Occupational safety assessment of biogenic urea nanofertilisers using in vitro pulmonary, and in vivo ocular models.

Nanomaterials (NMs) are now gaining popularity to be used in agriculture as fertilisers to reduce the dose of conventional fertilisers and enhance nutrient use efficiency. Urea has found its application as a conventional nitrogenous fertiliser since long, however, the nutrient use efficiency of the bulk form of urea is low due to issues related to ammonia volatilisation. This study proposes a biogenic synthesis route to develop urea nanoparticles that can be used as nano-fertiliser for better uptake and hence improved nutrient efficiency. Large scale production and widespread application of these nano-fertilisers to the agricultural fields will enhance the direct exposure to workers and farmers. Therefore, the occupational safety evaluation becomes critical. In this study, we report a new method for synthesis of urea nanoparticles (TNU, absolute size: 12.14 ± 7.79 nm) followed by nano-safety evaluation. Herein, the pulmonary and ocular compatibilities of TNU were investigated in vitro and in vivo respectively. The assay for cellular mitochondrial activity was carried out on human lung fibroblasts (WI-38) under varied TNU exposure concentrations up to 72 h. The acute biocompatibility effect, ocular irritation and sub-lethal effects were measured on New Zealand Rabbit. The results show that TNU do not exhibit any cytotoxicity and detrimental cell mitochondrial activity up to the highest tested concentration of 1000 µg/mL and 72 h of testing. The animal experiment results also show that neither acute nor sub-lethal toxic effects can be detected after TNU ocular instillation up to 21 days when tested up to environmentally relevant concentration of 15 µg/mL. These results suggest the occupational safety of biogenic urea nanoparticles and support its application as nanofertiliser.

Fertilizing benefits of biogenic phosphorous nanonutrients on Solanum lycopersicum in soils with variable pH.

Nanoformulations of Phosphorous (P) have recently been proposed as alternatives to P fertilizers. In this study, the fertilizing efficacies of P-based nanomaterials (NMs), nanohydroxyapatite (nHAP) and nanophosphorus (nP), were examined on Solanum lycopersicum (Pusa Rohini, Indian tomato) in growth room pot experiments. These NMs differed in their mode of synthesis, chemical composition, size and shape. Rock-phosphate (RP), phosphoric acid (PA) and di-ammonium phosphate (DAP) were included as bulk materials for comparison. Three varieties of artificial soils were included in the study, neutral (pH 7.2), acidic (pH 4.3) and basic (pH 9.8). The effects of the NMs on germination, plant growth, and P content were assessed at the 15th and 30th days after treatment. The results showed that P-based NMs enhance the overall germination and plant growth by increasing P levels in all types of soils for the tomato plants in comparison to the bulk P sources. Analysis using X-ray fluorescence revealed enhanced P content in the plants indicating the uptake of P-based NMs. Evaluation of H2O2, total phenolics and total flavonoids contents after NM treatment suggest that there is no stress caused due to the application of NMs to the plant. The results of this study indicate the beneficial role of P-based NMs as fertilizers at the early stages of plant development, which opens a scope for further investigation of underlying metabolic and molecular pathways and field trials.

Abiotic factors and aging alter the physicochemical characteristics and toxicity of Phosphorus nanomaterials to zebrafish embryos.

Nanoscale phosphorus (P)-based formulations are being investigated as potentially new fertilizers to overcome the challenges of conventional bulk P fertilizers in agriculture, including low efficacy rates and high application levels. After agricultural applications, the NMs may be released into aquatic environments and transform over time (by aging) or in the presence of abiotic factors such as natural organic matter or sunlight exposure. It is, therefore, important to investigate the physicochemical changes of NMs in environmentally realistic conditions and assess their potential acute and sublethal toxic effects on aquatic organisms. To investigate this, two separate studies were conducted: 1. the effects of 3-months aged P-based NMs on zebrafish embryos, and 2. the influence of humic acid (HA), UV exposure, or a combination of both on P-based NM toxicity in zebrafish embryos. Four different types of nanohydroxyapatites (nHAPs) and a nanophosphorus (nP) were included in the study. These NMs differed in their physicochemical properties, most prominently their shape and size. Environmental transformations were observed for P-based NMs due to aging or interaction with abiotic factors. The aging of the NMs increased the hydrodynamic diameter (HDD) of rod- and needle-shaped NMs and decreased the size of the platelet and spherical NMs, whereas interactions with HA and UV decreased the NMs' HDD. It was observed that no LC50 (survival) and IC50 (hatch and heart rates) were obtained when the zebrafish embryos were exposed to the aged NMs or when NMs were added in the presence of HA and UV. Overall, these results suggest that P-based NMs cause no acute toxicity and minimal sub-lethal toxicity to zebrafish embryos in environmentally realistic experimental conditions.

