Amphotericin B-loaded deformable lipid vesicles for topical treatment of cutaneous leishmaniasis skin lesions


Cutaneous leishmaniasis (CL), the most common clinical form of human leishmaniasis, is a non-fatal chronic and disabling disease characterized by erythema and nodular or ulcerative skin lesions that may cause permanent scars and disfigurement. Topical drug delivery represents a simple and efficacious approach to treat CL skin lesions. The association of drugs with nanocarrier systems enhances their permeation properties and increases the drug amount available in the dermis. Here, a deformable lipid vesicle (DLV) was optimized for the topical administration of Amphotericin B (AmB), with the aim of studying and understanding the advantages of this type of delivery system in the transport of a drug through the skin layers. AmB-DVL were characterized in terms of incorporation parameters, stability, and elasticity, and evaluated in vitro for their permeation properties, cytotoxicity, and anti-leishmanial activity. The AmB-DVL exhibited a translucent fluid gel-like aspect and a yellow color, a mean size of 132 nm (PdI ≤ 0.1), zeta potential values around zero (mV), and an AmB incorporation efficiency of 95%. Permeation and penetration assays suggest that AmB-DLV are suitable for topical administration since AmB was detected in the epidermal and dermal skin layers. AmB-DVL was able to reduce promastigote viability in a dose-dependent manner, as well as the number of intracellular amastigotes in THP-1 macrophages. Selectivity index (SI) value for AmB-DLV was considerably higher than that observed for free AmB. Results suggest that DLV may represent an attractive vehicle for dermal delivery of AmB and a new low-cost and safe therapeutic option in CL treatment.

Graphical abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Mears ER, Modabber F, Don R, Johnson GE. A review: the current in vivo models for the discovery and utility of new anti-leishmanial drugs targeting cutaneous leishmaniasis. PLoS Negl Trop Dis. 2015;9:1–23.

    Article  Google Scholar 

  2. 2.

    de Vries HJC, Reedijk SH, Schallig HDFH. Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol. 2015;16:99–109.

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    World Health Organization. Leishmaniasis: epidemiological situation. Accessed 20 Oct 2020.

  4. 4.

    Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R. Leishmaniasis: a review. F1000Res 2017;6:750.

  5. 5.

    Simoes S, Carvalheiro M, Gaspar MM, Simões S, Carvalheiro M, Gaspar MM. Lipid-based nanocarriers for cutaneous leishmaniais and buruli ulcer management. Curr Pharm Des. 2016;22:6577–86.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA. Cutaneous and mucocutaneous leishmaniasis: differential diagnosis, diagnosis, histopathology, and management. J Am Acad Dermatol. 2015;73:911–26.

    Article  PubMed  Google Scholar 

  7. 7.

    Cota GF, De Sousa MR, Fereguetti TO, Saleme PS, Alvarisa TK, Rabello A. The cure rate after placebo or no therapy in American cutaneous leishmaniasis: a systematic review and meta-analysis. PLoS One. 2016;11:1–15.

    CAS  Article  Google Scholar 

  8. 8.

    Monge-Maillo B, López-Vélez R. Miltefosine for visceral and cutaneous leishmaniasis: drug characteristics and evidence-based treatment recommendations. Clin Infect Dis. 2015;60:1398–404.

    Article  PubMed  Google Scholar 

  9. 9.

    Lopes R, Eleutério CV, Gonçalves LMD, Cruz MEM, Almeida AJ. Lipid nanoparticles containing oryzalin for the treatment of leishmaniasis. Eur J Pharm Sci. 2012;45:442–50.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Copeland NK, Aronson NE. Leishmaniasis: treatment updates and clinical practice guidelines review. Curr Opin Infect Dis. 2015;28:426–37.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Grogl M, Schuster BG, Ellis WY, Berman JD. Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (aminosidine) and gentamicin. J Parasitol. 2006;85:354.

    Article  Google Scholar 

  12. 12.

    Ameen M. Cutaneous leishmaniasis: advances in disease pathogenesis, diagnostics and therapeutics. Clin Exp Dermatol. 2010;35:699–705.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Mishra B, Singh R, Srivastava A, Tripathi V, Tiwari V. Fighting against leishmaniasis: search of alkaloids as future true potential anti-leishmanial agents. Mini-Reviews Med Chem. 2009;9:107–23.

    CAS  Article  Google Scholar 

  14. 14.

    WHO technical. Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases. World Health Organ Tech Rep Ser 2010:xii–xiii, 1–186.

  15. 15.

    Campos-Munoz L, Quesada-Cortes A, Martin-Diaz MA, Rubio-Flores C, de Lucas-Laguna R. Leishmania Braziliensis: report of a pediatric imported case with response to liposomal amphotericin B. Acta Anaesthesiol Scand. 2009;53:400–2.

