Nanodiagnostics in leishmaniasis: A new frontiers for early elimination.

Visceral leishmaniasis (VL) is still a major public health concern in developing countries having the highest outbreak and mortality potential. While the treatment of VL has greatly improved in recent times, the current diagnostic tools are limited for use in the post-elimination setting. Although conventional serological methods of detection are rapid, they can only differentiate between active disease in strict combination with clinical criteria, and thus are not sufficient enough to diagnose relapse patients. Therefore, there is a dire need for a portable, authentic, and reliable assay that does not require large space, specialized instrument facilities, or highly trained laboratory personnel and can be carried out in primary health care settings. Advances in the nanodiagnostic approaches have led to the expansion of new frontiers in the concerned area. The nanosized particles are blessed with an ability to interact one-on-one with the biomolecules because of their unique optical and physicochemical properties and high surface area to volume ratio. Biomolecular detection systems based on nanoparticles (NPs) are cost-effective, rapid, nongel, non-PCR, and nonculture based that provide fast, one-step, and reliable results with acceptable sensitivity and specificity. In this review, we discuss different NPs that are being used for the identification of molecular markers and other biomarkers, such as toxins and antigens associated with leishmaniasis. The most promising diagnostic approaches have been included in the article, and the ability of biomolecular recognition, advantages, and disadvantages have been discussed in detail to showcase the enormous potential of nanodiagnostics in human and veterinary medicine. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Diagnostic Tools > Biosensing.

Evaluation of Safety and Antileishmanial Efficacy of Amine Functionalized Carbon-Based Composite Nanoparticle Appended With Amphotericin B: An in vitro and Preclinical Study.

Visceral leishmaniasis (VL) has been a major health concern in the developing world, primarily affecting impoverished people. It is caused by a protozoan parasite Leishmania donovani and is characterized by immune dysfunction that can lead to deadly secondary infections. Several adverse side effects limit the existing treatment options; hence, the need of the hour is some drug option with high efficacy and no toxicity. To make targeted delivery of Amphotericin B (AmB), we have used amine-functionalized versions of carbon nanostructures, namely f-CNT and f-Graphene (f-Grap). The results with f-Grap-AmB, because of a much larger surface area, were expected to be better. However, the results obtained by us showed only marginal improvement (IC50 f-Grap-AmB; 0.0038 ± 0.00119 µg/mL). This is, in all likelihood, due to the agglomeration effect of f-Grap-AmB, which is invariably obtained with graphene. To resolve this issue, we have synthesized a graphene-CNT composite (graphene 70% and CNT 30% by weight). Because CNT is dispersed in between graphene sheets, the agglomeration effect is avoided, and our study suggests that the f-Composite-AmB (f-Comp-AmB) showed no toxicity against the murine J774A.1 macrophage cell line and did not induce any hepatic or renal toxicity in Swiss albino mice. The f-Comp-AmB also showed a remarkable elevation in the in vitro and in vivo antileishmanial efficacy in comparison to AmB and f-CNT-AmB or f-Grap-AmB in J774A.1 and Golden Syrian hamsters, respectively. Additionally, we have also observed that the percentage suppression of parasite replication in the spleen of the hamster was significantly higher in the f-Comp-AmB (97.79 ± 0.2375) treated group in comparison with the AmB (85.66 ± 1.164) treated group of hamsters. To conclude, f-Comp-AmB could be a safe and reliable therapeutic option over the other carbon-based nanoparticles (NPs), i.e., f-CNT-AmB, f-Grap-AmB, and conventional AmB.

Envisioning the innovations in nanomedicine to combat visceral leishmaniasis: for future theranostic application.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease affecting impoverished people of the developing world; and much effort has been spent on the early case detection and treatment. However, current diagnostics and treatment options are not sufficient for appropriate surveillance in VL elimination setting. Hence, there is a dire need to develop highly sensitive diagnostics and less toxic effective treatments for proper management of cases and to achieve the sustained disease elimination. Although, promising results have been observed with nanomedicines in leishmaniasis; there are great challenges ahead especially in translating this to clinical setting. This review provides updated progress of nanomedicines in VL, and discussed how these innovations and future directions play vital role in achieving VL elimination.

α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries.

