Encapsulation of Leflunomide (LFD) in a novel niosomal formulation facilitated its delivery to THP-1 monocytic cells and enhanced Aryl hydrocarbon receptor (AhR) nuclear translocation and activation

  • Mahsa Hasani
  • Neda Abbaspour Sani
  • Behnaz Khodabakhshi
  • Mehdi Sheikh Arabi
  • Saeed Mohammadi
  • Yaghoub YazdaniEmail author
Research article



Leflunomide (LFD) is an Aryl hydrocarbon receptor (AhR) agonist and immunomodulatory drug with several side effects. Niosomes are novel drug delivery systems used to reduce the unfavorable effects of drugs by enhancing their bioavailability, controlling their release and targeting specific sites.


Here, we prepared niosomal formulations of LFD, evaluated their properties and delivered to THP-1 monocytic cells to study the activation and nuclear translocation of AhR.


Four types of non-ionic surfactants were utilized to formulate niosomes by thin film hydration (TFH) method. Entrapment efficiency (EE %) of niosomes were quantified and dynamic light scattering (DLS) was performed. Transmission electron microscopy (TEM) was used to identify the morphology of LFD niosomes. Dialysis method was used to measure LFD release rate. MTS assay was adopted to examine the viability of the cells upon each treatment. The nuclear transfer of AhR was investigated by Immunocytochemistry (ICC). The mRNA expression of IL1β and CYP1A1 were evaluated using quantitative RT-PCR.


Span 60: cholesterol (1:1) showed the highest EE% (70.00 ± 6.24), largest particles (419.00 ± 4.16 nm) and the best uniformity with the lowest PDI (0.291 ± 0.007). TEM micrographs of Span 60 (1:1) nanoparticles showed conventional spherical vesicles with internal aqueous spaces. The release rate of LFD from Span 60 (1:1) vesicles was slower. Although the viability of LFD niosome-treated THP-1 cells was decreased, they were associated with lower cytotoxic effects compared with the free LFD counterparts. Both free and niosomal LFD treatments intensified the nuclear translocation of AhR. The mRNA expression of CYP1A1 was overexpressed while IL1β was downregulated in both free and niosomal LFD treated combinations.


LFD encapsulation in Span 60: cholesterol (1:1) niosomal formulation could be introduced as a suitable vehicle of transferring LFD to THP-1 cells, with minimal cytotoxic effects, enhancing the AhR nuclear translocation and activation and inducing immunomodulatory properties.

Graphical abstract

The Graphical abstract; it demonstrates the workflow of the study and summary of results in brief.


Aryl hydrocarbon receptor (AhR) Drug delivery Leflunomide (LFD) Niosome 



This article was derived from a thesis of M.Sc. degree in the field of Medical biotechnology (Grant Number: 960129002) at Gorgan School of Advanced Technologies in Medicine of Golestan University of Medical Sciences, Gorgan, Iran. We would like to thank Dr. Samadian, Mrs. Yousefi and Mrs. Haydari for their scientific and technical support.

Author contributions

MH: Acquisition of data, Analyses and interpretations of data, Manuscript drafting, Revision of the manuscript. NAS: Participation in data acquisition, analyses and interpretations. MSA: Participation in data acquisition. BK: Participation in data acquisition. SM: Study design and concept, participation in literature bibliography, data acquisition and analysis, manuscript drafting and critical revision of the manuscript. YY: Participation in study design and final revision of the manuscript. All authors read and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The study was approved by the Ethics committee of Golestan University of Medical Sciences (Code of Ethics: IR.GUOMS.REC.13950259).


