Boccafoschi, Francesca

Carmagnola, Irene

Chiono, Valeria

Ciardelli, Gianluca

Gentile, Piergiorgio

Georgiev, G.

Georgieva, V.

Ninov, M.

Pashkuleva, I.

Reis, R.

Tonda-Turo, C.

English

PORTO Publications Open Repository TOrino

Layer-by-Layer Coating of Photoactive Polymers for 
Biomedical Applications 
Valeria Chiono, Irene Carmagnola, Piergiorgio 
Gentile, Francesca Boccafoschi, Chiara Tonda-Turo, 
Gianluca Ciardelli 
Dept. of Mechanics 
Politecnico di Torino 
Torino, Italy  
e-mail: valeria.chiono@polito.it
George Georgiev, Momchil Ninov, Ventsislava 
Georgieva  
Laboratory of Water-Soluble Polymers, Polyelectrolytes and 
Biopolymers 
Faculty of Chemistry, University of Sofia 
Sofia, Bulgaria. 
Iva Pashkuleva, Rui Reis  
3B’s Research Group – Biomaterials, Biodegradables and 
Biomimetics  
Department of Polymer Engineering University of Minho 
Caldas das Taipas, Guimarães, Portugal 
and 
Institute for Biotechnology and Bioengineering 
PT Government Associated Laboratory, 
Caldas das Taipas, Guimarães, Portugal, Portugal. 
Abstract—The design of advanced, nanostructured materials at 
the molecular level is of tremendous interest for the scientific 
community because of their potential in several fields, including 
medicine, biology and tissue engineering. Layer-by-layer (LbL) 
assembly is a versatile technique for the realization of 
multilayered films with tailored characteristics at the nanometer 
scale. Photoactive nanostructured films were prepared by LbL 
assembly of photozymes: an amphiphilic zwitterionic copolymer, 
poly(sodium styrene sulphonate-stat-vinyl naphtalene-stat-3-
dimethyl(methacryloylethyl) ammonium propane sodium 
sulfonate) (PSSS-stat-VN-stat-DMPAS; code: ZI), and a cationic 
polyelectrolyte, chitosan-g-fluorescein (code: CHFL), on a 
crosslinked gelatin substrate. Although successful LbL assembly 
of photozymes was proven by the performed characterization, 
gelatin was not an optimal substrate for the coating, due to its low 
charge density and amphoteric nature. A regular growth of the 
layers was found after at least 6 layers were deposited. The 
macromolecules of the zwitterionic photozyme adopted a coiled 
micellar conformation in solution which was kept during the 
assembly. The obtained nanostructured films are promising 
candidates for carrying out efficient electron transfer process 
within them, responsible for the anti-microbial activity and the 
osteointegration ability of the coating.  
Keywords: chitosan-g-fluorescein; gelatin; layer-by-layer; 
poly(sodium styrene sulphonate-stat-vinyl naphtalene-stat-3-
dimethyl(methacryloylethyl) ammonium propane sodium 
sulfonate); photozymes. 
I. INTRODUCTION
Layer-by-layer (LbL) electrostatic self-assembly of charged 
polymers has been widely used as a versatile technique for the 
formation of multilayered thin films with a tailored structure 
and composition and wide range of electrical, magnetic, 
biological and optical properties [1, 2]. LbL self-assembly is 
based on the alternating exposure of a charged substrate to 
solutions of positively and negatively charged polyelectrolytes. 
A rinsing step is included between the two previously 
described adsorption processes, to remove excess as well as to 
prevent cross-contamination of the polyelectrolyte solutions. 
LbL technique allows obtaining of homogeneous monolayer 
structures and fine control of the coating properties. , 
Additionally, it is applicable to substrates of any shape, it is 
environmentally-friendly, it allows room temperature 
processing and low-cost manufacturing. The range of 
polyelectrolytes used in the LbL assembly is very broad, but 
the majority of the studies has been utilized  commercial 
homopolymers. There is a limited number of reports on 
amphiphilic polyelectrolytes [3, 4]. 
Photozymes are amphiphilic photoactive polymers, 
composed by fluorophore units such as xanthene dyes 
(fluorescein, rose bengal, eosin, eritrosin), naphthalene, 
carbazole, and hydrophilic units, containing strong electrolytes 
such as sulphonate groups or weak electrolytes such as the 
primary amino groups of chitosan chains [5-7]. Photozymes 
may be statistical, block or graft copolymers and they are 
generally water soluble. Aromatic chromophores undergo 
strong interactions, which induce the coiling of the 
macromolecules and the formation of hydrophobic domains 
surrounded by charged fragments of the chain. The 
hydrophobic packed domains may dissolve  hydrophobic guest 
molecules and play the role of nanoreactors with 
photochemical activity [7]. 
In this work, nanostructured multilayer photoactive films 
have been produced on crosslinked gelatin substrates via the 
