Synthesis, characterization, and mechanistic studies of a gold nanoparticle–amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity

Abstract

Background

Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity.

Methods

Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet–visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB.

Results

Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory T_h1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01).

Conclusion

GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

Keywords:

• gold nanoparticle
• amphotericin B
• antileishmanial
• macrophage uptake
• ergosterol
• immunostimulator

Acknowledgments

We acknowledge the financial assistance in research funding to Debabrata Mandal and PhD fellowship to Prakash Kumar, Pragya Prasanna, Saurabh Kumar, and Prasad Surendra Rajit from the Ministry of Chemicals and Fertilizers, Government of India.

Abbreviation list

GNP, gold nanoparticle; AmB, amphotericin B; LA, lipoic acid; PDI, polydispersity index; TEM, transmission electron microscopy; SAED, selected area electron diffraction; FT-IR, Fourier transform infrared spectroscopy; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, sulfo-N-hydroxysuccinamide; VL, visceral leishmaniasis; LD, Leishmania donovani; ERG, ergosterol; SOD, superoxide dismutase; NAC, N-acetyl l-cysteine; LDH, lactate dehydrogenase; IFN-γ, interferon-γ; RT, retention time.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Methods

Determination of reactive oxygen species (ROS) in promastigotes by fluorescent dye H₂DCFDA

Promastigotes (5×10⁶ cells/ml) were treated with GNP (2.5, 5 and 10 µg/ml), AmB/GL-AmB (0.05-0.2 µM) for 6 and 12 h. Amount of ROS accumulated in promastigotes were measured by fluorescent dye H₂DCFDA as described.¹

Determination of nitrites from amastigotes by Griess reagent

Reactive nitrogen species (RNS) and nitrites were measured by Griess reagent-based colorimetric assay at 540 nm from amastigote-infected macrophages.²

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Reverse transcription was performed using 1 μg of total RNA by cDNA synthesis kit (Roche, USA) according to the manufacturer's instruction. The synthesized cDNAs (from RNA of promastigotes) were amplified by PCR for specific genes viz. trypanothione reductase (TryR), superoxide dismutase (SOD), ascorbate peroxidase (APX), heat shock protein-70 (HSP-70), arginase-1, lanosterol-14-demethylase (Ldem), S-adenosyl-L-methionine:C-24-∆-sterol methyltransferase (SCMT), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA-R) and α-tubulin. The synthesized cDNAs (from RNA of amasitogote infected macrophages) were amplified by PCR for specific genes viz. inducible nitric oxide synthase (iNOS), IL-12, IL-10, IFN-γ and GAPDH. LD α-tubulin and macrophage GAPDH were used as loading control for promastigote and macrophage models, respectively. The PCR mixture (25 μl) contains 0.6 μM of forward and reverse primer, 0.5 mM of each dNTP, 2 mM MgCl₂, 0.5 μg of synthesized cDNA and 1 μl Taq polymerase. The sequence of PCR primers, annealing temperature and size of PCR products were shown in Table 1. The PCR was done for 28 cycles where each cycle had denaturation at 95°C for 30 sec, annealing (ranging from 55-62°C) for 30 sec and extension at 72°C for 45 sec. Samples were preheated at 95°C for 3 min before PCR. The products were run on 1.5% agarose gel, stained with ethidium bromide (0.5 μg/ml), and finally documented and quantified using the gel documentation system and associated Gene-tool software (Syngene, USA).

Isolation of total RNA from promastigotes and amastigote-infected macrophages

Parasites (5×10⁶ cells/well) were treated with GNP (5 μg/ml), AmB/GL-AmB (0.1 μM) for 6 h. Treated cells were centrifuged at 4200g for 10 min, washed thrice with PBS and then total RNA was isolated by addition of TRIZOL solution (Thermo Scientific, USA) as described. Pellet of RNA was air-dried, re-suspended in 100 µL of RNase-free water and treated with DNase I (1U/μl) at 37°C for 30 min. Digested RNA was loaded on RNeasy Mini Kit (Qiagen, USA) columns and RNA was eluted in 30 μl of RNase-free water. RNA quality was checked by gel electrophoresis and quantified by Nanodrop spectrophotometer (Thermo scientific, Nanodrop 2000, USA).

For isolation of RNA from macrophage-infected parasites, RAW 264.1 cells (1×10⁶/well) were grown in a 6 well plate and infected with 1×10⁷ parasites for 12 h. Non-phagocytic cells were washed out, fresh medium was added and then incubated further for 12 h. Parasite-infected macrophages were treated with GNP (10 μg/ml), AmB and GL-AmB (1 μM) for 6 h and then RNA was isolated from the cell pellet as described above.

Superoxide dismutase (SOD) activity assay

Promastigotes (5×10⁶ cells/ml) were treated with GNP (10 μg/ml), AmB/GL-AmB (0.2 μM) for 6 h and then harvested. Cells were lysed and SOD enzyme activity assay was performed as described.³ The enzyme activity was calculated as the percentage of inhibition in the reduction of nitro blue tetrazolium (NBT) measured at 560 nm. Reduction of untreated cells were considered 100%.

