Krishnendu Das

Graphene oxide in cetyltrimethylammonium bromide (CTAB) reverse micelle: A befitting soft nanocomposite for improving efficiency of surface-active enzymes

Herein, we report the successful inclusion of 2D allotrope of carbon, graphene oxide (GO) in cetyltrimethylammonium bromide (CTAB)/isooctane/n-hexanol/water reverse micelle without compromising the stability of water-in-oil (w/o) microemulsion. This newly developed self-assembled nanocomposites act as proficient host for surface-active enzymes, lipase, horseradish peroxidase (HRP), and soybean peroxidase (SBP). Lipase activity within GO-doped CTAB reverse micelles remarkably improved by 3.8-fold compared to that was observed in only CTAB reverse micelle (second-order rate constant, k2=433±7 cm3 g(-1) s(-1)). In case of GO-doped CTAB reverse micelle, the observed enzyme activity (k2=1653±11 cm3 g(-1) s(-1)) is till date the highest ever activity of lipase in CTAB w/o microemulsions. In case of HRP and SBP, the catalytic efficiency maximally increased up to 2.6-fold and 2.3-fold, respectively. Electrostatic attraction between cationic head group of CTAB and anionic surface of GO as well as intrinsic amphiphilic character of GO possibly resulted in the confinement of this 2D nanosheet at the interface of reverse micelles. Integration of GO at the interface augmented the interfacial space in vicinity of surface-active enzyme. This enlarged interface might have accommodated higher amount of substrate and lipase with flexibility in its conformation resulting in marked improvement in the enzyme activity. Interfacial localization of GO was established by fluorescence spectroscopy. In addition, change in secondary structure of lipase in presence of 2D carbon allotrope was substantiated by circular dichroism spectroscopy.

Amino acid functionalized blue and phosphorous-doped green fluorescent carbon dots as bioimaging probe

Amino acid functionalized carbon dots (CDs) were synthesized in a simple and cost effective bottom up approach. Citric acid was used as the source of the carbon core and three amino acids L-isoleucine, L-valine and glycine were used for the surface fabrication of CDs to produce CDiso, CDval and CDgly, respectively. Interestingly these CDs were found to fluoresce with a blue emission. Doping of phosphorus to these CDs (PCDs) tuned the photoemission properties and produced green emitting PCDs. The doping of phosphorous (P) to these CDs improved their fluorescence intensity as well as quantum yields. Both doped and non-doped CDs were characterized by spectroscopic and microscopic techniques. These highly stable CDs were biocompatible in nature and did not exhibit any photobleaching property over a long span of time even under UV exposure. Subsequently, these CDs were exploited as an excellent bioimaging probe. Importantly CDs and PCDs illuminated cells in two completely different spectral regions blue and green, respectively in accordance with their fluorescence spectral behaviour. Hence, amino acid functionalized carbon dots based bioimaging probes with different fluorescence characteristics were developed that are widely applicable for cellular imaging in both blue and green spectral regions.

Probing Enzyme Location in Water-in-Oil Microemulsion Using Enzyme–Carbon Dot Conjugates

This article delineates the formation and characterization of different enzyme-carbon dot conjugates in aqueous medium (pH = 7.0). We used soybean peroxidase (SBP), Chromobacterium viscosum (CV) lipase, trypsin, and cytochrome c (cyt c) for the formation of conjugate either with cationic carbon dot (CCD) or anionic carbon dot (ACD) depending on the overall charge of the protein at pH 7.0. These nanobioconjugates were used to probe the location of enzymes in water-in-oil (w/o) microemulsion. The size of the synthesized water-soluble carbon dots were of 2-3 nm with distinctive emission property. The formation of enzyme/protein-carbon dot conjugates in aqueous buffer was confirmed via fluorescence spectroscopy and zeta potential measurement, and the structural alteration of enzyme/protein was monitored by circular dichroism spectroscopy. Biocatalytic activities of protein/enzymes in conjugation with carbon dots were found to be decreased in aqueous phosphate buffer (pH 7.0, 25 mM). Interestingly, the catalytic activity of the nanobioconjugates of SBP, CV lipase, and cyt c did not reduce in cetyltrimethylammonium bromide (CTAB)-based reverse micelle. It indicates different localization of carbon dots and the enzymes inside the reverse micelle. The hydrophilic carbon dots always preferred to be located in the water pool of reverse micelle, and thus, enzyme must be located away from the water pool, which is the interface. However, in case of trypsin-carbon dot conjugate, the enzyme activity notably decreased in reverse micelle in the presence of carbon dot in a similar way that was observed in water. This implies that trypsin and carbon dots both must be located at the same place, which is the water pool of reverse micelle. Carbon dot induced deactivation was not observed for those enzymes which stay away from the water pool and localized at the interfacial domain while deactivation is observed for those enzymes which reside at the water pool. Thus, the location of enzymes in the microdomain of w/o microemulsion can be predicted by comparing the activity profile of enzyme-carbon dot conjugate in water and w/o microemulsion.

