Anti Microbial Activity of Chitosan Nanoparticles with Chlorhexidine- An In vitro Study
Main Article Content
Keywords
Endodontic Irrigants,Chitosan, Chitosan nanoparticles , Natural Irrigant, Antibacterial Activity, Biocompatibility, E. feacalis,Root Canal Treatment
Abstract
Introduction: Irrigants also play a crucial role in removing debris and smear layer from the root canal walls, in addition to antimicrobial activity which allows for better adhesion and penetration of root canal sealers and obturation materials (1). The most commonly used irrigants in endodontic treatment include sodium hypochlorite, chlorhexidine, and ethylenediaminetetraacetic acid (EDTA) [6]. However, each of these irrigants has its own limitations and potential side effects, such as cytotoxicity, allergic reactions, and dentin erosion (2). Therefore, the search for alternative irrigants that possess better antimicrobial activity and fewer side effects is ongoing in the field of endodontics.
Materials and Methods: The antimicrobial activity of the synthesised nano chitosan with chlorhexidine and Plain Chitosan with Chlorhexidine was evaluated using the agar well diffusion technique. Mueller Hinton agar plates were prepared and sterilised using an autoclave at 121°C for 15- 20 minutes. After sterilisation, the medium was poured on to the surface of sterile Petri plates and allowed to cool to room temperature. The bacterial suspension (E.faecalis) was spread evenly onto the agar plates using sterile cotton swabs. The wells were then filled with different concentrations of nanoparticles and plain chitosan solution. An antibiotic (e.g., Bacteria-Amoxyrite) was used as a standard. The plates were incubated at 37°C for 24 hours and 48 hours for bacterial cultures.
Results: Nanochitosan with chlorhexidine shows higher antimicrobial activity when compared to plain chitosan .Its activity increases with increase in dosage.10μl shows maximum antimicrobial efficacy. Increase in the time period showed increased antimicrobial efficacy. Antimicrobial efficacy at 10μl is comparable to positive control (sodium hypochlorite)
Conclusion: The irrigant nanochitosan with chlorhexidine showed better antibacterial efficacy than sodium hypochlorite and it can be used as an irrigant in endodontics. The several known advantages of this irrigant such as naturally available, non cytotoxic, biocompatible and low cost make it a good replacement of sodium hypochlorite as an irrigant.
References
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9. Fabian TM, Walker SE. Stability of sodium hypochlorite solutions. Am J Hosp Pharm. 1982 Jun;39(6):1016–7.
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11. Reeh ES, Messer HH. Long-term paresthesia following inadvertent forcing of sodium hypochlorite through perforation in maxillary incisor. Endod Dent Traumatol. 1989 Aug;5(4):200–3.
12. Lee LW, Lan WH, Wang GY. [A evaluation of chlorhexidine as an endosonic irrigan]. J Formos Med Assoc. 1990 Jun;89(6):491–7.
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14. Torabinejad M, Walton RE, Fouad A. Endodontics - E-Book: Principles and Practice. Elsevier Health Sciences; 2014. 496 p.
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16. Shabahang S, Aslanyan J, Torabinejad M. The substitution of chlorhexidine for doxycycline in MTAD: the antibacterial efficacy against a strain of Enterococcus faecalis. J Endod. 2008 Mar;34(3):288–90.
17. Afkhami F, Forghan P, Gutmann JL, Kishen A. Silver Nanoparticles and Their Therapeutic Applications in Endodontics: A Narrative Review. Pharmaceutics [Internet]. 2023 Feb 21;15(3). Available from: http://dx.doi.org/10.3390/pharmaceutics15030715
18. Betancourt P, Brocal N, Sans-Serramitjana E, Zaror C. Functionalized Nanoparticles Activated by Photodynamic Therapy as an Antimicrobial Strategy in Endodontics: A Scoping Review. Antibiotics (Basel) [Internet]. 2021 Sep 2;10(9). Available from: http://dx.doi.org/10.3390/antibiotics10091064
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20. Skoskiewicz-Malinowska K, Kaczmarek U, Malicka B, Walczak K, Zietek M. Application of Chitosan and Propolis in Endodontic Treatment: A Review. Mini Rev Med Chem. 2017;17(5):410–34.
