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Synthesis and characterization of chitosan nanoparticles and their inhibitory effects against Streptococcus mutans: role of concentration and exposure time.

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Title: Synthesis and characterization of chitosan nanoparticles and their inhibitory effects against Streptococcus mutans: role of concentration and exposure time.
Authors: Costa BP; Graduate Program in Dentistry, Universidade Federal da Paraíba (UFPB), Cidade Universitária, Castelo Branco III, João Pessoa, PB, 58051-900, Brazil., Reis RM; Departamento de Saúde Coletiva, Odontopediatria E Ortodontia, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (FOP/UNICAMP), Av. Limeira, 901, Areião, Piracicaba, SP, 13414-903, Brazil., de Castro Bernardino K; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil., Aguiar RT; Graduate Program in Dentistry, Universidade Federal da Paraíba (UFPB), Cidade Universitária, Castelo Branco III, João Pessoa, PB, 58051-900, Brazil., Carlo HL; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil., Dos Santos RL; Department of Dentistry, Universidade Federal de Juiz de Fora, Avenida Doutor Raimundo Rezende N. 330, Centro, Governador Valadares, Juiz de Fora, MG, 35012-140, Brazil., de Carvalho FG; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil. fabiola.carlo@ufu.br.
Source: Odontology [Odontology] 2026 Oct; Vol. 114 (4), pp. 1955-1965. Date of Electronic Publication: 2025 Nov 12.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: Springer-Verlag Tokyo Country of Publication: Japan NLM ID: 101134822 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1618-1255 (Electronic) Linking ISSN: 16181247 NLM ISO Abbreviation: Odontology Subsets: MEDLINE
Imprint Name(s): Original Publication: Tokyo : Springer-Verlag Tokyo, c2001-
MeSH Terms: Streptococcus mutans*/drug effects , Chitosan*/pharmacology , Chitosan*/chemical synthesis , Chitosan*/chemistry , Nanoparticles*/chemistry , Anti-Bacterial Agents*/pharmacology, Biofilms/drug effects ; Bacterial Adhesion/drug effects ; Microscopy, Electron, Transmission ; Spectroscopy, Fourier Transform Infrared ; Microbial Sensitivity Tests ; Time Factors
Abstract: This study aimed to synthesize and characterize chitosan nanoparticles suspension (ChNPs) and to evaluate their inhibitory effects on Streptococcus mutans (S. mutans) adherence and biofilm formation under different concentrations and exposure times. ChNPs were synthesized via ionic gelation using tripolyphosphate (TPP) and characterized by Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering. Antibacterial activity was investigated through inhibition zone tests, as well as minimum inhibitory and bactericidal concentration (MIC and MBC) assays. ChNPs at MIC, 2 × MIC, and 4 × MIC were tested against S. mutans, alongside 0.12% chlorhexidine (positive control) and 0.9% saline solution (negative control). In adherence and biofilm formation assays, solutions were applied for 2 h and 60 s, respectively. For mature biofilms, treatments were applied for 60 s every 24 h or continuously for 24 or 48 h. Inhibition percentage (%I) was quantified using crystal violet staining. Data were analyzed by one-way ANOVA and Tukey's test (α = 0.05). TEM images showed small spherical particles with diameter and zeta potential values of 75.8 nm and + 46.2 mV, respectively. MIC and MBC were 550,0 and 275,0 µg/mL, respectively. ChNPs inhibited 40% of bacterial adherence at all concentrations (p = 0.06). Mature biofilm inhibition ranged from 90 to 100% after continuous exposure for 24 or 48 h (p = 0.251). ChNPs at all concentrations and CHX 0.12% exhibited high %I (90-100%). These findings demonstrate that ChNPs effectively reduced S. mutans adherence and disrupted biofilm formation, with greater efficacy achieved through prolonged contact, highlighting their potential as bioactive agents for preventing or controlling cariogenic biofilms in oral healthcare.
(© 2025. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)
Competing Interests: Declarations. Conflict of interest: All authors declare that there is no conflict of interest regarding the publication of this article.
