Please cite this article in press as: V. Grumezescu, et al.

, MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-

co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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Applied Surface Science xxx (2014) xxxxxx
Contents lists available at ScienceDirect
Applied Surface Science
j our nal homepage: www. el sevi er . com/ l ocat e/ apsusc
MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric
acid-co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated
surfaces with anti-microbial properties
Valentina Grumezescu
a,b
, Alina Maria Holban
c
, Florin Iordache
d
, Gabriel Socol
a
,
George Dan Mogos anu
e
, Alexandru Mihai Grumezescu
b,
, Anton Ficai
b
,
Bogdan S tefan Vasile
b
, Roxana Trus c a
f
, Mariana Carmen Chiriuc
c
, Horia Maniu
d
a
Lasers Department, National Institute for Lasers, Plasma & Radiation Physics, P.O. Box MG-36, Magurele, Bucharest, Romania
b
Department of Science and Engineering of Oxidic Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnica
of Bucharest, 17 Polizu Street, 011061 Bucharest, Romania
c
Microbiology Immunology Department, Faculty of Biology, University of Bucharest, 13 Portocalelor Lane, Sector 5, 77206Bucharest, Romania
d
Institute of Cellular Biology and Pathology of Romanian Academy, Nicolae Simionescu, Department of Fetal and Adult StemCell Therapy, 8, B.P. Hasdeu,
Bucharest 050568, Romania
e
Department of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 PetruRares Street,
200349 Craiova, Romania
f
S.C. Metav-CD S.A., 31Rosetti Str., 020015 Bucharest, Romania
a r t i c l e i n f o
Article history:
Received 26 October 2013
Received in revised form20 January 2014
Accepted 21 January 2014
Available online xxx
Keywords:
MAPLE
Magnetite
Iron oxide nanostructure
Eugenol
S. aureus
Biolminhibition
Natural products
a b s t r a c t
This study reports the biological applications of a newly fabricated water dispersible nanostructure, based
on magnetite (Fe
3
O
4
) and eugenol (E), prepared in a well-shaped spherical form by precipitation method.
The presence of Fe
3
O
4
@E nanoparticles has been conrmed by transmission electron microscopy (TEM).
Nanoparticles have beenembedded into poly(3-hidroxybutyric acid-co-3-hidroxyvaleric acid)polyvinyl
alcohol (P(3HB-3HV)PVA) microspheres by oil-in-water emulsion technique. Functionalized P(3HB-
3HV)PVAFe
3
O
4
@E microspheres coatings have been fabricated by matrix assisted pulsed laser
evaporation (MAPLE). The coatings have been characterized by infrared microscopy (IRM) and scan-
ning electron microscopy (SEM). In vitro biolm formation by Staphylococcus aureus and Pseudomonas
aeruginosa was assessed by the viable cell counts technique. Nanomaterial biocompatibility has been
investigated by analyzing the phenotypic changes of cultured eukaryotic cells. Besides their excellent
anti-adherence and anti-biolm properties, the MAPLE coatings have the advantages of using bioactive
natural compounds, which are less toxic and easily biodegradable than current antibiotics. This approach
could be used as a successful alternative or adjuvant method to control and prevent microbial biolms
associated infections.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Recent progress in bio- and nanotechnology has led to the
expansion of a rich variety of novel materials, precisely manufac-
tured at nanoscale level, withgreat potential to improve traditional
medical treatments and therapies [13]. Nanobiotechnology prod-
ucts offers numerous applications in health care and biomedical
eld [46]. Nanobiomaterials based on magnetite are frequently
used in drug delivery [710], drug targeting [1115], cancer
therapy [16,17], modulation of microbial biolms [1820] or mag-
netic resonance imaging [21,22]. The preparation of magnetite

Corresponding author. Tel.: +40765349326.

E-mail address: grumezescu@yahoo.com(A.M. Grumezescu).
bionanostructures involves the understanding of the complex
interactions of the inorganic core with different biomolecules
[4,23,24]. Surface functionalized magnetite nanoparticles have
received increasing attention due to their improved properties
for biological applications [25]. Studies are currently focused on
developing appropriate strategies to improve the characteristics
of magnetite nanoparticles used for the inhibition of microbial
biolms.
