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Antimicrobial Effect of Chitosan

Nanoparticles on Streptococcus mutans


Biofilms
Luis E. Chvez de Paz, Anton Resin, Kenneth A. Howard,
Duncan S. Sutherland and Peter L. Wejse
Appl. Environ. Microbiol. 2011, 77(11):3892. DOI:
10.1128/AEM.02941-10.
Published Ahead of Print 15 April 2011.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2011, p. 38923895


0099-2240/11/$12.00 doi:10.1128/AEM.02941-10
Copyright 2011, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 11

Antimicrobial Effect of Chitosan Nanoparticles on


Streptococcus mutans Biofilms
Luis E. Chavez de Paz,1* Anton Resin,2 Kenneth A. Howard,2
Duncan S. Sutherland,2 and Peter L. Wejse3
Department of Oral Biology, Faculty of Odontology, Malmo
University, S-20506 Malmo
, Sweden1;
Interdisciplinary Nanoscience Center (iNANO), Faculty of Science, Aarhus University,
DK-8000 Aarhus C, Denmark2; and Global Ingredients R&D,
Arla Foods amba, Viby J, Denmark3

Nanoparticle complexes were prepared from chitosans of various molecular weights (MW) and degrees of
deacetylation (DD). The antimicrobial effect was assessed by the Live/Dead BacLight technique in conjunction
with confocal scanning laser microscopy (CSLM) and image analysis. Nanocomplexes prepared from chitosans
with high MW showed a low antimicrobial effect (20 to 25% of cells damaged), whereas those prepared from
low-MW chitosans showed high antimicrobial effect (>95% of cells damaged).
These chitosans were categorized into three groups: group A
(high DD and low MW), group B (high DD and high MW),
and group C (low DD and low MW).
Chitosan nanoparticles were created by ion gelation with
polyanionic sodium triphosphate (TPP) (Fig. 2a) (13). Since
one of the reported problems with chitosan preparations has
been its low solubility under neutral pH conditions, we further
developed the system to produce stable nanoparticle at neutral
pH. Nanoparticle assemblies were generally formulated by dissolving chitosan in acetic acid buffer (1 mg/ml) and by adding
100 l of this mixture drop by drop while stirring vigorously to
a premixed solution composed of 50 l of TPP solution (0.1%)
in water and 500 l of phosphate-buffered saline (PBS) buffer
(pH 7.4). The mass ratio of chitosan to TPP was kept constant
(2:1). The final pH of the solution was around 5 and was
adjusted to 7 by the addition of 2 M NaOH. The nanoparticle
solution was equilibrated at room temperature for an hour by
mixing. Particle size distribution and zeta potential were further characterized in a Malvern Zetasizer Nano ZS instrument
to measure particle size distributions and zeta potential in
different media. Scanning electron microscopy (SEM) (Fig. 2b)
was also used to visualize particles in deionized water before
mixing with media. The particle size distribution in water was
relatively narrow with some batch-to-batch variation. After
mixing the particles with media, the size distribution broadened and was between 20 and 1,000 nm showing a similar
profile for the different formulations.
Exposure of S. mutans biofilms to chitosan nanoparticles.
Biofilms were formed in the flow chamber system -Slide VI
(Integrated BioDiagnostics) as previously described (2). In
brief, 120 l of a washed suspension of Streptococcus mutans
UA159 in mid-exponential growth (optical density at 600 nm
[OD600] 0.4 0.1) were inoculated in the mini-flow chamber
slides and incubated in an atmosphere of 5% CO2 at 37C for
24 h under static conditions. Flow chambers were rinsed with
PBS to remove nonadherent cells. S. mutans biofilms were then
exposed to the different chitosan/TPP nanoparticle formulations diluted in Todd-Hewitt medium (BD Biosciences, Sweden) at a ratio of 1:1. Ratios of 1:4 and 1:10 were also tested.

