Novel nano-cubosomal chlorhexidine for endodontic irrigation: Development, characterization, and performance evaluation against E. faecalis and streptococcal biofilms in human extracted teeth

Sindhu U. Mukthamath; Ruchira Raychaudhuri; Mamatha Ballal; Kalyana C Pentapati; Krishnaraj Somayaji Shirur; Srinivas Mutalik

doi:10.7324/JAPS.2024.195780

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Abstract

Disinfection of the root canal system is one of the most complicated procedures and involves the use of a variety of equipment and techniques, irrigation regimens, and intracanal medications. “Cubosomes” (CUs) are among the few nanocarriers that are currently being explored for active drug delivery systems. A novel cubosomal chlorhexidine (CHX) for root canal irrigation was developed, and its release profile and antimicrobial efficacy against Enterococcus faecalis and streptococcal biofilms were evaluated. CUs were characterized for shape, particle size, polydispersity index, zeta potential, and drug entrapment. Drug release was determined using the dialysis method. Microbial analysis of live and dead bacteria was performed via confocal laser scanning microscopy (CLSM). The average particle size, polydispersity index, and zeta potential of the CHX CUs were 157 nm, 0.4, and 19 mV, respectively. The drug entrapment efficiency was approximately 47%. Drug release studies revealed that the cubosomal formulation released approximately 77% of the drug at the end of 6 hours, and complete release was detected at 24 hours. Confirmation of biofilm formation by CLSM and the percentage (%) survival of biofilm cells revealed that the Cubosomal formulation more effectively inhibited biofilm formation even after 48 hours. Both colony-forming unit and CLSM tests revealed that cubosomal CHX showed improved antimicrobial efficacy. The novel cubosomal CHX delivery system for endodontic irrigation was proven to be successful, as it improved the release of CHX, in turn improving its antimicrobial efficacy compared to that of regular CHX solution.

Keyword: Cubosomes chlorhexidine confocal laser scanning microscopy endodontic irrigation drug release

Citation:

Mukthamath SU, Raychaudhuri R, Ballal M, Chakravarthy K, Shirur KS, Mutalik S. Novel nano-cubosomal chlorhexidine for endodontic irrigation: Development, characterization, and performance evaluation against E. faecalis and streptococcal biofilms in human extracted teeth. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.195780