References: Int J Food Microbiol. 2010 Nov 15;144(1):51-63. (PMID: 20951455)
Appl Environ Microbiol. 2011 Jun;77(11):3892-5. (PMID: 21498764)
Anaerobe. 2013 Apr;20:27-31. (PMID: 23454497)
Cochrane Database Syst Rev. 2017 Mar 31;3:CD008676. (PMID: 28362061)
Cureus. 2024 Oct 4;16(10):e70810. (PMID: 39493032)
Carbohydr Res. 2004 Nov 15;339(16):2693-700. (PMID: 15519328)
Polymers (Basel). 2018 Apr 23;10(4):. (PMID: 30966497)
BMC Complement Altern Med. 2014 Dec 12;14:478. (PMID: 25495921)
Carbohydr Polym. 2014 Jan 30;101:1081-6. (PMID: 24299877)
Int J Dent. 2014;2014:982071. (PMID: 24648842)
J Dent Res. 1997 Feb;76(2):665-72. (PMID: 9062560)
Adv Drug Deliv Rev. 2010 Jan 31;62(1):83-99. (PMID: 19799949)
Int J Biomater. 2012;2012:632698. (PMID: 22829829)
Eur J Oral Sci. 2008 Oct;116(5):493-5. (PMID: 18821994)
Arch Oral Biol. 2007 Mar;52(3):290-4. (PMID: 17112460)
Molecules. 2016 Feb 17;21(2):. (PMID: 26901172)
Anaerobe. 2012 Jun;18(3):305-9. (PMID: 22561525)
Int J Mol Sci. 2023 Oct 17;24(20):. (PMID: 37894948)
Ann Clin Microbiol Antimicrob. 2014 Aug 20;13:41. (PMID: 25139679)
J Appl Oral Sci. 2021 Oct 08;29:e20210120. (PMID: 34644779)
AAPS PharmSciTech. 2006 Jun 02;7(2):E50. (PMID: 16796367)
J Bacteriol. 2010 Jun;192(12):3024-32. (PMID: 20233920)
Appl Environ Microbiol. 2002 Jun;68(6):2950-8. (PMID: 12039754)
Virulence. 2018 Jan 1;9(1):522-554. (PMID: 28362216)
J Appl Oral Sci. 2014 Sep-Oct;22(5):366-72. (PMID: 25466471)
PLoS One. 2014 Feb 13;9(2):e89059. (PMID: 24551218)
Caries Res. 2011;45(6):501-5. (PMID: 21912130)
J Dent. 2010 Jun;38 Suppl 1:S11-5. (PMID: 20621238)
Caries Res. 2013;47(4):318-24. (PMID: 23446436)
Grant Information: 458158/2014-3 Conselho Nacional de Desenvolvimento Científico e Tecnológico; APQ-02895-21 Fundação de Amparo à Pesquisa do Estado de Minas Gerais; CDS/PPM-00299-16 Fundação de Amparo à Pesquisa do Estado de Minas Gerais; FAPEMIG RED-00204-23 Fundação de Amparo à Pesquisa do Estado de Minas Gerais; 406840/2022-9 INCT Saúde Oral e Odontologia
Contributed Indexing: Keywords: Streptococcus mutans; Biofilms; Chitosan; Nanoparticles
Substance Nomenclature: 9012-76-4 (Chitosan)
0 (Anti-Bacterial Agents)
Entry Date(s): Date Created: 20251112 Date Completed: 20261002 Latest Revision: 20261008
Update Code: 20261008
DOI: 10.1007/s10266-025-01250-x
PMID: 41222863
Database: MEDLINE
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  Data: Synthesis and characterization of chitosan nanoparticles and their inhibitory effects against Streptococcus mutans: role of concentration and exposure time.
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  Data: <searchLink fieldCode="AU" term="%22Costa+BP%22">Costa BP</searchLink>; Graduate Program in Dentistry, Universidade Federal da Paraíba (UFPB), Cidade Universitária, Castelo Branco III, João Pessoa, PB, 58051-900, Brazil.<br /><searchLink fieldCode="AU" term="%22Reis+RM%22">Reis RM</searchLink>; Departamento de Saúde Coletiva, Odontopediatria E Ortodontia, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (FOP/UNICAMP), Av. Limeira, 901, Areião, Piracicaba, SP, 13414-903, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Castro+Bernardino+K%22">de Castro Bernardino K</searchLink>; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil.<br /><searchLink fieldCode="AU" term="%22Aguiar+RT%22">Aguiar RT</searchLink>; Graduate Program in Dentistry, Universidade Federal da Paraíba (UFPB), Cidade Universitária, Castelo Branco III, João Pessoa, PB, 58051-900, Brazil.<br /><searchLink fieldCode="AU" term="%22Carlo+HL%22">Carlo HL</searchLink>; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil.<br /><searchLink fieldCode="AU" term="%22Dos+Santos+RL%22">Dos Santos RL</searchLink>; Department of Dentistry, Universidade Federal de Juiz de Fora, Avenida Doutor Raimundo Rezende N. 330, Centro, Governador Valadares, Juiz de Fora, MG, 35012-140, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Carvalho+FG%22">de Carvalho FG</searchLink>; Department of Pediatric Dentistry-Dental School, Universidade Federal de Uberlândia-Campus Umuarama, Bloco UMU2G. Av. Pará, 1720, Umuarama, Uberlândia, MG, 38405-320, Brazil. fabiola.carlo@ufu.br.
