T.P. Yadav

Targeted killing of Leishmania donovani in vivo and in vitro with amphotericin B attached to functionalized carbon nanotubes

OBJECTIVES This study describes the antileishmanial efficacy of the novel drug formulation of amphotericin B (AmB) attached to functionalized carbon nanotubes (f-CNTs) and compares it with AmB. METHODS f-CNTs were prepared in a two-step chemical carboxylation and amidation process. The AmB was then attached to make f-CNT-AmB and its construction was confirmed by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). The cytotoxicity of the constructed compound, f-CNT-AmB, was assessed in vitro using the J774A.1 macrophage cell line and in vivo using healthy BALB/c mice. Antileishmanial activity of AmB and f-CNT-AmB was assessed in vitro using a macrophage (J774A.1 cell line) model of Leishmania donovani infection. Antileishmanial activity was assessed in vivo by comparing the parasite load of hamsters treated with a 5 day course of AmB, f-CNTs or f-CNT-AmB initiated at 30 days after infection with L. donovani parasites. RESULTS The FTIR spectroscopy and TEM data demonstrate the successful attachment of AmB to f-CNTs. The in vitro cytotoxicity of AmB, f-CNTs and f-CNT-AmB was measured by the cytotoxic concentration required to kill 50% of the cells: 0.48±0.06 μg/mL; 7.31±1.16 μg/mL; 0.66±0.17 μg/mL, respectively, in the J774A.1 cell line. The in vivo toxicity assessment of the compounds in BALB/c mice revealed no hepatic or renal toxicity. Against intracellular amastigotes the in vitro antileishmanial efficacy of f-CNT-AmB was significantly higher than that of AmB (IC50 0.00234±0.00075 μg/mL versus 0.03263±0.00123 μg/mL; P≤0.0001). The percentage inhibition of amastigote replication in hamsters treated with f-CNT-AmB was significantly more than that with AmB (89.85%±2.93% versus 68.97%±1.84%; P=0.0004). CONCLUSIONS The results of these experiments clearly demonstrate that f-CNT-AmB has significantly greater antileishmanial efficacy than AmB and had no significant cytotoxic effects.

Antileishmanial activity of nano-amphotericin B deoxycholate

OBJECTIVES The aim of the present study was to compare the efficacy of a nano form of amphotericin B deoxycholate with that of conventional amphotericin B deoxycholate for the treatment of visceral leishmaniasis. METHODS We have formulated nanoparticles (10-20 nM) from amphotericin B deoxycholate (1-2 microM) by applying high-pressure (150 argon) milling homogenization and have tested their efficacy in a J774A cell line and in hamsters. Parasite survival and tissue burden in spleen were evaluated for nano-amphotericin B and conventional amphotericin B. Both nano-amphotericin B and conventional amphotericin B were injected intraperitoneally at 5 mg/kg per day for 5 days. RESULTS The inhibition of amastigotes in the splenic tissue with nano-amphotericin B was significantly more than with conventional amphotericin B (92.18% versus 74.57%, P = 0.005). Similarly, the suppression of parasite replication in the spleen was also found to be significant (99.18% versus 97.17%, P = 0.05). In a cytotoxicity test, nano-amphotericin B against the J774A cell line had a CC(50) of 12.67 mg/L in comparison with 10.61 mg/L for amphotericin B, far higher than the doses used for ED(50). CONCLUSIONS Nanoparticles of amphotericin B had significantly greater efficacy than conventional amphotericin B. This formulation may have a favourable safety profile, and if production costs are low, it may prove to be a feasible alternative to conventional amphotericin B.

