Development and evaluation of miltefosine-loaded nanoemulsions on in vitro culture of Toxoplasma gondii

Document Type : Research Paper

Authors

1 Department of Parasitology and Mycology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

2 Department of Pharmaceutics, Faculty of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran

3 Department of Anatomy, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

10.22038/nmj.2024.75808.1842

Abstract

Objective(s): Toxoplasma gondii is a common parasite in the world. Pharmaceutical options for toxoplasmosis treatment are limited. Several studies have been conducted on the anti-infectious properties of miltefosine (MLF). We investigated the effectiveness of nanoemulsion miltefosine (NEM) in tachyzoites of T. gondii, RH strain.
Materials and Methods: Various NEM formulations were designed considering pseudo-ternary phase diagrams. Physicochemical properties of the developed nanoemulsions (NEs), including pH, polydispersity index (PDI), droplet size, and refractive index (RI) were evaluated. The considered formulation was analyzed for dilution and stability tests. MTT assay was performed on Vero cells for calculation CC50 and on Vero cells with RH strain tachyzoite for calculation IC50. Sulfadiazine (SDZ) and pyrimethamine (PYR) were positive controls. The trypan blue method was used to investigate the effect of drugs (NEM, MLF, SDZ, PYR) in reducing the number of infected Vero cells and reducing the intracellular proliferation of tachyzoites. Next, the viability of tachyzoites was measured in the tube in the direct vicinity of different drug concentrations.
Results: The final particle size of NEM was calculated to be 17.463 nm by DLS and TEM. The CC50 of NEM (75.7 µg/mL) indicated lower toxicity than the other drugs. IC50 obtained by trypan blue, MTT, and test tube methods showed that NEM (28.43 µg/mL) has a suitable IC50 against Toxoplasma tachyzoites.
Conclusion: The calculated selectivity index (SI) demonstrated that NEM (SI=2.66) is a more suitable drug candidate than the MLF and positive controls. The trypan blue assay indicated the excellent reduction effect of NEM on T. gondii intracellular proliferation rate and the number of infected cells. 
 

