PLGA submicron particles containing chlorhexidine, calcium and phosphorus inhibit Enterococcus faecalis infection and improve the microhardness of dentin

  • Wei Fan
  • Yanyun Li
  • Danfeng Liu
  • Qing Sun
  • Mengting Duan
  • Bing FanEmail author
Biomaterials Synthesis and Characterization Original Research
Part of the following topical collections:
  1. Biomaterials Synthesis and Characterization


Enterococcus faecalis (E. faecalis), a Gram-positive facultative anaerobe, is reported to take responsibility for a large portion of refractory root canal infections and root canal re-infections of human teeth. Chlorhexidine is a strong bactericide against E. faecalis but cannot infiltrate into dentinal tubules. On the other hand, a common negative effect of root canal medicaments is the decrease of dentin microhardness. In this study, poly(D,L-lactic-co-glycolide) (PLGA) submicron particles were applied as delivery carriers to load and release the chlorhexidine as well as calcium and phosphorus. The release profiles, antibacterial ability against E. faecalis, infiltration ability into dentinal tubules, biocompatibility and effects on dentin microhardness of these particles were investigated. Results revealed that encapsulated chemicals could be released in a sustained manner from the particles. The particles also exhibited excellent biocompatibility on MC3T3-E1 cells and significant antimicrobial property against E. faecalis. On dentin slices, the particles could be driven into dentinal tubules by ultrasonic activiation and inhibit E. faecalis colonization. Besides, dentin slices medicated with the particles displayed an increase in microhardness. In conclusion, PLGA submicron particles carrying chlorhexidine, calcium and phosphorus could be developed into a new intra-canal disinfectant for dental treatments.



This study was financially supported by the National Natural Science Foundation of China (Grant No. 81570969&81470732).