LbL method, using alternative layers of a stat-copolymer 
photozyme (forming pseudomicellar structures) and a graft-
copolymer photozyme (forming compact layers). These 
innovative nanostructured materials may have important 
798978-981-08-3694-8 (RPS) c© 2009 IEEE NANO Organizers
applications as multifunctional coatings for scaffolds for bone 
repair: the photosensitizing properties of the different types of 
photozymes are combined, leading to osteointegration by 
hydroxyapatite precipitation induced by the graft-copolymer 
photozyme and anti-microbial action promoted by the stat-
copolymer photozyme.  
II. MATERIALS AND METHODS 
A graft-copolymer photozyme, chitosan-g-fluorescein (code 
CHFL; 0.5 mol.% FL; produced from chitosan FG 90 with 
Mn:210±10 kDa and Mw/Mn: 2.17, Primex Ingredients ASA) 
and a stat-copolymer zwitterionic photozyme, poly(sodium 
styrene-stat-vinyl naphtalene-stat-sulphonate-co-3-
dimethyl(methacryloylethyl) ammonium propane sodium 
sulfonate) (PSSS-stat-VN-stat-DMAPS; code ZI; with 20 
mol.% VN, 70 mol.% SSA; 10 mol.% DMAPS with Mn: 53.2 
kDa and Mw/Mn: 2.34) were synthesized by copolymerization 
(AIBN, 60°C, DMSO) . The behavior of ZI photozyme 
macromolecules in water solutions with various concentration, 
pH and ion strength was studied by Dynamic Light Scattering, 
DLS and zeta potential measurements (Zetasizer Nano ZS). 
Before any applications, MTS (3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)  
cytotoxicity test was carried out on ZI and CHFL solutions 
using an osteoblast cell line SaOs-2 osteoblasts. The photo-
bioactivity of CHFL was studied by tests in simulated body 
fluid (SBF) under irradiation and in the dark (control). The 
photosensitizing activity of ZI was shown by standard tests 
using probe molecules [6]. Type A gelatin from porcine skin 
(code G) was supplied from Sigma-Aldrich. Genipin 
(Challenge Bioproducts) was used as a crosslinker for G. All 
solvents used were of analytical grade and used without further 
purification. Cast films on glass slides (1.2 cm diameter) were 
obtained from GP-crossliked G (code G_GP; 2.5 wt.% GP) [8] 
as a substrate for LbL assembly. LbL was realized by 
alternative incubation into 0.1% (w/v) photozyme solutions 
(pH 5.5) with intermediate washing steps (pH 4.5). The 
procedure was repeated and 10 layers were deposited. At the 
end, samples were further rinsed in bi-distilled water. LbL 
coatings on G_GP were characterized by scanning electron 
microscopy-energy dispersive x-ray  analysis (SEM-EDX; FEI 
QUANTA INSPECT 200 apparatus, equipped with EDAX 
Genesis Software), FTIR-ATR analysis (Perkin Elmer 
Spectrum One Spectrometer), UV-VIS spectroscopy (Perkin 
Elmer), static water contact angle (CAM 200 KSV Instrument). 
III. RESULTS 
DLS is a non-invasive well-established technique for 
measuring the size of molecules and particles typically in the 
submicron region. A bi-modal size distribution was determined 
for  the ZI aggregates  (with 40nm and 340nm medium size), 
regardless the ZI solution concentration (0.25-2.5 mg/ml). 
While the size of 40 nm is consistent with an isolated ZI 
macromolecule, the size of 340nm indicates the presence of 
macromolecular aggregates. This speculation was confirmed by 
the observation that  the quantity of the smaller aggregates 
increases at higher  ion strength of the solution.  ZP is one of 
the main forces that mediate inter-particle interactions. 
Particles with a high ZP of the same charge sign, either positive 
or negative, will repel each other. Conventionally a high ZP 
can be high in a positive or negative sense, i.e. <-30mV and 
>+30mV. For molecules and particles that are small enough, 
and of low enough density to remain in suspension, a high ZP 
will confer stability, i.e. the solution or dispersion will resist 
aggregation. ZP and DLS measurements confirmed the ability 
of ZI to assemble into stable nano-aggregates, with high ZP 
values (<-30 mV) even at elevate ion strength (2.5% KCl). 
However, a tendency for decreasing ZP was observed with 
increasing the photozyme concentration (0.25-2.5 mg/ml), 
which is a sign for possible decreasing stability of the 
dispersion in terms of aggregation abilities. ZP was only 
slightly affected by the solution pH in the 4-10 pH range. The 
solution ion strength highly influenced ZP, which decreased 
with increasing the ion strength (0-2.5% KCl). 
A previous work on LbL assembly of stat-copolymer 
photozymes has shown that their coiled micellar conformation 
is preserved in the layer-by-layer assembled film [9]. Here, the 
behavior of surface wettability of LbL coated films as a 
function of layer number could indirectly suggest a micellar 
assembly of ZI photozyme in the LbL coating layers (Fig. 1). 
