Fungal Infected Adipose Stem Cells: The Effects of Novel Lipo-Niosome Nanoparticles Loaded with Amphotericin B and Thymus Essential Oil

Document Type : Original Article


1 Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran

2 Department of Biomedical Engineering, Research Center for New Technologies in Life Science Engineering, University of Tehran, Tehran, Iran

3 School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran

4 Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam University Medical Centers-location Vumc and Academic

5 Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), The University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands


Objective: In this study, we aimed to develop new Lipo-niosomes based nanoparticles loaded with Amphotericin B
(AmB) and Thymus Essential Oil (TEO) and test their effectiveness in the treatment of fungal-infected human adipose stem cells (hASCs).
Materials and Methods: In this experimental study, optimal formulation of AmB and TEO loaded lipo-niosome (based on lipid-surfactant thin-film hydration method) was chemically, and biologically characterized. Therefore, encapsulation capacity, drug release, size, and the survival rate of cells with different concentrations of free and encapsulated AmB/ TEO were evaluated using the MTT method, and its antifungal activity was compared with conventional AmB.
Results: Lipo-Niosome containing Tween 60 surfactant: cholesterol: Dipalmitoyl phosphatidylcholine (DPPC): Polyethylene glycol (PEG) with a ratio of 20:40:60:3 were chosen as optimal formulation. Lipo-Niosomes entrapment efficiency was %94.15. The drug release rate after 24 hours was %52, %54, and %48 for Lipo-AmB, Lipo-TEO, and Lipo-AmB/TEO, respectively. Physical and chemical characteristics of the Lipo-Niosomes particles indicated size of 200 nm and a dispersion index of 0.32 with a Zeta potential of -24.56 mv. Furthermore, no chemical interaction between drugs and nano-carriers was observed. The cell viability of adipose mesenchymal stem cells exposed to 50 μg/ml of free AmB, free TEO, and free AmB/TEO was %13.4, %58, and %36.9, respectively. Whereas the toxicity of the encapsulated formulas of these drugs was %48.9, %70.8, and %58.3 respectively. The toxicity of nanoparticles was very low (%8.5) at this concentration. Fluorescence microscopic images showed that the antifungal activity of Lipo- AmB/TEO was significantly higher than free formulas of AmB, TEO, and AmB/TEO.
Conclusion: In this study, we investigated the efficacy of the TEO/AmB combination, in both free and encapsulatedniosomal form, on the growth of fungal infected-hASCs. The results showed that the AmB/TEO-loaded Lipo-Niosomes can be suggested as a new efficient anti-fungal nano-system for patients treated with hASCs.