An Oral Formulation of Amphotericin B Attached to Functionalized Carbon Nanotubes Is an Effective Treatment for Experimental Visceral Leishmaniasis

Amphotericin B (AmB), is a highly effective antileishmanial agent used as first-line treatment in different formulations in visceral leishmaniasis endemic areas of Bihar, India. However, parenteral infusion, prolonged hospitalization, and toxicity are major hurdles. Our previous work demonstrated the efficacy and stability of functionalized carbon nanotubes as a delivery mechanism for AmB. In this study, using the hamster model, we have shown that this novel formulation of AmB can be administered orally, resulting in 99% inhibition of parasite growth following a 5-day course at 15 mg/kg body weight.

Templated quasicrystalline molecular ordering.

Quasicrystals are materials with long-range ordering but no periodicity. We report scanning tunneling microscopy (STM) observations of quasicrystalline molecular layers on 5-fold quasicrystal surfaces. The molecules adopt positions and orientations on the surface consistent with the quasicrystalline ordering of the substrate. Carbon-60 adsorbs atop sufficiently separated Fe atoms on icosahedral Al-Cu-Fe to form a unique quasicrystalline lattice, whereas further C60 molecules decorate remaining surface Fe atoms in a quasi-degenerate fashion. Pentacene (Pn) adsorbs at 10-fold symmetric points around surface-bisected rhombic triacontahedral clusters in icosahedral Ag-In-Yb. These systems constitute the first demonstrations of quasicrystalline molecular ordering on a template.

An investigation on the transformation of the icosahedral phase in the Al-Fe-Cu system during mechanical milling and subsequent annealing

Abstract Icosahedral quasicrystalline material in the Al-Fe-Cu system was mechanically milled in an attritor ball mill (Szegvari attritor) for 1, 3, 6 and 10 h in dry air, at a speed of 400 rev min−1 and with a ball-to-powder ratio of 20 to 1. Structural transformations and the consequent phase evolutions during mechanical milling and subsequent heat treatments were studied by X-ray diffraction, differential thermal analysis (DTA) and transmission electron microscopy techniques. After milling for 1 h, the evolution of disordered B2 phase (a=0.29 nm) was observed to coexist with the parent icosahedral phase, whereas a microstructure consisting of nanosized B2 particles distributed in an amorphous matrix was observed after further milling (3–10 h). However, no sharp peak corresponding to the phase transformation was identified in DTA, but the microstructure of the powder milled for 10 h after the DTA experiment was found to transform to a mixture of icosahedral and B2 phases, where B2 appears to be the major phase in contrast with that in as-cast material. Isothermal heat treatment of powder mechanically milled at 850°C for 10 and 20 h led to complete transformation to a single B2 phase with a high degree of long-range ordering. The implication of these transformations will be discussed with reference to their relative stabilities among the competing phases during milling and subsequent heat treatment.

Strain-induced structural transformation of single-phase Al–Cu–Fe icosahedral quasicrystal during mechanical milling

A single-phase stable icosahedral quasicrystalline sample of high quality with the composition Al62.5Cu25Fe12.5 was produced by the spray forming technique. The material was further investigated by mechanical milling under an argon atmosphere to avoid oxidation during milling. At the initial stages of milling (within 5 h) a significant broadening of the diffraction peaks was observed, indicating a reduction of crystallite size and the introduction of lattice strain, which can be linked to phason strain of the quasilattice. Line broadening was noticed to increase with increasing milling time and in the material milled for longer time only a few broad diffraction peaks, which can be identified as a nanoscale bcc phase (i.e. disordered B2 phase, a ∼ 2.9 Å), were visible. At this stage the diffraction signals belonging to the quasicrystals were no longer observable, indicating a complete transformation of the quasicrystals into the bcc phase. Finally, the bcc phase formed during milling transformed back to the quasicrystalline phase during subsequent annealing treatment. The microhardness measured on the milled powders was found to decrease with increasing milling time, most likely as a consequence of the increased volume fraction of the ductile bcc phase. Attempts are made to rationalize the structural transformation.