Keywords


1.    Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965-1976.
2.    Montazeri M, Sharif M, Sarvi S, Mehrzadi S, Ahmadpour E, Daryani A. A Systematic Review of In vitro and In vivo Activities of Anti-Toxoplasma Drugs and Compounds (2006-2016). Front Microbiol. 2017;8:25.
3.    Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis. 1995;172(6):1561-1566.
4.    Villena I, Aubert D, Leroux B, Dupouy D, Talmud M, Chemla C, et al. Pyrimethamine-sulfadoxine treatment of congenital toxoplasmosis: follow-up of 78 cases between 1980 and 1997. Reims Toxoplasmosis Group. Scand J Infect Dis. 1998;30(3):295-300.
5.    Sahoo SK, Dilnawaz F, Krishnakumar S. Nanotechnology in ocular drug delivery. Drug Discov Today. 2008;13(3-4):144-151.
6.    Gupta A, Eral HB, Hatton TA, Doyle PS. Nanoemulsions: formation, properties and applications. Soft Matter. 2016;12(11):2826-4281.
7.    Ammar HO, Salama HA, Ghorab M, Mahmoud AA. Nanoemulsion as a potential ophthalmic delivery system for dorzolamide hydrochloride. AAPS PharmSciTech. 2009;10(3):808-819.
8.    Gutiérrez J, González C, Maestro A, Sole I, Pey C, Nolla J. Nanoemulsions: new applications and optimization of their preparation. Curr Opin Colloid Interface Sci. 2008;13(4):245–251.
9.    van Blitterswijk WJ, Verheij M. Anticancer alkylphospholipids: mechanisms of action, cellular sensitivity and resistance, and clinical prospects. Curr Pharm Des. 2008;14(21):2061-2074.
10.    Dorlo TP, Balasegaram M, Beijnen JH, de Vries PJ. Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother. 2012;67(11):2576-2597.
11.    Llull D, Rivas L, García E. In vitro bactericidal activity of the antiprotozoal drug miltefosine against Streptococcus pneumoniae and other pathogenic streptococci. Antimicrob Agents Chemother. 2007;51(5):1844-1848.
12.    Widmer F, Wright LC, Obando D, Handke R, Ganendren R, Ellis DH, Sorrell TC. Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis. Antimicrob Agents Chemother. 2006;50(2):414-421.
13.    Chugh P, Bradel-Tretheway B, Monteiro-Filho CM, Planelles V, Maggirwar SB, Dewhurst S, Kim B. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy. Retrovirology. 2008;5:11.
14.    Eissa MM, El-Azzouni MZ, Amer EI, Baddour NM. Miltefosine, a promising novel agent for schistosomiasis mansoni. Int J Parasitol. 2011;41(2):235-242.
15.    Blaha C, Duchêne M, Aspöck H, Walochnik J. In vitro activity of hexadecylphosphocholine (miltefosine) against metronidazole-resistant and -susceptible strains of Trichomonas vaginalis. J Antimicrob Chemother. 2006;57(2):273-278.
16.    Eissa MM, Amer EI. Giardia lamblia: A new target for miltefosine. Int J Parasitol. 2012;42(5):443-452.
17.    Seifert K, Duchêne M, Wernsdorfer WH, Kollaritsch H, Scheiner O, Wiedermann G, et al. Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica. Antimicrob Agents Chemother. 2001;45(5):1505-1510.
18.    Walochnik J, Duchêne M, Seifert K, Obwaller A, Hottkowitz T, Wiedermann G, et al. Cytotoxic activities of alkylphosphocholines against clinical isolates of Acanthamoeba spp. Antimicrob Agents Chemother. 2002;46(3):695-701.
19.    Polat ZA, Obwaller A, Vural A, Walochnik J. Efficacy of miltefosine for topical treatment of Acanthamoeba keratitis in Syrian hamsters. Parasitol Res. 2012;110(2):515-520.
20.    Schuster FL, Guglielmo BJ, Visvesvara GS. In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. J Eukaryot Microbiol. 2006;53(2):121-126.
21.    Garg R, Tremblay MJ. Miltefosine represses HIV-1 replication in human dendritic cell/T-cell cocultures partially by inducing secretion of type-I interferon. Virology. 2012;432(2):271-276.
22.    Ni Nyoman AD, Lüder CG. Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study. Apoptosis. 2013;18(6):664-680.
23.    World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
24.    Eissa MM, Barakat AM, Amer EI, Younis LK. Could miltefosine be used as a therapy for toxoplasmosis? Exp Parasitol. 2015;157:12-22.
25.    Mohammad Mehdi M, Seyed Mohsen F, Reza A. Brinzolamide-Loaded Nanoemulsions: In vitro release evaluation. Iran J Pharm Sci. 2016;12(3):75-93.