Recently, 10nm magnetite particles functionalized with nat-
ural products, as usnic acid exhibited an efcient antimicrobial
activity against planktonic and adherent cells, especially on Gram-
positive strains [26]. Magnetite nanoparticles functionalized with
different antibiotics exhibited an inhibitory activity on growth and
biolm formation of Staphylococcus aureus superior to that exhib-
ited by eachantibiotic alone [27]. Also, the magnetite nanoparticles
0169-4332/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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strongly inhibited the mature biolms development, as well as the
fungal adherence to the cellular substratum, suggesting that mag-
netic nanoparticles could be used for obtaining newmaterials with
anti-adherence properties [28].
The studied magnetic nanoparticles act as efcient anti-biolm
agents for combating microbial biolms on biomedical devices or
humantissues [29]. For this applicationit is alsoessential toprepare
micro and nanoparticles with controlled size and morphology.
Biolm-associated microbial infections represent a major cause
of mortality and morbidity among hospitalized patients worldwide
[30]. Even though we are still living in the era of antibiotics, their
extended and irrational use led to a signicantly decreased ef-
ciency of these chemotherapeutic compounds in both planktonic
and biolm-related infections [31]. The great resistance of biolm
embedded bacteria is due mainly to the protective matrix of the
complexmicrobial consortiumandalsotothe enhancedcell-to-cell
exchange of resistance genes and metabolism modulation within
biolms [32]. The major causes of persistent biolm-related infec-
tions are the Gram positive versatile bacterium S. aureus [33] and
the opportunistic Gramnegative pathogenPseudomonas aeruginosa
[34]. These bacteria are often encountered on the skin and mucosa
of healthy individuals, but may cause difcult to treat infections in
nosocomial environment. Severe staphylococcal and pseudomon-
ades associated infections are frequently reported for patients with
implanted prosthetic devices, since these bacteria have the ability
to successfully adhere and produce biolms on the surface of the
prosthetic material [31]. Apart of the ability to grow in biolms
withina susceptible host, S. aureus andP. aeruginosa are some of the
most resistant bacteria species, making most antibiotics infective
[31]. Therefore, novel therapeutic approaches, based on alternative
compounds with low potential for developing bacteria resistance
are of a high interest.
The aim of this study was to develop a new nanobioactive
surface prepared by MAPLE based on magnetite@eugenol and (3-
hidroxybutyric acid-co-3-hidroxyvaleric acid)polyvinyl alcohol
microspheres withgreat antibiolmproperties, whichmay be used
for developing novel in vitro biocompatible medical surfaces with
antimicrobial properties.
2. Materials and methods
2.1. Materials
Ferrous sulfate heptahydrate (FeSO
4
7H
2
O), ferric chloride
(FeCl
3
), ammonia (NH
3
), (3-hidroxybutyric acid-co-3-
hidroxyvaleric acid), polyvinyl alcohol and eugenol (E) were
purchased fromSigmaAldrich.
2.2. Preparation of Fe
3
O
4
@E
Fe
3
O
4
@E was prepared according to our recently published
paper [35].
2.3. Preparation of P(3HB-3HV)PVAFe
3
O
4
@E microspheres
P(3HB-3HV)PVAFe
3
O
4
@E microspheres were prepared using
a previously reported method [36,37]. 400mg P(3HB-3HV) were
solubilized in 3mL chloroform by sonication. The organic phase
was emulsied with a sonicator, model SONIC-1200WT fromMRC
for 5min, in ON/OFF steps of 5 and 3s with a limitation tempera-
ture of max 40

C in 5mL aqueous phase containing 2% (w/v) PVA

and 100mg Fe3O4@E. After sonication, the emulsion was added
in 100mL deionized water and stirred for 4h until the chloroform
was completely evaporated. The microsphere emulsion was cen-
trifuged at 6000rpm for 10min. The obtained microspheres were
washedfour times withdeionizedwater, collectedbyltration, and
thensubjectedtofreeze drying. P(3HB-3HV)PVAFe
3
O
4
@Emicro-
spheres were used to prepare the coatings by MAPLE technique.
