Characterization and evaluation of amine-modified graphene amphotericin B for the treatment of visceral leishmaniasis: in vivo and in vitro studies

Abstract

Amphotericin B (AmB) has been the first-line treatment for visceral leishmaniasis (VL), a neglected protozoan disease, especially in regions like Bihar, India, where resistance to antimonials is widespread. However, adverse drug reactions are a major limiting factor. We evaluated a novel formulation of AmB conjugated to amine-modified graphene (f-Gr) for safety and efficacy over conventional AmB. The f-Gr was prepared in a gentle one-step process of noncovalent (amine) functionalization with the help of amino acid L-cysteine. This f-Gr was further conjugated to AmB by peptide bond. The conjugate (f-Gr-AmB) was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. f-Gr-AmB was found to exhibit lesser cytotoxicity toward J774A.1 cells than AmB, and did not induce any hepatic or renal toxicity in Swiss albino mice. In vitro antileishmanial assay in J774A.1 cells showed significantly enhanced efficacy of f-Gr-AmB over AmB. Furthermore, percentage inhibition of amastigote replication in a hamster model of VL was significantly higher in the f-Gr-AmB treated group (87.8%) compared to the AmB group (70.4%). These results suggest that f-Gr-AmB could be a safe and effective alternative to conventional AmB in the treatment of VL.

Keywords:

• antileishmanial
• efficacy
• cytotoxicity
• macrophage
• Raman spectroscopy
• amastigote

Supplementary materials

Figure S1 SEM images of GS (A), f-Gr (B), and f-Gr-AmB (C).

Notes: (A) This figure shows clearly the typically flake like, slightly scrolled edged shapes of graphene sheet. (B) It is clearly visible from this figure that the small L-cysteine molecules are closely anchored on the surface of graphene sheets and held noncovalently (marked with arrows). (C) This figure illustrates the SEM image of f-Gr-AmB showing that a layer of drug was formed homogeneously (box area).
Abbreviations: SEM, scanning electron micrograph; GS, graphene sheets; f-Gr, amine-modified graphene; f-Gr-AmB, a novel amphotericin B formulation as a conjugate with f-Gr.

Figure S2 FTIR spectra of GS (A), f-Gr (B), and f-Gr-AmB (C).

Notes: Black arrows indicate the position of the peaks. (A) The absorption peak appearing at 1,585 cm⁻¹ in terms of the known results is attributed to the vibration of carbon skeleton of graphene sheet. (B) An additional peak is observed at 3,016 cm⁻¹ which is the stretching vibrations of the alkane group, while the presence of the peak at 1,475 cm⁻¹ can be assigned to N-H stretching vibrations of the amine group. (C) The peak at 1,019 cm⁻¹ is associated with the stretching vibration mode of the hydroxyl group of the carboxylic group present in AmB.
Abbreviations: AmB, amphotericin B; FTIR, Fourier transform infrared; GS, graphene sheets, f-Gr, amine-modified graphene; f-Gr-AmB, a novel amphotericin B formulation as a conjugate with f-Gr.

Acknowledgments

We would like to thank Dr Vijay Kumar Prajapati, Dr Paresh Kulkarni, and all research scholars of the Infectious Disease Research Laboratory, BHU for their kind help during the study, and Dr K Awsathi, Dr S Awasthi, and Dr H Raghuvanshi for the fruitful discussion on graphene synthesis. Shyam Lal Mudavath gives thanks to the University Grants Commission (UGC), New Delhi for providing a Junior Research Fellowship, and Mahe Talat is grateful to the Department of Science and Technology’s (DST’s) Women Scientists Scheme-A (WOS-A) for support.

This work was supported by DST (Unit on Nanoscience and Nanotechnology [UNANST], BHU) and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Tropical Medicine Research Center [TMRC] grant number P50AI074321). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosure

The authors report no conflicts of interest in this work.