Prospective Subunit Nanovaccine against Mycobacterium tuberculosis Infection─Cubosome Lipid Nanocarriers of Cord Factor, Trehalose 6,6' Dimycolate.

An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.

Self-assembled nanostructures of phosphomolybdate, nucleobase and metal ions synthesis and their in vitro cytotoxicity studies on cancer cell lines.

The ability of the multidentate nucleobases, adenine and thymine, to coordinate polyoxometalate and metal ions leading to the formation of self-assembled nanostructures and their strong cytotoxicity toward cancer cell lines have been demonstrated. A unique synthetic approach is developed to make a series of functional nanoscale hybrid materials consisting of nucleobases (adenine and thymine) and phosphomolybdic acid (PMA) through solid state chemical reaction and self-assembly process. Adenine was protonated through its ring nitrogen, while the ketone group in thymine was protonated during the addition of PMA to these nucleobases. The self-assembled nanostructures formed as a result of the electrostatic interaction between the protonated nucleobases and polyanionic PMA. To promote the base pairing between the nucleobases, chloroaurate ions and silver ions were added to each PMA/adenine and PMA/thymine nanostructures. The complexation between the nucleobases and the added metal ions was found to drive the formation of subsequent self-assembled nanostructures. All the materials were screened for their anticancer activity against breast (MDAMB-231) and prostate (PC-3) cancer cells, and non-cancerous keratinocyte cells HaCaT. PMA/adenine/[AuCl4]- and PMA/thymine/Ag+ nanostructures were found to have strong anti-cancer activity, while PMA/adenine/Ag+, PMA/thymine/[AuCl4]-, and PMA/pdenine, PMA/thymine nanostructures did not exhibit such activity. The unique redox properties of these materials and the self-assembly of the PMA and metal ions were the major factors responsible for the cytotoxicity. This unique approach of making functional nanomaterials incorporate the nucleobase, PMA and metal ions using solid state self-assembly and their anti-cancer applications are considered to be an effective approach for the development of inorganic nucleoside analogue bio-pharmaceutical agents.

Viability determination of Ascaris ova in raw wastewater: a comparative evaluation of culture-based, BacLight Live/Dead staining and PMA-qPCR methods.

Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.

Detection of helminth ova genera using in-situ biosynthesis of gold nanoparticles.

In this study, a presumptive colorimetric method was used to detect and differentiate the ova of two major soil transmitted helminths in wastewater, Ascaris and Trichuris. Gold nanoparticles were synthesised following the reduction of tetrachloroauric acid by the surface moiety of Ascaris suum, resulting in a colour change. In contrast there was no colour change with Trichuris suis indicating the absence of gold nanoparticle synthesis. Analysis of the ova using scanning electron microscopy (SEM) revealed that the synthesis of nanoparticles on the surface of ova was confirmed as gold nanoparticles (91 w/w %) by energy dispersive X-ray analysis (EDX). This study indicated that the surface moieties of helminth ova could be a potential target for ova detection and differentiation using the biosynthesis of nanoparticles by colorimetry methods. Three advantages: •Simple colorimetry based method requiring no sophisticated devices.•No trained personnel required.•Cost-effective and can be a potential candidate for biosensors.

Novel biocompatible poly(acrylamide)-grafted-dextran hydrogels: Synthesis, characterization and biomedical applications.

An electro-responsive PAAm-g-Dxt copolymer was synthesized and characterized by 1HNMR & FTIR spectroscopy, neutralization equivalent, elemental and thermogravimetric analysis to ascertain the grafting reaction. Further, we developed an electro-responsive transdermal drug delivery system (ETDS) utilizing PAAm-g-Dxt copolymer for rivastigmine tartarate delivery through skin. The ETDS were developed using drug-loaded PAAm-g-Dxt hydrogel as the reservoir, and cross-linked dextran-poly(vinyl alcohol) blend films as rate controlling membranes (RCM). In the absence of electrical stimuli, a small amount of drug was permeated from the ETDS, while in the presence of electrical stimuli, the drug permeability was increased. On application of electric stimulus, the flux was increased by 1.6 fold; drug permeability was enhanced when the strength of applied electric current was raised to 8 mA from 2 mA. The drug permeability characteristics studied under "on-off" stimuli suggested that there was faster drug permeation when electrical stimuli was 'on' and it decreased when electrical stimuli was 'off.' The histopathology study confirmed the altered skin structural integrity after application of electrical stimuli. Hence, the PAAm-g-Dxt based ETDS are useful for transdermal drug delivery triggered by an electric stimulus to deliver on-demand release of drug into systemic circulation.

