Chitosan-based nano-scaffolds as antileishmanial wound dressing in BALB/c mice treatment: Characterization and design of tissue regeneration

Document Type : Original Article


1 Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Parasitology and Mycology, School of Medicine, Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Anatomical Sciences, Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

4 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Department of Biochemistry and Biophysics, Education and Research Center of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran

6 Doctor of Veterinary Medicine (D.V.M.), Rasht, Iran

7 Virology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

8 Pathology Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Objective(s): Rapid healing of cutaneous leishmaniasis as one of the most important parasitic diseases leads to the decrease of scars and prevention of a great threat to the looks of the affected people. Today, the use of nano-scaffolds is rapidly increasing in tissue engineering and regenerative medicine with structures similar to the target tissue. Chitosan (CS) is a bioactive polymer with antimicrobial and accelerating features of healing wounds, which is commonly used in biomedicine. This study aimed to investigate the effects of CS/polyethylene oxide (PEO)/berberine (BBR) nanofibers on the experimental ulcers of Leishmania major in BALB/c mice.
Materials and Methods: CS/PEO/BBR nanofibers were prepared by the electrospinning method, and their morphology was examined by SEM, TEM, and AFM. Then, water absorption, stability, biocompatibility, porosity, and drug release from nano-scaffolds were explored. Afterward, 28 BALB/c mice infected with the parasite were randomly divided into control and experimental groups, and their wounds were dressed with the produced nano-scaffolds. Finally, the effect of nanobandage on the animals was investigated by macroscopic, histopathologic, and in vivo imaging examinations.
Results: The prepared nanofibers were completely uniform, cylindrical, bead-free, and biocompatible with an average diameter of 94±12 nm and had appropriate drug release. In addition, the reduced skin ulcer diameter (P=0.000), parasite burden (P=0.003), changes in the epidermis (P=0.023), and dermis (P=0.032) indicated significantly strong effectiveness of the produced nano-scaffolds against leishmania ulcers.
Conclusion: Studies showed that CS/PEO/BBR nanofibers have a positive effect on the rapid healing of leishmania ulcers. Future studies should focus on other chronic ulcers treatment.