Investigation into the trophic transfer and acute toxicity of phosphorus-based nano-agromaterials in Caenorhabditis elegans.

Biogenic phosphorus (P) based - nanomaterials (NMs) are currently being explored as nanofertilizers. In this study, the acute toxic effects and trophic transfer of multiple types of P-based NMs were examined on soil-dwelling nematode, Caenorhabditis elegans. The study involved four variants of nanohydroxyapatites (nHAPs) synthesized either via a biogenic or a chemical route and another NM, nanophosphorus (nP), biosynthesized from bulk rock phosphate (RP). The pristine NMs differed in their physicochemical properties with each possessing different shapes (biogenic nHAP: platelet-shaped, ˜35 nm; biogenic nP, ˜5-10 nm: dots; chemically synthesized nHAPs: spherical, ˜33 nm, rod, ˜80 nm and needle-shaped, ˜64 nm). The toxic effects of NMs' in C. elegans were assessed using survival, hatching and reproductive cycle as the key endpoints in comparison to bulk controls, calcium phosphate and RP. The interactions and potential uptake of fluorescent-tagged nHAP to E. coli OP50 and C. elegans were investigated using confocal microscopy. The transformation of NMs within the nematode gut was also explored using dynamic light scattering and electron microscopy. C. elegans exposed to all of the variants of nHAP and the nP had 88-100% survival and 82-100% hatch rates and insignificant effects on brood size as observed at the tested environmentally relevant concentrations ranging from 5 to 100 µg.mL-1. Confocal microscopy confirmed the interaction and binding of fluorescent-tagged nHAP with the surface of E. coli OP50 and their trophic transfer and internalization into C. elegans. Interestingly, there was only a small reduction in the hydrodynamic diameter of the nHAP after their uptake into C. elegans and the transformed NMs did not induce any additional toxicity as evident by healthy brood sizes after 72 h. This study provides key information about the environmental safety of agriculturally relevant P-based NMs on non-target species.

In Vitro Assessment of Core-Shell Micellar Nanostructures of Amphiphilic Cationic Polymer-Peptide Conjugates as Efficient Gene and Drug Carriers.

Design and development of biocompatible, biodegradable and stable dual delivery systems for drug and gene is the need of the hour. Here, we have designed a strategy to develop carrier systems consisting of above mentioned properties by (a) incorporating an unnatural amino acid in the peptide backbone, and b) conjugating a low molecular weight cationic polymer (polyethylenimine, PEI) for incorporating cationic charge. Using this strategy, we have synthesized a small series of Boc-FΔF-AH-polyethylenimine conjugates by varying the concentration of Boc-FΔF-aminohexanoic acid, viz., PP-1, PP-2 and PP-3. These conjugates self-assembled in aqueous medium to form micelles in the size range of ~144-205 nm with zeta potential ~ +7.9-14.2 mV bearing core-shell type of conformation. Positive surface of the micelles facilitated the binding of plasmid DNA as well as transportation inside the cells. The hydrophobic core of the nanostructures helped in the encapsulation of the hydrophobic drug molecule, which was then got released in a controlled manner. DNA complexes of the conjugates were not only found non-toxic but also exhibited higher transfection efficacy than the native polymer and Lipofectamine. Altogether, these nanostructures are capable of delivering a drug and a gene simultaneously in vitro and could be used as next-generation delivery agents.

Polydopamine -aminoglycoside nanoconjugates: Synthesis, characterization, antimicrobial evaluation and cytocompatibility.

Development of nanoparticle- and self-assembled nanomaterial-based therapeutics has become a rapidly growing area in the field of nanotechnology. One of the natural compounds, dopamine, presents as a neurotransmitter in the human brain serving as a messenger and deals with the behavioural responses, has provided an ideal platform through self-polymerization under aerobic conditions leading to the formation of a beneficial organic biopolymer, polydopamine (PDA). This polymer provides sufficient reactive functionalities, which can further be use to attach amine- or thiol-containing ligands to obtain conjugates. In the present study, self-polymerized polydopamine nanoparticles have been synthesized and tethered to aminoglycosides (AGs: Gentamicin, Kanamycin and Neomycin) through amino moieties to obtain PDA-AG nanoconjugates. These nanoconjugates are characterized by physicochemical techniques and evaluated for their antimicrobial potency against various bacterial strains including resistant ones. Simultaneously, cytocompatibility was also assessed for PDA-AG nanoconjugates. Of these three nanoconjugates (PDA-Gentamicin, PDA-Kanamycin and PDA-Neomycin), PDA-Kanamycin (PDA-K) nanoconjugate exhibited the highest activity against potent pathogens, least toxicity in human embryonic kidney (HEK 293) cells and intense toxic effects on human glioblastoma (U87) cells. Together, these results advocate the promising potential of these nanoconjugates to be used as potent antimicrobials in future applications.

