Microfluidic-Synthesized Chitosan Nanoparticles Loaded with Azithromycin: Impact on Toxoplasma gondii Tissue Cysts in Mouse Model
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Abstract
Background: We aimed to investigate the effect of chitosan nanoparticles loaded azithromycin on reducing the number of Toxoplasma gondii tissue cysts in the brain of a mouse model.
Methods: Chitosan nanoparticles and azithromycin loaded nanoparticles were synthesized using microfluidic system and characterized using dynamic light scattering (DLS) and TEM images. Forty BALB/c mice after infection with a cyst forming T. gondii strain, were divided into four groups daily receiving PBS, 10 mg/kg azithromycin, 10 mg/kg chitosan nanoparticles, and 10 mg/kg chitosan nanoparticles loaded azithromycin, respectively, for 10 days. Immediately after end of the treatment, the mice were sacrificed and the tissue cyst burden in their brain was investigated using an optical microscope and compared by ANOVA statistical test.
Results: The average particle size and dispersion index for chitosan nanoparticles were 193.66 nm and 0.43, and for nanoparticles containing azithromycin drug, they were 233.66 nm and 0.21, respectively. The amount of drug loading was 1.8% and the drug release was more than 90% after less than 48 hours. The stability of nanoparticles did not change significantly after 28 days of observation. Toxoplasma tissue cyst numbers obtained in a range of 1.48 to 1.95 in 10 ul brain suspension with no significant differences among the groups of treated mice.
Conclusion: The synthesis of chitosan nanoparticles loaded with azithromycin by microfluidic system could make the particles with more uniformity and stability and high loading of the drug with low cost and more convenient conditions.
1. Dubey JP. Toxoplasmosis–a waterborne zoonosis. Vet Parasitol. 2004;126(1-2):57-72.
2. Dubey J. Advances in the life cycle of Toxoplasma gondii. Int J Parasitol. 1998;28(7):1019-24.
3. Dubey JP. The history and life cycle of Toxoplasma gondii. In: Toxoplasma gondii. Elsevier; 2020. p. 1-19.
4. Dickerson FB, Stallings CR, Boronow JJ, Origoni AE, Yolken RH. A double-blind trial of adjunctive azithromycin in individuals with schizophrenia who are seropositive for Toxoplasma gondii. Schizophr Res. 2009;112(1-3):198-9.
5. Fuglewicz AJ, Piotrowski P, Stodolak A. Relationship between toxoplasmosis and schizophrenia: a review. Adv Clin Exp Med. 2017;26(6):1031-6.
6. Hrdá Š, Votýpka J, Kodym P, Flegr J. Transient nature of Toxoplasma gondii-induced behavioral changes in mice. J Parasitol. 2000;86(4):657-63.
7. Firouzeh N, Ziaali N, Sheibani V, et al. Chronic Toxoplasma gondii infection potentiates Parkinson’s disease course in mice model. Iran J Parasitol. 2021;16(4):527-537.
8. Gormley PD, Pavesio CE, Minnasian D, Lightman S. Effects of drug therapy on Toxoplasma cysts in an animal model of acute and chronic disease. Invest Ophthalmol Vis Sci. 1998;39(7):1171-5.
9. Walker MJ. Opportunistic infections studies update. NIAID AIDS Agenda. 1995:6-7.
10. Araujo F, Shepard R, Remington J. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii. Eur J Clin Microbiol Infect Dis. 1991;10:519-24.
11. Araujo FG, Guptill DR, Remington JS. Azithromycin, a macrolide antibiotic with potent activity against Toxoplasma gondii. Antimicrob Agents Chemother. 1988;32(5):755-7.
12. Araujo FG, Shepard RM, Remington JS. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii. Eur J Clin Microbiol Infect Dis. 1991;10(6):519-24.
13. Değerli K, Kilimcioğlu AA, Kurt O, Tamay AT, Ozbilgin A. Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey. Acta Trop. 2003;88(1):45-50.
14. Chang HR. The potential role of azithromycin in the treatment or prophylaxis of toxoplasmosis. Int J STD AIDS. 1996;7 Suppl 1:18-22.
15. Caprifico AE, Foot PJ, Polycarpou E, Calabrese G. Overcoming the blood-brain barrier: Functionalised chitosan nanocarriers. Pharmaceutics. 2020; 12(11):1013.
16. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003;55(3):329-47.
17. Assolini JP, Concato VM, Gonçalves MD, et al. Nanomedicine advances in toxoplasmosis: diagnostic, treatment, and vaccine applications. Parasitol Res. 2017;116:1603-15.
18. Briones E, Isabel Colino C, Lanao JM. Delivery systems to increase the selectivity of antibiotics in phagocytic cells. J Control Release. 2008;125(3):210-27.
19. Soheili S, Mandegar E, Moradikhah F, Doosti-Telgerd M, Javar HA. Experimental and numerical studies on microfluidic preparation and engineering of chitosan nanoparticles. J Drug Deliv Sci Technol. 2021;61:102268.
20. Chai Y, Wang Y, Li B, Qi W, Su R, He Z. Microfluidic synthesis of lignin/chitosan nanoparticles for the pH-responsive delivery of anticancer drugs. Langmuir. 2021;37(23):7219-26.
21. Cheraghipour K, Masoori L, Ezzatkhah F, et al. Effect of chitosan on Toxoplasma gondii infection: A systematic review. Parasite Epidemiol Control. 2020;11:e00189.
22. Etewa SE, El-Maaty DAA, Hamza RS, et al. Assessment of spiramycin-loaded chitosan nanoparticles treatment on acute and chronic toxoplasmosis in mice. J Parasit Dis. 2018;42:102-13.
23. El-Gendy AML, Mohammed MAA, Ghallab MMI, Aziz MOA, Ibrahim SM. Therapeutic effect of chitosan nanoparticles and metronidazole in treatment of experimentally giardiasis infected hamsters. Iran J Parasitol. 2021;16(1):32-42.
24. Hagras NA-e, Allam AF, Farag HF, et al. Successful treatment of acute experimental toxoplasmosis by spiramycin-loaded chitosan nanoparticles. Exp Parasitol. 2019;204:107717.
25. Teimouri A, Azami SJ, Keshavarz H, et al. Anti-Toxoplasma activity of various molecular weights and concentrations of chitosan nanoparticles on tachyzoites of RH strain. Int J Nanomedicine. 2018;13:1341-51.
26. Saraei M, Samadzadeh N, Khoeini J, et al. In vivo anti-Toxoplasma activity of aripiprazole. Iran J Basic Med Sci. 2015;18(9):938-41.
27. Saraei M, Ghaderi Y, Mosavi T, et al. The effect of fluphenazine and thioridazine on Toxoplasma gondii in vivo. Iran J Parasitol. 2016;11(2):226-231.
28. Mirhosseini M, Yazdani N, Dehghan Hamdan A. Investigation of antimicrobial properties of chitosan– ZnO nanocomposite. Razi J Med Sci. 2016;23(147):104-14.
29. Abd Elgawad H, Alhusseiny SM, Taman A, et al. Biological evaluation of newly synthesized quinoline–based compound PPQ-8 in acute and chronic toxoplasmosis: An experimental study. Exp Parasitol. 2019;206:107756.
30. Teimouri A, Motevalli Haghi A, Nateghpour M, et al. Antimalarial efficacy of low molecular weight chitosan against Plasmodium berghei infection in mice. J Vector Borne Dis. 2016;53(4):312-316.
31. Gaafar MR, Mady RF, . Diab RG, Shalaby ThI. Chitosan and silver nanoparticles: Promising anti-toxoplasma agents. Exp Parasitol. 2014; 143:30-38.
32. Rahimi Esboei B, Keighobadi M, Ziaei Hezarjaribi H, et al. Promising in vitro anti-Toxoplasma gondii effects of commercial chitosan. Infect Disord Drug Targets. 2021; 21(1):151-155.
33. Raouf M, Essa S, El Achy S, et al. Evaluation of Combined Ciprofloxacin and azithromycin free and nano formulations to control biofilm producing Pseudomonas aeruginosa isolated from burn wounds. Indian J Med Microbiol. 2021;39(1):81-7.
34. Jonassen H, Kjøniksen A-L, Hiorth M. Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Bio-macromolecules. 2012;13(11):3747-56.