1. Norouzinezhad F, Ghaffari F, Norouzinejad A, Kaveh F, Gouya MM. Cutaneous leishmaniasis in Iran: Results from an epidemiological study in urban and rural provinces. Asian Pac J Trop Biomed 2016;6:614-619.
2. Mahmoudzadeh‐Niknam H, Ajdary S, Riazi‐Rad F, Mirzadegan E, Rezaeian A, Khaze V, et al. Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran. Trop Med Int Health 2012;17:1335-1344.
3. Hoseini MHM, Moradi M, Alimohammadian MH, Shahgoli VK, Darabi H, Rostami A. Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection. J Parint 2016;65:99-104.
4. Saha P, Bhattacharjee S, Sarkar A, Manna A, Majumder S, Chatterjee M. Berberine chloride mediates its anti-leishmanial activity via differential regulation of the mitogen activated protein kinase pathway in macrophages. PLoS One 2011;6:1-8.
5. Zou K, Li Z, Zhang Y, Zhang H, Li B, Zhu W, et al. Advances in the study of berberine and its derivatives: a focus on anti-inflammatory and anti-tumor effects in the digestive system.  Acta Pharmacol Sin 2017;38:157–167.
6. Emamgholi A, Rahimi M, Kaka G, Sadraie SH, Najafi S. Presentation of a novel model of chitosan-polyethylene oxide-nanohydroxyapatite nanofibers together with bone marrow stromal cells to repair and improve minor bone defects. Iran J Basic Med Sci 2015;18:887-893.
7. Tchemtchoua VT, Atanasova G, Aqil A, Filee P, Garbacki N, Vanhooteghem O, et al. Development of a chitosan nanofibrillar scaffold for skin repair and regeneration. Biomacromolecules 2011;12:3194–3204.
8. Mohandas A, Deepthi S, Biswas R, Jayakumar R. Chitosan based metallic nanocomposite scaffolds as antimicrobial wound dressings. Bioact Mater 2018;3:267-277.
9. Kumar PT, Lakshmanan VK, Anilkumar TV, Ramya C, Reshmi P, Unnikrishnan AG, et al. Flexible and microporous chitosan hydrogel/nano ZnO composite bandages for wound dressing: In Vitro and In Vivo Evaluation. ACS Appl Mater Interfaces 2012;4:2618−2629.
10. Emamgholi A, Rahimi M, Seyyed Tabaei SJ, Ghorbani T, Ziai SA, Brouki Milan P, et al. Investigation of osteoblast-like cells cultured on nano-hydroxyapatite/chitosan based composite scaffold in the treatment of bone defects and limited mobility. Nanomed J 2019;6:167-175.
11. Rahimi M, Emamgholi A, Seyyed Tabaei SJ, Khodadoust M, Taghipour H, Jafari A. Perspectives of chitosan nanofiber/film scaffolds with bone marrow stromal cells in tissue engineering and wound dressing. Nanomed J 2019;6:27-34.    
12. Pebdeni AB, Sadri M, Pebdeni SB. Synthesis of chitosan/peo/silica nanofiber coating for controlled release of cefepime from implants. RSC Adv 2016;6:24418-24429.
13. Gryshkov O, Klyui NI, Temchenko VP, Kyselov VS, Chatterjee A, Belyaev AE, et al. Porous biomorphic silicon carbide ceramics coated with hydroxyapatite as prospective materials for bone implants. Mater Sci Eng C Mater Biol Appl 2016;68:143-52.
14. Schiffman JD, Schauer CL. One-step electrospinning of cross-linked chitosan fibers. Biomacromolecules 2007;8:2665-2667.
15. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol Pharm 2010;67:217-223.
16. Haghdoust S, Azizi M, Hajimollahoseini M, Bandehpour M, Mohsenimasooleh M, Yeganeh F. Direct fluorescent microscopy using Leishmania major expressing green fluorescent protein as a simple technique for quantifying parasite burden in experimental leishmaniasis. Iran J Parasitol 2020. [Accepted]    
17. Ghosh J, Bose M, Roy S, Bhattacharyya SN. Leishmania donovani targets dicer1 to downregulate miR-122, lower serum cholesterol, and facilitate murine liver infection. Cell Host Microbe 2013;13:277–288.
18. Akbari Bazm M, Khazaei M, Khazaei F, Naseri L. Nasturtium Officinale L. hydroalcoholic extract improved oxymetholone‐induced oxidative injury in mouse testis and sperm parameters. Andrologia 2019;51:e13294.
19. Azzi L, El-Alfy M, Martel C, Labrie F. Gender differences in mouse skin morphology and specific effects of sex steroids and dehydroepiandrosterone. J Investig Dermatol 2005;124:22-27.
20. Akbari Bazm M, Khazaei M, Ghanbari E, Naseri L. Protective effect of Vaccinium arctostaphylos L. fruit extract on gentamicin-induced nephrotoxicity in rats.  Comp Clin Path 2018;27:1327-1334.
21. Birdsall, TC, Kelly GS. Berberine: Therapeutic potential of an alkaloid found in several medicinal plants. Alt Med Rev 1997;2:94-103.
