Decisive Role of Polymer-Bovine Serum Albumin Interactions in Biofilm Substrates on "Philicity" and Extracellular Polymeric Substances Composition.

Formation of extracellular polymeric substances (EPS) is a crucial step for bacterial biofilm growth. The dependence of EPS composition on growth substrate and conditioning of the latter is thus of primary importance. We present results of studies on the growth of biofilms of two different strains each, of the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, on four polymers used commonly in indwelling medical devices ─polyethene, polypropylene, polycarbonate, and polytetrafluoroethylene─immersed in bovine serum albumin (BSA) for 24 h. The polymer substrates are studied before and after immersing in BSA for 9 and 24 h, using contact angle measurement (CAM) and field emission scanning electron microscopy (FE-SEM) to extract, respectively, the "philicity" φ (defined as -cos θ, where θ is the contact angle of the liquid on the solid at a particular temperature and ambient pressure) and spatial Hirsch parameter H (defined from the relation F(r) ∼ r2H, where F(r) is the mean squared density fluctuation at the sample surface). H = 0.5, <0.5, or >0.5 signifies no correlation, anticorrelation, and correlation, respectively. The substrates are seen to transform from large hydrophobicity to near amphiphilicity with the formation of a BSA conditioning surface layer, and the H-values distinguish the length scales of 100, 500, and 2000 nm, with the anticorrelation increasing with length scale. Biofilms of E. coli did not grow on bare PTFE and HDPE substrates. Biofilms grown on BSA-covered surfaces are studied with CAM, FE-SEM, Fourier transform infrared (FTIR), and surface-enhanced Raman spectroscopy (SERS). Both spectra and φ-values were independent of bacterial species but dependent on the polymer, while H-values show some bacterial variation. Thus, EPS composition and wetting properties of the corresponding bacterial biofilms seem to be decided by the interaction of the conditioning BSA layer with the specific polymer substrate.

Time-structuring in the evolution of 2D nanopatterns through interactions with substrate.

Hydrophobic dodecanethiol capped gold nanoparticles (AuNPs) are found to self-assemble into two-dimensional patterns in monolayers of amphiphiles spread at the air-water interface of a Langmuir trough. In this communication we investigate the role of the nanoparticle-monolayer (FNMA) and monolayer-monolayer (FMMA) lipophilic attraction in influencing morphology and dynamics of AuNP cluster patterns in fatty acid monolayers. FNMA and FMMA are progressively varied by changing n, where n is the number of -CH2 groups in the alkyl tails of the amphiphilic fatty acid (CH3(CH2)nCOOH) molecules forming the monolayer. Compressibility measurements on the pristine and nanoparticle-laden monolayers show that, while the compressibility of the pristine monolayer decreases with increasing n, pointing to a progressive increase in FMMA, the effect of nanoparticles (increase in compressibility or lowering of FMMA) is discernible only for 14 < n < 22. The corresponding pattern morphology, observed with a Brewster Angle Microscope (BAM) at an in-plane resolution of 450 nm for 6 hours, reveals that there are essentially three stages in pattern evolution, lamellae of Au nanoclusters spread over the fatty-acid monolayer background (the λ state) followed by a network of nanoclusters with high node density (the ν state) and finally rings (circular/elongated) of random sizes with very low node density (the ρ state), evolving from an initial unsegregated state, without appreciable change in the average nanoparticle number density over the field of view. Increasing FNMA alongwith FMMA is found to shift a certain state to later times, thus playing the role of a viscous drag and introducing a delay in the timeline. The mean square fluctuation of BAM intensity remains flat and then decays as f(ξ) = ξ(2H) over smaller length scales, where ξ is the spatial separation and H the Hurst exponent. The study of f(ξ) over time reveals the growth of a sub-diffusive regime (H < 0.5) at the intermediate length scale, in almost all the films coinciding with the emergence of the ρ state. The growth of this sub-diffusive regime is slower for stronger FNMA and FMMA, the interactions thus acting as control parameters in dictating the time structure of the spatio-temporal patterns.

