital camera.
The number of cells was determined using the
image analysis software NIS-Elements AR 3.0.
The viability of cultivated cells was measured on Vi-
Cell XR (Beckman Coulter) using the trypan blue dye
exclusion method. The samples were rinsed in PBS,
inserted to 12-well plates with trypsin and placed in
the thermostat for 7 min. After that DMEMwas added
to the samples in well plates. The cells in suspension
(1 ml) were then transferred to cuvettes and placed in
the automated Vi-Cell device.
Results and discussion
Characterisation of CNPs
Figure 3 presents the results on the total surface area
(black round points) and pore volumes (red squared
points) of CNPs determined by BET and BJH ana-
lyses before and after amino-compounds grafting.
The grafting of amino-compounds causes a decrease
in the total surface area from 1059 m2 g−1 and pore
volume from 0.602 cm3 g−1 (for unmodified CNPs) to
the lower values depending on used amino-com-
pounds. This decrease indicates the coverage of sur-
face and also of pores by grafted compounds.
It is clear that the smallest molecules of ED tend to
bind predominantly in the pores and only small
molecules graft on the CNPs surface (the pore
volume decreases dramatically to the value of
0.160 cm3 g−1). On the other hand, the longer mole-
cules of TET are grafted preferably on the surface
with the minimum molecules into the pores (pore
volume decreases to the value of 0.420 cm3 g−1).
The branched molecules of TMAE also fill up the
pores (pore volume decreases to the value of
0.169 cm3 g−1) and even due to the steric effect
their concentration on the surface is the lowest. The
grafting of individual amino-compounds is schema-
tically shown inside Figure 3. These results were
confirmed by XPS and electrokinetic determinations.
These results are also added in Figure 3. While the
highest amount of N was observed for TET indicating
the richest coverage of the surface with amino-com-
pounds, the concentration of N for TMAE is only
4.4%. Also the zeta potential indicates the presence of
amino-compounds on the CNPs. While the zeta
potential of pristine CNPs equals to the negative
value of −10.5 mV, zeta potential of all amino-grafted
CNPs changed to the positive values and the most
positive values were obtained also for TET (21.3 mV)
indicating the highest amount of amino-groups on
the surface, while for ED zeta potential is only
15.9 mV indicating the grafting of ED not only on
the CNPs surface but preferably to the pores.
MATERIALS TECHNOLOGY 379
Characterisation of CNPs grafted polymers
One widely accepted advanced ‘trend’ in tissue engineer-
ing is the creation of surface that promotes cell
colonisation. For this application, it is necessary to adjust
not only chemical composition but also surface wettabil-
ity [44]. The effect of plasma treatment on the PLLA and
Figure 3. Results of BET (surface area, black points) and BJH (pore volume, red points) analyses electrokinetic analysis (zeta
potential) and XPS surface elements C (1s), O (1s) and N (1s) determination of the pristine (pristine CNPs) and functionalised
CNPs (ethylenediamine, ED; triethylenetetramine, TET and tris[2-(methylamino)ethyl]amine, TMAE). Inside the figure, there is a
schematic illustration of grafting of individual amino-compounds on the surface and into the pores of the CNPs.
Figure 4. Water contact angle measurements of different PLLA and PHBV samples: etched in the water solution of hydrochloric
acid (/HCl) and CNPs grafted (/ED;/TET;/TMAE).
380 N. SLEPIČKOVÁ KASÁLKOVÁ ET AL.
the PHBV samples were described in detail elsewhere
[28]. Contact anglemeasurement was performed on both
biopolymer samples and they exhibit similar trends, see
Figure 4. In comparison with pristine biopolymer films
(CAPLLA = 71.3 ± 1.1° and CAPHBV = 64.5 ± 0.9°) etching
of those biopolymer films in the water solution of hydro-
chloric acid after plasma exposure causes significant
hydrofilisation of the polymer surface for both powers
of plasma (3 and 8W). This is caused primarily by newly
formed oxygen groups on the surface after plasma treat-
ment and subsequently after washing out fragments of
plasma degraded biopolymer by the solution of hydro-
chloric acid [43]. On the other hand, CA values of CNPs
grafted biopolymers increases for both PLLA and PHBV
substrates. In case of PLLA CA values of grafted sub-
strates (/ED,/TET,/TMAE) treated in 3 W plasma
discharge are above that of pristine polymer. Using
higher power of the plasma discharge, i.e. 8 W causes
the surface of such treated biopolymer to bemore hydro-
philic. The same trend can be observed with PHBV albeit
to a lesser extent. The individual differences between
individual substrates treated with 3 and 8 W plasma
discharge are very small for PHBV. In some cases, i.e.
plasma treated PHBV grafted with TET are the differ-
ences so small that they are in the margin of error.
AFM scans of pristine biopolymers can be seen in
Figure 5. Both biopolymers exhibit low surface rough-
ness, only 3.9 nm (for PLLA) and 3.2 nm (for PHBV).
Grafting of CNPs onto the surface causes a slight
increase of surface roughness as can be seen in Figure
6. Both biopolymers show completely changed surface
morphology after graftingof CNPs compared to pristine
Ra = 3.9 nm
PLLA
Ra = 3.2 nm
PHBV
Figure 5. AFM scans of pristine PLLA and PHBV samples. Ra represents the average surface roughness and is given in
nanometers.
Ra=12.3 nm
PLLA/3/ED
Ra=11.4 nm
PHBV/3/ED
Ra=9.1 nm
PLLA/8/ED
Ra=12.9 nm
PHBV/8/ED
Figure 6. AFM scans of PLLA and PHBV samples treated by 3 and 8 W plasma and subsequently grafted with ED. Ra represents
the average surface roughness and is given in nanometers. Plasma treatment time for all modified samples was 120 s.
