Synergistic Effects of Cold Atmospheric Multiple Plasma Jet and Amphotericin B on Leishmania major: An In-Vitro study
Abstract
Background: This study aimed to assess the In vitro effects of Cold Atmospheric Multiple Plasma Jet (CAMPJ) on Leishmania major.
Methods: A plasma jet device was designed in the Department of Chemical Engineering, Shiraz University, Shiraz, Iran, incorporating a high-purity air supply, an air flow controller, a DC power supply, a 9-10 Farad capacitor, an oscilloscope, and a cold plasma reactor. The CAMPJ was applied to L. major promastigotes and amastigotes under various plasma conditions, including different flow rates, voltages, and exposure times. The effectiveness of CAMPJ was compared to amphotericin B and a combination of both therapies. Viability and cytotoxicity were assessed on L. major and macrophage cell lines, using MAT assay, while apoptosis was quantified through flow cytometry.
Results: The optimal experimental conditions were identified as 2 million L. major promastigotes in 500 μL of culture, a flow rate of 500 mL/min, a voltage of 8.6 kV, a distance of 1 cm, and an exposure time of 15 minutes. The CAMPJ showed limited cytotoxicity to macrophage cells. The CAMPJ treatment significantly reduced the viability of L. major and induced apoptosis. CAMPJ-amphotericin B combination treatment significantly increased the treatment efficacy, when compared with the CAMPJ alone or negative controls.
Conclusion: CAMPJ, alone or in combination with amphotericin B, effectively induces apoptosis in L. major. CAMPJ might be a promising alternative or adjunct therapy for cutaneous leishmaniasis, warranting further investigation in animal models and clinical settings.
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14. Moldgy A, Nayak G, Aboubakr HA, et al. Inactivation of virus and bacteria using cold atmospheric pressure air plasmas and the role of reactive nitrogen species. J Phys D App Phys. 2020;53:434004.
15. Kazemi M, Taghvaei H. A novel multi array dielectric barrier discharge plasma gas diffuser for wastewater treatment: The role of reactive species. Sep Purif Technol. 2021;260:118236.
16. Bulson JM, Liveris D, Derkatch I, et al. Non‐thermal atmospheric plasma treatment of onychomycosis in an In vitro human nail model. Mycoses. 2020;63:225-232.
17. Colagar AH, Alavi O, Sohbatzadeh F. Lipid peroxidation of Escherichia coli by triplet non-thermal atmospheric pressure plasma jet. Chiang Mai J Sci. 2017;44:678-687.
18. Att RR, Al Haideri H, Murbat HH. Influence of cold atmospheric plasma on Acinetobacter baumannii. Baghdad Sci J. 2019; 16:151-161.
19. Ali AH, Murbet HH, ALhmeedi IA, et al. Studying the effect of physical plasma on the blood using digital image processing. IJCSMC. 2015;4
20. Heslin C, Boehm D, Milosavljevic V, et al. Quantitative assessment of blood coagulation by cold atmospheric plasma. Plasma Med. 2014; 4(1-4): 153-163
21. Laroussi M. Plasma medicine: A brief introduction. Plasma. 2018;1:47-60.
22. Hoffmann C, Berganza C, Zhang J. Cold atmospheric plasma: Methods of production and application in dentistry and oncology. Med Gas Res. 2013;3:21.
23. Shemer A, Daniel R, Kassem R, Geffen Y, Galili E. Cold sub‐atmospheric and atmospheric pressure plasma for the treatment of Trichophyton rubrum onychomycosis: An in‐vitro study. Dermatol Ther. 2020;33:e14084.
24. Zhao YM, Ojha S, Burgess C, Sun DW, Tiwari B. Inactivation efficacy and mechanisms of plasma activated water on bacteria in planktonic state. J Appl Microbiol. 2020;129:1248-1260.
25. Bekeschus S, Seebauer C, Wende K, Schmidt A. Physical plasma and leukocytes - immune or reactive? Biol Chem. 2018, 19;400(1):63-75.
26. Lux J, Dobiáš R, Kuklová I, et al. Inactivation of dermatophytes causing onychomycosis and its therapy using non-thermal plasma. J Fungi (Basel). 2020;6:214.