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Reference

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2. Johnson EM, Flannagan SE, Sedgley CM. Coaggregation interactions between oral and endodontic Enterococcus faecalis and bacterial species isolated from persistent apical periodontitis. J Endod. 2006 Oct 1;32(10):946-50. https://doi.org/10.1016/j.joen.2006.03.023
3. Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Eur J Oral Sci. 1981;89(4):321-8. https://doi.org/10.1111/j.1600-0722.1981.tb01689.x
4. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009;42(4):288-302. https://doi.org/10.1111/j.1365-2591.2008.01540.x
5. Usman N, Baumgartner JC, Marshall JG. Influence of instrument size on root canal debridement. J Endod. 2004 Feb 1;30(2):110-2. https://doi.org/10.1097/00004770-200402000-00012
6. Loesche WJ. Chemotherapy of dental plaque infections. Oral Sci Rev. 1976 Jan 1;9:65-107.
7. Yan H, Yang H, Li K, Yu J, Huang C. Effects of chlorhexidine-encapsulated mesoporous silica nanoparticles on the anti-biofilm and mechanical properties of glass ionomer cement. Molecules. 2017;22(7):1225. https://doi.org/10.3390/molecules22071225
8. Seneviratne CJ, Leung KCF, Wong CH, Lee SF, Li X, Leung PC, et al. Nanoparticle-encapsulated chlorhexidine against oral bacterial biofilms. PLoS One. 2014;9(8):e103234. https://doi.org/10.1371/journal.pone.0103234
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10. Gomes B, Souza SFC, Ferraz CCR, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J. 2003;36(4):267-75. https://doi.org/10.1046/j.1365-2591.2003.00634.x
11. Haseeb R, Lau M, Sheah M, Montagner F, Quiram G, Palmer K, et al. Synthesis and characterization of new chlorhexidine-containing nanoparticles for root canal disinfection. Materials. 2016;9(6):452. https://doi.org/10.3390/ma9060452
12. Wei Y, Zhang J, Zheng Y, Gong Y, Fu M, Liu C, et al. Cubosomes with surface cross-linked chitosan exhibit sustained releaseand bioavailability enhancement for vinpocetine. RSC Adv. 2019;9(11):6287-98. https://doi.org/10.1039/C8RA10302J
13. Mohsen AM, Younis MM, Salama A, Darwish AB. Cubosomes as a potential oral drug delivery system for enhancing the hepatoprotective effect of coenzyme Q10. J Pharm Sci. 2021;110(7):2677-86. https://doi.org/10.1016/j.xphs.2021.02.007
14. Beyth N, Redlich M, Harari D, Friedman M, Steinberg D. Effect of sustained-release chlorhexidine varnish on Streptococcus mutans and Actinomyces viscosus in orthodontic patients. Am J Orthod Dentofacial Orthop. 2003;123(3):345-8. https://doi.org/10.1067/mod.2003.19
15. Çetin EÖ, Buduneli N, Atl?han E, K?r?lmaz L. In vitro studies on controlled-release cellulose acetate films for local delivery of chlorhexidine, indomethacin, and meloxicam. J Clin Periodontol. 2004;31(12):1117-21. https://doi.org/10.1111/j.1600-051X.2004.00620.x
16. Elgindy NA, Mehanna MM, Mohyeldin SM. Self-assembled nano-architecture liquid crystalline particles as a promising carrier for progesterone transdermal delivery. Int J Pharm. 2016;501(1-2):167-79. https://doi.org/10.1016/j.ijpharm.2016.01.049
17. Shreya AB, Managuli RS, Menon J, Kondapalli L, Hegde AR, Avadhani K, et al. Nano-transfersomal formulations for transdermal delivery of asenapine maleate: in vitro and in vivo performance evaluations. J Liposome Res. 2016;26(3):221-32. https://doi.org/10.3109/08982104.2015.1098659
18. Rarokar NR, Saoji SD, Raut NA, Taksande JB, Khedekar PB, Dave VS. Nanostructured cubosomes in a thermoresponsive Depot system: an alternative approach for the controlled delivery of docetaxel. AAPS PharmSciTech. 2016 Apr 1;17(2):436-45. https://doi.org/10.1208/s12249-015-0369-y
19. Vitkov L, Hannig M, Krautgartner WD, Herrmann M, Fuchs K, Klappacher M, et al. Ex vivo gingival-biofilm consortia. Lett Appl Microbiol. 2005;41(5):404-11. https://doi.org/10.1111/j.1472-765X.2005.01787.x
20. Jagielski J, Przysiecka ?, Flak D, Diak M, Pietralik-Moli?ska Z, Kozak M, et al. Comprehensive and comparative studies on nanocytotoxicity of glyceryl monooleate- and phytantriol-based lipid liquid crystalline nanoparticles. J Nanobiotechnol. 2021 Jun 3;19(1):168. https://doi.org/10.1186/s12951-021-00913-5
21. Hinton TM, Grusche F, Acharya D, Shukla R, Bansal V, Waddington LJ, et al. Bicontinuous cubic phase nanoparticle lipid chemistry affects toxicity in cultured cells. Toxicol Res. 2014 Jan 1;3(1):11-22. https://doi.org/10.1039/C3TX50075F
