Biomimetically engineered Amphotericin B nano-aggregates circumvent toxicity constraints and treat systemic fungal infection in experimental animals

Abstract

Biomimetic synthesis of nanoparticles offers a convenient and bio friendly approach to fabricate complex structures with sub-nanometer precision from simple precursor components. In the present study, we have synthesized nanoparticles of Amphotericin B (AmB), a potent antifungal agent, using Aloe vera leaf extract. The synthesis of AmB nano-assemblies (AmB-NAs) was established employing spectro-photometric and electron microscopic studies, while their crystalline nature was established by X-ray diffraction. AmB-nano-formulation showed much higher stability in both phosphate buffer saline and serum and exhibit sustained release of parent drug over an extended time period. The as-synthesized AmB-NA possessed significantly less haemolysis as well as nephrotoxicity in the host at par with Ambisome^®, a liposomized AmB formulation. Interestingly, the AmB-NAs were more effective in killing various fungal pathogens including Candida spp. and evoked less drug related toxic manifestations in the host as compared to free form of the drug. The data of the present study suggest that biomimetically synthesized AmB-NA circumvent toxicity issues and offer a promising approach to eliminate systemic fungal infections in Balb/C mice.

Figure 1

(A) Aloe vera leaf extract induces biomimetic synthesis of AmB-NAs: Effect of various parameters on biomimetic synthesis of AmB-NA (i) Time kinetics of AmB-NA synthesis (ii) UV-VIS spectra of AmB-NA to monitor interaction of 1 mM AmB solution with varying volume of Aloe vera leaf extract (iii) Effect of increasing concentration of AmB on biomimetic synthesis of AmB-NA as revealed by UV-VIS spectroscopy. (B) Biomimetically synthesized AmB-NA exhibits superaggregated assembly pattern: Fluorescence emission (Em) spectra of AmB-NA as well as Fungizone at (i) 408 nm (ii) 250 nm (iii) 325 nm. (C) The characteristic functional groups of parent AmB compound remained intact in as synthesized AmB-NA: (i) FTIR absorption spectra of AmB formulations (ii) Structure of AmB.

Figure 2

Size distribution and morphology of AmB-NA: (A) Representative TEM image of optimized AmB-NA synthesized by employing 5 ml of Aloe Vera leaf extract mixed with 5 ml of 1 mM AmB solution. (B) The average diameter of AmB-NA determined by DLS measurements was 95 ± 12 nm and 104 ± 9 nm for AmB-NA3 and AMB-NA5. (C) Particle size distribution of AmB-NA assessed by photon correlation spectroscopy. (D) XRD pattern of AmB-NA. (E) Effect of change in concentration of (i) AmB (ii) Aloe vera leaf extract and (iii) reaction time on the size of the AmB-NA.

Figure 3

AmB-NA releases monomeric AmB in sustained manner over extended time period: Various AmB-NA formulations were incubated in (A) sterile 20 mM phosphate buffer saline (pH 7.4) (B) Serum. (C) Histidine Buffer. The amount of AmB released at various time points was analysed spectrophotometrically at 405 nm. Each time point represents an average of three runs ± SD.

Figure 4

In vitro toxicity tests: (A) Hemolysis caused by AmB-NA upon their interaction with human RBCs. The extent of damage incurred to blood erythrocytes by AmB-NA was measured as percentage lysis of total erythrocytes (B) Intracellular K⁺ leakage incurred by human RBCs upon exposure to various AmB formulations. (C) Dose-response effects of AmB nano-formulation on cytotoxicity against (a) HEK-293cells (b) J774A.1 cells. The cells were exposed to two forms of AmB-NA for 24 hr. MTT values were normalized to the control untreated cells. Data are reported as means ± standard deviation of quadruplet. Fungizone, AmBisome and pure AmB were taken as controls. (D) Representative photomicrograph of J774A.1 cell line treated with (i) 25 µg/mL, (ii) 50 µg/mL, (iii) 100 µg/mL and (iv) 200 µg/mL concentration of AmB-NA for 24 hr. (E) LDH release induced by various AmB formulations, as a function of membrane damage in macrophage J774 A.1. The LDH release was detected by measuring the absorbance of colored complex at 500 nm. Triton X-100 (0.1%) was used as a positive control. Fungizone, AmBisome and pure AmB powder used in preparation of the complex were taken as controls. Data represented are pooled from three different experiments. Each data point is an average ± SD. Aloe vera leaf extract showed no change in any of the parameters tested (data not shown).

Figure 5

AmB-NA formulation demonstrated strong antifungal activity against fungal pathogen: (A) Representative time–kill curve plot for C. albicans in the presence of Fungizone, AmBisome or AmB-NA at 4 × MIC. Wells containing no antibiotic were taken as control. Assays were performed in triplicate. Each result is representative of at least three separate experiments. Values represent mean ± SD.

Figure 6

Potential of AmB-NA in treatment of systemic Candida albicans infection in experimental animals. Residual fungal load in the vital organs of experimental animals after treatment with AmB-NA formulation. (A) Fungal load in spleen. (B) Fungal load in Kidney (C) Fungal load in liver. [Group I versus Group III (p value < 0.001); Group I versus Group IV (p value < 0.001); Group I versus Group V (p value < 0.001); Group III (Lip-AmB) versus Group V (AmB-NA5) (p value < 0.05)]. (D) Survival rate of animals after exposure to infection followed by treatment with AmB-NAs. Group I versus Group III P value < 0.001; Group I versus Group IV P value < 0.001; Group I versus Group V P value < 0.001; Group III (Lip-AmB) versus Group V (AmB-NA5) P value < 0.05.