Iron chelator Deferoxamine protects human neuroblastoma cell line SH-SY5Y from 6-Hydroxydopamine-induced apoptosis and autophagy dysfunction.

BACKGROUND: Intracellular iron involves in Fenton's reaction-mediated Hydroxyl radical (OH·) generation by reacting with the neurotoxic agent 6-Hydroxydopamine (6-OHDA) autoxidation derivative Hydrogen Peroxide (H2O2). Several studies have been conducted so far on the neuroprotective activities of the iron chelator Deferoxamine (DFO) but little or no clear evidence about the underlying cellular mechanism is available. METHODS: The present study was conducted on Human neuroblastoma cell line SH-SY5Y in the absence or presence of 6-OHDA or H2O2 and / or DFO. Following incubation, cell viability assay, intracellular reactive oxygen species (ROS) determination, flow cytometric quantification of apoptotic cells followed by nuclear staining, intracellular tracking of transfected fusion construct of microtubule-associated protein 1B-light chain with Green fluorescent protein - Red fluorescent protein (LC3B-GFP-RFP reporters) and immunocytochemistry of intracellular Cathepsin protein by confocal microscopy, were conducted. In addition, western blotting was carried out to detect expressions of apoptotic and autophagy related proteins. RESULTS: This study confirmed the neuroprotective potential of DFO by inhibiting 6-OHDA-mediated cell death and ROS generation. Reduced percentage of apoptotic cells and appearance of altered nuclei architecture followed by a reduced expression of cleaved PARP (Poly-ADP-ribose Polymerase) and cleaved Caspase-3 were observed upon DFO treatment against 6-OHDA, and as well as against H2O2 in SH-SY5Y cell lines. Besides, DFO induced the intracellular autophagolysosome formation (red puncta) rather than autophagosome (yellow puncta) only. Thereafter it was observed that DFO restored the expression of intracellular lysosomal protease Cathepsin and reduced the expression of the LC3-II. CONCLUSION: Taken together, this study clearly demonstrated that the anti-Fenton activity of DFO inhibited apoptosis and caused blockade in ALP or autophagy dysfunction in SH-SY5Y cell lines. These outcomes further suggest that DFO provides neuroprotection by inhibiting apoptosis and inducing the progression of Autophagy- lysosomal pathway (ALP).

A new method for biological synthesis of agriculturally relevant nanohydroxyapatite with elucidated effects on soil bacteria.

The study describes a novel and environment friendly route of biosynthesis of nanohydroxyapatite (nHAP). Bacillus licheniformis mediated synthesis of nHAP has been carried out with different phosphate concentrations (2%, 5%, 10% and 20% w/v) of potassium dihydrogen orthophosphate monobasic (K2HPO4). The synthesis is supported by a two-step mechanism - (i) solubilization of P by organic acids extracellularly secreted by the bacterial strain and (ii) gelation of P and Ca. The nHAP particles were characterized using electron microscopy and XRD analysis. Powdered crystalline particles with a size range of 30 ± 5 nm were obtained with shape and size dependent on phosphate concentrations. The particles showed no adverse effect on plant growth-promoting bacteria. Evaluation of nHAP prepared by this route with 2% P source provides scope for a wide range of applications, especially as a nano-fertilizer.

Role of Endocrine-Disrupting Engineered Nanomaterials in the Pathogenesis of Type 2 Diabetes Mellitus.