  1. 1.
    Fragoso YD, Brooks JBB. Leflunomide and teriflunomide: altering the metabolism of pyrimidines for the treatment of autoimmune diseases. Expert Rev Clin Pharmacol. 2015;8(3):315–20.CrossRefPubMedGoogle Scholar
  2. 2.
    Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol. 1999;93(3):198–208. Scholar
  3. 3.
    Cutolo M, Sulli A, Ghiorzo P, Pizzorni C, Craviotto C, Villaggio B. Anti-inflammatory effects of leflunomide on cultured synovial macrophages from patients with rheumatoid arthritis. Ann Rheum Dis. 2003;62(4):297–302.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    O'Donnell EF, Saili KS, Koch DC, Kopparapu PR, Farrer D, Bisson WH, et al. The anti-inflammatory drug leflunomide is an agonist of the aryl hydrocarbon receptor. PLoS One. 2010;5(10):e13128.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Baban B, Liu JY, Mozaffari MS. Aryl hydrocarbon receptor agonist, leflunomide, protects the ischemic-reperfused kidney: role of Tregs and stem cells. Am J Physiol Regul Integr Comp Physiol. 2012;303(11):R1136–R46.CrossRefPubMedGoogle Scholar
  6. 6.
    Yazdani Y, Sadeghi H, Alimohammadian M, Andalib A, Moazen F, Rezaei A. Expression of an innate immune element (mouse hepcidin-1) in baculovirus expression system and the comparison of its function with synthetic human hepcidin-25. Iran J Pharm Res. 2011;10(3):559–68.PubMedPubMedCentralGoogle Scholar
  7. 7.
    Jutooru I, Chadalapaka G, Safe S. Aryl hydrocarbon receptor ligands: toxic, biochemical, and therapeutic effects. Endocrine toxicology. 3rd ed. Boca Raton, Florida, United States: CRC Press; 2016. p. 201–21.Google Scholar
  8. 8.
    Vondracek J, Umannova L, Machala M. Interactions of the aryl hydrocarbon receptor with inflammatory mediators: beyond CYP1A regulation. Curr Drug Metab. 2011;12(2):89–103.CrossRefPubMedGoogle Scholar
  9. 9.
    Tilg H. Cruciferous vegetables: prototypic anti-inflammatory food components. Clin Phytosci. 2015;1(1):10.CrossRefGoogle Scholar
  10. 10.
    Pund S, Pawar S, Gangurde S, Divate D. Transcutaneous delivery of leflunomide nanoemulgel: mechanistic investigation into physicomechanical characteristics, in vitro anti-psoriatic and anti-melanoma activity. Int J Pharm. 2015;487(1):148–56.CrossRefPubMedGoogle Scholar
  11. 11.
    Waddad AY, Abbad S, Yu F, Munyendo WL, Wang J, Lv H, et al. Formulation, characterization and pharmacokinetics of Morin hydrate niosomes prepared from various non-ionic surfactants. Int J Pharm. 2013;456(2):446–58.CrossRefPubMedGoogle Scholar
  12. 12.
    Hong M, Zhu S, Jiang Y, Tang G, Pei Y. Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin. J Control Release. 2009;133(2):96–102.CrossRefPubMedGoogle Scholar
  13. 13.
    Moghassemi S, Hadjizadeh A. Nano-niosomes as nanoscale drug delivery systems: an illustrated review. J Control Release. 2014;185:22–36.CrossRefPubMedGoogle Scholar
  14. 14.
    Ruckmani K, Sankar V. Formulation and optimization of zidovudine niosomes. AAPS PharmSciTech. 2010;11(3):1119–27.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Uchegbu IF, Duncan R. Niosomes containing N-(2-hydroxypropyl) methacrylamide copolymer-doxorubicin (PK1): effect of method of preparation and choice of surfactant on niosome characteristics and a preliminary study of body distribution. Int J Pharm. 1997;155(1):7–17.CrossRefGoogle Scholar
  16. 16.