Contact angles of coated surfaces were similar to the value 
measured for G_GP up to the 2nd layer which is consistent 
with the sensibility of this surface characterization technique. 
Then, the contact angles increased for the 3
rd 
and the 4
th
 coating 
layers. The observed changes can be associated not only with 
changes of the surface chemistry but also with the increased 
roughness generated during the coating process.  
40
45
50
55
60
65
70
75
80
0 1 2 3 4 5 6 7 8 9 10
Layer number
C
o
n
ta
ct
 a
n
g
le
 (
°)
Figure 1. Contact angle as a function of the layer number. Reported data are 
the average values whereas the bars are the standard deviation (n=5).  
After the 4th layer, contact angle displayed alternate values 
suggesting the beginning of the layer-by-layer assembly. 
Literature data report that contact angles of LbL films generally 
oscillate between the values for pure components. A similar 
result was not found for the photozyme coatings: in this case, 
the contact angle of each layer varied between 65°-70° which is 
only higher than the CHFL contact angle (60°) and a lower 
value (around 55°), which is much higher than the typical ZI 
contact angle (around 18°). The assembly of ZI photozyme into 
nano-aggregates (pseudomicelles) on the corresponding layers 
produced nano-roughness on the surface which could be the 
cause for the increased surface wettability of the ZI layers. 
Atomic force microscopy measures are in progress to study the 
ZI morphology onto each layer.  
9th IEEE Conference on Nanotechnology 2009 799
Moreover, contact angle measurements are sensitive to both 
surface nano- and micro-roughness. SEM analysis showed an 
increase of surface roughness with increasing the number of 
layers (Fig. 2). On the other hand, as LbL coatings are expected 
to have nanometer thickness, it was not possible to distinguish 
the deposited layers by SEM images of the fractured surfaces.  
Figure 2. SEM images of surfaces of LbL coated G_GP with alternate 
ZI/CHFL layers: (a) 1 layer; (b) 7 layers. 
FTIR-ATR analysis showed changes in the chemical 
characteristics of the surfaces of coated G_GP cast films as 
compared to the control, particularly in the 1400-900 cm
-1
wavenumber range. The main absorption bands of ZI 
photozyme were present in the FTIR-ATR spectra of the coated 
surfaces, precisely asymmetric (1125 and 1180 cm
-1
) and 
symmetric (1036 cm
-1
) stretching of O=S=O groups and S-O-C 
stretching (1009 cm
-1
). On the other hand, the CH-FL 
absorption bands were not detected in the FTIR-ATR spectra of 
coated G_GP. Similar results have been reported for 
multilayers of poly(styrene-sulphonate)/chitosan [10], for 
which FITR-ATR analysis only evidenced the absorption bands 
of the poly(syrenesulphonate) polyanion.  
EDX analysis allows the investigation of a surface layer 
with a micrometer thickness. In our case, when the analysis 
was performed on the upper surface of coated cast films, EDX 
spectra included the elemental composition of both the G_GP 
substrate and the coatings. EDX spectra of the upper surfaces 
of coated G_GP showed the presence of S due to the occurred 
deposition of the anionic photozyme (ZI). The peak due to S 
was particularly intense on surface agglomerates. In 
conclusion, both FTIR-ATR and EDX analyses confirmed that 
ZI was successfully assembled onto G_GP films.  
On the other hand, UV-VIS spectroscopy performed on 
LbL coated films showed the typical absorbance peak of FL 
dye at around 500 nm, which intensity increased with 
increasing the layer number (Fig. 3) and the characteristic 
absorption peak of G_GP at around 605-610 nm. 
Figure 3. UV-VIS spectra of CHFL, G-GP and LbL coated G_GP with 
alternate ZI/CHFL layers.   
CHFL absorption peak was not detected for a number of 
layers lower than 8, probably due to the low intensity of the 
CHFL peak which was masked by the absorption of G_GP.  
Several cytotoxicity tests, such as MTS or MTT, are well 
accepted for an initial screening of a material biocompatibility 
Citotoxicity  tests using osteoblasts cell line (SaOs-2) were 
performed with pure photozymes . The MTS results  for ZI are 
presented in Fig. 4. ZI solutions in culture medium did not 
show any cytotoxic behavior against SaOs-2 osteoblast like 
cells. A slight decrease of cell viability with increasing the 
material concentration was observed. However, the cell 
viability calculated for all the samples was above 80%, which 
indicates a good biocompatibility of the photozyme.  
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.0
63
 m
g/m
l
0.2
5 m
g/m
l
95
,17
%
94
,23
%
91
,06
%
80
,86
%
15,74%
100%
+ -
CONTROL
0.1
25
mg
/m
l
*
0.5
mg
/m
l
O
.D
 