27. Tanzooei AM, Karimi J, Taghvaei H. Exploring non-thermal plasma technology for microalgae removal. Ecotoxicol Environ Saf. 2024;286:117127.
28. Esmaeilifallah M, Khanahmad H, Ghayour Z, et al. Evaluation of the antileishmanial effect of polyclonal antibodies and cationic antimicrobial peptides. Pathog Glob Health. 2023;117(4):366-380.
29. Rahnama V, Motazedian MH, Mohammadi-Samani S, et al. Artemether-loaded nanostructured lipid carriers: preparation, characterization, and evaluation of In vitro effect on Leishmania major. Res Pharm Sci. 2021;16(6):623-633.
30. Khalili S, Mohebali M, Ebrahimzadeh E, et al. Antimicrobial activity of an antimicrobial peptide against amastigote forms of Leishmania major. Vet Res Forum. 2018;9(4):323-328.
31. Nadaes NR, Silva da Costa L, Santana RC, et al. DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis. Front Cell Infect Microbiol. 2020;10:247.
32. Alanyalı FS, Algı O. Cytotoxic and Apoptotic Impacts of Ceranib-2 on RAW 264.7 Macrophage Cells. Anticancer Agents Med Chem. 2023;23(20):2183-2188.
33. Flynn PB, Graham WG, Gilmore BF. Acinetobacter baumannii biofilm biomass mediates tolerance to cold plasma. Lett Appl Microbiol. 2019;68:344-349.
34. Svarnas P, Spiliopoulou A, Koutsoukos PG, et al. Acinetobacter baumannii deactivation by means of dbd-based helium plasma jet. Plasma. 2019;2:77-90.
35. Daeschlein G, Scholz S, Emmert S, et al. Plasma medicine in dermatology: Basic antimicrobial efficacy testing as prerequisite to clinical plasma therapy. Plasma Med. 2012;2:33-69.
36. Lokajová E, Julák J, Khun J, et al. Inactivation of dermatophytes causing onychomycosis using non-thermal plasma as a prerequisite for therapy. J Fungi. 2021;7:715.
37. Daeschlein G, Scholz S, von Woedtke T, et al. In vitro killing of clinical fungal strains by low-temperature atmospheric-pressure plasma jet. IEEE Trans Plasma Sci. 2010;39:815-821.
38. Julák J, Scholtz V. The potential for use of non-thermal plasma in microbiology and medicine. Epidemiol Mikrobiol Imunol. 2020;69(1):29-37.
39. Fridman G, Shereshevsky A, Jost MM, et al. Floating electrode dielectric barrier discharge plasma in air promoting apoptotic behavior in melanoma skin cancer cell lines. Plasma Chem Plasma Process. 2007;27:163-176.
40. Lin AG, Xiang B, Merlino DJ, Baybutt TR, et al. Non-thermal plasma induces immunogenic cell death in vivo in murine ct26 colorectal tumors. Oncoimmunology. 2018;7:e1484978.
41. Moszczyńska J, Roszek K, Wiśniewski M. Non-thermal plasma application in medicine—focus on reactive species involvement. Int J Mol Sci. 2023;24:12667.
42. Panngom K, Baik K, Nam M, et al. Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis. 2013;4:e642.
43. Isbary G, Morfill G, Schmidt H, et al. A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients. Br J Dermatol. 2010;163:78-82.
44. Scholtz V, Pazlarova J, Souskova H, et al. Nonthermal plasma—a tool for decontamination and disinfection. Biotechnol Adv. 2015; 33(6 Pt 2):1108-19.
2. Pereira S, Pita-Pereira D, Araujo-Pereira T, et al. First molecular detection of Leishmania infantum in sergentomyia minuta (diptera, psychodidae) in Alentejo, southern Portugal. Acta Trop. 2017;174:45-48.
3. Sabzevari S, Teshnizi SH, Shokri A, Bahrami F, Kouhestani F. Cutaneous leishmaniasis in Iran: A systematic review and meta-analysis. Microb Pathog. 2021;152:104721.
4. Borba JV, Silva AC, Ramos PI, et al. Unveiling the kinomes of Leishmania infantum and L. braziliensis empowers the discovery of new kinase targets and antileishmanial compounds. Comput Struct Biotechnol J. 2019;17:352-361.