2.4. MAPLE target preparation and experimental details
MAPLE targets were prepared by freezing for 30min at
liquid nitrogen temperature a suspension of 1% (w/v) P(3HB-
3HV)PVAFe
3
O
4
@Emicrospheres inDMSO. The radiationof a KrF*
(=248nmand
FWHM
=25ns) laser source COMPexPro205model,
Lambda Physics-Coherent impinged the frozen targets at a laser
unce of (300500) mJ/cm
2
and a repetition rate of 15Hz. A laser
beamhomogenizer was used to improve the energy distribution of
the laser spot. During the deposition, the target was rotated with
0.4Hz to avoid the heating and drilling of target. All depositions
wereconductedat roomtemperatureintoabackgroundpressureof
0.1Pa and a target-substrate separation distance of 5cmby apply-
ing (45,000160,000) subsequent laser pulses. During deposition,
the MAPLE target was kept at lowtemperature by continuos liquid
nitrogen cooling. The coatings were deposited onto glass, both side
polished (100) silicon for IRM, SEM, and biological assays. Prior to
introduction inside the deposition chamber, the substrates were
successively cleaned into an ultrasonic bath with acetone, ethanol
anddeionizedwater for 15min, driedinajet of highpuritynitrogen.
3. Characterization
3.1. TEM
The transmission electron microscopy (TEM) images were
obtained on nely powdered samples using a Tecnai
TM
G2 F30 S-
TWIN high resolution transmission electron microscope from FEI
Company (OR, USA) equipped with SAED. The microscope oper-
ated in transmission mode at 300kV with TEMpoint resolution of
2

A and line resolution of 1

A. The Fe
3
O
4
@E was dispersed into pure
ethanol and ultrasonicated for 15min. After that, diluted sample
was poured onto a holey carbon-coated copper grid and left to dry
before TEManalysis.
3.2. IRM
IR mapping were recorded on a Nicolet iN10 MX FT-IR
Microscope with MCT liquid nitrogen cooled detector in the mea-
surement range 4000700cm
1
. Spectral collection was made in
reection mode at 4cm
1
resolution. For each spectrum, 32 scans
were co-added and converted to absorbance using OmincPicta
software (Thermo Scientic). Approximately 600 spectra were
analyzed for each coating and dropcast. Four absorptions peaks
known as being characteristics for the PLGA-PVA microspheres
were selected as spectral markers of magnetic microspheres pres-
ence in the prepared coatings.
3.3. SEM
SEManalysis was performedona FEI electronmicroscope, using
secondary electron beams with energies of 30keV, on samples cov-
ered with a thin gold layer.
3.4. Cell viability and uorescence microscopy
Human endothelial cells (EAhy926 cell line) were grown in
DMEM culture medium containing 10% FBS, and 1% penicillin and
neomycin (Sigma Aldrich, St. Louis, MO, USA). For cell prolifera-
tion and viability assay a kit with two standardized solutions (I
and II) was used (CellTiter 96 Non-Radioactive Cell Proliferation
Assay, Promega, Madison, USA). EAhy926 cells were seeded in 96-
well plate at a density of 510
3
cells/well, in DMEM medium,
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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Fig. 1. TEMimage (a) and SAED pattern (b) of Fe
3
O
4
@E.
supplemented with 10% foetal bovine serum (FBS). The cultures
were incubated with the microsphere coatings for 2472h, while
the controls were represented by endothelial cells grown in the
same culture conditions, but on bare substrates. Cell proliferation
assay was performed in triplicates, according to manufacturers
guidelines, at different time intervals. Briey, 15l of Promega Kit
Solution I was added in each well and incubated for 4h. Further-
more, afterwards 100l of Promega Kit Solution II was added in
the 96-well plate and incubated for another hour and spectropho-
tometry mesurements were performed at 570nmusing Mithras LB
940 (Berthold Technology, Germany).
REDCMTPXuorophore (Life Technologies, Invitrogen, USA) is a
cell tracker for long-termtracing of living cells. The REDCMTPXdye
was added in the culture mediumat a nal concentration of 5M,
incubated 30min for allowing the dye to penetrate through the
cells. Furthermore, the cells were washed with posphate buffered
saline (PBS) and visualized by uorescence microscopy. The nuclei
were counterstained with DAPI (1mg/ml). The photomicrographs
were taken by a digital camera driven by the software Axio-Vision
4.6 (Carl Zeiss).