Compliance with ethical standards

The teeth samples used for making dentin slices were collected under the approval of ethics committee of School and Hospital of Stomatology, Wuhan University.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Ricucci D, Siqueira JF Jr., Bate AL, Pitt Ford TR. Histologic investigation of root canal-treated teeth with apical periodontitis: a retrospective study from twenty-four patients. J Endod. 2009;35:493–502.CrossRefGoogle Scholar
  2. 2.
    Penas PP, Mayer MP, Gomes BP, Endo M, Pignatari AC, Bauab KC, et al. Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections. J Endod. 2013;39:858–64.CrossRefGoogle Scholar
  3. 3.
    Sakamoto M, Siqueira JF Jr, Rôças IN, Benno Y. Molecular analysis of the root canal microbiota associated with endodontic treatment failures. Oral Microbiol Immunol. 2008;23:275–81.CrossRefGoogle Scholar
  4. 4.
    Ran S, Wang J, Jiang W, Zhu C, Liang J. Assessment of dentinal tubule invasion capacity of Enterococcus faecalis under stress conditions ex vivo. Int Endod J. 2015;48:362–72.CrossRefGoogle Scholar
  5. 5.
    Kayaoglu G, Ørstavik D. Virulence factors of Enterococcus faecalis relationship to endodontic disease. Crit Rev Oral Biol Med. 2004;15:308–20.CrossRefGoogle Scholar
  6. 6.
    Karpiński TM, Szkaradkiewicz AK. Chlorhexidine–pharmaco-biological activity and application. Eur Rev Med Pharmacol Sci. 2015;19:1321–6.Google Scholar
  7. 7.
    Martins Justo A, Abreu da Rosa R, Santini MF, Cardoso Ferreira MB, Pereira JR, Hungaro Duarte MA, et al. Effectiveness of final irrigant protocols for debris removal from simulated canal irregularities. J Endod. 2014;40:2009–14.CrossRefGoogle Scholar
  8. 8.
    Soares Ade J, Lins FF, Nagata JY, Gomes BP, Zaia AA, Ferraz CC, et al. Pulp revascularization after root canal decontamination with calcium hydroxide and 2% chlorhexidine gel. J Endod. 2013;39:417–20.CrossRefGoogle Scholar
  9. 9.
    Ebru-Tirali R, Bodur H, Ece G In vitro antimicrobial activity of sodium hypochlorite, chlorhexidine gluconate and octenidine dihydrochloride in elimination of microor-ganisms within dentinal tubules of primary and permanent teeth. Med Oral Patol Oral Cir Bucal 2012:e517-22.Google Scholar
  10. 10.
    Hasheminia S, Farhad AR, Saatchi M, Rajabzadeh M. Synergistic antibacterial activity of chlorhexidine and hydrogen peroxide against Enterococcus faecalis. J Oral Sci. 2013;55:275–80.CrossRefGoogle Scholar
  11. 11.
    Mir M, Ahmed N, Rehman AU. Recent applications of PLGA based nanostructures in drug delivery. Colloids Surf B Biointerfaces. 2017;159:217–31.CrossRefGoogle Scholar
  12. 12.
    Kashi TS, Eskandarion S, Esfandyari-Manesh M, Marashi SM, Samadi N, Fatemi SM, et al. Improved drug loading and antibacterial activity of minocycline-loaded PLGA nanoparticles prepared by solid/oil/water ion pairing method. Int J Nanomed. 2012;7:221–34.Google Scholar
  13. 13.
    Matoba T, Egashira K. Nanoparticle-mediated drug delivery system for cardiovascular disease. Int Heart J. 2014;55:281–6.CrossRefGoogle Scholar
  14. 14.
    Yallapu MM, Gupta BK, Jaggi M, Chauhan SC. Fabrication of curcumin encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer cells. J Colloid Interface Sci. 2010;351:19–29.CrossRefGoogle Scholar
  15. 15.
    Kuo YC, Yu HW. Surface coverage of didecyl dimethylammonium bromide on poly(lactide-co-glycolide) nanoparticles. Colloids Surf B Biointerfaces. 2011;84:253–8.CrossRefGoogle Scholar
  16. 16.
    Cunha-Azevedo EP, Silva JR, Martins OP, Siqueira-Moura MP, Bocca AL, Felipe MS, et al. In vitro antifungal activity and toxicity of itraconazole in DMSA-PLGA nanoparticles. J Nanosci Nanotechnol. 2011;11:2308–14.CrossRefGoogle Scholar
  17. 17.
    Simon-Yarza T, Tamayo E, Benavides C, Lana H, Formiga FR, Grama CN, et al. Functional benefits of PLGA particulates carrying VEGF and CoQ10 in an animal of myocardial ischemia. Int J Pharm. 2013;454:784–90.CrossRefGoogle Scholar
  18. 18.
    Jiang X, Lin H, Jiang D, Xu G, Fang X, He L, et al. Co-delivery of VEGF and bFGF via a PLGA nanoparticle-modified BAM for effective contracture inhibition of regenerated bladder tissue in rabbits. Sci Rep. 2016;6:20784.CrossRefGoogle Scholar
  19. 19.
    Zhu H, Chen H, Zeng X, Wang Z, Zhang X, Wu Y, et al. Co-delivery of chemotherapeutic drugs with vitamin E TPGS by porous PLGA nanoparticles for enhanced chemotherapy against multi-drug resistance. Biomaterials. 2014;35:2391–400.CrossRefGoogle Scholar
  20. 20.
    Aslantas EE, Buzoglu HD, Altundasar E, Serper A. Effect of EDTA, sodium hypochlorite, and chlorhexidine gluconate with or without surface modifiers on dentin microhardness. J Endod. 2014;40:876–9.CrossRefGoogle Scholar
  21. 21.
    Yoldaş O, Doğan C, Seydaoğlu G. The effect of two different calcium hydroxide combinations on root dentine microhardness. Int Endod J. 2004;37:828–31.CrossRefGoogle Scholar
  22. 22.
    Cruz-Filho AM, Sousa-Neto MD, Savioli RN, Silva RG, Vansan LP, Pecora JD. Effect of chelating solutions on the microhardness of root canal lumen dentin. J Endod. 2011;37:358–62.CrossRefGoogle Scholar
  23. 23.
    Alcala-Alcala S, Benitez-Cardoza CG, Lima-Munoz EJ, Pinon-Segundo E, Quintanar-Guerrero D. Evaluation of a combined drug-delivery system for proteins assembled with polymeric nanoparticles and porous microspheres; characterization and protein integrity studies. Int J Pharm. 2015;489:139–47.CrossRefGoogle Scholar
  24. 24.
    Farokhi M, Mottaghitalab F, Shokrgozar MA, Ai J, Hadjati J, Azami M. Bio-hybrid silk fibroin/calcium phosphate/PLGA nanocomposite scaffold to control the delivery of vascular endothelial growth factor. Mater Sci Eng C Mater Biol Appl. 2014;35:401–10.CrossRefGoogle Scholar