Evaluation of plasmid DNA stability against ultrasonic shear stress and its in vitro delivery efficiency using ionic liquid [Bmim][PF6].

The hydrophobic ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF6] forms nanostructures with negatively charged plasmid DNA through electrostatic interactions. The formation of plasmid DNA/IL nanostructures was confirmed by measuring the zeta potential of plasmid DNA as well as plasmid DNA/IL nanostructures. The zeta potential of the nanostructures was positive, although plasmid DNA is negatively charged. The positive zeta potential is due to the complexation between plasmid DNA and positively charged ionic liquid [Bmim][PF6]. The ability of ionic liquid [Bmim][PF6] to protect plasmid DNA against ultrasonic shear stress was also investigated using an agarose gel electrophoretic assay and showed that ionic liquid stabilizes plasmid DNA against ultrasonication. The plasmid DNA and plasmid DNA/IL nanostructures were subjected to ultrasonic shear stress for different time periods and the biological functionality of pristine plasmid DNA (i.e., expression of the eGFP gene) as well as the self-assembled nanostructures was investigated in vitro using three different cell lines, COS7, HEK293 and HeLa. Ionic liquid [Bmim][PF6] protected the plasmid DNA against ultrasonic shear stress and also enhanced gene transfection efficiency in vitro. Furthermore, the cytotoxicity of ionic liquid [Bmim][PF6] was assayed in vitro using all three cell lines and the toxicity was very low. Therefore, the ionic liquid [Bmim][PF6] stabilizes plasmid DNA against ultrasonic shear stress and also enhances its in vitro delivery efficiency.

Microfluidic Synthesis of Nanoparticles and their Biosensing Applications.

We present an extensive overview of the evolution and progress made in the field of microstructures and nanostructures preparation using microfluidic techniques in recent times. A microfluidic system creates particles that are within a narrow range of shape and size distribution. It enables controlling the shape, size and composition of nanomaterials (NMs) for various applications. A brief evaluation of the advantages of both droplet-based and continuous flow synthesis of nanoparticles (NPs) is discussed in detail and compared with the traditional wet chemical batch synthesis approach. Due to increasing applications of biosensing, nanobiotechnology, nanomedicine and diagnostics devices, special attention should be paid to metal NPs developed through microfluidic routes.

Charge-switchable gold nanoparticles for enhanced enzymatic thermostability.

This study illustrates a facile strategy for efficient immobilization of enzymes on a metal nanoparticle surface. The strategy proposed here enables the enzymatic activity to be retained while increasing the enzyme thermostability. It is demonstrated that the use of a zwitterionic amino acid tyrosine as a reducing and capping agent to synthesise gold nanoparticles allows efficient immobilization of phytase enzyme through charge-switchable electrostatic interactions. The detailed kinetic and thermodynamic studies reveal that the proposed enzyme immobilization strategy improves the overall quality of phytase by reducing the activation energy required for substrate hydrolysis and broadening the temperature window in which immobilized enzyme is able to operate. The outcomes of this study indicate that the underlying zwitterionic nature of 20 natural amino acids along with significant variability in their isoelectric points and hydropathy indices as well the ability of some of the amino acids to reduce metal ions is likely to offer significant opportunities for tailoring nano-bio interfaces in a rational manner for a range of biological applications.

Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF6]: Enhanced Efficiency in Bacterial Gene Transformation.