22. Karami Z, Hamidi M. Cubosomes: remarkable drug delivery potential. Drug Discov Today. 2016;21(5):789-801. https://doi.org/10.1016/j.drudis.2016.01.004
23. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci. 2002;6(4):319-27. https://doi.org/10.1016/S1359-0286(02)00117-1
24. Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J Control Release. 2002;82(1):105-14. https://doi.org/10.1016/S0168-3659(02)00127-X
25. Onnainty R, Onida B, Páez P, Longhi M, Barresi A, Granero G. Targeted chitosan-based bionanocomposites for controlled oral mucosal delivery of chlorhexidine. Int J Pharm. 2016;509(1-2):408-18. https://doi.org/10.1016/j.ijpharm.2016.06.011
26. Garala K, Joshi P, Shah M, Ramkishan A, Patel J. Formulation and evaluation of periodontal in situ gel. Int J Pharm Investig. 2013;3(1):29. https://doi.org/10.4103/2230-973X.108961
27. Tartari T, Wichnieski C, Bachmann L, Jafelicci Jr M, Silva RM, Letra A, et al. Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine. Int Endod J. 2018;51(12):1420-33. https://doi.org/10.1111/iej.12960
28. Mahendra A, Koul M, Upadhyay V, Dwivedi R. Comparative evaluation of antimicrobial substantivity of different concentrations of chlorhexidine as a root canal irrigant: an in vitro study. J Oral Biol Craniofacial Res. 2014;4(3):181-5. https://doi.org/10.1016/j.jobcr.2014.11.005
29. Akram Z, Aati S, Ngo H, Fawzy A. pH-dependent delivery of chlorhexidine from PGA grafted mesoporous silica nanoparticles at resin-dentin interface. J Nanobiotechnol. 2021;19(1):1-16. https://doi.org/10.1186/s12951-021-00788-6
30. Tirali RE, Turan Y, Akal N, Karahan ZC. In vitro antimicrobial activity of several concentrations of NaOCl and Octenisept in elimination of endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol. 2009;108(5):e117-20. https://doi.org/10.1016/j.tripleo.2009.07.012
31. Haenni S, Schmidlin PR, Mueller B, Sener B, Zehnder M. Chemical and antimicrobial properties of calcium hydroxide mixed with irrigating solutions. Int Endod J. 2003;36(2):100-5. https://doi.org/10.1046/j.1365-2591.2003.00629.x
32. Oliveira C, Ferreira CJO, Sousa M, Paris JL, Gaspar R, Silva BFB, et al. A versatile nanocarrier-cubosomes, characterization, and applications. Nanomaterials. 2022 Jan;12(13):2224. https://doi.org/10.3390/nano12132224
33. Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J. 2006 Dec 1;32(3):112-5. https://doi.org/10.1111/j.1747-4477.2006.00033.x
34. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin. J Endod. 2008;34(7):850-4. https://doi.org/10.1016/j.joen.2008.04.006
35. Kapoor K, Pandit V, Nagaich U. Development and characterization of sustained release methotrexate loaded cubosomes for topical delivery in rheumatoid arthritis. Int J Appl Pharm. 2020;12(3):33-9. https://doi.org/10.22159/ijap.2020v12i3.36863
36. 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(9):1065-72. https://doi.org/10.1177/0022034516656135
37. Jain K, Agarwal P, Jain S, Seal M, Adlakha T. Alexidine versus chlorhexidine for endodontic irrigation with sodium hypochlorite. Eur J Dent. 2018;12(03):398-402. https://doi.org/10.4103/ejd.ejd_180_17
38. Gomes B, Ferraz CCR, Vianna ME, Berber VB, Teixeira FB, Souza?Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J. 2001;34(6):424-8. https://doi.org/10.1046/j.1365-2591.2001.00410.x
39. Zapata RO, Bramante CM, de Moraes IG, Bernardineli N, Gasparoto TH, Graeff MS, et al. Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin. J Endod. 2008;34(10):1198-201. https://doi.org/10.1016/j.joen.2008.07.001
40. Koburger T, Hübner NO, Braun M, Siebert J, Kramer A. Standardized comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother. 2010;65(8):1712-9. https://doi.org/10.1093/jac/dkq212
41. Cherian B, Gehlot PM, Manjunath MK. Comparison of the antimicrobial efficacy of octenidine dihydrochloride and chlorhexidine with and without passive ultrasonic irrigation-an in vitro study. J Clin Diagn Res JCDR. 2016 Jun;10(6):ZC71-77. https://doi.org/10.7860/JCDR/2016/17911.8021
42. Salah S, Mahmoud AA, Kamel AO. Etodolac transdermal cubosomes for the treatment of rheumatoid arthritis: ex vivo permeation and in vivo pharmacokinetic studies. Drug Deliv. 2017;24(1):846-56. https://doi.org/10.1080/10717544.2017.1326539
43. Shahadeh M, Jabban MO, Altaki Z. Evaluation of antimicrobial effectiveness of two endodontic irrigation solutions on the microbial reduction of Enterococcus faecalis in infected root canals (in vitro study). Int Dent Med J Adv Res. 2015;1(1):1-6. https://doi.org/10.15713/ins.idmjar.33