5. Fata A, Moghaddas E, Rezee A, Abdali A, Jarahi L, Shamsian A. Epidemiological study of cutaneous leishmaniasis and identification of etiological species. J Mazandaran Univ Med Sci. 2018;27:123-131.
6. Almeida-Souza F, Calabrese KDS, Abreu-Silva AL, Cardoso F. Leishmania parasites: Epidemiology, immunopathology and hosts. BoD–Books on Demand; 2024.
7. Hajjaran H, Saberi R, Borjian A, et al. The geographical distribution of human cutaneous and visceral leishmania species identified by molecular methods in Iran: A systematic review with meta-analysis. Front Public Health. 2021;9:661674.
8. Norouzinezhad F, Ghaffari F, Norouzinejad A, et al. Cutaneous leishmaniasis in Iran: Results from an epidemiological study in urban and rural provinces. Asian Pac J Trop Biomed. 2016;6:614-619.
9. Sundar S, Singh B. Emerging therapeutic targets for treatment of leishmaniasis. Expert Opin Ther Targets. 2018;22(6):467-486.
10. World Health Organization. Global leishmaniasis update, 2006-2015: A turning point in leishmaniasis surveillance. Wkly Epidemiol Rec. 2017;92:557-565.
11. Madusanka RK, Silva H, Karunaweera ND. Treatment of Cutaneous Leishmaniasis and Insights into Species-Specific Responses: A Narrative Review. Infect Dis Ther. 2022 Apr;11(2):695-711.
12. Kong MG, Kroesen G, Morfill G, et al. Plasma medicine: An introductory review. New J Phys. 2009;11:115012.
13. Yazdani Z, Pasandi MS, Golpour M, et al. Effect of cold atmospheric plasma on changing of biomolecular structures involved in apoptosis pathways of melanoma cancer. Skin Res Technol. 2024;30(1):e13544.
14. Moldgy A, Nayak G, Aboubakr HA, et al. Inactivation of virus and bacteria using cold atmospheric pressure air plasmas and the role of reactive nitrogen species. J Phys D App Phys. 2020;53:434004.
15. Kazemi M, Taghvaei H. A novel multi array dielectric barrier discharge plasma gas diffuser for wastewater treatment: The role of reactive species. Sep Purif Technol. 2021;260:118236.
16. Bulson JM, Liveris D, Derkatch I, et al. Non‐thermal atmospheric plasma treatment of onychomycosis in an In vitro human nail model. Mycoses. 2020;63:225-232.
17. Colagar AH, Alavi O, Sohbatzadeh F. Lipid peroxidation of Escherichia coli by triplet non-thermal atmospheric pressure plasma jet. Chiang Mai J Sci. 2017;44:678-687.
18. Att RR, Al Haideri H, Murbat HH. Influence of cold atmospheric plasma on Acinetobacter baumannii. Baghdad Sci J. 2019; 16:151-161.
19. Ali AH, Murbet HH, ALhmeedi IA, et al. Studying the effect of physical plasma on the blood using digital image processing. IJCSMC. 2015;4
20. Heslin C, Boehm D, Milosavljevic V, et al. Quantitative assessment of blood coagulation by cold atmospheric plasma. Plasma Med. 2014; 4(1-4): 153-163
21. Laroussi M. Plasma medicine: A brief introduction. Plasma. 2018;1:47-60.
22. Hoffmann C, Berganza C, Zhang J. Cold atmospheric plasma: Methods of production and application in dentistry and oncology. Med Gas Res. 2013;3:21.
23. Shemer A, Daniel R, Kassem R, Geffen Y, Galili E. Cold sub‐atmospheric and atmospheric pressure plasma for the treatment of Trichophyton rubrum onychomycosis: An in‐vitro study. Dermatol Ther. 2020;33:e14084.
24. Zhao YM, Ojha S, Burgess C, Sun DW, Tiwari B. Inactivation efficacy and mechanisms of plasma activated water on bacteria in planktonic state. J Appl Microbiol. 2020;129:1248-1260.
25. Bekeschus S, Seebauer C, Wende K, Schmidt A. Physical plasma and leukocytes - immune or reactive? Biol Chem. 2018, 19;400(1):63-75.
