chotosan 2

7/21/2019 chotosan 2

1/5

Published Ahead of Print 15 April 2011.10.1128/AEM.02941-10.

2011, 77(11):3892. DOI:Appl. Environ. Microbiol.Duncan S. Sutherland and Peter L. WejseLuis E. Chvez de Paz, Anton Resin, Kenneth A. Howard,

BiofilmsNanoparticles on Streptococcus mutansAntimicrobial Effect of Chitosan

http://aem.asm.org/content/77/11/3892Updated information and services can be found at:

These include:REFERENCES

http://aem.asm.org/content/77/11/3892#ref-list-1This article cites 13 articles, 3 of which can be accessed free at:

CONTENT ALERTS

morearticles cite this article),Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders:http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

onSeptember30,2014byguest

http://aem.asm.org/

Downloadedfrom


http://aem.asm.org/

Downloadedfrom
http://aem.asm.org/content/77/11/3892http://aem.asm.org/content/77/11/3892http://aem.asm.org/content/77/11/3892#ref-list-1http://aem.asm.org/content/77/11/3892#ref-list-1http://aem.asm.org/cgi/alertshttp://aem.asm.org/cgi/alertshttp://journals.asm.org/site/misc/reprints.xhtmlhttp://journals.asm.org/site/subscriptions/http://journals.asm.org/site/misc/reprints.xhtmlhttp://journals.asm.org/site/misc/reprints.xhtmlhttp://journals.asm.org/site/subscriptions/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://journals.asm.org/site/subscriptions/http://journals.asm.org/site/misc/reprints.xhtmlhttp://aem.asm.org/cgi/alertshttp://aem.asm.org/content/77/11/3892#ref-list-1http://aem.asm.org/content/77/11/3892


2/5

APPLIED ANDENVIRONMENTALMICROBIOLOGY, June 2011, p. 38923895 Vol. 77, No. 110099-2240/11/$12.00 doi:10.1128/AEM.02941-10Copyright 2011, American Society for Microbiology. All Rights Reserved.

Antimicrobial Effect of Chitosan Nanoparticles onStreptococcus mutansBiofilms

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

Received 16 December 2010/Accepted 1 April 2011

Nanoparticle complexes were prepared from chitosans of various molecular weights (MW) and degrees ofdeacetylation (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 chitosanswith high MW showed a low antimicrobial effect (20 to 25% of cells damaged), whereas those prepared fromlow-MW chitosans showed high antimicrobial effect (>95% of cells damaged).

Oral biofilm communities are naturally formed on toothsurfaces and are associated with diseases such as caries, guminflammation (gingivitis), and degradation of periodontal tis-sues (periodontitis) (5, 6). These diseases are mainly caused bythose biofilm organisms that exhibit phenotypic traits capableof surviving adverse environmental conditions. For example,Streptococcus mutansundergoes an acid tolerance response toadapt and survive the acidic conditions provoked by excesssugar 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 prod-ucts, such as dentifrices and mouthwashes, rely on targeting

these highly adaptable oral organisms and blocking their keymechanisms of phenotypic variation. One major step forwardin achieving this goal has been the development of antimicro-bial systems that could effortlessly diffuse across all biofilmstructures (4). For this purpose, there has been increasinginterest in developing nanoscale systems to be used as biolog-ical carriers within biofilms. Of special interest are those nano-scale systems developed from natural polymers, e.g., chitosan(10). Chitosan is obtained by deacetylation of chitin and is usedin biomedical applications due to its high biocompatibility andantimicrobial properties (8). In the present study, we formu-lated nanoparticles from different commercially available chi-tosans (with different molecular weights [MW] and degrees ofdeacetylation [DD]) by ion gelation with polyanionic sodiumtriphosphate (TPP) and studied their penetrative antimicrobialeffect on 24-h-old biofilms ofS. mutans.

Formulation of chitosan nanoparticles.The first step was toformulate nanoparticles using various chitosans with differentdegrees of deacetylation and molecular weights (Bioneer A/Sand NovaMatrix, Norway). Figure 1 shows 9 different chitosansused in this study distributed according to their DD and MW.

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 withpolyanionic sodium triphosphate (TPP) (Fig. 2a) (13). Sinceone of the reported problems with chitosan preparations hasbeen its low solubility under neutral pH conditions, we furtherdeveloped the system to produce stable nanoparticle at neutralpH. Nanoparticle assemblies were generally formulated by dis-solving chitosan in acetic acid buffer (1 mg/ml) and by adding100 l of this mixture drop by drop while stirring vigorously toa 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 wasadjusted to 7 by the addition of 2 M NaOH. The nanoparticlesolution was equilibrated at room temperature for an hour bymixing. Particle size distribution and zeta potential were fur-ther characterized in a Malvern Zetasizer Nano ZS instrumentto measure particle size distributions and zeta potential indifferent media. Scanning electron microscopy (SEM) (Fig. 2b)was also used to visualize particles in deionized water beforemixing with media. The particle size distribution in water wasrelatively narrow with some batch-to-batch variation. Aftermixing the particles with media, the size distribution broad-

