Subhabrata Maiti

In situ synthesized Ag nanoparticle in self-assemblies of amino acid based amphiphilic hydrogelators: development of antibacterial soft nanocomposites

The present work reports the development of a new class of antibacterial soft-nanocomposites by in situ synthesis of silver nanoparticle (AgNP) within the supramolecular self-assemblies of amino acid (tryptophan/tyrosine) based amphiphilic hydrogelators. Interestingly, the nanoparticle synthesis does not require the use of any external reducing/stabilizing agents. The nanocomposites were characterized by UV-vis spectra, transmission electron microscopy (TEM) images, X-ray diffraction spectroscopy (XRD) and thermo gravimetric analysis (TGA). Encouragingly, these soft nanocomposites showed excellent antibacterial activity against both Gram-positive and Gram-negative bacteria whereas the amphiphiles alone were lethal only toward Gram-positive bacteria. Judicious combination of bactericidal AgNP within the self-assemblies of inherently antibacterial amphiphilic gelators led to the development of soft nanocomposites effective against both type of bacteria. The head group charge and structure of the amphiphiles were altered to investigate their important role on the synthesis and stabilization of AgNP and also in modulating the antibacterial activity of the nanocomposites. The antibacterial activities of soft nanocomposites comprising amphiphiles with cationic head group were found to be more efficient than the anionic soft nanocomposites. Interestingly, these nanocomposites have shown considerable biocompatibility to mammalian cell, NIH3T3. Furthermore, the well-known tissue engineering scaffold, agar-gelatin film infused with these soft nanocomposites allowed normal growth of mammalian cells on its surface while being lethal toward both Gram-positive and Gram-negative bacteria.

Covalently functionalized single-walled carbon nanotubes at reverse micellar interface: a strategy to improve lipase activity.

The present work reports covalent functionalization of single-walled carbon nanotubes (f-SWNTs) to introduce hydrophilicity to the otherwise amphiphobic nanotubes. The charge and spacer length of the functional moiety were varied by using quaternized ethylene diamine, 6-aminocaproate, quaternized (ethylenedioxy)bis(ethylamine), and a poly(ethylene glycol) (PEG) unit (f-SWNT-1 to f-SWNT-4, respectively). These f-SWNTs with varying degrees of hydrophilicity were incorporated within cetyltrimethyl ammonium bromide (CTAB) reverse micelles to develop stable self-assembled nanohybrids. An optimum hydrophilicity on the SWNT surface led to interfacial localization of f-SWNTs resulting in the augmentation of space at the interface. A surface-active enzyme, lipase, localized at this enhanced interface of f-SWNT-containing CTAB reverse micelles exhibited significant activation (2.5-fold) compared to that in the absence of the nanoconstructs. This improvement in lipase activity was mainly due to the smooth occupancy of lipase and also presumably because of the increase in the concentrations of both substrate and the enzyme at the augmented interface. Interestingly, the f-SWNTs that activate lipase in reverse micelles deactivate the same enzyme in water. The dispersion of f-SWNTs in water and its matching integration at the interface of reverse micelles were confirmed through transmission electron microscopic (TEM) investigations. The interfacial localization of these nanoconstructs was also established from the distinct fluorescence behavior of a hydrophobic fluorescent probe, fluorescein isothiocyanate (FITC), adsorbed onto the f-SWNT surface. In concurrence with the observed lipase activity, the corresponding changes in the enzyme conformation within f-SWNTs integrated reverse micelle as well as in aqueous medium were studied by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy.

Refining hydrogelator design: soft materials with improved gelation ability, biocompatibility and matrix for in situ synthesis of specific shaped GNP

Despite the continuous surge in the development of new supramolecular gels, the prediction of a gelators structure still remains elusive. It is also imperative to consolidate the existing inventory of gelators and devise ways to make the gels functional. In the present work, L-phenylalanine based poor (C-16) or non-gelating (C-12 tail) amphiphiles were converted to excellent gelators with the simple incorporation of N-terminal protected amino acid/dipeptide at the end of the alkyl tail. More than 6-fold enhancement in gelation efficiency was observed for amino acid/dipeptides incorporated at the tail of amphiphile in comparison to the corresponding unmodified alkyl tail. Interestingly, amphiphile with the tertiary butyloxycarbonyl (Boc) protected amino acid at the tail had better gelation ability than the amphiphile with the aromatic Fmoc (N-fluorenyl-9-methoxycarbonyl) protecting group. Spectroscopic investigations (XRD and FTIR) revealed that the modification at the tail compels the amphiphiles to take a different course of self-assembly than that adopted by their predecessors (alkyl tailed gelator, C-16). For example, in the case of the amphiphile having a dipeptide at the tail, formation of β-sheet structure through anti-parallel arrangement between the molecules results in notable improvement in its gelation ability. Most importantly, these tail modified amphiphiles were capable of in situ synthesis of gold nanoparticles (GNPs) of specific shape without the help of any external reducing agents in the newly developed soft materials. The biocompatibility of hydrogels is also crucial for their prolific biomedicinal functions. MTT assay showed dramatic improvement in the biocompatibility of the tail modified hydrogelators towards mammalian cells in comparison to the amphiphiles having no amino acid at the tail.