Water-in-oil microemulsion doped with gold nanoparticle decorated single walled carbon nanotube: Scaffold for enhancing lipase activity

The present work reports the development of water-in-oil (w/o) microemulsion doped with newly designed nanocomposite comprising of gold nanoparticle (GNP) decorated single walled carbon nanotube (SWNT). This nanocomposite included cationic reverse micelle was used to boost the catalytic activity of a surface-active enzyme, Chromobacterium viscosum lipase (CV lipase). SWNT was non-covalently dispersed using cetyltrimethylammonium bromide (CTAB), cetylalaninetrimethylammonium chloride (CATAC) while GNP was synthesized by reduction of HAuCl4 with reducing/stabilizing agent trisodium citrate. Counterion exchange between cationic SWNT dispersing agent and anionic capping agent of GNP led to the formation of GNP decorated SWNT (SWNT-GNP) nanocomposite. This newly developed SWNT-GNP included CTAB reverse micelle was characterized by several microscopic and spectroscopic techniques. Interfacially located SWNT-GNP included w/o microemulsion (confirmed from biphasic and fluorescence experiment) was used as a proficient host for enhancing the catalytic activity of lipase. Lipase activity within this self-assembled soft nanocomposite improved up to 3.9-fold (second order rate constant, k2=1694±16 cm(3) g(-1) s(-1)) compared to standard CTAB reverse micelle (k2=433±7 cm(3) g(-1) s(-1)). In case of cetyltripropyl ammonium bromide (CTPAB) based reverse micelle, the observed lipase activity improved to k2=2036±11 cm(3) g(-1) s(-1) in the presence of SWNT-GNP composite. Notably, this catalytic activity of lipase within SWNT-GNP included reverse micelle was till date the highest activity found in any w/o microemulsion. The attainment of flexibility in enzyme conformation at the augmented interface was verified using circular dichroism (CD) spectroscopy.

Striking improvement in peroxidase activity of cytochrome c by modulating hydrophobicity of surface-functionalized gold nanoparticles within cationic reverse micelles.

This work demonstrates a remarkable enhancement in the peroxidase activity of mitochondrial membrane protein cytochrome c (cyt c) by perturbing its tertiary structure in the presence of surface-functionalised gold nanoparticles (GNPs) within cetyltrimethylammonium bromide (CTAB) reverse micelles. The loss in the tertiary structure of cyt c exposes its heme moiety (which is buried inside in the native globular form), which provides greater substrate (pyrogallol and H(2)O(2)) accessibility to the reactive heme residue. The surfactant shell of the CTAB reverse micelle in the presence of co-surfactant (n-hexanol) exerted higher crowding effects on the interfacially bound cyt c than similar anionic systems. The congested interface led to protein unfolding, which resulted in a 56-fold higher peroxidase activity of cyt c than that in water. Further perturbation in the proteins structure was achieved by doping amphiphile-capped GNPs with varying hydrophobicities in the water pool of the reverse micelles. The hydrophobic moiety on the surface of the GNPs was directed towards the interfacial region, which induced major steric strain at the interface. Consequently, interaction of the protein with the hydrophobic domain of the amphiphile further disrupted its tertiary structure, which led to better opening up of the heme residue and, thereby, superior activity of the cyt c. The cyt c activity in the reverse micelles proportionately enhanced with an increase in the hydrophobicity of the GNP-capping amphiphiles. A rigid cholesterol moiety as the hydrophobic end group of the GNP strikingly improved the cyt c activity by up to 200-fold relative to that found in aqueous buffer. Fluorescence studies with both a tryptophan residue (Trp59) of the native protein and the sodium salt of fluorescein delineated the crucial role of the hydrophobicity of the GNP-capping amphiphiles in improving the peroxidase activity of cyt c by unfolding its tertiary structure within the reverse micelles.