26.    Shafiq S, Shakeel F. Stability and self-nanoemulsification efficiency of ramipril nanoemulsion containing labrasol and plurol oleique. Clinical Research and Regulatory Affairs. 2010 Mar 1;27(1):7-12.
27.    Butani D, Yewale C, Misra A. Amphotericin B topical microemulsion: Formulation, characterization and evaluation. Colloids Surf B Biointerfaces. 2014;116:351-358.
28.    Ebrahimzadeh MA, Taheri MM, Ahmadpour E, Montazeri M, Sarvi S, Akbari M, Daryani A. Anti-Toxoplasma effects of methanol extracts of feijoa sellowiana, quercus castaneifolia, and allium paradoxum. J Pharmacopuncture. 2017;20(3):220-226.
29.    Khamesipour F, Razavi SM, Hejazi SH, Ghanadian SM. In vitro and in vivo Anti-Toxoplasma activity of Dracocephalum kotschyi essential oil. Food Sci Nutr. 2021;9(1):522-531.
30.    Montazeri M, Emami S, Asgarian-Omran H, Azizi S, Sharif M, Sarvi S, et al. In vitro and in vivo evaluation of kojic acid against Toxoplasma gondii in experimental models of acute toxoplasmosis. Exp Parasitol. 2019;200:7-12.
31.    Coelho AC, Trinconi CT, Costa CH, Uliana SR. In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis. PLoS Negl Trop Dis. 2014;8(7):e2999.
32.    Barbosa BF, Gomes AO, Ferro EA, Napolitano DR, Mineo JR, Silva NM. Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models. Vet Parasitol. 2012;187(1-2):44-52.
33.    Shah J, Nair AB, Jacob S, Patel RK, Shah H, Shehata TM, Morsy MA. Nanoemulsion based vehicle for effective ocular delivery of moxifloxacin using experimental design and pharmacokinetic study in rabbits. Pharmaceutics. 2019;11(5).
34.    Fouad SA, Basalious EB, El-Nabarawi MA, Tayel SA. Microemulsion and poloxamer microemulsion-based gel for sustained transdermal delivery of diclofenac epolamine using in-skin drug depot: in vitro/in vivo evaluation. Int J Pharm. 2013;453(2):569-578.
35.    Garcia CR, Malik MH, Biswas S, Tam VH, Rumbaugh KP, Li W, Liu X. Nanoemulsion delivery systems for enhanced efficacy of antimicrobials and essential oils. Biomater Sci. 2022;10(3):633-653.
36.    Chavhan SS, Petkar KC, Sawant KK. Simvastatin nanoemulsion for improved oral delivery: Design, characterisation, in vitro and in vivo studies. J Microencapsul. 2013;30(8):771-779.
37.    Altaani BM, Almaaytah AM, Dadou S, Alkhamis K, Daradka MH, Hananeh W. Oral delivery of teriparatide using a nanoemulsion system: Design, in vitro and in vivo evaluation. Pharm Res. 2020;37(4):80.
38.    Laxmi M, Bhardwaj A, Mehta S, Mehta A. Development and characterization of nanoemulsion as carrier for the enhancement of bioavailability of artemether. Artif Cells Nanomed Biotechnol. 2015;43(5):334-344.
39.    Nemati S, Mohammad Rahimi H, Hesari Z, Sharifdini M, Jalilzadeh Aghdam N, Mirjalali H, Zali MR. Formulation of Neem oil-loaded solid lipid nanoparticles and evaluation of its anti-Toxoplasma activity. BMC Complement Med Ther. 2022;22(1):122.
40.    Sanfelice R, Bortoleti B, Tomiotto-Pellissier F, Silva TF, Bosqui LR, Nakazato G, et al. Biogenic silver nanoparticles (AgNp-Bio) reduce Toxoplasma gondii infection and proliferation in HeLa cells, and induce autophagy and death of tachyzoites by apoptosis-like mechanism. Acta Trop. 2021;222:106070.
41.    Azami SJ, Amani A, Keshavarz H, Najafi-Taher R, Mohebali M, Faramarzi MA, et al. Nanoemulsion of atovaquone as a promising approach for treatment of acute and chronic toxoplasmosis. Eur J Pharm Sci. 2018;117:138-146.
42.    Adeyemi OS, Murata Y, Sugi T, Kato K. Inorganic nanoparticles kill Toxoplasma gondii via changes in redox status and mitochondrial membrane potential. Int J Nanomedicine. 2017;12:1647-1661.
43.    Arruda da Silva Sanfelice R, Silva TF, Tomiotto-Pellissier F, Bortoleti B, Lazarin-Bidóia D, Scandorieiro S, et al. Biogenic silver nanoparticles reduce Toxoplasma gondii infection and proliferation in RAW 264.7 macrophages by inducing tumor necrosis factor-alpha and reactive oxygen species production in the cells. Microbes Infect. 2022;24(5):104971.
44.    Quan JH, Gao FF, Ismail H, Yuk JM, Cha GH, Chu JQ, Lee YH. Silver nanoparticle-induced apoptosis in ARPE-19 cells is inhibited by Toxoplasma gondii pre-infection through suppression of NOX4-dependent ROS generation. Int J Nanomedicine. 2020;15:3695-3716.