3.5. Microbial biolmassay
Biolm formation was assessed using 6 multi-well plates
(Nunc), in a static model for monospecic biolm development.
Coated and uncoated glass substrata were distributed in the plates
containing 2mL of microbial inoculum diluted to 10
4
10
5
CFU/ml
in Luria Broth. Samples were incubated for 24h at 37

C. Biolms
formation was assessed after 24h, 48h and 72h by viable cell
counts assay [38].
After 24h incubation time the culture medium was removed
and the the samples were washed with sterile PBS, in order to
remove the unattached bacteria. Coated and uncoated substrates
were placed in fresh medium and incubated for other additional
24h, 48h and 72h. After the incubation, the samples were gently
washed with sterile PBS to remove the non-adherent cells and
placed in 1.5mL microcentrifuge tubes containing 750L PBS. In
order to disperse biolm cells into the suspension, the samples
were vigorously mixed by vortexing for 30s and sonicated for 10s.
Serial ten-fold dilutions were achieved and plated on LB Agar for
viable cell counts assay. Experiments were performed in triplicate
and repeated in three separate occasions [39,40].
4. Results and discussions
The synthesized magnetite nanoparticles are shown Fig. 1. It
can be noticed that the particles are nanometric size (less than
10nm) with homogenous particles size distribution. Also, it can be
observed that the particles have a quasi-spherical shape without
aggregation. Structural information from Fe
3
O
4
@E was obtained
from SAED analysis. The diffraction rings of the Fe
3
O
4
@E (Fig. 1b)
indicate that the prepared nanostructures is polycrystalline, and
the labeled indices match with the lattice facets of cubic mag-
netite (Fe
3
O
4
) without the presence of any other crystalline phases
[41].
Fig. 2. Second derivate IR mappings and IR spectra of dropcast surface: intensity distribution of (a) 2973cm
1
(CH
3
stretch), (b) 1728cm
1
(carbonyl group), (c) 1279cm
1
(C H), (d) 1056cm
1
(C O stretch).
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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Fig. 3. Second derivate IR mappings and IR spectra of coating (F =300mJ/cm
2
) surface: intensity distribution of (a) 2973cm
1
(CH
3
stretch), (b) 1728cm
1
(carbonyl group),
(c) 1279cm
1
(C H), (d) 1056cm
1
(C O stretch).
Fig. 4. Second derivate IR mappings and IR spectra of coating (F =400mJ/cm
2
) surface: intensity distribution of (a) 2973cm
1
(CH
3
stretch), (b) 1728cm
1
(carbonyl group),
(c) 1279cm
1
(C H), (d) 1056cm
1
(C O stretch).
Figs. 25 represent the second derivative infrared micrographs
of P(3HB-3HV)PVAFe
3
O
4
@E microspheres coatings prepared by
MAPLE at laser uencies of 300, 400 and 500mJ/cm
2
, respectively.
Absorbance intensities of IR spectra maps are proportional to color
changes starting with blue (the lowest intensity) and gradually
increasing through green, yellow to nally red (the highest inten-
sity) [42].
Analyzing the IR spectra and IR mapping for each sample
deposited at different laser uences and the corresponding
dropcast coating, we selected a laser uence of 300mJ/cm
2
as a
compromise between the deposition rate and the preservation
of the chemical structure of the constituent compounds. Higher
laser uences (400 and 500mJ/cm
2
) altered the integrity of the
monitored functional groups (Figs. 4 and 5).
Morphological examination of coatings prepared at
F =300mJ/cm
2
using scanning electron microscopy showed
that the microparticles into the coatings were uniformly dis-
tributed with binding tendency, but without forming compact
Fig. 5. Second derivate IR mappings and IR spectra of coating (F =500mJ/cm
2
) surface: intensity distribution of (a) 2973cm
1
(CH
3
stretch), (b) 1728cm
1
(carbonyl group),
(c) 1279cm
1
(C H), (d) 1056cm
1
(C O stretch).
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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Fig. 6. SEMmicrographs of P(3HB-3HV)PVAFe
3
O
4
@E microspheres coatings.