l-Phenylalanine-Tethered, Naphthalene Diimide-Based, Aggregation-Induced, Green-Emitting Organic Nanoparticles

The present article delineates the formation of green fluorescent organic nanoparticle through supramolecular aggregation of naphthalene diimide (NDI)-based, carboxybenzyl-protected, l-phenylalanine-appended bola-amphiphile, NDI-1. The amphiphilic molecule is soluble in DMSO, and, with gradual addition of water within the DMSO solution, the amphiphile starts to self-assemble via H-type aggregation to form spherical nanoparticles. These self-assembly of NDI-1 in the presence of a high amount of water exhibited aggregation-induced emission (AIE) through excimer formation. Notably, in the presence of 99% water content, the amphiphile forms spherical aggregated nanoparticles as confirmed from microscopic investigations and dynamic light scattering study. Interestingly, the emission maxima of molecularly dissolved NDI-1 (weak blue fluorescence) red-shifted upon aggregation with increase in water concentration and led to the formation of green-emitting fluorescent organic nanoparticles (FONPs) at 99% water content. These green-emitting FONPs were utilized in cell imaging as well as for efficient transportation of anticancer drug curcumin inside mammalian cells.

Hydrophobically Tailored Carbon Dots toward Modulating Microstructure of Reverse Micelle and Amplification of Lipase Catalytic Response.

This article delineates the modulation of microstructure of cationic reverse micelle utilizing hydrophobically modified carbon dots (CDs) with varying surface functionalizations. Citric acid was used as the source of the carbon core, and Na-salt of glycine, glycine, Na-salt of 11-aminoundecanoic acid, 11-aminoundecanoic acid, and n-hexadecylamine were used for the surface fabrication of CDs to produce CD 1s, CD 1a, CD 2s, CD 2a, and CD 3, respectively. All these CDs having dimension of 5-7 nm were characterized by spectroscopic and microscopic techniques. The hydrodynamic diameter of cetyltrimethylammonium bromide (CTAB) reverse micelle (CTAB/isooctane/n-hexanol/water) at z ([cosurfactant]/[surfactant]) = 6.4 and W0 ([water]/[surfactant]) = 44 is around 15-20 nm. Interestingly, the size of the water-in-oil (w/o) microemulsions dramatically increased up to 120-200 nm upon doping hydrophobic surface functionalized CD 2a and CD 3. This is possibly due to change in the micellar exchange dynamics and reorganization of the micellar aggregates via hydrophobic interaction between surfactant (CTAB) tail and hydrophobic surface modifier of the carbon dots. However, no alteration in the size of reverse micelles was noted in the presence of carbon dots CD 1s, CD 1a, and CD 2s. Spectroscopic and microscopic investigations confirmed that the hydrophobic CD 2a and CD 3 were localized at the interface of reverse micelles whereas CD 1s, CD 1a, and CD 2s were possibly located in the water pool (away from interface). The activity of Chromobacterium viscosum lipase encapsulated within CD 3 and CD 2a doped significantly large CTAB reverse micelles showed remarkable improvement (3.7-fold and 3.4-fold) in its catalytic response. However, hydrophilic carbon dots CD 1s and CD 2s as well as moderately hydrophobic CD 1a had no significant effect on the microstructure of reverse micelles as well as on the lipase activity.

An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin

Herein we report the design and development of α5β1 integrin‐specific noncovalent RGDK–lipopeptide‐functionalized single‐walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide‐tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT)‐ and lactate dehydrogenase (LDH)‐based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non‐cytotoxic nature of these RGDK–lipopeptide–SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against αvβ3, αvβ5, and α5β1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3‐labeled oligonucleotides, Cy3‐oligo) to B16F10 cells selectively via α5β1 integrin. Notably, the nanovectors failed to deliver the Cy3‐oligo to NIH3T3 cells. The RGDK–SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)‐bearing C57BL/6J mice showed tumor‐selective accumulation of curcumin intravenously administered via RGDK–lipopeptide–SWNT nanovectors.