Академический Документы
Профессиональный Документы
Культура Документы
These include:
REFERENCES
CONTENT ALERTS
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.
* 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.
3893
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-
3894
VEZ
CHA
DE
PAZ ET AL.
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
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.
REFERENCES
1. Cha
vez de Paz, L. E. 2009. Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms.
Appl. Environ. Microbiol. 75:17341739.
2. Cha
vez de Paz, L. E., I. R. Hamilton, and G. Svensa
ter. 2008. Oral bacteria
in biofilms exhibit slow reactivation from nutrient deprivation. Microbiology
154:19271938.
3. Hamilton, I. R., and N. D. Buckley. 1991. Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol. Immunol. 6:6571.
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.
4. Hetrick, E. M., J. H. Shin, H. S. Paul, and M. H. Schoenfisch. 2009. Antibiofilm efficacy of nitric oxide-releasing silica nanoparticles. Biomaterials
30:27822789.
5. Marsh, P. D. 2004. Dental plaque as a microbial biofilm. Caries Res. 38:
204211.
6. Marsh, P. D. 2005. Dental plaque: biological significance of a biofilm and
community life-style. J. Clin. Periodontol. 32(Suppl. 6):715.
7. Nasti, A., et al. 2009. Chitosan/TPP and chitosan/TPP-hyaluronic acid nanoparticles: systematic optimisation of the preparative process and preliminary
biological evaluation. Pharm. Res. 26:19181930.
8. Rabea, E. I., M. E. Badawy, C. V. Stevens, G. Smagghe, and W. Steurbaut.
2003. Chitosan as antimicrobial agent: applications and mode of action.
Biomacromolecules 4:14571465.
3895
9. Senel, S., et al. 2000. Chitosan films and hydrogels of chlorhexidine gluconate for oral mucosal delivery. Int. J. Pharm. 193:197203.
10. Sinha, V. R., et al. 2004. Chitosan microspheres as a potential carrier for
drugs. Int. J. Pharm. 274:133.
11. Svensa
ter, G., U. B. Larsson, E. C. Greif, D. G. Cvitkovitch, and I. R.
Hamilton. 1997. Acid tolerance response and survival by oral bacteria. Oral
Microbiol. Immunol. 12:266273.
12. Van Houte, J., C. Sansone, K. Joshipura, and R. Kent. 1991. Mutans streptococci and non-mutans streptococci acidogenic at low pH, and in vitro
acidogenic potential of dental plaque in two different areas of the human
dentition. J. Dent. Res. 70:15031507.
13. Zhang, H., M. Oh, C. Allen, and E. Kumacheva. 2004. Monodisperse chitosan
nanoparticles for mucosal drug delivery. Biomacromolecules 5:24612468.