Oral biofilm communities are naturally formed on tooth


surfaces and are associated with diseases such as caries, gum
inflammation (gingivitis), and degradation of periodontal tissues (periodontitis) (5, 6). These diseases are mainly caused by
those biofilm organisms that exhibit phenotypic traits capable
of surviving adverse environmental conditions. For example,
Streptococcus mutans undergoes an acid tolerance response to
adapt and survive the acidic conditions provoked by excess
sugar intake (3, 11). This important phenotypic trait of S.
mutans is directly linked with caries development (5, 12).
Therefore, novel approaches for developing oral care products, such as dentifrices and mouthwashes, rely on targeting
these highly adaptable oral organisms and blocking their key
mechanisms of phenotypic variation. One major step forward
in achieving this goal has been the development of antimicrobial systems that could effortlessly diffuse across all biofilm
structures (4). For this purpose, there has been increasing
interest in developing nanoscale systems to be used as biological carriers within biofilms. Of special interest are those nanoscale systems developed from natural polymers, e.g., chitosan
(10). Chitosan is obtained by deacetylation of chitin and is used
in biomedical applications due to its high biocompatibility and
antimicrobial properties (8). In the present study, we formulated nanoparticles from different commercially available chitosans (with different molecular weights [MW] and degrees of
deacetylation [DD]) by ion gelation with polyanionic sodium
triphosphate (TPP) and studied their penetrative antimicrobial
effect on 24-h-old biofilms of S. mutans.
Formulation of chitosan nanoparticles. The first step was to
formulate nanoparticles using various chitosans with different
degrees of deacetylation and molecular weights (Bioneer A/S
and NovaMatrix, Norway). Figure 1 shows 9 different chitosans
used in this study distributed according to their DD and MW.

* Corresponding author. Mailing address: Department of Oral Biology, Faculty of Odontology, Malmo
University, SE-20506 Malmo
,
Sweden. Phone: 46 40 6658659. Fax: 46 40 929359. E-mail: luis.chavez
.de.paz@mah.se.

Published ahead of print on 15 April 2011.


3892

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Received 16 December 2010/Accepted 1 April 2011

VOL. 77, 2011

ANTIMICROBIAL EFFECT OF CHITOSAN ON S. MUTANS BIOFILMS

3893

A solution of TPP (0.1%) in PBS was used as control. Flow


cells were incubated for 2 h in an atmosphere of 5% CO2 at
37C. Antimicrobial activity was assessed using the Live/Dead
BacLight bacterial viability kit for microscopy (Invitrogen Ltd.,
Paisley, United Kingdom). The fluorescence from stained cells
was viewed using an inverted confocal scanning laser microscope, Eclipse TE2000 (Nikon, Tokyo, Japan), where 10 randomly selected image stack sections were imaged in each biofilm sample. Image stack sections were composed of 10 images,
each taken with a variation of 2 m along the z position.
Images were acquired with a 60 oil immersion objective and
digitalized by the software EZ-C1 v.3.40 build 691 (Nikon,
Tokyo, Japan) at a resolution of 512 by 512 pixels and with a
zoom factor of 1.0, giving a final pixel resolution of 0.42 m/
pixel. Individual biofilm images covered an area of 0.05 mm2
per field of view. Experiments were done in three triplicate sets
of biofilms with each set having its own unique total cell count.
Confocal scanning laser microscopy (CSLM) images were analyzed to produce information of the total biofilm population
as well as the independent subpopulations represented by red
and green fluorescent colors by using the bioImage_L software
program (1). The structure and spatial differences in green
(viable) and red (damaged) biofilm subpopulations were characterized by three-dimensional analysis where parameters such
as biovolume (m3), substratum coverage (reported as a percentage), and spatial thickness (m) were calculated.
Large effect of low-molecular-weight chitosans. When the
biofilms were 24 h old, they were observed to have average
substratum coverage of 44.7% 3.1%, mean total biovolume
of 6.2 104 0.4 104 m3, mean thickness of 16.4 1.1
m, and 96.4% 2.3% of its total population with intact cell
membranes (cells that were stained green when the Live/Dead
BacLight bacterial viability kit was used). After the application
of the chitosan nanoparticles, no significant difference in the
structure of biofilms was found. However, the effect of the
tested nanoparticle formulations gave strong differences in
the level of cell membrane damage (cells that stained red when
the Live/Dead BacLight bacterial viability kit was used) compared with the negative control (TPP) (see representative
three-dimensional [3D] biofilm reconstructions in Fig. 3). The

FIG. 2. Formation of the chitosan-tripolyphosphate complex by


ionotropic gelation. (a) Schematic illustration of the chitosan-TPP
complex and (b) SEM image. Bar, 200 nm.