A two-dimensional network of Au nanoclusters on water surface: example of facile control of nanospace.

We report isotherm and Brewster Angle Microscopy (BAM) studies on mixed monolayers of Stearic Acid (StA) and Dodecanethiol-capped gold nanoparticles (AuNPs) (10% by weight) at the air-water interface. The surface pressure-specific molecular area isotherm for the mixed monolayer shows small hysteresis as well as a lowering of the slope change in going from L2-L2' and L2'-S phases, indicating lesser number of monolayer phases for the mixed film relative to a pristine StA monolayer. The BAM study shows formation of a two-dimensional, stable, reproducible, and extensive network of nanoparticle clusters with near-circular pores, in the 5-20 mN/m surface pressure range. This assembly of nanospaces has a pore area peak at approximately 75 microm2, width approximately 120 microm2 and can be disassembled and reassembled simply by changing the surface pressure.

Nanoparticle-induced morphological transition of Bombyx mori nucleopolyhedrovirus: a novel method to treat silkworm grasserie disease.

Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed α structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe 'roughening' of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09±6.61% after 96 h. In this group, 61.04±8.03% larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases.

Comparative analysis of stability and toxicity profile of three differently capped gold nanoparticles for biomedical usage.

Nowadays gold nanoparticle (GNP) is increasingly being used in drug delivery and diagnostics. Here we have reported a comparative analysis of detailed stability and toxicity (in vitro and in vivo) profile of three water soluble spherical GNPs, having nearly similar size, but the surfaces of which were modified with three different capping materials aspartic acid (GNPA), trisodium citrate dihydrate (GNPC) or bovine serum albumin (GNPB). Spectral analyses on the stability of these GNPs revealed that depending on the nature of capping agents, GNPs behave differently at different environmental modalities like wide range of pH, high salt concentrations, or in solutions and buffers of biological usage. GNPB was found to be extremely stable, where capped protein molecule successfully maintained its secondary structure and helicity on the nanoparticle, whereas colloidal stability of GNPA was most susceptible to altered conditions. In vitro cytotoxicity of these nanoparticle formulations in vitro were determined by water soluble tetrazolium and lactate dehydrogenase assay in human fibroblast cell line (MRC-5) and acute oral toxicity was performed in murine model system. All the GNPs were non-toxic to MRC-5 cells. GNPC had slight hepatotoxic and nephrotoxic responses. Hepatotoxicity was also evident for GNPA treatment. Present study established that there is a correlation between capping material and stability together with toxicity of nanoparticles. GNPB was found to be most biocompatible among the three GNPs tested.

Preparation of serum capped silver nanoparticles for selective killing of microbial cells sparing host cells.

Following access into the cell, colloidal silver nanoparticles exhibit generalized cytotoxic properties, thus appear as omnipotent microbicidal, but not suitable for systemic use unless are free of toxic effects on host cells. The AgNP-Serum-18 when prepared from silver nitrate, using dextrose as reducing and group-matched homologous serum as a stabilizing agent, selective endocytosis, and oxidative stress-dependent bio-functional damages to the host are mostly eliminated. For their bio-mimicking outer coat, there is the least possibility of internalization into host cells or liberation of excess oxidants in circulation following interaction with erythrocytes or vascular endothelial cells. The presence of infection-specific antibodies in the serum can make such nano-conjugates more selective. A potent antimicrobial action and a wide margin of safety for mammalian cells in comparison with very similar PVA-capped silver nanoparticles have been demonstrated by the in-vitro challenge of such nanoparticles on different microbes, human liver cell-line, and in-vivo study on mice model. This may open up wide-range therapeutic prospects of colloidal nanoparticles.

Nanoparticle surface as activation site.