(49
0 
nm
)
Figure 4. Viability of SaOs-2 cells cultured for 24 h in solutions of ZI 
photozyme at different concentrations.  
Similarly, CHFL dissolved in culture medium at 0.01-2 
mg/ml concentrations was found not to be cytotoxic towards 
osteoblast-like cells.  
400 440 480 520 560 600 640 680 720 760 800,0
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
1,1
A 
CH-FL
10
9
8
6
2
G_GP
(a)
(b)
800 9th IEEE Conference on Nanotechnology 2009
CHFL was also found to have photo-bioactivity, i.e. the 
ability to deposit HA crystals under visible light irradiation in a 
suitable ion medium (SBF) (Fig. 5).  
Figure 5. SEM image of the surface of CH-FL cast films after incubation in 
SBF for 14 days under irradiation. 
This property was probably a consequence of light 
excitation of CHFL, leading to singlet oxygen formation, 
variation of the material surface charge and electrostatic 
attraction of the SBF solution ions with HA nucleation and 
further growth. However, the study of the mechanisms for the 
deposition of HA is in progress. 
The ability of the ZI photozyme towards singlet oxygen 
formation was also demonstrated by a UV-VIS spectroscopy  
using probe molecules, such as perylene or methylene blue: this 
property could be exploited for tailored photochemical 
reactions, e.g. with the purpose to obtain materials able to kill 
bacterials [11], increasing the well-known anti-fouling 
properties of zwitterionic polymers.  
IV. CONCLUSIONS 
The layer-by-layer deposition of photoactive molecules 
(photozymes) was demonstrated by an accurate physico-
chemical characterization. Further studies are in progress to 
analyze the morphology of the layers at the nano-scale. The 
studied LbL films showed interesting properties for future 
applications as coatings on scaffolds (or prostheses) to be 
implanted for bone regeneration purposes. The alternate 
deposition of ZI and CHFL photozymes allows the 
combination of their properties for the obtainment of materials 
with decreased risk of infection (due to ZI antimicrobial and 
antifouling properties) and able to stimulate the light-
switchable deposition of HA (due to CHFL photo-bioactivity).  
ACKNOWLEDGMENT
This work was supported by the European Union within the 
project “Photonanotech, Photozyme Nanoparticle Applications 
For Water Purification, Textile Finishing, Photodynamic 
Biomineralization And Biomaterial Coating” (Contract 
Number: 033168). 
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9th IEEE Conference on Nanotechnology 2009 801


Alternating bioactivity of multilayer thin films assembled from charged derivatives of chitosan”,

Biomedical Applications of Layer-by-Layer Assembly:

Electrostatic layer-by-layer self assembly of poly(carboxybetaine)s: role of zwitterions in film growth,”

Genipin-crosslinked chitosan/gelatin blends for biomedical applications”,

Layer-by layer assembly of gelatin,”

Multilayer Thin Films Sequential Assembly of Nanocomposite Materials,”

Nanostructured micellar films formed via layer-by-layer deposition of photoactive polymer”,

Photoactive modified chitosan” Biomacromolecules, doi 10.1021/bm800141v,

Polymer catalysts for important photoelectron transfer reactions,”

Porphyrin-based light-activated antimicrobial materials,”

Studies on the antenna effect in polymer molecules. 25. Photozymes with rose Bengal chromophores,”

Layer-by-Layer Coating of Photoactive Polymers for Biomedical Applications

Layer-by-Layer Coating of Photoactive Polymers for Biomedical Applications

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