    Article  Google Scholar 

  16. 16.

    Kumara R, Pandeya K, Dasa V, Yousuf Ansaria M, Dasa P, Sahoo GC. PLGA-PEG Encapsulated sitamaquine nanoparticles drug delivery system against Leishmania donovani. J Sci Innov Res JSIR. 2014;3:85–90.

    Google Scholar 

  17. 17.

    World Health Organization. Leishmaniasis: diagnosis, detection and surveillance. Accessed 21 Oct 2020.

  18. 18.

    Kaur L, Jain SK, Manhas RK, Sharma D. Nanoethosomal formulation for skin targeting of amphotericin B: an in vitro and in vivo assessment. J Liposome Res. 2015;25:294–307.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Tevyashova AN, Olsufyeva EN, Solovieva SE, Printsevskaya SS, Reznikova MI, Trenin AS, et al. Structure-antifungal activity relationships of polyene antibiotics of the amphotericin B group. Antimicrob Agents Chemother. 2013;57:3815–22.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Readio JD, Bittman R. Equilibrium binding of amphotericin B and its methyl ester and borate complex to sterols. Biochim Biophys Acta - Biomembr. 1982;685:219–24.

    CAS  Article  Google Scholar 

  21. 21.

    Wijnant G, Van Bocxlaer K, Yardley V, Harris A, Murdan S, Croft SL. Relation between skin pharmacokinetics and efficacy in ambisome treatment of murine cutaneous leishmaniasis. Antimicrob Agents Chemother 2018;62:1–9.

  22. 22.

    Akbari M, Oryan A, Hatam G. Application of nanotechnology in treatment of leishmaniasis: A Review. Acta Trop. 2017;172:86–90.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Davidson RN, Croft SL, Scott A, Maini M, Moody AH, Bryceson AD. Liposomal amphotericin B in drug-resistant visceral leishmaniasis. Lancet. 1990;337:1061–2.

    Article  Google Scholar 

  24. 24.

    El Maghraby GMM, Williams AC, Barry BW. Skin delivery of 5-fluorouracil from ultradeformable and standard liposomes in-vitro. J Pharm Pharmacol. 2001;53:1069–77.

    Article  PubMed  Google Scholar 

  25. 25.

    Elsayed MMA, Abdallah OY, Naggar VF, Khalafallah NM. Lipid vesicles for skin delivery of drugs: reviewing three decades of research. Int J Pharm. 2007;332:1–16.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Tran T-NT. Cutaneous drug delivery: an update. J Investig Dermatology Symp Proc 2013;16:S67–9.

  27. 27.

    Nastiti CMRR, Ponto T, Abd E, Grice JE, Benson HAE, Roberts MS. Topical nano and microemulsions for skin delivery. Pharmaceutics. 2017;9:1–25.

    CAS  Article  Google Scholar 

  28. 28.

    NIH. Accessed 7 Jan 2021.

  29. 29.

    López L, Vélez I, Asela C, Cruz C, Alves F, Robledo S, et al. A phase II study to evaluate the safety and efficacy of topical 3% amphotericin B cream (Anfoleish) for the treatment of uncomplicated cutaneous leishmaniasis in Colombia. PLoS Negl Trop Dis 2018;12:e0006653.

  30. 30.

    Cevc G, Blume G. Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. BBA - Biomembr. 1992;1104:226–32.

    CAS  Article  Google Scholar 

  31. 31.

    Bahrami F, Harandi AM, Rafati S. Biomarkers of cutaneous leishmaniasis. Front Cell Infect Microbiol. 2018;8:1–8.

    CAS  Article  Google Scholar 

  32. 32.

    Cevc G. Lipid vesicles and other colloids as drug carriers on the skin. Adv Drug Deliv Rev. 2004;56:675–711.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    El Zaafarany GM, Awad GAS, Holayel SM, Mortada ND. Role of edge activators and surface charge in developing ultradeformable vesicles with enhanced skin delivery. Int J Pharm. 2010;397:164–72.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Bahia APCO, Azevedo EG, Ferreira LAM, Frézard F. New insights into the mode of action of ultradeformable vesicles using calcein as hydrophilic fluorescent marker. Eur J Pharm Sci. 2010;39:90–6.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Perez AP, Altube MJ, Schilrreff P, Apezteguia G, Celes FS, Zacchino S, et al. Topical amphotericin B in ultradeformable liposomes: formulation, skin penetration study, antifungal and antileishmanial activity in vitro. Colloids Surfaces B Biointerfaces. 2016;139:190–8.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Rouser G, Fleischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970;5:494–6.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Van Bocxlaer K, Yardley V, Murdan S, Croft SL. Drug permeation and barrier damage in Leishmania-infected mouse skin. J Antimicrob Chemother. 2016;71:1578–85.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Gaspar DP, Faria V, Gonçalves LMD, Taboada P, Remuñán-López C, Almeida AJ. Rifabutin-loaded solid lipid nanoparticles for inhaled antitubercular therapy: physicochemical and in vitro studies. Int J Pharm. 2016;497:199–209.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Marto J, Vitor C, Guerreiro A, Severino C, Eleutério C, Ascenso A, et al. Ethosomes for enhanced skin delivery of griseofulvin. Colloids Surfaces B Biointerfaces. 2016;146:616–23.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    OECD. Guidance Notes on Dermal Absorption 2011. Available at:

  41. 41.