Fig. 7. Human endothelial cells (EAhy926 cell line) metabolism revealed by MTT
method, after growing on control vs. thin coating surfaces for 24h, 48h, 72h.
aggregates at the surface microspheres diameter was between
500nmand 2m(Fig. 6).
Nanobiomaterials offer great promises in biomedical eld.
However, not all potential health hazards are understood. The
usefulness of MAPLE fabricated microspheres for anti-infectious
therapies as well as their potential negativesideeffects has not been
reported yet. Our results demonstrate that the obtained coatings
proved great biocompatibility. Cell viability assay showed that in
the presence of P(3HB-3HV)PVAFe
3
O
4
@E coatings, endothelial
cells are viable, moreover at 24h after incubation cell proliferation
was increased. Viability of endothelial cells at 48h, respectively
72h was maintained compared to control, suggesting that this
type of structures based on magnetite and eugenol are highly
Fig. 9. Graphic representation of viable cell counts analysis after removing P.
aeruginosa biolm embedded cells at 24h, 48h and 72h post infection (P(3HB-
3HV)PVAFe
3
O
4
@E coatings vs. uncoated control).
biocompatiblefor theinvitrotestedendothelial cell model. Further-
more, uorescence microscopy analysis of endothelial cells labeled
with RED CMTPX cell tracker has conrmed the viability of these
cells after 5 days in the presence of P(3HB-3HV)PVAFe
3
O
4
@E.
Inside the cells, the RED CMTPX dye reacts with tiols group, in a
glutathione S-transferase-mediated reaction, and is transformed
into a cell-impermeant uorescent dye (577602nm), thus the
presence of uorescent cells is related to viable endothelial cells
(Figs. 7 and 8).
Microbiology results demonstrated that the nanocoated bioac-
tive samples exhibited a great antimicrobial efcacy, inhibiting
the microbial adherence and biolm development of S. aureus
Fig. 8. Human endothelial cells (EAhy926 cell line) after ve days of growth on (a) control; (b) coated slides.
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
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Fig. 10. Graphic representation of viable cell counts analysis after removing S.
aureus biolm embedded cells at 24h, 48h and 72h post infection (P(3HB-
3HV)PVAFe
3
O
4
@E coatings vs. uncoated control).
and P. aeruginosa tested strains, being a successful candidate for
developing novel alternative antimicrobial strategies. Pseudomon-
adal biolm development was signicantly impaired in the rst
stages of development, but the effect of the nano-modiedmaterial
decreased during time (Fig. 9).
Onthe other handthe testedbioactive material exhibiteda great
inhibitory effect against S. aureus biolmdevelopment at the three
time points tested (Fig. 10). This effect may be explained by the
fact that eugenol has a greater antimicrobial effect against Gram-
positive, than on Gram-negative strains.
5. Conclusions
Our results proved that the obtained microspheres coatings,
combining the excellent structural properties of the iron oxide
nanoparticles and the antimicrobial properties of eugenol, rep-
resent a novel and successful alternative for inhibiting microbial
adhesion and biolmformation on medical devices and other clin-
ically relevant materials and surfaces. Furthermore, this material
proved great biocompatibility, being a successful candidate for
developing novel anti-biolmstrategies.
Acknowledgement
This work was supported by the Romanian National Author-
ity for Scientic Research, CNCS-UEFISCDI, project number
PCCA153/02.07.2012.
References
[1] R. Cristescu, C. Popescu, A.C. Popescu, S. Grigorescu, L. Duta, I.N. Mihailescu,
G. Caraene, R. Albulescu, L. Albulescu, A. Andronie, I. Stamatin, A. Ionescu, D.
Mihaiescu, T. Buruiana, D.B. Chrisey, Functionalized polyvinyl alcohol deriva-
tives thin lms for controlled drug release and targeting systems: MAPLE
deposition and morphological, chemical and in vitro characterization, Appl.
Surf. Sci. 255 (2009) 56005604.
[2] R. Cristescu, C. Popescu, A.C. Popescu, S. Grigorescu, L. Duta, I.N. Mihailescu,
A. Andronie, I. Stamatin, O.S. Ionescu, D. Mihaiescu, T. Buruiana, D.B. Chrisey,
Laser processing of polyethylene glycol derivative and block copolymer thin
lms, Appl. Surf. Sci. 255 (2009) 56055610.