The immense surface-to-volume (S/V) ratio in nanoparticles leads to large surface energy density. These high densities play the role of sites for activities that are not triggered in bulk materials. Here we present some examples of such distinctive activities taking place at nanoparticle surfaces. Our first example involves the morphological changes in silkworm (Bombyx mori L.) nuclear polyhedrosis virus (BmNPV) brought about by lipophilic amorphous silica nanoparticles (LASN). Microscopy studies show that nanoparticles severely alter the structure of the virus envelope by a 'deflation' of the viral polyhedron and formation of elongated structures. The second example shows the spatial variation in aggregation potential with temperature, for dodecanethiol-capped Au nanoparticles on an amorphous polystyrene film surface. We find that on increasing the temperature from 32 degrees C to 50 degrees C the aggregating potential becomes almost completely confined to the film surface, whereas going over to 100 degrees C the confining potential is overcome and out-of-plane growth takes place. A tentative and qualitative explanation has been attempted.

Morphology and structural evolution in cobalt stearate Langmuir-Blodgett films.

Structural evolution of cobalt stearate (CoSt) Langmuir-Blodgett (LB) films show 2D layer-by-layer or Frank van der Merwe type growth, with in-plane defect-free morphology and bidentate bridging metal ion headgroup coordination, as observed from both Atomic Force Microscopy (AFM) and X-ray Reflectivity (XRR) studies. Difference in headgroup structure of first monolayer with that of subsequent bilayers is observed from XRR studies. However, AFM study of preformed cobalt stearate (CoStp) LB films did not show such defect-free growth in spite of having same metal ion coordination, as seen from its bulk Fourier Transform Infra-red (FTIR) spectra. Increased interaction between two methyl groups of adjacent hydrocarbon tails in case of CoSt deposition was indicated from the observed lengthening of C-H bonds in these. Along with metal ion headgroup coordination, supramolecular tail-tail interaction is proposed to play a key role in defect free multilayer formation.

Self-assembly of a two-dimensional Au-nanocluster superlattice and its photoluminescence spectra.

A solution of dodecanethiol-capped Au nanoparticles (diameter approximately 2 nm), prepared through reduction of hydrogen tetrachloroaurate, was coated on a patterned cross-linked polydimethylsiloxane (PDMS) film (thickness > or =1 microm) comprising array of square pillars, by either spin- or dip coating. Drying in ambient conditions for about 5 days resulted in the self-assembly of a superlattice (300 nm x 300 nm x 90 nm) of monodisperse nanoparticle clusters for the dip-coated samples, the clusters occupying the interstitial locations between four adjacent pillars on the polymer surface. Initial patterning of the PDMS is found to give rise to new photoluminescence bands in the near ultraviolet and visible regions, which the nanoclusters quench.

Nanoparticle-virus complex shows enhanced immunological effect against baculovirus.

Insects protect themselves from majority of infections by a non-specific innate immune system (present in both vertebrates and invertebrates). Bombyx mori nuclear polyhedrosis virus (BmNPV), a baculovirus, causing the deadly grasserie disease is a scourge to silkworm industry and we report here the first success in combating this disease with the help of a nanosilica-virus complex. Hydrophobic aluminium silicate nanoparticles were mixed with live BmNPV in vitro. This mixture was injected into one day old 5th instar silkworm larvae (into the hemocoel at the third abdominal spiracle) before challenging the larvae with live BmNPV via a second injection. This led to substantial enhancement of longevity in the diseased silkworm larvae and 35 +/- 5.3% larvae completed their lifecycle (i.e., formed normal pupae and enclosed as moth). On the other hand, 100% larvae infected with BmNPV alone died within 36 hours. The larvae treated with nanoparticles before infection had a longer lifespan but all of them eventually succumbed, not a single larva metamorphosed to adult stage. Results suggest two pathways of host protective response--one mediated by nanoparticlealone and the second, more important, via non-specific innate immunological mechanism. AFM and confocal studies show that nanoparticles alter 3-D molecular structure of the virus envelope. Possibly this exhibits novel potent epitope(s) which stimulate(s) anti-viral machinery in infected silkworm larvae. SDS-PAGE results suggest that 39 KDa viral protein is the major target of the nanoparticles.