ened and was between 20 and 1,000 nm showing a similarprofile 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). Inbrief, 120 l of a washed suspension ofStreptococcus mutansUA159 in mid-exponential growth (optical density at 600 nm[OD600] 0.4 0.1) were inoculated in the mini-flow chamberslides and incubated in an atmosphere of 5% CO2at 37C for24 h under static conditions. Flow chambers were rinsed withPBS to remove nonadherent cells.S. mutansbiofilms were thenexposed to the different chitosan/TPP nanoparticle formula-tions diluted in Todd-Hewitt medium (BD Biosciences, Swe-den) at a ratio of 1:1. Ratios of 1:4 and 1:10 were also tested.

* Corresponding author. Mailing address: Department of Oral Bi-ology, Faculty of Odontology, Malmo University, SE-20506 Malmo,Sweden. Phone: 46 40 6658659. Fax: 46 40 929359. E-mail: [email protected].

Published ahead of print on 15 April 2011.

3892


http://aem.asm.org/

Downloadedfrom
http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/


3/5

A solution of TPP (0.1%) in PBS was used as control. Flowcells were incubated for 2 h in an atmosphere of 5% CO

2at

37C. Antimicrobial activity was assessed using the Live/DeadBacLight bacterial viability kit for microscopy (Invitrogen Ltd.,Paisley, United Kingdom). The fluorescence from stained cellswas viewed using an inverted confocal scanning laser micro-scope, Eclipse TE2000 (Nikon, Tokyo, Japan), where 10 ran-domly selected image stack sections were imaged in each bio-film 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 anddigitalized 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 setsof biofilms with each set having its own unique total cell count.Confocal scanning laser microscopy (CSLM) images were an-alyzed to produce information of the total biofilm populationas well as the independent subpopulations represented by redand green fluorescent colors by using the bioImage_L softwareprogram (1). The structure and spatial differences in green(viable) and red (damaged) biofilm subpopulations were char-acterized by three-dimensional analysis where parameters suchas biovolume (m3), substratum coverage (reported as a per-centage), and spatial thickness (m) were calculated.

Large effect of low-molecular-weight chitosans. When thebiofilms were 24 h old, they were observed to have averagesubstratum coverage of 44.7% 3.1%, mean total biovolumeof 6.2 104 0.4 104 m3, mean thickness of 16.4 1.1m, and 96.4% 2.3% of its total population with intact cellmembranes (cells that were stained green when the Live/DeadBacLight bacterial viability kit was used). After the applicationof the chitosan nanoparticles, no significant difference in thestructure of biofilms was found. However, the effect of thetested nanoparticle formulations gave strong differences inthe level of cell membrane damage (cells that stained red whenthe Live/Dead BacLight bacterial viability kit was used) com-pared with the negative control (TPP) (see representativethree-dimensional [3D] biofilm reconstructions in Fig. 3). The

ratio that the chitosan nanoparticles were mixed in the me-dium did not have an effect on their antimicrobial activity (datanot shown).

The representative nanoparticle preparations comprising

chitosans with low molecular weights, groups A and C, showedthe highest antimicrobial activity at the various depths of thebiofilms formed by S. mutans (95% of total cells damaged).These results indicated that the differences in the levels ofdeacetylation in group A (high DD) and group C (low DD) didnot seem to affect the antimicrobial activity of the chitosannanoparticles. This lack of influence of the deacetylation levelmay be due to free amino groups being neutral at pH 7. Thismay explain why our results differ from results in other studiesthat found a positive correlation between the levels of deacety-lation and the antimicrobial effect of chitosan at a lower pH(7). In contrast, we observed a clear tendency between themolecular weight of the chitosan and the effect on the mem-

brane integrity ofS. mutans with the lower-molecular-weightchitosans showing the highest effect (groups A and C) and withprogressively decreased effect on membrane integrity forhigher molecular weights (group B) (Table 1). Chitosan nano-particles from group B showed a scattered effect in damagingthe cell membrane of cells (20 to 25% of cells damaged) mainlyat the upper levels of the biofilms (15 m). At the levelscloser to the substrate (4 m), chitosan nanoparticlesshowed a slight effect in cell membrane damage (5%). We donot have a clear picture of the mechanism of the molecularweight effect on chitosan antimicrobial activity in nanopar-ticles. The heterogeneous distribution of cell membrane dam-age at the upper levels of the biofilms and the lack of effect atthe substratum levels when using high-MW formulations, how-

FIG. 2. Formation of the chitosan-tripolyphosphate complex byionotropic gelation. (a) Schematic illustration of the chitosan-TPPcomplex and (b) SEM image. Bar, 200 nm.

FIG. 1. Categorization of the different chitosan subtypes based ontheir molecular size (kDa) and degree of deacetylation.