MATERIALS TECHNOLOGY 381
substrates. On all ED grafted samples can be seen similar
structures created by CNPs, i.e ED grafting on the bio-
polymer surface.
In vitro tests of cytocompatibility and viability of
CNPs grafted polymers
Cytocompatibility was studied on both biopolymers
and on various types of samples: (i) pristine, (ii) plasma
treated and (iii) CNPs grafted substrates. For cytocom-
patibility testing were chosen substrates that have been
treated in 8Wplasma discharge for 120 s [37]. In Figure
7 we can see a number of adhered a proliferated VSMCs
on pristine, plasma treated and CNPs grafted substrates.
After plasma treatment, there is increment in the num-
ber of proliferated cells for both biopolymers. However,
in the case of plasma treated PHBV, there is also a
significant deviation that is caused by uneven coverage
of different parts of the samples. After grafting of CNPs
there is also an increase in the number of cultivated
cells. For PLLA, the increase is lower for grafted samples
than on those treated only in the plasma discharge.
Figure 7. The number of VSMCs for different cultivation periods (first, third and sixth day) on PLLA and PHBV samples: pristine
(PLLA), plasma treated (/120), and CNPs grafted (/ED,/TET,/TMAE). Plasma exposure time for all modified samples was 120 s and
power 8 W.
Figure 8. The viability of VSMCs for different cultivation periods (first, third and sixth day) on PLLA and PHBV samples: pristine
(PLLA), plasma treated (/120), and CNPs grafted (/ED,/TET;/TMAE). Plasma exposure time for all modified samples was 120 s and
power 8 W.
382 N. SLEPIČKOVÁ KASÁLKOVÁ ET AL.
However, those samples show also decreased deviation
in cell number; therefore, cells aremore homogeneously
spread on the surface of such modified substrates. The
number of cultivated cells on the surface of CNPs
grafted PLLA are similar for all three types of CNPs. A
similar trend can be seen with PHBV samples.
However, there is a significant decrease of deviations
for CNPs grafted substrates in comparison with plasma
treated substrates.
Another significant parameter in cytocompatibility
testing is cell viability, see Figure 8. Viability of cells
cultured on pristine substrates was very high for PHBV
and acceptable for PLLA. However, the amount of
cultivated cells was so small that the relatively high cell
viability is of small importance. Plasma treated sub-
strates differed in cell viability for both biopolymers.
Plasma treated PLLA exhibits good values of cell viabi-
lity; however, plasma-treated PHBV showed cell
PLLA/ED 
PHBV/TMAE
PHBV
PHBV/ED
PLLA 
PLLA/TET PHBV/TET
PLLA/TMAE 
Figure 9. Fluorescence microscopy images of VSMCs adhered (first day) and proliferated (sixth day) on various PLLA and PHBV
samples: pristine (PLLA, PHBV), plasma treated (/120), and CNPs grafted (/ED;/TET;/TMAE). Plasma treatment time for all
modified samples was 120 s and power 8 W.
MATERIALS TECHNOLOGY 383
viability of only 63% (sixth day). In the case of grafted
PLLA substrates, they show similar cell viability for all
types of CNPs on the sixth day of cultivation. In com-
parison with pristine PHBV substrate grafted with
CNPs they show an increase of cell viability. The cell’s
attachment to modified PHBV may be less strong than
to PLLA and the trypsinisation process may cause
damage to some already loosen cells.
As can be seen on the photograph in Figure 9 cells
cultivated on CNPs grafted substrates are fairly homo-
geneously spread on the sample surface. They exhibit
correct physiological size and shape. From the photo-
graphs is evident that PHBV grafted with ED is more
densely covered in comparison with PHBV grafted with
TMAE, the more branched amine functionalised CNPs.
In case of PLLA, there is visible the similar coverage of
cells on each type of grafted sample.
Conclusion
We studied surface properties and cytocompatibility of
biopolymer films (PLLA and PHBV) grafted with three
types of amine functionalised carbon nanoparticles using
two different powers of plasma discharge. BET analysis
(surface area, pore volume) and electrokinetic analyses of
CNPs confirmed the successful grafting of tested amino-
compounds on the surface and also into the pores (espe-
cially for ED and TMAE). The highest amount of amino-
groups on the surface were analysed on CNPs grafted
with TET. Contact angle measurements show significant
differences in CA values of CNPs grafted PLLA after
both plasma discharge powers (i.e. 3 and 8 W).
Contrary for PHBV there were no significant differences
between both used plasma discharges prior to CNPs
grafting. There are changes in surface morphology and
surface roughness consistent with CNPs grafting. Cell
adhesion and proliferation were studied using VSMCs.
The results suggest that CNPs have a positive effect on
cell cultivation. All used cytocompatibility tests suggest
that cells cultivated on grafted substrates are spread
homogeneously on substrates’ surface and they exhibit
correct physiological features such as size and morphol-
ogy. In overall conclusion CNPs grafted PLLA substrates
evinced more positive cytocompatibility results, i.e. high
cell viability than CNPs grafted PHBV.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Grant Agency of Health
Ministry no. 15-33018A.
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MATERIALS TECHNOLOGY 385
	Abstract
	Introduction
	Experimental
	Material, plasma treatment, carbon nanoparticles activation and their grafting
	Used analytical methods
	Cell culture, adhesion and proliferation and viability
	Results and discussion
	Characterisation of CNPs
	Characterisation of CNPs grafted polymers
	In vitro tests of cytocompatibility and viability of CNPs grafted polymers
	Conclusion
	Disclosure statement
	Funding
	References