[3] A.M. Grumezescu, E. Andronescu, V. Grumezescu, C. Bleotu, C. Saviuc, D.E.
Mihaiescu, C.M. Chiriuc, Biocompatible magnetic hollow silica microspheres
for drug delivery, Curr. Org. Chem. 17 (2013) 10291033.
[4] S. Theerdhalaa, D. Bahadur, S. Vitta, N. Perkas, Z. Zhong, A. Gedanken,
Sonochemical stabilization of ultrane colloidal biocompatible magnetite-
nanoparticles using amino acid, l-arginine, for possible bio applications,
Ultrason. Sonochem. 17 (2010) 730737.
[5] I. Asghari, F. Esmaeilzadeh, Formationof ultranedeferasiroxparticles viarapid
expansion of supercritical solution (RESS process) using Taguchi approach, Int.
J. Pharm. 433 (2012) 149156.
[6] M. Gary-Bobo, O. Hocine, D. Brevet, M. Maynadier, L. Raehm, S. Richeter, V. Cha-
rasson, B. Loock, A. Morre, P. Maillard, M. Garcia, J.O. Durand, Cancer therapy
improvement with mesoporous silica nanoparticles combining targeting, drug
delivery and PDT, Int. J. Pharm. 423 (2012) 509515.
[7] A.M. Grumezescu, E. Andronescu, A. Ficai, D. Ficai, K.S. Huang, I. Gheorghe, M.C.
Chiriuc, Water soluble magnetic biocomposite with potential applications for
the antimicrobial therapy, Biointerface Res. Appl. Chem. 2 (2012) 469475.
[8] A.M. Grumezescu, A.M. Holban, E. Andronescu, A. Ficai, C. Bleotu, M.C. Chi-
riuc, Water dispersible metal oxide nanobiocomposite as a potentiator of
the antimicrobial activity of kanamycin, Lett. Appl. Nanobioscience 1 (2012)
7782.
[9] A.M. Grumezescu, A. Holban, E. Andronescu, A. Ficai, C. Bleotu, M.C. Chiriuc,
Microbiological applications of a newwater dispersible magnetic nanobiocom-
posite, Lett. Appl. Nanobioscience 1 (2012) 8390.
[10] D.E. Mihaiescu, M. Horja, I. Gheorghe, A. Ficai, A.M. Grumezescu, C. Bleotu,
C.M. Chiriuc, Water soluble magnetite nanoparticles for antimicrobial drugs
delivery, Lett. Appl. Nanobioscience 1 (2012) 4549.
[11] A.M. Grumezescu, A. Ficai, D. Ficai, G. Prdean, M.C. Chiriuc, Polymeric magnetic
silica microspheres as a drug loader for antimicrobial delivery substances, Dig.
J. Nanomater. Bios. 7 (2012) 18911896.
[12] A.M. Grumezescu, E. Andronescu, A. Ficai, C. Bleotu, D.E. Mihaiescu, M.C. Chi-
riuc, Synthesis, characterization and in vitro assessment of the magnetic
chitosan-carboxymethylcellulosebiocomposite interactions with the prokar-
yotic and eukaryotic cells, Int. J. Pharm. 436 (2012) 771777.
[13] E. Andronescu, A.M. Grumezescu, A. Ficai, I. Gheorghe, M. Chiriuc, D.E. Mihai-
escu, V. Lazar, In vitro efcacy of antibiotic magnetic dextran microspheres
complexes agains Staphylococcus aures and Pseudomonas aeruginosa strains,
Biointerface Res. Appl. Chem. 2 (2012) 332338.
[14] C. Huang, Z. Tang, Y. Zhou, X. Zhou, Y. Jin, D. Li, Y. Yang, S. Zhou, Magnetic
micelles as a potential platformfor dual targeted drug delivery in cancer ther-
apy, Int. J. Pharm. 429 (2012) 113122.
[15] M. Jansch, P. Stumpf, C. Graf, E. Rhl, R.H. Mller, Adsorption kinetics of plasma
proteins on ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles,
Int. J. Pharm. 428 (2012) 125133.
[16] A.M. Grumezescu, E. Andronescu, A. Ficai, C.H. Yang, K.S. Huang, B.S. Vasile, G.