    Akbarzadeh A, Rezaei-Sadabady R, Davaran S, Joo SW, Zarghami N, Hanifehpour Y, et al. Liposome: classification, preparation, and applications. Nanoscale Res Lett. 2013;8(1):102.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Daigneault M, Preston JA, Marriott HM, Whyte MK, Dockrell DH. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One. 2010;5(1):e8668.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yazdani Y, Keyhanvar N, Kalhor HR, Rezaei A. Functional analyses of recombinant mouse hepcidin-1 in cell culture and animal model. Biotechnol Lett. 2013;35(8):1191–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Roohi A, Yazdani Y, Khoshnoodi J, Jazayeri SM, Carman WF, Chamankhah M, et al. Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens. World J Gastroenterol. 2006;12(33):5368–74.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Danaei M, Dehghankhold M, Ataei S, Hasanzadeh Davarani F, Javanmard R, Dokhani A, et al. Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. Pharmaceutics. 2018;10(2):57.CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Bera B, editor. Nanoporous silicon prepared by vapour phase strain etch and sacrificial technique. Proceedings of the International Conference on Microelectronic Circuit and System (Micro), Kolkata, India; 2015.Google Scholar
  23. 23.
    Tibbitt MW, Dahlman JE, Langer R. Emerging frontiers in drug delivery. J Am Chem Soc. 2016;138(3):704–17.CrossRefPubMedGoogle Scholar
  24. 24.
    Abdelbary G, El-gendy N. Niosome-encapsulated gentamicin for ophthalmic controlled delivery. AAPS PharmSciTech. 2008;9(3):740–7.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Nebert DW, Dalton TP, Okey AB, Gonzalez FJ. Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. J Biol Chem. 2004;279(23):23847–50.CrossRefPubMedGoogle Scholar
  26. 26.
    Murray IA, Patterson AD, Perdew GH. Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat Rev Cancer. 2014;14(12):801–14.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Faust D, Kletting S, Ueberham E, Dietrich C. Aryl hydrocarbon receptor-dependent cell cycle arrest in isolated mouse oval cells. Toxicol Lett. 2013;223(1):73–80.CrossRefPubMedGoogle Scholar
  28. 28.
    Stockinger B, Meglio PD, Gialitakis M, Duarte JH. The aryl hydrocarbon receptor: multitasking in the immune system. Annu Rev Immunol. 2014;32:403–32.CrossRefPubMedGoogle Scholar
  29. 29.
    Li EK, Tam LS, Tomlinson B. Leflunomide in the treatment of rheumatoid arthritis. Clin Ther. 2004;26(4):447–59.CrossRefPubMedGoogle Scholar
  30. 30.
    Raja NR, Pillai G, Udupa N, Chandrashekar G. Anti-inflammatory activity of niosome encapsulated diclofenac sodium in arthritic rats. Indian J Pharmacol. 1994;26(1):46.Google Scholar
  31. 31.
    Guinedi AS, Mortada ND, Mansour S, Hathout RM. Preparation and evaluation of reverse-phase evaporation and multilamellar niosomes as ophthalmic carriers of acetazolamide. Int J Pharm. 2005;306(1):71–82.CrossRefPubMedGoogle Scholar
  32. 32.
    Marwa A, Omaima S, Hanaa E-G, Mohammed A-S. Preparation and in-vitro evaluation of diclofenac sodium niosomal formulations. Int J Pharm Sci Res. 2013;4(5):1757–65.Google Scholar
  33. 33.
    Zaki RM, Ali AA, El Menshawe SF, Bary AA. Formulation and in vitro evaluation of diacerein loaded niosomes. Int J Pharm Pharm Sci. 2014;6(Suppl 2):515–21.Google Scholar
  34. 34.
    Hu W, Sorrentino C, Denison MS, Kolaja K, Fielden MR. Induction of Cyp1a1 is a non-specific biomarker of aryl hydrocarbon receptor activation: results of large scale screening of pharmaceuticals and toxicants in vivo and in vitro. Mol Pharmacol. 2007;71(6):1475–86.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Mahsa Hasani
    • 1
  • Neda Abbaspour Sani
    • 1
  • Behnaz Khodabakhshi
    • 2
  • Mehdi Sheikh Arabi
    • 3
  • Saeed Mohammadi
    • 2
    • 4
  • Yaghoub Yazdani
    • 2
    • 4
    Email author
  1. 1.Department of Medical Biotechnology, School of Advanced Technologies in MedicineGolestan University of Medical SciencesGorganIran
  2. 2.Infectious Diseases Research CenterGolestan University of Medical SciencesGorganIran
  3. 3.Medical Cellular and Molecular Research CenterGolestan University of Medical SciencesGorganIran
  4. 4.Stem Cell Research CenterGolestan University of Medical SciencesGorganIran

Personalised recommendations