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23. Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod. 2008 Dec;34(12):1515–20.
24. Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm. J Endod. 2014 Feb;40(2):285–90.
25. Roshdy NN, Kataia EM, Helmy NA. Assessment of antibacterial activity of 2.5% NaOCl, chitosan nano-particles against contaminating root canals with and without diode laser irradiation: an study. Acta Odontol Scand. 2019 Jan;77(1):39–43.
26. Jana S, Jana S. Functional Chitosan: Drug Delivery and Biomedical Applications. Springer Nature; 2020. 489 p.
27. Gupta A. Chitosan Nanoparticles. Arcler Press; 2017.
28. Pereira P. Chitosan Nanoparticles for Biomedical Applications. 2010. 76 p.
29. Goud S, Aravelli S, Dronamraju S, Cherukuri G, Morishetty P. Comparative Evaluation of the Antibacterial Efficacy of Aloe Vera, 3% Sodium Hypochlorite, and 2% Chlorhexidine Gluconate Against Enterococcus faecalis: An In Vitro Study. Cureus. 2018 Oct 22;10(10):e3480.
30. Agrawal V, Rao MR, Dhingra K, Gopal VR, Mohapatra A, Mohapatra A. An in vitro comparison of antimicrobial effcacy of three root canal irrigants-BioPure MTAD, 2% chlorhexidine gluconate and 5.25% sodium hypochlorite as a final rinse against E. faecalis. J Contemp Dent Pract. 2013 Sep 1;14(5):842–7.
31. Del Carpio-Perochena A, Kishen A, Felitti R, Bhagirath AY, Medapati MR, Lai C, et al. Antibacterial Properties of Chitosan Nanoparticles and Propolis Associated with Calcium Hydroxide against Single- and Multispecies Biofilms: An In Vitro and In Situ Study. J Endod. 2017 Aug;43(8):1332–6.
32. De Deus G, Silva EJN, Souza E, Versiani MA, Zuolo M. Shaping for Cleaning the Root Canals: A Clinical-Based Strategy. Springer Nature; 2021. 376 p.
33. Retamozo B, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. Minimum contact time and concentration of sodium hypochlorite required to eliminate Enterococcus faecalis. J Endod. 2010 Mar;36(3):520–3.
34. Kesar S, Bhatti MS. Chlorination of secondary treated wastewater with sodium hypochlorite (NaOCl): An effective single alternate to other disinfectants. Heliyon. 2022 Nov;8(11):e11162.
35. Ravinanthanan M, Hegde MN, Shetty V, Kumari S, Al Qahtani FN. A Comparative Evaluation of Antimicrobial Efficacy of Novel Surfactant-Based Endodontic Irrigant Regimen’s on. Contemp Clin Dent. 2022 Sep 24;13(3):205–10.
36. Del Carpio-Perochena A, Bramante CM, Duarte MAH, de Moura MR, Aouada FA, Kishen A. Chelating and antibacterial properties of chitosan nanoparticles on dentin. Restor Dent Endod. 2015 Aug;40(3):195–201.
37. Hong L, Luo SH, Yu CH, Xie Y, Xia MY, Chen GY, et al. Functional Nanomaterials and Their Potential Applications in Antibacterial Therapy. Pharm Nanotechnol. 2019;7(2):129–46.
38. Arias-Moliz MT, Ordinola-Zapata R, Baca P, Ruiz-Linares M, García García E, Hungaro Duarte MA, et al. Antimicrobial activity of Chlorhexidine, Peracetic acid and Sodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms. Int Endod J. 2015 Dec;48(12):1188–93.