35. Habibizadeh M, Nadri S, Fattahi A, et al. Surface modification of neurotrophin-3 loaded PCL/chitosan nanofiber/net by alginate hydrogel microlayer for enhanced biocompatibility in neural tissue engineering. J Biomed Mater Res A. 2021;109(11):2237-54.
36. Dumas JL, Chang R, Mermillod B, et al. Evaluation of the efficacy of prolonged administration of azithromycin in a murine model of chronic toxoplasmosis. J Antimicrob Chemother. 1994;34(1):111-8.
37. Asgari Q, Keshavarz H, Shojaee S, et al. In vitro and in vivo potential of RH strain of Toxoplasma gondii (Type I) in tissue cyst forming. Iran J Parasitol. 2013;8(3):367-75.
38. Salimi M, Shojaee S, Keshavarz H, Mohebali M. Cyst formation from virulent RH strain of Toxoplasma gondii tachyzoite: in vitro cultivation. Iran J Parasitol. 2016;11(1):81-5.
39. Djurković-Djaković O, Milenković V, Nikolić A, et al. Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii. J Antimicrob Chemother. 2002;50(6):981-7.
40. Ferreira RA, Oliveira AB, Ribeiro MF, et al. Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1, 4-naphthoquinone alone or combined with sulfadiazine. Exp Parasitol. 2006; 113(2):125-9.
41. Shu H, Jiang L. Effect of garlicin and minocycline on the cyst formation of Toxoplasma gondii in mice. Chinese J Zoonoses. 2002;18(1):100-1.
42. Ferguson D, Huskinson-Mark J, Araujo F, Remington J. An ultrastructural study of the effect of treatment with atovaquone in brains of mice chronically infected with the ME49 strain of Toxoplasma gondii. Int J Exp Pathol. 1994;75(2):111-6.
43. Rutaganira FU, Barks J, Dhason MS, et al. Inhibition of calcium dependent protein kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii. J Med Chem. 2017;60(24):9976-89.
44. Mahmoud DM, Mahmoud MS, Ezz-El-Din HM, et al. Artesunate effect on RH virulent and ME49 non-virulent strains of Toxoplasma gondii: in vitro and in vivo experimental studies. Sci Parasitol. 2016;17:83-92.
45. Chew WK, Segarra I, Ambu S, Mak JW. Significant reduction of brain cysts caused by Toxoplasma gondii after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis. Antimicrob Agents Chemother. 2012;56(4):1762-8.
46. Ghaffarifar F, Pour MA, Sharifi Z, Asl AD, Al-Kawaz E. The effect of vitamin D3 alone and mixed with IFN-γ on tachyzoites of Toxoplasma gondii (RH strain) proliferation and nitric oxide (NO) production in infected macrophages of BALB/C mice. Iran J Parasitol. 2010;5(3):48-56.
2. Dubey J. Advances in the life cycle of Toxoplasma gondii. Int J Parasitol. 1998;28(7):1019-24.
3. Dubey JP. The history and life cycle of Toxoplasma gondii. In: Toxoplasma gondii. Elsevier; 2020. p. 1-19.
4. Dickerson FB, Stallings CR, Boronow JJ, Origoni AE, Yolken RH. A double-blind trial of adjunctive azithromycin in individuals with schizophrenia who are seropositive for Toxoplasma gondii. Schizophr Res. 2009;112(1-3):198-9.
5. Fuglewicz AJ, Piotrowski P, Stodolak A. Relationship between toxoplasmosis and schizophrenia: a review. Adv Clin Exp Med. 2017;26(6):1031-6.
6. Hrdá Š, Votýpka J, Kodym P, Flegr J. Transient nature of Toxoplasma gondii-induced behavioral changes in mice. J Parasitol. 2000;86(4):657-63.
7. Firouzeh N, Ziaali N, Sheibani V, et al. Chronic Toxoplasma gondii infection potentiates Parkinson’s disease course in mice model. Iran J Parasitol. 2021;16(4):527-537.
8. Gormley PD, Pavesio CE, Minnasian D, Lightman S. Effects of drug therapy on Toxoplasma cysts in an animal model of acute and chronic disease. Invest Ophthalmol Vis Sci. 1998;39(7):1171-5.