1. Hedayati MT, Khodavaisy S, Alialy M, Omran SM, Habibi MR. Invasive aspergillosis in intensive care unit patients in Iran. Acta Medica (Hradec Kralove). 2013; 56(2): 52-56.
2. da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells. 2008; 26(9): 2287-2299.
3. Otto WR, Green AM. Fungal infections in children with haematologic malignancies and stem cell transplant recipients. Br J Haematol. 2020; 189(4): 607-624.
4. Bays DJ, Thompson GR 3rd. Fungal infections of the stem cell transplant recipient and hematologic malignancy patients. Infect Dis Clin North Am. 2019; 33(2): 545-566.
5. Pagano L, Caira M, Nosari A, Van Lint MT, Candoni A, Offidani M, et al. Fungal infections in recipients of hematopoietic stem cell transplants: results of the SEIFEM B-2004 study--Sorveglianza Epidemiologica Infezioni Fungine Nelle Emopatie Maligne. Clin Infect Dis. 2007; 45(9): 1161-1170.
6. Schneemann M, Schaffner A. Host defense mechanism in aspergillus fumigatus infections. Contrib Microbiol. 1999; 2: 57-68.
7. Singh N, Limaye AP, Forrest G, Safdar N, Muñoz P, Pursell K, et al. Combination of voriconazole and caspofungin as primary therapy for invasive aspergillosis in solid organ transplant recipients: a prospective, multicenter, observational study. Transplantation. 2006; 81(3): 320-326.
8. Yazgi M, Awad D, Jreikous B. Screening of the antifungal activity of plant Mentha longifolia crude extracts against two fungi Alternaria citri and Fusarium moniliforme. J Entomol Zool Stud. 2015; 3(2): 359-364.
9. Arias JL. Nanotechnology and drug delivery. Nanoplatforms in drug delivery. 1st ed. CRC Press; 2014.
10. Kamiński DM. Recent progress in the study of the interactions of amphotericin B with cholesterol and ergosterol in lipid environments. Eur Biophys J. 2014; 43(10-11): 453-467.
11. Diezi TA, Kwon G. Amphotericin B/sterol co-loaded PEG-phospholipid micelles: effects of sterols on aggregation state and hemolytic activity of amphotericin B. Pharm Res. 2012; 29(7): 1737-1744.
12. Walsh TJ, Finberg RW, Arndt C, Hiemenz J, Schwartz C, Bodensteiner D, et al. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. National institute of allergy and infectious diseases mycoses study group. N Engl J Med. 1999; 340(10): 764-771.
13. Mostafavi M, Sharifi I, Farajzadeh S, Khazaeli P, Sharifi H, Pourseyedi E, et al. Niosomal formulation of amphotericin B alone and in combination with glucantime: In vitro and in vivo leishmanicidal effects. Biomed Pharmacother. 2019; 116: 108942.
14. Alam M, Dwivedi V, Khan AA, Mohammad O. Efficacy of niosomal formulation of diallyl sulfide against experimental candidiasis in Swiss albino mice. Nanomedicine (Lond). 2009; 4(7): 713-724.
15. Haque F, Sajid M, Cameotra SS, Battacharyya MS. Anti-biofilm activity of a sophorolipid-amphotericin B niosomal formulation against Candida albicans. Biofouling. 2017; 33(9): 768-779.
16. Kopermsub P, Mayen V, Warin C. Potential use of niosomes for encapsulation of nisin and EDTA and their antibacterial activity enhancement. Int Food Res. 2011; 44(2): 605-612.
17. Sharma V, Anandhakumar S, Sasidharan M. Self-degrading niosomes for encapsulation of hydrophilic and hydrophobic drugs: An efficient carrier for cancer multi-drug delivery. Mater Sci Eng C Mater Biol Appl. 2015; 56: 393-400. 18. Zhou Y, Chen Y, Wang S, Qin F, Wang L. MSCs helped reduce scarring in the cornea after fungal infection when combined with anti-fungal treatment. BMC Ophthalmol. 2019; 19(1): 226.
19. Higashi N, Sunamoto J. Endocytosis of poly(ethylene oxide)-modified liposome by human lymphoblastoid cells. Biochim Biophys Acta. 1995; 1243(3): 386-392.
20. McCarthy J, Inkielewicz-Stępniak I, Corbalan JJ, Radomski MW. Mechanisms of toxicity of amorphous silica nanoparticles on human lung submucosal cells in vitro: protective effects of fisetin. Chem Res Toxicol. 2012; 25(10): 2227-2235.