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  Data: <searchLink fieldCode="JN" term="%22101134822%22">Odontology</searchLink> [Odontology] 2026 Oct; Vol. 114 (4), pp. 1955-1965. <i>Date of Electronic Publication: </i>2025 Nov 12.
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  Data: <searchLink fieldCode="MM" term="%22Streptococcus+mutans%22">Streptococcus mutans*</searchLink>/<searchLink fieldCode="MM" term="%22Streptococcus+mutans+drug+effects%22">drug effects</searchLink> <br /><searchLink fieldCode="MM" term="%22Chitosan%22">Chitosan*</searchLink>/<searchLink fieldCode="MM" term="%22Chitosan+pharmacology%22">pharmacology</searchLink> <br /><searchLink fieldCode="MM" term="%22Chitosan%22">Chitosan*</searchLink>/<searchLink fieldCode="MM" term="%22Chitosan+chemical+synthesis%22">chemical synthesis</searchLink> <br /><searchLink fieldCode="MM" term="%22Chitosan%22">Chitosan*</searchLink>/<searchLink fieldCode="MM" term="%22Chitosan+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Nanoparticles%22">Nanoparticles*</searchLink>/<searchLink fieldCode="MM" term="%22Nanoparticles+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Anti-Bacterial+Agents%22">Anti-Bacterial Agents*</searchLink>/<searchLink fieldCode="MM" term="%22Anti-Bacterial+Agents+pharmacology%22">pharmacology</searchLink><br /><searchLink fieldCode="MH" term="%22Biofilms%22">Biofilms</searchLink>/<searchLink fieldCode="MH" term="%22Biofilms+drug+effects%22">drug effects</searchLink> ; <searchLink fieldCode="MH" term="%22Bacterial+Adhesion%22">Bacterial Adhesion</searchLink>/<searchLink fieldCode="MH" term="%22Bacterial+Adhesion+drug+effects%22">drug effects</searchLink> ; <searchLink fieldCode="MH" term="%22Microscopy%2C+Electron%2C+Transmission%22">Microscopy, Electron, Transmission</searchLink> ; <searchLink fieldCode="MH" term="%22Spectroscopy%2C+Fourier+Transform+Infrared%22">Spectroscopy, Fourier Transform Infrared</searchLink> ; <searchLink fieldCode="MH" term="%22Microbial+Sensitivity+Tests%22">Microbial Sensitivity Tests</searchLink> ; <searchLink fieldCode="MH" term="%22Time+Factors%22">Time Factors</searchLink>
– Name: Abstract
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  Group: Ab
  Data: This study aimed to synthesize and characterize chitosan nanoparticles suspension (ChNPs) and to evaluate their inhibitory effects on Streptococcus mutans (S. mutans) adherence and biofilm formation under different concentrations and exposure times. ChNPs were synthesized via ionic gelation using tripolyphosphate (TPP) and characterized by Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering. Antibacterial activity was investigated through inhibition zone tests, as well as minimum inhibitory and bactericidal concentration (MIC and MBC) assays. ChNPs at MIC, 2 × MIC, and 4 × MIC were tested against S. mutans, alongside 0.12% chlorhexidine (positive control) and 0.9% saline solution (negative control). In adherence and biofilm formation assays, solutions were applied for 2 h and 60 s, respectively. For mature biofilms, treatments were applied for 60 s every 24 h or continuously for 24 or 48 h. Inhibition percentage (%I) was quantified using crystal violet staining. Data were analyzed by one-way ANOVA and Tukey's test (α = 0.05). TEM images showed small spherical particles with diameter and zeta potential values of 75.8 nm and + 46.2 mV, respectively. MIC and MBC were 550,0 and 275,0 µg/mL, respectively. ChNPs inhibited 40% of bacterial adherence at all concentrations (p = 0.06). Mature biofilm inhibition ranged from 90 to 100% after continuous exposure for 24 or 48 h (p = 0.251). ChNPs at all concentrations and CHX 0.12% exhibited high %I (90-100%). These findings demonstrate that ChNPs effectively reduced S. mutans adherence and disrupted biofilm formation, with greater efficacy achieved through prolonged contact, highlighting their potential as bioactive agents for preventing or controlling cariogenic biofilms in oral healthcare.<br /> (© 2025. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)
– Name: Abstract
  Label: Competing Interests
  Group: Ab
  Data: Declarations. Conflict of interest: All authors declare that there is no conflict of interest regarding the publication of this article.