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1. Antunes HS, Rôças IN, Alves FR, Siqueira Jr JF. Total and specific bacterial levels in the apical root canal system of teeth with post-treatment apical periodontitis. J Endod. 2015;41(7):1037-42. https://doi.org/10.1016/j.joen.2015.03.008

2. Johnson EM, Flannagan SE, Sedgley CM. Coaggregation interactions between oral and endodontic Enterococcus faecalis and bacterial species isolated from persistent apical periodontitis. J Endod. 2006 Oct 1;32(10):946-50. https://doi.org/10.1016/j.joen.2006.03.023

3. Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Eur J Oral Sci. 1981;89(4):321-8. https://doi.org/10.1111/j.1600-0722.1981.tb01689.x

4. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009;42(4):288-302. https://doi.org/10.1111/j.1365-2591.2008.01540.x

5. Usman N, Baumgartner JC, Marshall JG. Influence of instrument size on root canal debridement. J Endod. 2004 Feb 1;30(2):110-2. https://doi.org/10.1097/00004770-200402000-00012

6. Loesche WJ. Chemotherapy of dental plaque infections. Oral Sci Rev. 1976 Jan 1;9:65-107.

7. Yan H, Yang H, Li K, Yu J, Huang C. Effects of chlorhexidine-encapsulated mesoporous silica nanoparticles on the anti-biofilm and mechanical properties of glass ionomer cement. Molecules. 2017;22(7):1225. https://doi.org/10.3390/molecules22071225

8. Seneviratne CJ, Leung KCF, Wong CH, Lee SF, Li X, Leung PC, et al. Nanoparticle-encapsulated chlorhexidine against oral bacterial biofilms. PLoS One. 2014;9(8):e103234. https://doi.org/10.1371/journal.pone.0103234

9. Tronstad L, Barnett F, Cervone F. Periapical bacterial plaque in teeth refractory to endodontic treatment. Dent Traumatol. 1990;6(2):73-7. https://doi.org/10.1111/j.1600-9657.1990.tb00394.x

10. Gomes B, Souza SFC, Ferraz CCR, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J. 2003;36(4):267-75. https://doi.org/10.1046/j.1365-2591.2003.00634.x

11. Haseeb R, Lau M, Sheah M, Montagner F, Quiram G, Palmer K, et al. Synthesis and characterization of new chlorhexidine-containing nanoparticles for root canal disinfection. Materials. 2016;9(6):452. https://doi.org/10.3390/ma9060452

12. Wei Y, Zhang J, Zheng Y, Gong Y, Fu M, Liu C, et al. Cubosomes with surface cross-linked chitosan exhibit sustained releaseand bioavailability enhancement for vinpocetine. RSC Adv. 2019;9(11):6287-98. https://doi.org/10.1039/C8RA10302J

13. Mohsen AM, Younis MM, Salama A, Darwish AB. Cubosomes as a potential oral drug delivery system for enhancing the hepatoprotective effect of coenzyme Q10. J Pharm Sci. 2021;110(7):2677-86. https://doi.org/10.1016/j.xphs.2021.02.007