ratio that the chitosan nanoparticles were mixed in the medium did not have an effect on their antimicrobial activity (data
not shown).
The representative nanoparticle preparations comprising
chitosans with low molecular weights, groups A and C, showed
the highest antimicrobial activity at the various depths of the
biofilms formed by S. mutans (95% of total cells damaged).
These results indicated that the differences in the levels of
deacetylation in group A (high DD) and group C (low DD) did
not seem to affect the antimicrobial activity of the chitosan
nanoparticles. This lack of influence of the deacetylation level
may be due to free amino groups being neutral at pH 7. This
may explain why our results differ from results in other studies
that found a positive correlation between the levels of deacetylation and the antimicrobial effect of chitosan at a lower pH
(7). In contrast, we observed a clear tendency between the
molecular weight of the chitosan and the effect on the membrane integrity of S. mutans with the lower-molecular-weight
chitosans showing the highest effect (groups A and C) and with
progressively decreased effect on membrane integrity for
higher molecular weights (group B) (Table 1). Chitosan nanoparticles from group B showed a scattered effect in damaging
the cell membrane of cells (20 to 25% of cells damaged) mainly
at the upper levels of the biofilms (15 m). At the levels
closer to the substrate (4 m), chitosan nanoparticles
showed a slight effect in cell membrane damage (5%). We do
not have a clear picture of the mechanism of the molecular
weight effect on chitosan antimicrobial activity in nanoparticles. The heterogeneous distribution of cell membrane damage at the upper levels of the biofilms and the lack of effect at
the substratum levels when using high-MW formulations, how-

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FIG. 1. Categorization of the different chitosan subtypes based on


their molecular size (kDa) and degree of deacetylation.

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APPL. ENVIRON. MICROBIOL.

ever, indicate a lower diffusive potential than low-MW formulations which affected the membrane integrity of cells all across
the biofilm. It is also possible that the lower-molecular-weight
nanoparticles have a systematically reduced number of TPP
molecules available per molecule and may thus be more susceptible to disaggregation in the biofilm. Once homogenously
distributed across the biofilms, chitosan particles can directly
interact with bacterial cells. Different mechanisms of interaction with bacteria have been proposed for chitosans (9). One
proposed mechanism is based on the interaction between positively charged chitosan molecules and negatively charged microbial cell membranes leading to the leakage of proteinaceous
and other intracellular constituents; however, at pH 7, free
chitosan and the outer chitosan in the nanoparticle complexes
are expected to be neutral, but chitosan within the particle may
retain its charge. A second proposed mechanism is based on

TABLE 1. Antimicrobial effect of different groups


of chitosan nanoparticlesa

binding of chitosan with microbial DNA, in turn interfering


with mRNA and protein synthesis, which clearly requires entry
of the chitosan into the cell.
Concluding remarks. This study showed antimicrobial activity of chitosan nanoparticles at neutral pH to have a strong
trend toward higher activity of particles formed from lower-MW
chitosans. Furthermore, the effect of low-molecular-weight formulations affected the cell membrane integrity of S. mutans in
a homogenous manner across the entire biofilm. It is expected
that this system will greatly improve the uniform delivery of
chitosan formulated as nanoparticles through biofilm structures at neutral pH, and combined with other compounds, it
could aid targeting of strongly adaptable organisms in complex
biofilm systems.
This work was carried out within the ProSURF platform (ProteinBased Functionalisation of Surfaces), which is funded by the Danish
National Advanced Technology Foundation.
We are grateful to Jrgen Kjems (Interdisciplinary Nanoscience
Center, Aarhus University) for assistance in formulating the chitosan
nanoparticles.

% of damaged biofilm cells (mean SEM)


Level (distance
from substratum)

Upper (20 m)
Middle (15 m)
Low (2 m)
a
b

Group A
(low MW,
high DD)b

Group B
(high MW,
high DD)

Group C
(low MW,
low DD)

95.5 0.8
94.6 1.1
96.1 3

21.4 1.2
7.5 1
1.2 0.6

94.9 1
93.6 1.7
96.7 2.3

The values for the control (no chitosan nanoparticles) were 1 for all levels.
MW, molecular weight; DD, degree of deacetylation.

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FIG. 3. Antimicrobial effect of different chitosan-tripolyphosphate complexes in S. mutans biofilms. 3D biofilm reconstructions show results
with the Live/Dead stain (green [viable cells] and red [damaged cells]) at different depths of the biofilms. The units on the axes are micrometers.

VOL. 77, 2011

ANTIMICROBIAL EFFECT OF CHITOSAN ON S. MUTANS BIOFILMS

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