α-Amylase is imperative for starch and its deriviatized industries. Functionalized graphene sheets were tailored and optimized as scaffold for α-amylase immobilization using Response Surface Methodology based on Box-Behnken design, with an overall immobilization efficiency of 85.16%. Analysis of variance provided adequacy to the mathematical model for further studies. Native and immobilized functionalized graphene were characterized using transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Wheat α-amylase conjugated with functionalized graphene sheets were visually evident on transmission and scanning micrographs while the FTIR spectra showed interplay of various chemical interactions and bonding, during and after immobilization. Optimum pH and optimum temperature for immobilized enzyme though remained unchanged but showed broader range whereas Km showed a slight decrease (1.32 mg/mL). It also showed enhanced thermal and storage stability and retained 73% residual activity after 10 uses. These ensemble of properties and non-toxic nature of functionalized graphene, makes it viable to be absorbed commercially in starch processing industries.

Functionalized graphene sheets as immobilization matrix for Fenugreek ß-amylase: enzyme kinetics and stability studies.

ß-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek ß-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries.

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

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.

Cicer α-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications.

Cicer α-galactosidase was immobilized onto functionalized graphene with immobilization efficiency of 84% using response surface methodology (Box-Behnken design). The immobilized enzyme had higher thermal stability than the soluble one, attractive for industrial applications. Immobilization of the enzyme lowered the Km to 1/3rd compared to the soluble enzyme. Raffinose family oligosaccharides (RFOs) are mainly responsible for flatulence by taking soybean derived food products. The immobilized enzyme can be used effectively for the hydrolysis of RFOs. After ten successive runs, the immobilized enzyme still retained approximately 60% activity, with soybean RFOs. The easy availability of enzyme source, ease of its immobilization on matrices, non-toxicity, increased stability of immobilized enzyme and effective hydrolysis of RFOs increase the Cicer α-galactosidase application in food processing industries.

Immobilization of ß-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications.

BACKGROUND: ß-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction. METHODOLOGY/PRINCIPAL FINDINGS: Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E(a)) remains unchanged whereas optimum pH and K(m) were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene. SIGNIFICANCE: Immobilized ß-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.

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.

Response surface analysis of nano-ureases from Canavalia ensiformis and Cajanus cajan.

Ureases isolated from leguminous sources, Canavalia ensiformis and Cajanus cajan were immobilized onto gold nanoparticles (nano-ureases). Optimization of the urease immobilization was carried using response surface methodology based on Central Composite Design. Immobilization efficiency of nano-urease from C. ensiformis and C. cajan were found to be 215.10% and 255.92%, respectively. The methodology adopted has deviation of 2.56% and 3.01% with respect to experimental values in case of C. ensiformis and C. cajan, respectively. Nano-urease from C. cajan has broad physico-chemical parameters with pH optimum from 7.1 to 7.3 and temperature optimum from 50 to 70°C. Nano-urease from C. ensiformis has sharp pH and temperature optima at 7.3 and 70°C, respectively. Fourier transform infra-red spectroscopy has revealed involvement of groups viz. amino, glycosyl moiety, etc. in urease immobilization onto gold nano-particles. Transmission and scanning electron micrographs revealed that arrangement of urease onto gold nano-particles from C. ensiformis was uniform while it was localized in case of C. cajan. Nano-urease from C. ensiformis has higher specificity and catalysis toward urea as compared to nano-urease from C. cajan. Nano-ureases from both sources are equally stable for 6 months under dried conditions and can be used for 10 washes.

Scalable synthesis of aligned carbon nanotubes bundles using green natural precursor: neem oil.

Practical application of aligned carbon nanotubes (ACNTs) would have to be determined by a matter of its economical and large-scale preparation. In this study, neem oil (also named Margoaa oil, extracted from the seeds of the neem--Azadirachta indica) was used as carbon source to fabricate the bundles of ACNTs. ACNTs have been synthesized by spray pyrolysis of neem oil and ferrocene mixture at 825°C. The major components of neem oil are hydrocarbon with less amount of oxygen, which provided the precursor species in spray pyrolysis growth of CNTs. The bundles of ACNTs have been grown directly inside the quartz tube. The as-grown ACNTs have been characterized through Raman spectroscopy, scanning and transmission electron microscopic (SEM/TEM) techniques. SEM images reveal that the bundles of ACNTs are densely packed and are of several microns in length. High-resolution TEM analysis reveals these nanotubes to be multi-walled CNTs. These multi-walled CNTs were found to have inner diameter between 15 and 30 nm. It was found that present technique gives high yield with high density of bundles of ACNTs.