Effect of Ti addition on the quasicrystalline phase formation and indentation characteristics of Zr69.5Al7.5Cu12Ni11 alloy

The crystallisation behaviour and indentation characteristics of melt-spun (Zr69.5Al7.5Cu12Ni11)100− x Ti x (x = 0–16 at. %) metallic glasses was investigated. Differential scanning calorimetry traces revealed changes in crystallisation behaviour with addition of Ti. Formation of a nanoquasicrystalline phase by annealing the glass was found up to x = 12. Further increase of Ti content gives rise to formation of a Zr2Ni-type crystalline phase. In addition to this, the size of nanoquasicrystals decreases with increasing addition of Ti. The load-dependent hardness behaviour of the metallic glasses and nanoquasicrystal–glass composites is reported in detail. The value of the yield strength of the materials was estimated with the help of hardness values based on the empirical correlations available in literature. The formation of shear bands around the indentation periphery was observed. It was found that microstructural and morphological changes alter the indentation characteristics of glasses as well as composites formed after controlled crystallisation.

Effects of ball milling and annealing of an alloy in the Al-Fe-Cu system: Implications for phase equilibria

An icosahedral quasicrystalline alloy in the Al-Fe-Cu system has been mechanically milled in a high-energy ball mill (Szegvari attritor) for 1, 3, 6 and 10 h. Samples were characterized by X-ray diffraction and transmission electron microscopy. The evolution of nanosize crystallites of the disordered B2 phase (bcc; a = 0.29 nm), coexisting with either the parent icosahedral phase or an amorphous phase, occurs during milling. Isothermal heat treatment of milled powder at various temperatures (200, 500, 600, 700, 800 and 850°C) leads in all cases, except at 200°C, to the transformation from disordered B2 and amorphous phases to an ordered B2 phase with a high degree of long-range ordering. The maximum degree of superlattice ordering was found after isothermal treatment at 800oC. The implications of these results are discussed with reference to phase equilibria existing between crystalline and quasicrystalline phases in the Al-Fe-Cu system.

NANOINDENTATION STUDIES OF METALLIC GLASSES AND NANOQUASICRYSTAL–GLASS COMPOSITES IN Zr–Al (Ga)–Cu–Ni ALLOYS

In this presentation, we report the indentation behavior of Zr69.5Al7.5−xGaxCu12Ni11 (x = 0 and 1.5 at.%) metallic glasses as well as nanoquasicrystal–glass composites. The minor alloying addition in Zr-based glassy alloys greatly influences their mechanical properties. The nanoindentation tests were carried out to study the load (P) versus displacement (h) curves for the determination of reduced modulus as well as their hardness behavior. The formation of shear bands around the indentation periphery has been investigated. The evidence of the pile up was observed. The P − h curve for as-synthesized melt-spun ribbons displayed the presence of displacement burst, which are known as pop-ins. It has been concluded that Ga substitution leads to the improvement of the mechanical behavior of Zr–Al (Ga)–Cu–Ni alloys.

Nanocrystallization and structural correlation in quasicrystalline and crystalline phases during mechanical milling

Abstract The main objective of the proposed work is to investigate the formation of nanoquasicrystalline and related nanocrystalline phases in Al—Cu—Fe system during mechanical milling at the initial stage of milling. The mechanical milling of a quasicrystalline Al65Cu20Fe15 alloy was performed in a high-energy ball mill (Szegvari attritor) by varying milling time from 2.5 min to 60 min under liquid hexane medium at the speed of 400 rpm with a ball to powder ratio of 40 : 1. X-ray diffraction was carried out for evaluating the lattice strain, lattice parameters and crystallite sizes of the milled sample. It was found that the evolution of monoclinic Al13Fe4 phase from Al65Cu20Fe15 quasicrystalline phase occurred after 2.5 min of milling, which was further confirmed by transmission electron microscopy. After 5 min of ball milling nano size disordered B2 phase (bcc, a = 0.29 nm) was also found. The systematic analysis of the XRD patterns for evolving lattice parameter, sequential strain suggests that the lattice strain during milling has been quite considerable.