Nanotechnology has enabled the development of innovative technologies and products for several industrial sectors. Their unique physicochemical and size-dependent properties make the engineered nanomaterials (ENMs) superior for devising solutions for various research and development sectors, which are otherwise unachievable by their bulk forms. However, the remarkable advantages mediated by ENMs and their applications have also raised concerns regarding their possible toxicological impacts on human health. The actual issue stems from the absence of systematic data on ENM exposure-mediated health hazards. In this direction, a comprehensive exploration on the health-related consequences, especially with respect to endocrine disruption-related metabolic disorders, is largely lacking. The reasons for the rapid increase in diabetes and obesity in the modern world remain largely unclear, and epidemiological studies indicate that the increased presence of endocrine disrupting chemicals (EDCs) in the environment may influence the incidence of metabolic diseases. Functional similarities, such as mimicking natural hormonal actions, have been observed between the endocrine-disrupting chemicals (EDCs) and ENMs, which supports the view that different types of NMs may be capable of altering the physiological activity of the endocrine system. Disruption of the endocrine system leads to hormonal imbalance, which may influence the development and pathogenesis of metabolic disorders, particularly type 2 diabetes mellitus (T2DM). Evidence from many in vitro, in vivo and epidemiological studies, suggests that ENMs generally exert deleterious effects on the molecular/hormonal pathways and the organ systems involved in the pathogenesis of T2DM. However, the available data from several such studies are not congruent, especially because of discrepancies in study design, and therefore need to be carefully examined before drawing meaningful inferences. In this review, we discuss the outcomes of ENM exposure in correlation with the development of T2DM. In particular, the review focuses on the following sub-topics: (1) an overview of the sources of human exposure to NMs, (2) systems involved in the uptake of ENMs into human body, (3) endocrine disrupting engineered nanomaterials (EDENMs) and mechanisms underlying the pathogenesis of T2DM, (4) evidence of the role of EDENMs in the pathogenesis of T2DM from in vitro, in vivo and epidemiological studies, and (5) conclusions and perspectives.

Enzyme sensitive smart inulin-dehydropeptide conjugate self-assembles into nanostructures useful for targeted delivery of ornidazole.

Molecular self-assembly of biodegradable amphiphilic polymers allows rational design of biocompatible nanomaterials for drug delivery. Use of substituted polysaccharides for such applications offers the ease of design and synthesis, and provides higher biofunctionality and biocompatibility to nanomaterials. The present work focuses on the synthesis, characterization and potential biomedical applications of self-assembled polysaccharide-based materials. We demonstrated that the synthesized amphiphilic inulin self-assembled in aqueous medium into nanostructures with average size in the range of 146-486nm and encapsulated hydrophobic therapeutic molecule, ornidazole. Hydrophophic dehydropeptide was conjugated with inulin via a biocompatible ester linkage. Dehydrophenylalanine, an unusual amino acid, was incorporated in the peptide to make it stable at a broader range of pH as well as against proteases. The resulting core-shell type of nanostructures could encapsulate ornidazole in the hydrophobic core and released it in a controlled fashion. By taking the advantage of inulin, which gets degraded in the colon by colonic bacteria, the effect of enzyme, inulinase, present in the microflora of the large intestine, on inulin-peptide degradation followed by drug release has been studied. Altogether, small peptide conjugated to inulin offers novel scaffold for the future design of nanostructures with potential applications in the field of targeted drug delivery.

Mussel-inspired polydopamine-polyethylenimine conjugated nanoparticles as efficient gene delivery vectors for mammalian cells.

Efficient delivery of DNA to cells is the primary concern to address the objective of gene therapy. Many attempts have been made to develop polymeric carriers for gene delivery. To have an efficient carrier, it is vital to understand the properties of the vector for better stability, transfection efficiency and minimal toxicity. Branched polyethylenimine (bPEI) has been considered as the 'gold standard' for gene delivery but suffers a major drawback of exhibiting high cytotoxicity. Here, we have attempted to develop a mussel-derived polymer, polydopamine (PDA), conjugated polyethylenimine nanoparticles in such a way that the toxic nature of bPEI is suppressed by the conversion of free primary amine groups to secondary and tertiary amines. Keeping the amount of PDA fixed, varying amounts of bPEIs of different molecular weights (25, 10 and 1.8kDa) were conjugated via Michael addition and/or Schiff base. A trend in hydrodynamic size of the conjugated nanoparticles was observed in the range from 160 to 300nm and zeta potential from +12-30mV in the projected three series, viz., (i) PDA1-25bPEI0.5, PDA1-25bPEI1, PDA1-25bPEI2; (ii) PDA1-10bPEI0.5, PDA1-10bPEI1, PDA1-10bPEI2; and (iii) PDA1-1.8bPEI0.5, PDA1-1.8bPEI1, PDA1-1.8bPEI2. A visible trend in the DNA condensation ability and buffering capacity was also noticed. Further, cell cytotoxicity assays revealed that pDNA complexes of PDA-bPEI nanoparticles were non-toxic to mammalian cells and these complexes exhibited several folds higher transfection efficiency than the complexes of native bPEIs as demonstrated by fluorescence measurements and flow cytometry. Altogether, the results advocate the promising potential of these conjugated nanoparticles for future in vivo applications.