  25. 25.
    Priyadarshini BM, Mitali K, Lu TB, Handral HK, Dubey N, Fawzy AS. PLGA nanoparticles as chlorhexidine-delivery carrier to resin-dentin adhesive interface. Dent Mater. 2017;33:830–46.CrossRefGoogle Scholar
  26. 26.
    Fan W, Li Y, Sun Q, Ma T, Fan B. Calcium-silicate mesoporous nanoparticles loaded with chlorhexidine for both anti- Enterococcus faecalis and mineralization properties. J Nanobiotechnology. 2016;14:72.CrossRefGoogle Scholar
  27. 27.
    Priyadarshini BM, Selvan ST, Lu TB, Xie H, Neo J, Fawzy AS. Chlorhexidine nanocapsule drug delivery approach to the resin-dentin interface. J Dent Res. 2016;95:1065–72.CrossRefGoogle Scholar
  28. 28.
    Heard DD, Ashworth RW. The colloidal properties of chlorhexidine and its interaction with some macromolecules. J Pharm Pharmac. 1968;20:505–12.CrossRefGoogle Scholar
  29. 29.
    Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J. 2007;52:S64–82.CrossRefGoogle Scholar
  30. 30.
    Vasita R, Katti DS. Structural and functional characterization of proteins adsorbed on hydrophilized polylactide-co-glycolide microfibers. Int J Nanomed. 2012;7:61–71.Google Scholar
  31. 31.
    Allahyari M, Mohabati R, Amiri S, Esmaeili Rastaghi AR, Babaie J, Mahdavi M, et al. Synergistic effect of rSAG1 and rGRA2 antigens formulated in PLGA microspheres in eliciting immune protection against Toxoplasama gondii. Exp Parasitol. 2016;170:236–46.CrossRefGoogle Scholar
  32. 32.
    Lu JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn. 2009;9:325–41.CrossRefGoogle Scholar
  33. 33.
    Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials. 2000;21:2475–90.CrossRefGoogle Scholar
  34. 34.
    Alqahtani S, Simon L, Astete CE, Alayoubi A, Sylvester PW, Nazzal S, et al. Cellular uptake, antioxidant and antiproliferative activity of entrapped alpha-tocopherol and gamma-tocotrienol in poly (lactic-co-glycolic) acid (PLGA) and chitosan covered PLGA nanoparticles (PLGA-Chi). J Colloid Interface Sci. 2015;445:243–51.CrossRefGoogle Scholar
  35. 35.
    Trif M, Florian PE, Roseanu A, Moisei M, Craciunescu O, Astete CE, et al. Cytotoxicity and intracellular fate of PLGA and chitosan-coated PLGA nanoparticles in Madin-Darby bovine kidney (MDBK) and human colorectal adenocarcinoma (Colo 205) cells. J Biomed Mater Res A. 2015;103:3599–611.CrossRefGoogle Scholar
  36. 36.
    Qian Y, Zhou X, Sun H, Yang J, Chen Y, Li C, et al. Biomimetic domain-active electrospun scaffolds facilitating bone regeneration synergistically with antibacterial efficacy for bone defects. ACS Appl Mater Interfaces. 2018;10:3248–59.CrossRefGoogle Scholar
  37. 37.
    Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92:446–50.CrossRefGoogle Scholar
  38. 38.
    Li YC, Kuan YH, Lee SS, Huang FM, Chang YC. Cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro. Environ Toxicol. 2014;29:452–8.CrossRefGoogle Scholar
  39. 39.
    Prado M, Silva EJ, Duque TM, Zaia AA, Ferraz CC, Almeida JF, et al. Antimicrobial and cytotoxic effects of phosphoric acid solution compared to other root canal irrigants. J Appl Oral Sci. 2015;23:158–63.CrossRefGoogle Scholar
  40. 40.
    Arias-Moliz MT, Ferrer-Luque CM, Espigares-Rodriguez E, Liebana-Urena J, Espigares-Garcia M. Bactericidal activity of phosphoric acid, citric acid, and EDTA solutions against Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;106:e84–9.CrossRefGoogle Scholar
  41. 41.
    Hennequin M, Douillard Y. Effects of citric acid treatment on the Ca, P and Mg. J Clin Peridontol. 1995;22:550–7.CrossRefGoogle Scholar
  42. 42.
    Dogan H, Calt S. Effects of chelating agents and sodium hypochlorite on mineral content of root dentin. J Endod. 2001;27:578–80.CrossRefGoogle Scholar
  43. 43.
    Ari H, Erdemir A. Effects of endodontic irrigation solutions on mineral content of root canal dentin using ICP-AES technique. J Endod. 2005;31:187–9.CrossRefGoogle Scholar
  44. 44.
    Rotstein I, Dankner E, Goldman A, Heling I, Stabholz A, Zalkind M. Histochemical analysis of dental hard tissues following bleaching. J Endod. 1996;22:23–5.CrossRefGoogle Scholar
  45. 45.
    Oliveira LD, Carvalho CA, Nunes W, Valera MC, Camargo CH, Jorge AO. Effects of chlorhexidine and sodium hypochlorite on the microhardness of root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104:e125–8.CrossRefGoogle Scholar
  46. 46.
    Marcelino APM, Bruniera JF, Rached-Junior FA, Silva SRCd, Messias DC. Impact of chemical agents for surface treatments on microhardness and flexural strength of root dentin. Braz Oral Res. 2014;28:1–6.CrossRefGoogle Scholar
  47. 47.
    de Oliveira DP, Teixeira EC, Ferraz CC, Teixeira FB. Effect of intracoronal bleaching agents on dentin microhardness. J Endod. 2007;33:460–2.CrossRefGoogle Scholar
  48. 48.
    Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutions on the microhardness and the roughness of root canal dentin. J Endod. 2004;30:792–5.CrossRefGoogle Scholar
  49. 49.
    Yassen GH, Vail MM, Chu TG, Platt JA. The effect of medicaments used in endodontic regeneration on root fracture and microhardness of radicular dentine. Int Endod J. 2013;46:688–95.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wei Fan
    • 1
  • Yanyun Li
    • 1
  • Danfeng Liu
    • 1
  • Qing Sun
    • 1
  • Mengting Duan
    • 1
  • Bing Fan
    • 1
    Email author
  1. 1.The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of StomatologyWuhan UniversityWuhanPeople’s Republic of China

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