14. Beyth N, Redlich M, Harari D, Friedman M, Steinberg D. Effect of sustained-release chlorhexidine varnish on Streptococcus mutans and Actinomyces viscosus in orthodontic patients. Am J Orthod Dentofacial Orthop. 2003;123(3):345-8. https://doi.org/10.1067/mod.2003.19

15. Çetin EÖ, Buduneli N, Atl?han E, K?r?lmaz L. In vitro studies on controlled-release cellulose acetate films for local delivery of chlorhexidine, indomethacin, and meloxicam. J Clin Periodontol. 2004;31(12):1117-21. https://doi.org/10.1111/j.1600-051X.2004.00620.x

16. Elgindy NA, Mehanna MM, Mohyeldin SM. Self-assembled nano-architecture liquid crystalline particles as a promising carrier for progesterone transdermal delivery. Int J Pharm. 2016;501(1-2):167-79. https://doi.org/10.1016/j.ijpharm.2016.01.049

17. Shreya AB, Managuli RS, Menon J, Kondapalli L, Hegde AR, Avadhani K, et al. Nano-transfersomal formulations for transdermal delivery of asenapine maleate: in vitro and in vivo performance evaluations. J Liposome Res. 2016;26(3):221-32. https://doi.org/10.3109/08982104.2015.1098659

18. Rarokar NR, Saoji SD, Raut NA, Taksande JB, Khedekar PB, Dave VS. Nanostructured cubosomes in a thermoresponsive Depot system: an alternative approach for the controlled delivery of docetaxel. AAPS PharmSciTech. 2016 Apr 1;17(2):436-45. https://doi.org/10.1208/s12249-015-0369-y

19. Vitkov L, Hannig M, Krautgartner WD, Herrmann M, Fuchs K, Klappacher M, et al. Ex vivo gingival-biofilm consortia. Lett Appl Microbiol. 2005;41(5):404-11. https://doi.org/10.1111/j.1472-765X.2005.01787.x

20. Jagielski J, Przysiecka ?, Flak D, Diak M, Pietralik-Moli?ska Z, Kozak M, et al. Comprehensive and comparative studies on nanocytotoxicity of glyceryl monooleate- and phytantriol-based lipid liquid crystalline nanoparticles. J Nanobiotechnol. 2021 Jun 3;19(1):168. https://doi.org/10.1186/s12951-021-00913-5

21. Hinton TM, Grusche F, Acharya D, Shukla R, Bansal V, Waddington LJ, et al. Bicontinuous cubic phase nanoparticle lipid chemistry affects toxicity in cultured cells. Toxicol Res. 2014 Jan 1;3(1):11-22. https://doi.org/10.1039/C3TX50075F

22. Karami Z, Hamidi M. Cubosomes: remarkable drug delivery potential. Drug Discov Today. 2016;21(5):789-801. https://doi.org/10.1016/j.drudis.2016.01.004

23. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci. 2002;6(4):319-27. https://doi.org/10.1016/S1359-0286(02)00117-1

24. Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J Control Release. 2002;82(1):105-14. https://doi.org/10.1016/S0168-3659(02)00127-X

25. Onnainty R, Onida B, Páez P, Longhi M, Barresi A, Granero G. Targeted chitosan-based bionanocomposites for controlled oral mucosal delivery of chlorhexidine. Int J Pharm. 2016;509(1-2):408-18. https://doi.org/10.1016/j.ijpharm.2016.06.011

26. Garala K, Joshi P, Shah M, Ramkishan A, Patel J. Formulation and evaluation of periodontal in situ gel. Int J Pharm Investig. 2013;3(1):29. https://doi.org/10.4103/2230-973X.108961

27. Tartari T, Wichnieski C, Bachmann L, Jafelicci Jr M, Silva RM, Letra A, et al. Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine. Int Endod J. 2018;51(12):1420-33. https://doi.org/10.1111/iej.12960