39. Darcey J, Jawad S, Taylor C, Roudsari RV, Hunter M. Modern Endodontic Principles Part 4: Irrigation. Dent Update. 2016 Jan-Feb;43(1):20–2, 25–6, 28–30 passim.
40. Rôças IN, Provenzano JC, Neves MAS, Siqueira JF Jr. Disinfecting Effects of Rotary Instrumentation with Either 2.5% Sodium Hypochlorite or 2% Chlorhexidine as the Main Irrigant: A Randomized Clinical Study. J Endod. 2016 Jun;42(6):943–7.
41. Del Carpio-Perochena AE, Bramante CM, Duarte MAH, Cavenago BC, Villas-Boas MH, Graeff MS, et al. Biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. J Endod. 2011 Aug;37(8):1134–8.
42. Neelakantan P, Grotra D, Sharma S. Retreatability of 2 mineral trioxide aggregate-based root canal sealers: a cone-beam computed tomography analysis. J Endod. 2013 Jul;39(7):893–6.
43. Aldhuwayhi S, Mallineni SK, Sakhamuri S, Thakare AA, Mallineni S, Sajja R, et al. Covid-19 Knowledge and Perceptions Among Dental Specialists: A Cross-Sectional Online Questionnaire Survey. Risk Manag Healthc Policy. 2021 Jul 7;14:2851–61.
44. Sheriff KAH, Ahmed Hilal Sheriff K, Santhanam A. Knowledge and Awareness towards Oral Biopsy among Students of Saveetha Dental College [Internet]. Vol. 11, Research Journal of Pharmacy and Technology. 2018. p. 543. Available from: http://dx.doi.org/10.5958/0974-360x.2018.00101.4
45. Markov A, Thangavelu L, Aravindhan S, Zekiy AO, Jarahian M, Chartrand MS, et al. Mesenchymal stem/stromal cells as a valuable source for the treatment of immune-mediated disorders. Stem Cell Res Ther. 2021 Mar 18;12(1):192.
46. Jayaraj G, Ramani P, Herald J. Sherlin, Premkumar P, Anuja N. Inter-observer agreement in grading oral epithelial dysplasia – A systematic review [Internet]. Vol. 27, Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology. 2015. p. 112–6. Available from: http://dx.doi.org/10.1016/j.ajoms.2014.01.006
47. Paramasivam A, Priyadharsini JV, Raghunandhakumar S, Elumalai P. A novel COVID-19 and its effects on cardiovascular disease. Hypertens Res. 2020 Jul;43(7):729–30.
48. Li Z, Veeraraghavan VP, Mohan SK, Bolla SR, Lakshmanan H, Kumaran S, et al. Apoptotic induction and anti-metastatic activity of eugenol encapsulated chitosan nanopolymer on rat glioma C6 cells via alleviating the MMP signaling pathway [Internet]. Vol. 203, Journal of Photochemistry and Photobiology B: Biology. 2020. p. 111773. Available from: http://dx.doi.org/10.1016/j.jphotobiol.2019.111773
49. Gan H, Zhang Y, Zhou Q, Zheng L, Xie X, Veeraraghavan VP, et al. Zingerone induced caspase-dependent apoptosis in MCF-7 cells and prevents 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in experimental rats. J Biochem Mol Toxicol. 2019 Oct;33(10):e22387.
50. Dua K, Wadhwa R, Singhvi G, Rapalli V, Shukla SD, Shastri MD, et al. The potential of siRNA based drug delivery in respiratory disorders: Recent advances and progress. Drug Dev Res. 2019 Sep;80(6):714–30.
51. Mohan M, Jagannathan N. Oral field cancerization: an update on current concepts. Oncol Rev. 2014 Mar 17;8(1):244.
2. Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of NaOCl on vital tissue. J Endod. 1985 Dec;11(12):525–8.
3. Zehnder M, Kosicki D, Luder H, Sener B, Waltimo T. Tissue-dissolving capacity and antibacterial effect of buffered and unbuffered hypochlorite solutions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002 Dec;94(6):756–62.
4. Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod. 2003 May;29(5):334–7.
5. Naenni N, Thoma K, Zehnder M. Soft tissue dissolution capacity of currently used and potential endodontic irrigants. J Endod. 2004 Nov;30(11):785–7.
6. Siqueira JF Jr, Magalhães KM, Rôças IN. Bacterial reduction in infected root canals treated with 2.5% NaOCl as an irrigant and calcium hydroxide/camphorated paramonochlorophenol paste as an intracanal dressing. J Endod. 2007 Jun;33(6):667–72.
7. Virtej A, MacKenzie CR, Raab WHM, Pfeffer K, Barthel CR. Determination of the performance of various root canal disinfection methods after in situ carriage. J Endod. 2007 Aug;33(8):926–9.
8. Clarkson RM, Moule AJ, Podlich HM. The shelf-life of sodium hypochlorite irrigating solutions. Aust Dent J. 2001 Dec;46(4):269–76.
9. Fabian TM, Walker SE. Stability of sodium hypochlorite solutions. Am J Hosp Pharm. 1982 Jun;39(6):1016–7.
10. Hülsmann M, Hahn W. Complications during root canal irrigation--literature review and case reports. Int Endod J. 2000 May;33(3):186–93.
11. Reeh ES, Messer HH. Long-term paresthesia following inadvertent forcing of sodium hypochlorite through perforation in maxillary incisor. Endod Dent Traumatol. 1989 Aug;5(4):200–3.
12. Lee LW, Lan WH, Wang GY. [A evaluation of chlorhexidine as an endosonic irrigan]. J Formos Med Assoc. 1990 Jun;89(6):491–7.
13. Southard SR, Drisko CL, Killoy WJ, Cobb CM, Tira DE. The effect of 2% chlorhexidine digluconate irrigation on clinical parameters and the level of Bacteroides gingivalis in periodontal pockets. J Periodontol. 1989 Jun;60(6):302–9.
14. Torabinejad M, Walton RE, Fouad A. Endodontics - E-Book: Principles and Practice. Elsevier Health Sciences; 2014. 496 p.
15. Fedorowicz Z, Sequeira P. Efficacy of sodium hypochlorite and chlorhexidine against Enterococcus faecalis--a systematic review. J Appl Oral Sci. 2009 May-Jun;17(3):ii.
16. Shabahang S, Aslanyan J, Torabinejad M. The substitution of chlorhexidine for doxycycline in MTAD: the antibacterial efficacy against a strain of Enterococcus faecalis. J Endod. 2008 Mar;34(3):288–90.
17. Afkhami F, Forghan P, Gutmann JL, Kishen A. Silver Nanoparticles and Their Therapeutic Applications in Endodontics: A Narrative Review. Pharmaceutics [Internet]. 2023 Feb 21;15(3). Available from: http://dx.doi.org/10.3390/pharmaceutics15030715
18. Betancourt P, Brocal N, Sans-Serramitjana E, Zaror C. Functionalized Nanoparticles Activated by Photodynamic Therapy as an Antimicrobial Strategy in Endodontics: A Scoping Review. Antibiotics (Basel) [Internet]. 2021 Sep 2;10(9). Available from: http://dx.doi.org/10.3390/antibiotics10091064
19. Raura N, Garg A, Arora A, Roma M. Nanoparticle technology and its implications in endodontics: a review. Biomater Res. 2020 Dec 4;24(1):21.
20. Skoskiewicz-Malinowska K, Kaczmarek U, Malicka B, Walczak K, Zietek M. Application of Chitosan and Propolis in Endodontic Treatment: A Review. Mini Rev Med Chem. 2017;17(5):410–34.
21. Kishen A. Nanotechnology in Endodontics: Current and Potential Clinical Applications. Springer; 2015. 199 p.
22. DaSilva L, Finer Y, Friedman S, Basrani B, Kishen A. Biofilm formation within the interface of bovine root dentin treated with conjugated chitosan and sealer containing chitosan nanoparticles. J Endod. 2013 Feb;39(2):249–53.
23. Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod. 2008 Dec;34(12):1515–20.
24. Wu D, Fan W, Kishen A, Gutmann JL, Fan B. Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm. J Endod. 2014 Feb;40(2):285–90.
25. Roshdy NN, Kataia EM, Helmy NA. Assessment of antibacterial activity of 2.5% NaOCl, chitosan nano-particles against contaminating root canals with and without diode laser irradiation: an study. Acta Odontol Scand. 2019 Jan;77(1):39–43.
26. Jana S, Jana S. Functional Chitosan: Drug Delivery and Biomedical Applications. Springer Nature; 2020. 489 p.
27. Gupta A. Chitosan Nanoparticles. Arcler Press; 2017.
28. Pereira P. Chitosan Nanoparticles for Biomedical Applications. 2010. 76 p.
29. Goud S, Aravelli S, Dronamraju S, Cherukuri G, Morishetty P. Comparative Evaluation of the Antibacterial Efficacy of Aloe Vera, 3% Sodium Hypochlorite, and 2% Chlorhexidine Gluconate Against Enterococcus faecalis: An In Vitro Study. Cureus. 2018 Oct 22;10(10):e3480.
30. Agrawal V, Rao MR, Dhingra K, Gopal VR, Mohapatra A, Mohapatra A. An in vitro comparison of antimicrobial effcacy of three root canal irrigants-BioPure MTAD, 2% chlorhexidine gluconate and 5.25% sodium hypochlorite as a final rinse against E. faecalis. J Contemp Dent Pract. 2013 Sep 1;14(5):842–7.
31. Del Carpio-Perochena A, Kishen A, Felitti R, Bhagirath AY, Medapati MR, Lai C, et al. Antibacterial Properties of Chitosan Nanoparticles and Propolis Associated with Calcium Hydroxide against Single- and Multispecies Biofilms: An In Vitro and In Situ Study. J Endod. 2017 Aug;43(8):1332–6.
32. De Deus G, Silva EJN, Souza E, Versiani MA, Zuolo M. Shaping for Cleaning the Root Canals: A Clinical-Based Strategy. Springer Nature; 2021. 376 p.
33. Retamozo B, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. Minimum contact time and concentration of sodium hypochlorite required to eliminate Enterococcus faecalis. J Endod. 2010 Mar;36(3):520–3.
34. Kesar S, Bhatti MS. Chlorination of secondary treated wastewater with sodium hypochlorite (NaOCl): An effective single alternate to other disinfectants. Heliyon. 2022 Nov;8(11):e11162.
35. Ravinanthanan M, Hegde MN, Shetty V, Kumari S, Al Qahtani FN. A Comparative Evaluation of Antimicrobial Efficacy of Novel Surfactant-Based Endodontic Irrigant Regimen’s on. Contemp Clin Dent. 2022 Sep 24;13(3):205–10.
36. Del Carpio-Perochena A, Bramante CM, Duarte MAH, de Moura MR, Aouada FA, Kishen A. Chelating and antibacterial properties of chitosan nanoparticles on dentin. Restor Dent Endod. 2015 Aug;40(3):195–201.
37. Hong L, Luo SH, Yu CH, Xie Y, Xia MY, Chen GY, et al. Functional Nanomaterials and Their Potential Applications in Antibacterial Therapy. Pharm Nanotechnol. 2019;7(2):129–46.
38. Arias-Moliz MT, Ordinola-Zapata R, Baca P, Ruiz-Linares M, García García E, Hungaro Duarte MA, et al. Antimicrobial activity of Chlorhexidine, Peracetic acid and Sodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms. Int Endod J. 2015 Dec;48(12):1188–93.
39. Darcey J, Jawad S, Taylor C, Roudsari RV, Hunter M. Modern Endodontic Principles Part 4: Irrigation. Dent Update. 2016 Jan-Feb;43(1):20–2, 25–6, 28–30 passim.