21. Manke A, Wang L, Rojanasakul Y. Mechanisms of nanoparticleinduced oxidative stress and toxicity. Biomed Res Int. 2013; 2013: 942916.
22. Rosenholm JM, Sahlgren C, Lindén M. Towards multifunctional, targeted drug delivery systems using mesoporous silica nanoparticles-- opportunities & challenges. Nanoscale. 2010; 2(10): 1870-1883.
23. Heyes J, Hall K, Tailor V, Lenz R, MacLachlan I. Synthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug delivery. J Control Release. 2006; 112(2): 280-290.
24. Haghiroalsadat F, Azhdari M, Oroojalian F, Omidi M, Azimzadeh M. The chemical assessment of seed essence of three native medicinal plants of Yazd Province (Bunium Premium, Cuminum Cyminum, Trachyspermum Copticum) and the comparison of their antioxidant properties. Journal of Shahid Sadoughi University of Medical Sciences and Health Services. 2015; 2(6): 1592-1603.
25. Raposio E, Bertozzi N. isolation of ready-to-use adipose-derived stem cell (ASC) pellet for clinical applications and a comparative overview of alternate methods for ASC isolation. Curr Protoc Stem Cell Biol. 2017; 41: 1F.17.1-1F.17.12.
26. Blanco MT, Pérez-Giraldo C, Blanco J, Morán FJ, Hurtado C, Gómez-García AC. In vitro studies of activities of some antifungal agents against candida albicans ATCC 10231 by the turbidimetric method. Antimicrob Agents Chemother. 1992; 36(4): 898-901.
27. Upton A, Kirby KA, Carpenter P, Boeckh M, Marr KA. Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin Infect Dis. 2007; 44(4): 531-540.
28. Naderinezhad S, Amoabediny G, Haghiralsadat F. Co-delivery of hydrophilic and hydrophobic anticancer drugs using biocompatible pH-sensitive lipid-based nano-carriers for multidrug-resistant cancers. RSC Adv. 2017; 7(48): 30008-30019.
29. Gagoś M, Arczewska M. FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol. Eur Biophys J. 2012; 41(8): 663-673.
30. Jain S, Valvi PU, Swarnakar NK, Thanki K. Gelatin coated hybrid lipid nanoparticles for oral delivery of amphotericin B. Mol Pharm. 2012; 9(9): 2542-2553.
31. Silva L, Coutinho A, Fedorov A, Prieto M. Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin. A fluorescence study. Biophys J. 2006; 90(10): 3625-3631.
32. Moen MD, Lyseng-Williamson KA, Scott LJ. Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive fungal infections. Drugs. 2009; 69(3): 361-392.
33. Tollemar J, Höckerstedt K, Ericzon BG, Sundberg B, Ringdén O. Fungal prophylaxis with AmBisome in liver and bone marrow transplant recipients: results of two randomized double-blind studies. Transplant Proc. 1994; 26(3): 1833.
34. Kume H, Yamazaki T, Abe M, Tanuma H, Okudaira M, Okayasu I. Increase in aspergillosis and severe mycotic infection in patients with leukemia and MDS: comparison of the data from the annual of the pathological autopsy cases in Japan in 1989, 1993 and 1997. Pathol Int. 2003; 53(11): 744-750.
35. Marr KA, Seidel K, White TC, Bowden RA. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J Infect Dis. 2000; 181(1): 309-316.
36. Cordonnier C, Mohty M, Faucher C, Pautas C, Robin M, Vey N, et al. Safety of a weekly high dose of liposomal amphotericin B for prophylaxis of invasive fungal infection in immunocompromised patients: PROPHYSOME Study. Int J Antimicrob Agents. 2008; 31(2): 135-141.
37. Wadhwa S, Paliwal R, Paliwal SR, Vyas SP. Nanocarriers in ocular drug delivery: an update review. Curr Pharm Des. 2009; 15(23): 2724-2750.
38. Gurudevan S, Francis AP, Jayakrishnan A. Amphotericin B-albumin conjugates: synthesis, toxicity and anti-fungal activity. Eur J Pharm Sci. 2018; 115: 167-174.
39. Kim J, Sudbery P. Candida albicans, a major human fungal pathogen. J Microbiol. 2011; 49(2): 171-177.
40. Li WR, Shi QS, Dai HQ, Liang Q, Xie XB, Huang XM, et al. Antifungal activity, kinetics and molecular mechanism of action of garlic oil against Candida albicans. Sci Rep. 2016; 6: 22805.