Results

Determination of ROS in promastigotes

Amount of ROS produced by GL-AmB was ~2.4, ~2.1 and ~1.6 fold higher than AmB at 0.05, 0.1 and 0.2 μM doses, respectively (P < 0.001, Figure S4A). Interestingly, at IC₅₀ dose for both GNP (2.5 μg/ml) and AmB (0.05 μM) amount of ROS produced was ~2 fold higher for GNP than AmB. Therefore, generation of oxidative stress by citrate-reduced GNPs is one of the possible reason for its antileishmanial efficacy. Amount of ROS produced by GNP, AmB and GL-AmB in promastigotes can be reduced to the basal level by pre-incubating the reaction with 1 mM NAC. Although a wide range of concentrations of AmB and GL-AmB were used in most experiments, results were shown only for those concentrations which had shown significant differences between AmB and GL-AmB-treated samples.

Determination of RNS in promastigotes

Amount of RNS produced in macrophages was also higher (~1.5 fold) for GL-AmB-treated cells than AmB-treated cells at all indicated doses (Figure S4B). Also, RNS produced by GNP and AmB is almost comparable. Therefore, production of higher oxidative stress is one of the possible mechanism of increased antileishmanial efficacy of GL-AmB compared to AmB.

SOD Assay

Activity of SOD was ~1.9 fold reduced in lysates which were prepared from 0.2 μM of GL-AmB-treated cells in comparison to AmB-treated cells after 6 h.

Supplementary materials

Figure S1 HPLC chromatogram of AmB (0.2 mg/ml) (A) and synthesized GL-AmB (amount equivalent to 0.2 mg/ml of AmB used in synthesis) (B). AmB standard curve based on HPLC determination (C).

Abbreviation: AmB, amphotericin B.

Figure S2 Zeta potential diagram of GNP, GL and GL-AmB (A). Tabular representation of measured average diameter and PDI of NPs by DLS (B).

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle; PDI, polydispersity index.

Figure S3 X-ray diffraction (XRD) of crystalline GL-AmB characterized by the presence of four peaks corresponding to standard Bragg reflections. The diffraction peak of 38.2° relates to (111), 47.7° relates to (200), 65.3° relates to (220), and 77.1° relates to (311) facets of the face centre cubic (FCC) crystal.

Figure S4 Activity of NPs and AmB after 72 h treatment against LD promastigotes in vitro (A) and against intracellular amastigotes ex vivo (C). Comparative efficacy for AmB and GL-AmB against promastigote (C) and amastigotes (D).

Note: *P<0.05; ***0.01<P<0.001.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle.

Figure S5 Activity of NPs and AmB after 96 h treatment against LD promastigotes in vitro (A) and against intracellular amastigotes ex vivo (C). Comparative efficacy for AmB and GL-AmB against promastigote (C) and amastigotes (D).

Note: *P<0.05; **0.05<P<0.01; ***0.01<P<0.001.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle.

Figure S6 Cyto-toxicity assay on THP-1 cells after 72 h treatment with NPs and AmB (A). Hemolysis assay on human RBCs after 4 h treatment with NPs and AmB (C). DMSO (0.1%) used as negative control. Comparative efficacy of AmB and GL-AmB in cytotoxicity (B) and hemolysis (D) assay.

Note: *P<0.05; **0.05<P<0.01; ***0.01<P<0.001.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle.

Figure S7 In vitro activity of AmB and NPs after 48 h treatment against C. albicans (A). Comparative efficacy of AmB and GL-AmB against C. albicans (B).

Note: **0.05<P<0.01; ***0.01<P<0.001.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle.

Figure S8 Measurement of ROS produced by promastigotes after treatment with NPs and AmB for 6 h with or without 1 mM NAC (A). Measurement of RNS produced by amastigotes after measuring absorbance of Griess reagent at 540 nm under similar treatment conditions for NPs and AmB with or without 0.1 mM DPI (B).

Note: *P<0.05; **0.05<P<0.01; ***0.01<P<0.001.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle.

Figure S9 Protein carbonyl content was determined by DNPH-based spectrophotometric method from parasites treated with NPs and AmB after 6 h (A) and 12 h (B). Lipid peroxidation assay for promastigotes after treatment with NPs and AmB for 6 h with or without 1 mM NAC (C).

Note: *P<0.05; **0.05<P<0.01.

Abbreviations: AmB, amphotericin B; GL, GNP-lipoic acid product; GNP, gold nanoparticle; NS, not significant.

Figure S10 Semi-quantitative PCR for LD specific genes after treatment of parasites with AmB and NPs for 6 h (A) where -tubulin was used as loading control. SOD activity assay with LD cell lysates after treatment with AmB and NPs for 6 h (B) where activity of untreated cells were considered 100%. Semi-quantitative PCR for macrophage iNOS after treatment with macrophage-infected parasites with AmB and NPs for 6 h (C).

Note: **0.05<P<0.01.

Abbreviations: AmB, amphotericin B; APX, ascorbate peroxidase; GL, GNP-lipoic acid product; GNP, gold nanoparticle; HSP, heat shock protein; iNOS, inducible nitric oxidase.; SCMT, S-adenosyl-L-methionine:C-24-∆-sterol methyltransferase; SOD, superoxide dismutase; TryR, trypanathione reductase.