40. Rôças IN, Provenzano JC, Neves MAS, Siqueira JF Jr. Disinfecting Effects of Rotary Instrumentation with Either 2.5% Sodium Hypochlorite or 2% Chlorhexidine as the Main Irrigant: A Randomized Clinical Study. J Endod. 2016 Jun;42(6):943–7.
41. Del Carpio-Perochena AE, Bramante CM, Duarte MAH, Cavenago BC, Villas-Boas MH, Graeff MS, et al. Biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. J Endod. 2011 Aug;37(8):1134–8.
42. Neelakantan P, Grotra D, Sharma S. Retreatability of 2 mineral trioxide aggregate-based root canal sealers: a cone-beam computed tomography analysis. J Endod. 2013 Jul;39(7):893–6.
43. Aldhuwayhi S, Mallineni SK, Sakhamuri S, Thakare AA, Mallineni S, Sajja R, et al. Covid-19 Knowledge and Perceptions Among Dental Specialists: A Cross-Sectional Online Questionnaire Survey. Risk Manag Healthc Policy. 2021 Jul 7;14:2851–61.
44. Sheriff KAH, Ahmed Hilal Sheriff K, Santhanam A. Knowledge and Awareness towards Oral Biopsy among Students of Saveetha Dental College [Internet]. Vol. 11, Research Journal of Pharmacy and Technology. 2018. p. 543. Available from: http://dx.doi.org/10.5958/0974-360x.2018.00101.4
45. Markov A, Thangavelu L, Aravindhan S, Zekiy AO, Jarahian M, Chartrand MS, et al. Mesenchymal stem/stromal cells as a valuable source for the treatment of immune-mediated disorders. Stem Cell Res Ther. 2021 Mar 18;12(1):192.
46. Jayaraj G, Ramani P, Herald J. Sherlin, Premkumar P, Anuja N. Inter-observer agreement in grading oral epithelial dysplasia – A systematic review [Internet]. Vol. 27, Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology. 2015. p. 112–6. Available from: http://dx.doi.org/10.1016/j.ajoms.2014.01.006
47. Paramasivam A, Priyadharsini JV, Raghunandhakumar S, Elumalai P. A novel COVID-19 and its effects on cardiovascular disease. Hypertens Res. 2020 Jul;43(7):729–30.
48. Li Z, Veeraraghavan VP, Mohan SK, Bolla SR, Lakshmanan H, Kumaran S, et al. Apoptotic induction and anti-metastatic activity of eugenol encapsulated chitosan nanopolymer on rat glioma C6 cells via alleviating the MMP signaling pathway [Internet]. Vol. 203, Journal of Photochemistry and Photobiology B: Biology. 2020. p. 111773. Available from: http://dx.doi.org/10.1016/j.jphotobiol.2019.111773
49. Gan H, Zhang Y, Zhou Q, Zheng L, Xie X, Veeraraghavan VP, et al. Zingerone induced caspase-dependent apoptosis in MCF-7 cells and prevents 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in experimental rats. J Biochem Mol Toxicol. 2019 Oct;33(10):e22387.
50. Dua K, Wadhwa R, Singhvi G, Rapalli V, Shukla SD, Shastri MD, et al. The potential of siRNA based drug delivery in respiratory disorders: Recent advances and progress. Drug Dev Res. 2019 Sep;80(6):714–30.
51. Mohan M, Jagannathan N. Oral field cancerization: an update on current concepts. Oncol Rev. 2014 Mar 17;8(1):244.
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Jun 6, 2023
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Anjali Sankar, Sindhu Ramesh, S.Rajeshkumar, & Nishitha Arun. (2023). Anti Microbial Activity of Chitosan Nanoparticles with Chlorhexidine- An In vitro Study. Journal of Population Therapeutics and Clinical Pharmacology, 30(14), 41–48. https://doi.org/10.47750/jptcp.2023.30.14.006
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