VOL. 77, 2011 ANTIMICROBIAL EFFECT OF CHITOSAN ON S. MUTANS BIOFILMS 3893


http://aem.asm.org/

Downloadedfrom


4/5

ever, indicate a lower diffusive potential than low-MW formu-lations which affected the membrane integrity of cells all across

the biofilm. It is also possible that the lower-molecular-weightnanoparticles have a systematically reduced number of TPPmolecules available per molecule and may thus be more sus-ceptible to disaggregation in the biofilm. Once homogenouslydistributed across the biofilms, chitosan particles can directlyinteract with bacterial cells. Different mechanisms of interac-tion with bacteria have been proposed for chitosans (9). Oneproposed mechanism is based on the interaction between pos-itively charged chitosan molecules and negatively charged mi-crobial cell membranes leading to the leakage of proteinaceousand other intracellular constituents; however, at pH 7, freechitosan and the outer chitosan in the nanoparticle complexesare expected to be neutral, but chitosan within the particle mayretain its charge. A second proposed mechanism is based on

binding of chitosan with microbial DNA, in turn interferingwith mRNA and protein synthesis, which clearly requires entry

of the chitosan into the cell.Concluding remarks.This study showed antimicrobial activ-ity of chitosan nanoparticles at neutral pH to have a strongtrend toward higher activity of particles formed from lower-MWchitosans. Furthermore, the effect of low-molecular-weight for-mulations affected the cell membrane integrity ofS. mutansina homogenous manner across the entire biofilm. It is expectedthat this system will greatly improve the uniform delivery ofchitosan formulated as nanoparticles through biofilm struc-tures at neutral pH, and combined with other compounds, itcould aid targeting of strongly adaptable organisms in complexbiofilm systems.

This work was carried out within the ProSURF platform (Protein-Based Functionalisation of Surfaces), which is funded by the DanishNational Advanced Technology Foundation.

We are grateful to Jrgen Kjems (Interdisciplinary NanoscienceCenter, Aarhus University) for assistance in formulating the chitosannanoparticles.

REFERENCES

1. Chavez de Paz, L. E. 2009. Image analysis software based on color segmen-tation for characterization of viability and physiological activity of biofilms.Appl. Environ. Microbiol.75:17341739.

2. Chavez de Paz, L. E., I. R. Hamilton, and G. Svensater.2008. Oral bacteriain biofilms exhibit slow reactivation from nutrient deprivation. Microbiology154:19271938.

3. Hamilton, I. R., and N. D. Buckley.1991. Adaptation by Streptococcus mu-tansto acid tolerance. Oral Microbiol. Immunol. 6:6571.

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

TABLE 1. Antimicrobial effect of different groupsof chitosan nanoparticlesa

Level (distancefrom substratum)

% of damaged biofilm cells (mean SEM)

Group A(low MW,high DD)b

Group B(high MW,high DD)

Group C(low MW,low DD)

Upper (20 m) 95.5 0.8 21.4 1.2 94.9 1Middle (15 m) 94.6 1.1 7.5 1 93.6 1.7Low (2 m) 96.1 3 1.2 0.6 96.7 2.3

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

3894 CHA VEZ DE PAZ ET AL. A PPL. ENVIRON. MICROBIOL.


http://aem.asm.org/

Downloadedfrom


5/5

4. Hetrick, E. M., J. H. Shin, H. S. Paul, and M. H. Schoenfisch. 2009. Anti-biofilm efficacy of nitric oxide-releasing silica nanoparticles. Biomaterials30:27822789.

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 andcommunity life-style. J. Clin. Periodontol. 32(Suppl. 6):715.

7. Nasti, A., et al. 2009. Chitosan/TPP and chitosan/TPP-hyaluronic acid nano-particles: 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.Biomacromolecules4:14571465.

9. Senel, S., et al. 2000. Chitosan films and hydrogels of chlorhexidine gluco-nate for oral mucosal delivery. Int. J. Pharm. 193:197203.

10. Sinha, V. R., et al. 2004. Chitosan microspheres as a potential carrier fordrugs. Int. J. Pharm. 274:133.

11. Svensater, G., U. B. Larsson, E. C. Greif, D. G. Cvitkovitch, and I. R.Hamilton.1997. Acid tolerance response and survival by oral bacteria. OralMicrobiol. Immunol. 12:266273.

12. Van Houte, J., C. Sansone, K. Joshipura, and R. Kent. 1991. Mutans strep-tococci and non-mutans streptococci acidogenic at low pH, and in vitro

acidogenic potential of dental plaque in two different areas of the humandentition. J. Dent. Res. 70:15031507.

13. Zhang, H., M. Oh, C. Allen, and E. Kumacheva.2004. Monodisperse chitosannanoparticles for mucosal drug delivery. Biomacromolecules 5:24612468.

VOL. 77, 2011 ANTIMICROBIAL EFFECT OF CHITOSAN ON S. MUTANS BIOFILMS 3895


http://aem.asm.org/

Downloadedfrom

chotosan 2

Documents