9. Walker MJ. Opportunistic infections studies update. NIAID AIDS Agenda. 1995:6-7.
10. Araujo F, Shepard R, Remington J. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii. Eur J Clin Microbiol Infect Dis. 1991;10:519-24.
11. Araujo FG, Guptill DR, Remington JS. Azithromycin, a macrolide antibiotic with potent activity against Toxoplasma gondii. Antimicrob Agents Chemother. 1988;32(5):755-7.
12. Araujo FG, Shepard RM, Remington JS. In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii. Eur J Clin Microbiol Infect Dis. 1991;10(6):519-24.
13. Değerli K, Kilimcioğlu AA, Kurt O, Tamay AT, Ozbilgin A. Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey. Acta Trop. 2003;88(1):45-50.
14. Chang HR. The potential role of azithromycin in the treatment or prophylaxis of toxoplasmosis. Int J STD AIDS. 1996;7 Suppl 1:18-22.
15. Caprifico AE, Foot PJ, Polycarpou E, Calabrese G. Overcoming the blood-brain barrier: Functionalised chitosan nanocarriers. Pharmaceutics. 2020; 12(11):1013.
16. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003;55(3):329-47.
17. Assolini JP, Concato VM, Gonçalves MD, et al. Nanomedicine advances in toxoplasmosis: diagnostic, treatment, and vaccine applications. Parasitol Res. 2017;116:1603-15.
18. Briones E, Isabel Colino C, Lanao JM. Delivery systems to increase the selectivity of antibiotics in phagocytic cells. J Control Release. 2008;125(3):210-27.
19. Soheili S, Mandegar E, Moradikhah F, Doosti-Telgerd M, Javar HA. Experimental and numerical studies on microfluidic preparation and engineering of chitosan nanoparticles. J Drug Deliv Sci Technol. 2021;61:102268.
20. Chai Y, Wang Y, Li B, Qi W, Su R, He Z. Microfluidic synthesis of lignin/chitosan nanoparticles for the pH-responsive delivery of anticancer drugs. Langmuir. 2021;37(23):7219-26.
21. Cheraghipour K, Masoori L, Ezzatkhah F, et al. Effect of chitosan on Toxoplasma gondii infection: A systematic review. Parasite Epidemiol Control. 2020;11:e00189.
22. Etewa SE, El-Maaty DAA, Hamza RS, et al. Assessment of spiramycin-loaded chitosan nanoparticles treatment on acute and chronic toxoplasmosis in mice. J Parasit Dis. 2018;42:102-13.
23. El-Gendy AML, Mohammed MAA, Ghallab MMI, Aziz MOA, Ibrahim SM. Therapeutic effect of chitosan nanoparticles and metronidazole in treatment of experimentally giardiasis infected hamsters. Iran J Parasitol. 2021;16(1):32-42.
24. Hagras NA-e, Allam AF, Farag HF, et al. Successful treatment of acute experimental toxoplasmosis by spiramycin-loaded chitosan nanoparticles. Exp Parasitol. 2019;204:107717.
25. Teimouri A, Azami SJ, Keshavarz H, et al. Anti-Toxoplasma activity of various molecular weights and concentrations of chitosan nanoparticles on tachyzoites of RH strain. Int J Nanomedicine. 2018;13:1341-51.
26. Saraei M, Samadzadeh N, Khoeini J, et al. In vivo anti-Toxoplasma activity of aripiprazole. Iran J Basic Med Sci. 2015;18(9):938-41.
27. Saraei M, Ghaderi Y, Mosavi T, et al. The effect of fluphenazine and thioridazine on Toxoplasma gondii in vivo. Iran J Parasitol. 2016;11(2):226-231.
28. Mirhosseini M, Yazdani N, Dehghan Hamdan A. Investigation of antimicrobial properties of chitosan– ZnO nanocomposite. Razi J Med Sci. 2016;23(147):104-14.
29. Abd Elgawad H, Alhusseiny SM, Taman A, et al. Biological evaluation of newly synthesized quinoline–based compound PPQ-8 in acute and chronic toxoplasmosis: An experimental study. Exp Parasitol. 2019;206:107756.
30. Teimouri A, Motevalli Haghi A, Nateghpour M, et al. Antimalarial efficacy of low molecular weight chitosan against Plasmodium berghei infection in mice. J Vector Borne Dis. 2016;53(4):312-316.