Voicu, D.E. Mihaiescu, C. Bleotu, Magnetic nanouid with antitumoral proper-
ties, Lett. Appl. Nanobioscience 1 (2012) 5660.
[17] X. Chen, H. Lv, M. Ye, S. Wang, E. Ni, F. Zeng, C. Cao, F. Luo, J. Yan, Novel super-
paramagnetic iron oxide nanoparticles for tumor embolization application:
preparation, characterization and double targeting, Int. J. Pharm. 426 (2012)
248255.
[18] A.M. Grumezescu, M.C. Chiriuc, C. Saviuc, V. Grumezescu, R. Hristu, D. Mihai-
escu, G.A. Stanciu, E. Andronescu, Hybrid nanomaterial for stabilizing the
antibiolmactivityof Eugeniacarryophyllataessential oil, IEEETrans. Nanobio-
science 11 (2012) 360365.
[19] A.M. Grumezescu, C. Saviuc, C.M. Chiriuc, R. Hristu, D.E. Mihaiescu, P.
Balaure, G. Stanciu, V. Lazar, Inhibitory activity of Fe
3
O
4
/oleic acid/usnic
acidcore/shell/extra-shell nanouid on S. aureus biolm development, IEEE
Trans. Nanobioscience 10 (2011) 269274.
[20] C. Saviuc, A.M. Grumezescu, M.C. Chiriuc, C. Bleotu, G. Stanciu, R. Hristu, D.E.
Mihaiescu, V. Lazar, In vitro methods for the study of microbial biolms, Bioin-
terface Res. Appl. Chem. 1 (2011) 31.
[21] Y. Ling, K. Wei, F. Zou, S. Zhong, Temozolomide loaded PLGA-based super-
paramagnetic nanoparticles for magnetic resonance imaging and treatment
of malignant glioma, Int. J. Pharm. 430 (2012) 266275.
[22] D.J. Lee, G.Y. Park, K.T. Oh, N.M. Oh, D.S. Kwag, Y.S. Youn, Y.T. Oh, J.W. Park,
E.S. Lee, Multifunctional poly(lactide-co-glycolide) nanoparticles for lumines-
cence/magnetic resonance imaging and photodynamic therapy, Int. J. Pharm.
434 (2012) 257263.
[23] M. Furlana, J. Klugeb, M. Mazzottib, M. Lattuada, Preparation of biocompatible
magnetitePLGA composite nanoparticles using supercritical uid extraction
of emulsions, J. Supercrit. Fluids 54 (2010) 348356.
[24] A. Masoudi, H.R.M. Hosseini, M.A. Shokrgozar, R. Ahmadi, M.A. Oghabian, The
effect of poly(ethylene glycol) coating on colloidal stability of superparamag-
netic iron oxide nanoparticles as potential MRI contrast agent, Int. J. Pharm.
433 (2012) 129141.
[25] R. Abu Mukh-Qasem, A. Gedanken, Sonochemical synthesis of stable hydrosol
of Fe
3
O
4
nanoparticles, J. Colloid Interface Sci. 284 (2005) 489494.
[26] A.M. Grumezescu, A.I. Cotar, E. Andronescu, A. Ficai, C.D. Ghitulica, V.
Grumezescu, B.S. Vasile, M.C. Chiriuc, In vitro activity of the new water
dispersible Fe
3
O
4
@usnic acidnanostructure against planktonic andsessile bac-
terial cells, J. Nanoparticle Res. 15 (2013) 1766.
[27] A.I. Cotar, A.M. Grumezescu, K.S. Huang, C.M. Chiriuc, R. Radulescu, Mag-
netite nanoparticles inuence the efcacy of antibiotics against biolm
embedded Staphylococcus aureus cells, Biointerface Res. Appl. Chem. 3 (2013)
559565.
[28] M.C. Chiriuc, A.M. Grumezescu, C. Saviuc, R. Hristu, V. Grumezescu, C. Bleotu,
G. Stanciu, D.E. Mihaiescu, E. Andronescu, V. Lazar, R. Radulescu, Magnetic
nanoparticles for controlling invitrofungal biolms, Curr. Org. Chem. 17(2013)
10231028.
[29] M.C. Chiriuc, A.M. Grumezescu, E. Andronescu, A. Ficai, A.I. Cotar, V.