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.

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.

GNP confinement at the interface of cationic reverse micelles: influence in improving the lipase activity

The present work reports thiol-assisted confinement of gold nanoparticles (GNPs) at the interface of reverse micelles with the aim to enhance the interfacial area and thereby the efficiency of surface-active Chromobacterium viscosum lipase. The strong gold capping ability of optimally hydrophobic thiols (1-dodecanethiol and 1,6-hexanedithiol) was aptly utilized to pull GNPs (∼3–5 nm) from the water pool to the oil/water interface of cetyltrimethylammonium bromide (CTAB) reverse micelles. These small sized GNPs were fitted at the microscopic interface of CTAB reverse micelles possibly because of the comparable thickness of the interface (∼1–2 nm) to that of the GNP diameter. Lipase solubilized within this augmented interface enjoys a flexible conformation, which resulted in the improvement of its activity (∼2.5 fold) with respect to only CTAB microemulsion. The activity of lipase within CTAB reverse micelles was thoroughly studied in the presence of mono and dithiols with varying chain length, where a greater improvement in activity was observed with dithiols. Bidentate ligand property of dithiols led to firm localization of higher number of GNPs at the interface which enhanced the total space in vicinity of enzyme at the interfacial domain. Fitting fusion of small sized GNPs within CTAB reverse micellar interface was confirmed by microscopic and spectroscopic studies. Smooth localization of lipase at the enhanced interface was also confirmed from the improvement in its secondary structure (α-helical content) in circular dichroism spectroscopic analysis. Interestingly, large sized GNPs (∼8 and 20 nm) were found to be well fitted at the interface of bigger head group-containing surfactants, cetyltriethylammonium bromide (CTEAB) and cetyltripropylammonium bromide (CTPAB). The hydrolytic efficiency of lipase in 1,6-hexanedithiol included GNP (∼20–25 nm)-doped CTPAB reverse micelles improved by ∼3.4 fold compared to that observed in only CTAB.

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.

Unmodified “GNP–Oligonucleotide” Nanobiohybrids: A Simple Route for Emission Enhancement of DNA Intercalators

We present herein a simple method for enhancing the emission of DNA intercalators in homogeneous nanobiohybrids of unlabeled oligonucleotides and unmodified gold nanoparticles (GNPs). Pristine single-stranded DNA (ss-DNA) has been wrapped around unmodified GNPs to induce metal-enhanced fluorescence (MEF) of DNA intercalators, such as ethidium bromide and propidium iodide. The thickness of the ss-DNA layer on the gold nanosurface determines the extent of MEF, since this depends on the position of the intercalator in relation to the metal surface. Presumably, at a suitable thickness of this DNA layer, more of the intercalator is localized at the optimum distance from the nanoparticle to give rise to MEF. Importantly, no external spacer or coating agent was needed to induce the MEF effect of the GNPs. The concentration ratios of Au to DNA in the nanohybrids, as well as the capping agents applied to the GNPs, play key roles in enhancing the emission of the intercalators. The dimensions of both components of the nanobiohybrids, that is, the size of the GNPs and the length of the oligonucleotide, have considerable influences on the emission enhancement of the intercalators. Emission intensity increased with increasing size of the GNPs and length of the oligonucleotide only when the DNA efficiently wrapped the nanoparticles. An almost 100 % increment in the quantum yield of ethidium bromide was achieved with the GNP-DNA nanobiohybrid compared with that with DNA alone (in the absence of GNP), and the fluorescence emission was enhanced by 50 % even at an oligonucleotide concentration of 2 nM. The plasmonic effect of the GNPs in the emission enhancement was also established by the use of similar nanobioconjugates of ss-DNA with nonmetallic carbon nanoparticles and TiO(2) nanoparticles, with which no increase in the fluorescence emission of ethidium bromide was observed.