22. Soto J, Hernandez N, Mejia H, Grogl M, Berman J. Successful treatment of new world cutaneous leishmaniasis with a combination of topical paromomycin/methylbenzethonium chloride and injectable meglumine antimoniate. Clin Infect Dis 1995;20:47-51.
23. Cho YW, Cho YN, Chung SH, Yoo G, Ko SW. Water-soluble chitin as a wound healing accelerator. Biomaterials 1999;20:2139-2145.
24. Loke WK, Lau SK, Yong LL, Khor E, Sum CK. Wound dressing with sustained antimicrobial capability. J Biomed Mater Res B 2000;53:8-17.
25. Lee YM, Kim SS, Park MH, Song KW, Sung YK, Kang IK. Beta-chitin-based wound dressing containing silver sulfurdiazine. J Mater Sci Mater Med 2000;11:817-823.
26. Ishihara M, Nakanishi K, Ono K, Sato M, Kikuchi M, Saito Y, et al. Photo cross linkable chitosan as a dressing for wound occlusion and accelerator in healing process. Biomaterials 2002;23: 833-840.
27. Mi FL, Wu YB, Shyu SS, Chao AC, Lai  JY, Su CC. Asymmetric chitosan membranes prepared by dry/wet phase separation: A new type of wound dressing for controlled antibacterial release. J Membrane Sci 2003;212:237-254.
28. Han SS. Topical formulations of watersoluble chitin as a wound healing assistant evaluation on open wounds using a rabbit ear model. Fiber Polym 2005;6:219-223.
29. Mattioli-Belmonte M, Zizzi A, Lucarini G, Giantomassi F, Biagini G, Tucci G, et al. Chitin nanofibrils linked to chitosan glycolate as spray, gel and gauze preparations for wound repair. J Bioact Compat Polym 2007;22:525-538.
30. Gholipour-Kanani A, Bahrami SH, Joghataie M, Samadikuchaksaraei A. Nanofibrous scaffolds based on poly(caprolactone)/chitosan/poly vinyl alcohol) blend for skin tissue engineering. JIPST 2013;26:159-170.
31. Ignatova M, Manolova N, Rashkov I. Novel antibacterial fibers of quaternized chitosan and poly (vinyl pyrrolidone) prepared by electrospinning. Eur Polym J 2007;43:1112-1122.
32. Mirzaei E, Faridi-Majidi R, Shokrgozar MA, Asghari Paskiabi F. Genipin cross-linked electrospun chitosanbased nanofibrous mat as tissue engineering scaffold. Nanomed J 2014;1:137-146.
33. Zhou Y, Yang D, Chen X, Xu Q, Lu F, Nie J. Electrospun watersoluble carboxyethyl chitosan /poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration. Biomacromolecules 2007;9:349-354.
34. Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001;22:331-336.
35. Ueno H, Yamada H, Tanaka I, Kaba N, Matsuura M, Okumura M,  et al. Accelerating effects of chitosan for healing at early phase of experimental openwound in dogs. Biomaterials 1999;20:1407-1414.
36. Boucard N, Viton C, Agay D, Mari E, Roger T, Chancerelle Y. The use of physical hydrogels of chitosan for skin regeneration following third-degree burns. Biomaterials 2007;28:3478-3488.
37. Nascimento EG, Sampaio TB, Medeiros AC, Azevedo EP. Evaluation of chitosan gel with 1% silver sulfadiazine as an alternative for burn wound treatment in rats. Acta Cir Bras 2009;24:460-465.
38. Sezer AD, Hatipoğlu F, Cevher E, Oğurtan Z, Baş AL, Akbuğa J. Chitosan film containing fucoidan as a wound dressing for dermal burn healing: preparation and in vitro/in vivo evaluation. AAPS Pharm Sci Tech  2007;8:1-8.
39. Hamilton V, Yuan Y, Rigney DA, Puckett AD, Ong JL, Yang Y, et al. Characterization of chitosan films and effects on fibroblast cell attachment and proliferation. J Mater Sci - Mater Med 2006;17:1373-1381.
40. Kojima K, Okamoto Y, Kojima K, Miyatake K, Fujise H, Shigemasa Y, et al. Effects of chitin and chitosan on collagen synthesis in wound healing. J Vet Med Sci 2004;66:1595-1598.
41. Ribeiro MP, Espiga A, Silva D, Baptista P, Henriques J, Ferreira C, et al. Development of a new chitosan hydrogel for wound dressing. Wound Repair Regen 2009;17:817-824.
42. Dai T, Tanaka M, Huang Y-Y, Hamblin MR. Chitosan preparations for wounds and  burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther 2011;9:857-879.
43. Rodríguez-Vázquez M, Vega-Ruiz B, Ramos-Zúñiga R, Saldaña-Koppel DA, Quiñones-Olvera LF. Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine. BioMed Res Int 2015;2015:1-15.
44. Muzzarelli RAA, Morganti P, Morganti G, Palombo P, Palombo M, Biagini G, et al. Chitin nanofibrils/chitosan glycolate composites as wound medicaments. Carbohydr Polym 2007;70:274-284.