Grumezescu, E. Bezirtzoglou, V. Lazar, R. Radulescu, Water dispersible mag-
netite nanoparticles inuence the efcacy of antibiotics against planktonic and
biolmembedded Enterococcus faecalis cells, Anaerobe 22 (2013) 1419.
[30] A.S. Lynch, G.T. Robertson, Bacterial and fungal biolm infections, Annu. Rev.
Med. 59 (2008) 415428.
[31] J.W. Costerton, L. Montanaro, C.R. Arciola, Biolm in implant infections: its
production and regulation, Int. J. Artif. Organs 28 (2005) 10621068.
[32] T.F.C. Mah, G.A. OToole, Mechanisms of biolm resistance to antimicrobial
agents, Trends Microbiol. (2001) 9.
Please cite this article in press as: V. Grumezescu, et al., MAPLE fabricated magnetite@eugenol and (3-hidroxybutyric acid-
co-3-hidroxyvaleric acid)polyvinyl alcohol microspheres coated surfaces with anti-microbial properties, Appl. Surf. Sci. (2014),
http://dx.doi.org/10.1016/j.apsusc.2014.01.126
ARTICLE IN PRESS
G Model
APSUSC-27134; No. of Pages 7
V. Grumezescu et al. / Applied Surface Science xxx (2014) xxxxxx 7
[33] R.A. Brady 1, G.A. OMay, J.G. Leid, M.L. Prior, J.W. Costerton, M.E. Shirtliff,
Resolution of staphylococcus aureus biolm infection using vaccination and
antibiotic treatment, Infect. Immun. 79 (2011) 17971803.
[34] D. Lebeaux, A. Chauhan, O. Rendueles, C. Beloin, Fromin vitro to in vivo models
of bacterial biolm-related infections, Pathogens 2 (2013) 288356.
[35] A.M. Grumezescu, B.S. Vasile, A.M. Holban, Eugenol functionalized magnetite
nanostructures used for antimicrobial therapy, Lett. Appl. Nanobiosci. 2 (2013)
120.
[36] T.V.P. Doan, W. Couet, J.C. Olivier, Formulation and in vitro characterization
of inhalable rifampicin-loaded PLGA microspheres for sustained lung delivery,
Int. J. Pharm. 414 (2011) 112117.
[37] C. Hu, H. Feng, C. Zhu, Preparation and characterization of rifampicin-PLGA
microspheres/sodiumalginateinsitugel combinationdeliverysystem, Colloids
Surf. B 95 (2012) 162169.
[38] C. Saviuc, A.M. Grumezescu, E. Oprea, V. Radulescu, L. Dascalu, C. Chiriuc, M.
Bucur, O. Banu, V. Lazar, Antifungal activity of some vegetal extracts on Candida
biolms developed on inert substratum, Biointerface Res. Appl. Chem. 1 (2011)
15.
[39] C. Saviuc, A.M. Grumezescu, A. Holban, C. Chiriuc, D. Mihaiescu, V. Lazar,
Hybrid nanostructurated material for biomedical applications, Biointerface
Res. Appl. Chem. 1 (2011) 64.
[40] I. Anghel, V. Grumezescu, E. Andronescu, G.A. Anghel, A.M. Grumezescu, D.E.
Mihaiescu, M.C. Chiriuc, Protective effect of magnetite nanoparticle/Salvia
ofcinalis essential oil hybridnanobiosystemagainst fungal colonizationonthe
Provox voice section prosthesis, Dig. J. Nanomater. Bios. 7 (2012) 12051212.
[41] W. Jiang, Y. Wu, B. He, X. Zeng, K. Lai, Z. Gu, Effect of sodiumoleate as a buffer
on the synthesis of superparamagnetic magnetite colloids, J. Colloid Interface
Sci. 347 (2010) 17.
[42] A.M. Grumezescu, E. Andronescu, A.M. Holban, A. Ficai, D. Ficai, G. Voicu, V.
Grumezescu, P.C. Balaure, M.C. Chiriuc, Water dispersible cross-linked mag-
netic chitosan beads for increasing the antimicrobial efciency of different
therapeutic agents, Int. J. Pharm. 454 (2013) 233240.