31. Gaafar MR, Mady RF, . Diab RG, Shalaby ThI. Chitosan and silver nanoparticles: Promising anti-toxoplasma agents. Exp Parasitol. 2014; 143:30-38.
32. Rahimi Esboei B, Keighobadi M, Ziaei Hezarjaribi H, et al. Promising in vitro anti-Toxoplasma gondii effects of commercial chitosan. Infect Disord Drug Targets. 2021; 21(1):151-155.
33. Raouf M, Essa S, El Achy S, et al. Evaluation of Combined Ciprofloxacin and azithromycin free and nano formulations to control biofilm producing Pseudomonas aeruginosa isolated from burn wounds. Indian J Med Microbiol. 2021;39(1):81-7.
34. Jonassen H, Kjøniksen A-L, Hiorth M. Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Bio-macromolecules. 2012;13(11):3747-56.
35. Habibizadeh M, Nadri S, Fattahi A, et al. Surface modification of neurotrophin-3 loaded PCL/chitosan nanofiber/net by alginate hydrogel microlayer for enhanced biocompatibility in neural tissue engineering. J Biomed Mater Res A. 2021;109(11):2237-54.
36. Dumas JL, Chang R, Mermillod B, et al. Evaluation of the efficacy of prolonged administration of azithromycin in a murine model of chronic toxoplasmosis. J Antimicrob Chemother. 1994;34(1):111-8.
37. Asgari Q, Keshavarz H, Shojaee S, et al. In vitro and in vivo potential of RH strain of Toxoplasma gondii (Type I) in tissue cyst forming. Iran J Parasitol. 2013;8(3):367-75.
38. Salimi M, Shojaee S, Keshavarz H, Mohebali M. Cyst formation from virulent RH strain of Toxoplasma gondii tachyzoite: in vitro cultivation. Iran J Parasitol. 2016;11(1):81-5.
39. Djurković-Djaković O, Milenković V, Nikolić A, et al. Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii. J Antimicrob Chemother. 2002;50(6):981-7.
40. Ferreira RA, Oliveira AB, Ribeiro MF, et al. Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1, 4-naphthoquinone alone or combined with sulfadiazine. Exp Parasitol. 2006; 113(2):125-9.
41. Shu H, Jiang L. Effect of garlicin and minocycline on the cyst formation of Toxoplasma gondii in mice. Chinese J Zoonoses. 2002;18(1):100-1.
42. Ferguson D, Huskinson-Mark J, Araujo F, Remington J. An ultrastructural study of the effect of treatment with atovaquone in brains of mice chronically infected with the ME49 strain of Toxoplasma gondii. Int J Exp Pathol. 1994;75(2):111-6.
43. Rutaganira FU, Barks J, Dhason MS, et al. Inhibition of calcium dependent protein kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii. J Med Chem. 2017;60(24):9976-89.
44. Mahmoud DM, Mahmoud MS, Ezz-El-Din HM, et al. Artesunate effect on RH virulent and ME49 non-virulent strains of Toxoplasma gondii: in vitro and in vivo experimental studies. Sci Parasitol. 2016;17:83-92.
45. Chew WK, Segarra I, Ambu S, Mak JW. Significant reduction of brain cysts caused by Toxoplasma gondii after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis. Antimicrob Agents Chemother. 2012;56(4):1762-8.
46. Ghaffarifar F, Pour MA, Sharifi Z, Asl AD, Al-Kawaz E. The effect of vitamin D3 alone and mixed with IFN-γ on tachyzoites of Toxoplasma gondii (RH strain) proliferation and nitric oxide (NO) production in infected macrophages of BALB/C mice. Iran J Parasitol. 2010;5(3):48-56.
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| Issue | Vol 20 No 1 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Toxoplasma gondii Tissue cyst Azithromycin Chitosan Nanoparticles | ||
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How to Cite
1.
Heidari MM, Fazaeli A, Nadri S, Torabi N, Saraei M. Microfluidic-Synthesized Chitosan Nanoparticles Loaded with Azithromycin: Impact on Toxoplasma gondii Tissue Cysts in Mouse Model. Iran J Parasitol. 2025;20(1):54-64.