28. Mahendra A, Koul M, Upadhyay V, Dwivedi R. Comparative evaluation of antimicrobial substantivity of different concentrations of chlorhexidine as a root canal irrigant: an in vitro study. J Oral Biol Craniofacial Res. 2014;4(3):181-5. https://doi.org/10.1016/j.jobcr.2014.11.005

29. Akram Z, Aati S, Ngo H, Fawzy A. pH-dependent delivery of chlorhexidine from PGA grafted mesoporous silica nanoparticles at resin-dentin interface. J Nanobiotechnol. 2021;19(1):1-16. https://doi.org/10.1186/s12951-021-00788-6

30. Tirali RE, Turan Y, Akal N, Karahan ZC. In vitro antimicrobial activity of several concentrations of NaOCl and Octenisept in elimination of endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol. 2009;108(5):e117-20. https://doi.org/10.1016/j.tripleo.2009.07.012

31. Haenni S, Schmidlin PR, Mueller B, Sener B, Zehnder M. Chemical and antimicrobial properties of calcium hydroxide mixed with irrigating solutions. Int Endod J. 2003;36(2):100-5. https://doi.org/10.1046/j.1365-2591.2003.00629.x

32. Oliveira C, Ferreira CJO, Sousa M, Paris JL, Gaspar R, Silva BFB, et al. A versatile nanocarrier-cubosomes, characterization, and applications. Nanomaterials. 2022 Jan;12(13):2224. https://doi.org/10.3390/nano12132224

33. Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J. 2006 Dec 1;32(3):112-5. https://doi.org/10.1111/j.1747-4477.2006.00033.x

34. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin. J Endod. 2008;34(7):850-4. https://doi.org/10.1016/j.joen.2008.04.006

35. Kapoor K, Pandit V, Nagaich U. Development and characterization of sustained release methotrexate loaded cubosomes for topical delivery in rheumatoid arthritis. Int J Appl Pharm. 2020;12(3):33-9. https://doi.org/10.22159/ijap.2020v12i3.36863

36. 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(9):1065-72. https://doi.org/10.1177/0022034516656135

37. Jain K, Agarwal P, Jain S, Seal M, Adlakha T. Alexidine versus chlorhexidine for endodontic irrigation with sodium hypochlorite. Eur J Dent. 2018;12(03):398-402. https://doi.org/10.4103/ejd.ejd_180_17

38. Gomes B, Ferraz CCR, Vianna ME, Berber VB, Teixeira FB, Souza?Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J. 2001;34(6):424-8. https://doi.org/10.1046/j.1365-2591.2001.00410.x

39. Zapata RO, Bramante CM, de Moraes IG, Bernardineli N, Gasparoto TH, Graeff MS, et al. Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin. J Endod. 2008;34(10):1198-201. https://doi.org/10.1016/j.joen.2008.07.001

40. Koburger T, Hübner NO, Braun M, Siebert J, Kramer A. Standardized comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother. 2010;65(8):1712-9. https://doi.org/10.1093/jac/dkq212

41. Cherian B, Gehlot PM, Manjunath MK. Comparison of the antimicrobial efficacy of octenidine dihydrochloride and chlorhexidine with and without passive ultrasonic irrigation-an in vitro study. J Clin Diagn Res JCDR. 2016 Jun;10(6):ZC71-77. https://doi.org/10.7860/JCDR/2016/17911.8021

42. Salah S, Mahmoud AA, Kamel AO. Etodolac transdermal cubosomes for the treatment of rheumatoid arthritis: ex vivo permeation and in vivo pharmacokinetic studies. Drug Deliv. 2017;24(1):846-56. https://doi.org/10.1080/10717544.2017.1326539

43. Shahadeh M, Jabban MO, Altaki Z. Evaluation of antimicrobial effectiveness of two endodontic irrigation solutions on the microbial reduction of Enterococcus faecalis in infected root canals (in vitro study). Int Dent Med J Adv Res. 2015;1(1):1-6. https://doi.org/10.15713/ins.idmjar.33