The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 µL of IL ([Bmim][PF6]) and 1 µg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

Facile approach for the dispersion of regenerated cellulose in aqueous system in the form of nanoparticles.

This study reports a facile method to disperse cellulose in deionized water, wherein a critical condition of regenerated cellulose is discovered, where it completely disperses up to a maximum of 5 g L(-1) concentration in deionized water with the help of ultrasonication. The dispersed cellulose is characterized by TEM and DLS, the latter among which shows 200 nm hydrodynamic radii of cellulose nanoparticles dispersed in deionized water. FTIR analysis of dispersed cellulose reveals that dispersed cellulose losses its crystallinity during regeneration and dispersion step employed in this study. The dispersed cellulose reported in this study is able to form free-standing, transparent films, which were characterized by SEM, XRD, TGA, EDX, and FTIR spectroscopy and show resistance against dissolution in water. Additionally, the dispersed cellulose is able to undergo at least three times faster enzymatic hydrolysis in comparison to pristine microcrystalline cellulose under similar reaction conditions. The dispersed cellulose reported here could be a better material for reinforcement, preparation of hydrogels, and drug delivery applications under physiological environment.

Self-assembled histidine acid phosphatase nanocapsules in ionic liquid [BMIM][BF4] as functional templates for hollow metal nanoparticles.

We report the biomacromolecular self-assembly of histidine acid phosphatase (HAP), an enzyme of significant biomedical and industrial importance, in the ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF(4)]). The spontaneous self-assembly of HAP enzyme in [BMIM][BF(4)] results in the formation of HAP nanocapsules. The HAP enzyme molecules were found to retain their enzymatic activity after the self-assembly process, which enabled us to utilize self-assembled HAP capsules as self-catalyzing templates for the synthesis of a range of hollow metal nanoparticles (Au, Ag, Pd, and Ni) without employing any additional reducing agent. The hollow metal nanospheres with HAP encapsulated within their cavity were found to retain enzymatic activity for at least up to four cycles, as demonstrated in the case of Au-coated HAP capsules as the model system.

Cationic amino acids specific biomimetic silicification in ionic liquid: a quest to understand the formation of 3-D structures in diatoms.

The intricate, hierarchical, highly reproducible, and exquisite biosilica structures formed by diatoms have generated great interest to understand biosilicification processes in nature. This curiosity is driven by the quest of researchers to understand nature's complexity, which might enable reproducing these elegant natural diatomaceous structures in our laboratories via biomimetics, which is currently beyond the capabilities of material scientists. To this end, significant understanding of the biomolecules involved in biosilicification has been gained, wherein cationic peptides and proteins are found to play a key role in the formation of these exquisite structures. Although biochemical factors responsible for silica formation in diatoms have been studied for decades, the challenge to mimic biosilica structures similar to those synthesized by diatoms in their natural habitats has not hitherto been successful. This has led to an increasingly interesting debate that physico-chemical environment surrounding diatoms might play an additional critical role towards the control of diatom morphologies. The current study demonstrates this proof of concept by using cationic amino acids as catalyst/template/scaffold towards attaining diatom-like silica morphologies under biomimetic conditions in ionic liquids.

Self-assembled enzyme capsules in ionic liquid [BMIM][BF4] as templating nanoreactors for hollow silica nanocontainers.

Most of the self-assembly studies have hitherto explored the aqueous media as fluid phase for self-assembly of amphiphilic biomacromolecules, wherein architectural modification of biomolecules is generally a prerequisite for self-assembly of modified biomolecules. We demonstrate for the first time that ionic liquids can act as nonaqueous designer solvents to self-assemble amphiphilic biomacromolecules without requiring their prior modification. To this end, we show that enzyme (phytase) molecules self-assembled in the presence of an appropriate ionic liquid, resulting in the formation of enzyme capsules. Phytase capsules synthesized using this approach were further used as templating nanoreactors for the synthesis of enzyme-containing hollow silica nanocontainers. In situ immobilized phytase enzyme in the silica nanocontainers, when subjected to enzyme-reusability application, establishes them as excellent reusable biocatalysts.