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.

Synthesis and optical properties of different CuO (ellipsoid, ribbon and sheet like) nanostructures.

We report the Synthesis of different CuO (ellipsoid, ribbon and sheet like) nanostructures in a solution phase with high yield at low cost bysimple reduction of aqueous solution of copper nitrate with alkaline solution of sodium hydroxide (NaOH). The morphology of the synthesized nanostructures is significantly influenced by the feedingconcentration of alkaline NaOH solution. Cu(OH)2 nanomaterials can be readily obtained by the reduction of Cu(NO3)2 solution with NaOH solution and these synthesized materials obtained atdifferent molar concentration of NaOH solution get transformed into different nanostructures ofCuO by subsequent heat treatment at 80 degrees C for half an hour. Nanoellipsoid, nanoribbon and nanosheet like structures were obtained after heating the copper nitrate solution reduced with 0.25 M, 0.50 M, 0.75 M and 1 M concentrated NaOH solution respectively. Optical absorption spectra and corresponding band gap calculation showed that these nanomaterials have higher band gap than their bulk materials.

Synthesis and characterization of different metal oxide nanostructures by simple electrolysis based oxidation of metals.

We report the Synthesis of different metal oxide (Cu2O, SnO2, Fe3O4 and PbO2) nanostructures by simple electrolysis based oxidation of metals (Cu, Sn, Fe and Pb). We have utilized the two electrode set up for the electrolysis and used different metal electrodes as anode and platinum as cathode. The synthesized nanomaterials were delaminated in the electrolyte. The microstructural characterization of synthesized materials in electrolytes after electrolysis at different electrode potentials revealed that the nanostructures strongly depend on the applied voltage between the electrodes. Various nanostructures (nanothreads, nanowires, nanocubes, nanotetrapods and hexagons-like) of metal oxides have been synthesized by this method. In case of copper electrode we have found nanothreads and nanowires of cuprous oxide. Tin electrode resulted nanothreads, nanotetrapod and nanocube like structures of tin oxide. Iron electrode resulted, nanowire like structures of iron oxide and lead sheet transformed into hexagon like and six petals like structures of lead oxide.

Lactose nano-probe optimized using response surface methodology.

A lactose nano-probe has been developed by immobilization of PsBGAL onto gold nanoparticles (AuNps). It is helpful for severe lactose intolerants for quality check of lactose hydrolyzed milk and estimation of hidden lactose present in variety of food products. Optimization of PsBGAL immobilization onto AuNps using spacer arm (cysteamine-glutaraldehyde) was carried out by response surface methodology (Box-Behnken design). The process has led to immobilization of enzyme onto AuNps with an efficiency of 140.81%. AuNp-PsBGAL was characterized using transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized enzyme showed broad temperature and pH optima and a significant enhancement in catalytic efficiency (V(max)/K(m)) with respect to soluble PsBGAL. AuNp-PsBGAL was stable under dried conditions than wet conditions for 6 months with loss of 10.2% and 87.53%, respectively. It has reusability of over five batchwise uses, with almost no loss in activity. Hill's coefficient was found to be 1.71 corresponding to lactose concentration ranging from 0.1% to 2.0%.

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.

Synthesis of copper nanoparticles by electrolysis of DNA utilizing copper as sacrificial anode.

Copper nanoparticles have been synthesized by anodic oxidation through a simple electrolysis process employing de-oxy ribonucleic acid (DNA) as electrolyte. Platinum was taken as cathode and copper as anode. The applied voltage was 4 V and the electrolysis was performed for duration of 1 h. The copper nanoparticles were prepared in situ from the electron beam irradiation on residues of electrolyte consisting of DNA and copper particles: DNA (Cu) complexes. The size of the nanoparticles ranges between 5-50 nm. A tentative explanation has been given for the formation of copper nanoparticles.