26. Lux J, Dobiáš R, Kuklová I, et al. Inactivation of dermatophytes causing onychomycosis and its therapy using non-thermal plasma. J Fungi (Basel). 2020;6:214.
27. Tanzooei AM, Karimi J, Taghvaei H. Exploring non-thermal plasma technology for microalgae removal. Ecotoxicol Environ Saf. 2024;286:117127.
28. Esmaeilifallah M, Khanahmad H, Ghayour Z, et al. Evaluation of the antileishmanial effect of polyclonal antibodies and cationic antimicrobial peptides. Pathog Glob Health. 2023;117(4):366-380.
29. Rahnama V, Motazedian MH, Mohammadi-Samani S, et al. Artemether-loaded nanostructured lipid carriers: preparation, characterization, and evaluation of In vitro effect on Leishmania major. Res Pharm Sci. 2021;16(6):623-633.
30. Khalili S, Mohebali M, Ebrahimzadeh E, et al. Antimicrobial activity of an antimicrobial peptide against amastigote forms of Leishmania major. Vet Res Forum. 2018;9(4):323-328.
31. Nadaes NR, Silva da Costa L, Santana RC, et al. DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis. Front Cell Infect Microbiol. 2020;10:247.
32. Alanyalı FS, Algı O. Cytotoxic and Apoptotic Impacts of Ceranib-2 on RAW 264.7 Macrophage Cells. Anticancer Agents Med Chem. 2023;23(20):2183-2188.
33. Flynn PB, Graham WG, Gilmore BF. Acinetobacter baumannii biofilm biomass mediates tolerance to cold plasma. Lett Appl Microbiol. 2019;68:344-349.
34. Svarnas P, Spiliopoulou A, Koutsoukos PG, et al. Acinetobacter baumannii deactivation by means of dbd-based helium plasma jet. Plasma. 2019;2:77-90.
35. Daeschlein G, Scholz S, Emmert S, et al. Plasma medicine in dermatology: Basic antimicrobial efficacy testing as prerequisite to clinical plasma therapy. Plasma Med. 2012;2:33-69.
36. Lokajová E, Julák J, Khun J, et al. Inactivation of dermatophytes causing onychomycosis using non-thermal plasma as a prerequisite for therapy. J Fungi. 2021;7:715.
37. Daeschlein G, Scholz S, von Woedtke T, et al. In vitro killing of clinical fungal strains by low-temperature atmospheric-pressure plasma jet. IEEE Trans Plasma Sci. 2010;39:815-821.
38. Julák J, Scholtz V. The potential for use of non-thermal plasma in microbiology and medicine. Epidemiol Mikrobiol Imunol. 2020;69(1):29-37.
39. Fridman G, Shereshevsky A, Jost MM, et al. Floating electrode dielectric barrier discharge plasma in air promoting apoptotic behavior in melanoma skin cancer cell lines. Plasma Chem Plasma Process. 2007;27:163-176.
40. Lin AG, Xiang B, Merlino DJ, Baybutt TR, et al. Non-thermal plasma induces immunogenic cell death in vivo in murine ct26 colorectal tumors. Oncoimmunology. 2018;7:e1484978.
41. Moszczyńska J, Roszek K, Wiśniewski M. Non-thermal plasma application in medicine—focus on reactive species involvement. Int J Mol Sci. 2023;24:12667.
42. Panngom K, Baik K, Nam M, et al. Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis. 2013;4:e642.
43. Isbary G, Morfill G, Schmidt H, et al. A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients. Br J Dermatol. 2010;163:78-82.
44. Scholtz V, Pazlarova J, Souskova H, et al. Nonthermal plasma—a tool for decontamination and disinfection. Biotechnol Adv. 2015; 33(6 Pt 2):1108-19.
| Files | ||
| Issue | Vol 20 No 3 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Leishmaniasis Cold atmospheric mul-tiple plasma jet (CAMPJ) Leishmania major Apoptosis Amphotericin B | ||
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How to Cite
1.
Rezaee E, Taghavi H, Hatam G, Zomorodian K, Sarkari B. Synergistic Effects of Cold Atmospheric Multiple Plasma Jet and Amphotericin B on Leishmania major: An In-Vitro study. Iran J Parasitol. 2025;20(3):444-453.