    Khadir F, Shaler CR, Oryan A, Rudak PT, Mazzuca DM, Taheri T, et al. Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin. PLoS Negl Trop Dis. 2018;12:e0006701.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Jain SK, Sahu R, Walker LA, Tekwani BL. A parasite rescue and transformation assay for antileishmanial screening against intracellular Leishmania donovani amastigotes in THP1 human acute monocytic leukemia cell line. J Vis Exp 2012:1–14.

  43. 43.

    Lanza JS, Pomel S, Loiseau PM, Frézard F. Recent advances in amphotericin B delivery strategies for the treatment of leishmaniases. Expert Opin Drug Deliv. 2019;16:1063–79.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Faustino C, Pinheiro L. Lipid systems for the delivery of amphotericin B in antifungal therapy. Pharmaceutics. 2020;12:29.

    CAS  Article  PubMed Central  Google Scholar 

  45. 45.

    Cevc G, Schätzlein A, Richardsen H. Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements. Biochim Biophys Acta - Biomembr 2002;1564:21–30.

  46. 46.

    Peralta MF, Guzmán ML, Pérez AP, Apezteguia GA, Fórmica ML, Romero EL, et al. Liposomes can both enhance or reduce drugs penetration through the skin. Sci Rep. 2018;8:1–11.

    CAS  Article  Google Scholar 

  47. 47.

    Goyal P, Goyal K, Vijaya Kumar SG, Singh A, Katare OP, Mishra DN. Liposomal drug delivery systems—clinical applications. Acta Pharm. 2005;55:1–25.

    CAS  PubMed  Google Scholar 

  48. 48.

    Volmer AA, Szpilman AM, Carreira EM. Synthesis and biological evaluation of amphotericin B derivatives. Nat Prod Rep. 2010;27:1329–49.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Dar MJ, Din FU, Khan GM. Sodium stibogluconate loaded nano-deformable liposomes for topical treatment of leishmaniasis: Macrophage as a target cell. Drug Deliv. 2018;25:1595–606.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Esteves MA, Fragiadaki I, Lopes R, Scoulica E, Cruz MEM. Synthesis and biological evaluation of trifluralin analogues as antileishmanial agents. Bioorganic Med Chem. 2010;18:274–81.

    CAS  Article  Google Scholar 

  51. 51.

    De Muylder G, Ang KKH, Chen S, Arkin MR, Engel JC, McKerrow JH. A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit. PLoS Negl Trop Dis. 2011;5:e1253.

    Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Ascenso A, Salgado A, Euletério C, Praça FG, Bentley MVLB, Marques HC, et al. In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles. Eur J Pharm Biopharm. 2014;88:48–55.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Gupta S, Nishi. Visceral leishmaniasis: experimental models for drug discovery. Indian J Med Res 2011;133:27–39.

  54. 54.

    Carvalheiro M, Esteves MA, Santos-Mateus D, Lopes RM, Rodrigues MA, Eleutério CV, et al. Hemisynthetic trifluralin analogues incorporated in liposomes for the treatment of leishmanial infections. Eur J Pharm Biopharm. 2015a;93:346–52.

    CAS  Article  PubMed  Google Scholar 

Download references


This work was partially funded by Fundação para a Ciência e a Tecnologia (FCT) through the program UID/DTP/04138/2019 and UIDB/04138/2020.

Author information




Conceptualization: Manuela Carvalheiro; Methodology: Jennifer Vieira, Catarina Faria-Silva, Joana Marto, Manuela Carvalheiro, Sandra Simões; Software: Catarina Faria-Silva; Analysis: Manuela Carvalheiro, Sandra Simões; Original draft preparation: Jennifer Vieira; Review and editing: Manuela Carvalheiro, Sandra Simões, Catarina Faria-Silva. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Sandra Simões.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Carvalheiro, M., Vieira, J., Faria-Silva, C. et al. Amphotericin B-loaded deformable lipid vesicles for topical treatment of cutaneous leishmaniasis skin lesions. Drug Deliv. and Transl. Res. (2021).

Download citation


  • Cutaneous leishmaniasis
  • Topical formulations
  • Drug delivery
  • Deformable lipid vesicles
  • Amphotericin B