– Name: Ref
  Label: References
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  Data: Int J Food Microbiol. 2010 Nov 15;144(1):51-63. (PMID: <searchLink fieldCode="PM" term="%2220951455%22">20951455)</searchLink><br />Appl Environ Microbiol. 2011 Jun;77(11):3892-5. (PMID: <searchLink fieldCode="PM" term="%2221498764%22">21498764)</searchLink><br />Anaerobe. 2013 Apr;20:27-31. (PMID: <searchLink fieldCode="PM" term="%2223454497%22">23454497)</searchLink><br />Cochrane Database Syst Rev. 2017 Mar 31;3:CD008676. (PMID: <searchLink fieldCode="PM" term="%2228362061%22">28362061)</searchLink><br />Cureus. 2024 Oct 4;16(10):e70810. (PMID: <searchLink fieldCode="PM" term="%2239493032%22">39493032)</searchLink><br />Carbohydr Res. 2004 Nov 15;339(16):2693-700. (PMID: <searchLink fieldCode="PM" term="%2215519328%22">15519328)</searchLink><br />Polymers (Basel). 2018 Apr 23;10(4):. (PMID: <searchLink fieldCode="PM" term="%2230966497%22">30966497)</searchLink><br />BMC Complement Altern Med. 2014 Dec 12;14:478. (PMID: <searchLink fieldCode="PM" term="%2225495921%22">25495921)</searchLink><br />Carbohydr Polym. 2014 Jan 30;101:1081-6. (PMID: <searchLink fieldCode="PM" term="%2224299877%22">24299877)</searchLink><br />Int J Dent. 2014;2014:982071. (PMID: <searchLink fieldCode="PM" term="%2224648842%22">24648842)</searchLink><br />J Dent Res. 1997 Feb;76(2):665-72. (PMID: <searchLink fieldCode="PM" term="%229062560%22">9062560)</searchLink><br />Adv Drug Deliv Rev. 2010 Jan 31;62(1):83-99. (PMID: <searchLink fieldCode="PM" term="%2219799949%22">19799949)</searchLink><br />Int J Biomater. 2012;2012:632698. (PMID: <searchLink fieldCode="PM" term="%2222829829%22">22829829)</searchLink><br />Eur J Oral Sci. 2008 Oct;116(5):493-5. (PMID: <searchLink fieldCode="PM" term="%2218821994%22">18821994)</searchLink><br />Arch Oral Biol. 2007 Mar;52(3):290-4. (PMID: <searchLink fieldCode="PM" term="%2217112460%22">17112460)</searchLink><br />Molecules. 2016 Feb 17;21(2):. (PMID: <searchLink fieldCode="PM" term="%2226901172%22">26901172)</searchLink><br />Anaerobe. 2012 Jun;18(3):305-9. (PMID: <searchLink fieldCode="PM" term="%2222561525%22">22561525)</searchLink><br />Int J Mol Sci. 2023 Oct 17;24(20):. (PMID: <searchLink fieldCode="PM" term="%2237894948%22">37894948)</searchLink><br />Ann Clin Microbiol Antimicrob. 2014 Aug 20;13:41. (PMID: <searchLink fieldCode="PM" term="%2225139679%22">25139679)</searchLink><br />J Appl Oral Sci. 2021 Oct 08;29:e20210120. (PMID: <searchLink fieldCode="PM" term="%2234644779%22">34644779)</searchLink><br />AAPS PharmSciTech. 2006 Jun 02;7(2):E50. (PMID: <searchLink fieldCode="PM" term="%2216796367%22">16796367)</searchLink><br />J Bacteriol. 2010 Jun;192(12):3024-32. (PMID: <searchLink fieldCode="PM" term="%2220233920%22">20233920)</searchLink><br />Appl Environ Microbiol. 2002 Jun;68(6):2950-8. (PMID: <searchLink fieldCode="PM" term="%2212039754%22">12039754)</searchLink><br />Virulence. 2018 Jan 1;9(1):522-554. (PMID: <searchLink fieldCode="PM" term="%2228362216%22">28362216)</searchLink><br />J Appl Oral Sci. 2014 Sep-Oct;22(5):366-72. (PMID: <searchLink fieldCode="PM" term="%2225466471%22">25466471)</searchLink><br />PLoS One. 2014 Feb 13;9(2):e89059. (PMID: <searchLink fieldCode="PM" term="%2224551218%22">24551218)</searchLink><br />Caries Res. 2011;45(6):501-5. (PMID: <searchLink fieldCode="PM" term="%2221912130%22">21912130)</searchLink><br />J Dent. 2010 Jun;38 Suppl 1:S11-5. (PMID: <searchLink fieldCode="PM" term="%2220621238%22">20621238)</searchLink><br />Caries Res. 2013;47(4):318-24. (PMID: <searchLink fieldCode="PM" term="%2223446436%22">23446436)</searchLink>
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      – SubjectFull: Biofilms drug effects
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