Periodicities in the roughness and biofilm growth on glass substrate with etching time: Hydrofluoric acid etchant.

Adherence of the microorganism to submerged solid surfaces leads to biofilm formation. Biofilm formation modifies the surfaces in favor of bacteria facilitating the survival of the bacteria under different stressed conditions. On the other hand, the formation of biofilm has a direct adverse economic impact in various industries and more importantly in medical practices. This adherence is the reason for the failure of many indwelling medical devices. Surface biofilm adhesion is the key to biofilm growth and stability. Hence this adhesion needs to be substantially lowered to inhibit biofilm stability. Both chemical and physical properties of the surface influence biofilm formation and modulating these properties can control this formation. In this study, we have investigated the effect of Hydrofluoric acid (HF), at a specific concentration as an etchant, on the surface morphology of substrates and the growth of biofilms of Pseudomonas aeruginosa. and Staphylococcus aureus. We find that the bacterial counts on the etched surfaces undergo a periodic increase and decrease. This, on one hand, shows the close correlation between the biofilm growth and the particular roughness scale, and on the other hand, explains the existing contradictory results regarding the effects of etching on substrate roughness and biofilm growth. We propose a simple model of a sequence of hole formation, hole expansion and etching away of the hole walls to form a new, comparatively smooth surface, coupled with the preferential accumulation of bacteria at the hole edges, to explain these periodicities.

Chain-length dependence of lipophilic force: comparison with the two-body van der Waals' force.

Hydrophobic dodecanethiol capped Au nanoparticles (AuNPs) form two-dimensional patterns in monolayers of amphiphilic fatty acids ([Formula: see text]) at the air water interface. An interplay between the various lipophilic interactions, in turn, decides the NP cluster size, where stronger NP-monolayer and monolayer-monolayer attraction in fatty acid monolayers with longer tail length oppose nanoparticle aggregation resulting in a decrease in cluster size in both in-plane and out-of-plane directions. The decrease in the in-plane cluster size is steepest for 14 < n < 18, n being the total number of C atoms in the fatty acid, and levels off for higher fatty acids and cannot be explained on the basis of the two body van der Waals pair potential atleast at initial phases of pattern formation. The potential can be used only at later times, closer to stability.

Two-dimensional self-organization of gold nanoparticles on supramolecular aggregates.

Alkanethiol-capped gold (Au) nanoparticles, dispersed in toluene and spin-coated on atactic polystyrene films that are themselves spin-coated on fused quartz plates, self-assemble into a characteristic two-dimensional pattern-a network of the Au-thiol nano-composite separating polystyrene 'islands' of dimensions around 200 nm. This pattern changes on varying the concentration of the nanoparticles in the coating solution, evolving into the network from isolated nanoparticle clusters, and then going over to a distribution of 'pinholes' on a surface covered by the nano-composite, resembling a partially wetted surface with near-full coverage. Study of the pristine polystyrene surface reveals a pattern of low-cohesion regions separating 'islands' of polystyrene, where the latter are known to be supramolecular aggregates. It is suggested that these regions play the role of low-resistance channels to the flow of the nano-composite during spin coating.

Critical behavior of a two-dimensional complex fluid: Macroscopic and mesoscopic views.

Liquid disordered (L_{d}) to liquid ordered (L_{o}) phase transition in myristic acid [MyA, CH_{3}(CH_{2})_{12}COOH] Langmuir monolayers was studied macroscopically as well as mesoscopically to locate the critical point. Macroscopically, isotherms of the monolayer were obtained across the 20^{∘}C-38^{∘}C temperature (T) range and the critical point was estimated, primarily from the vanishing of the order parameter, at ≈38^{∘}C. Mesoscopically, domain morphology in the L_{d}-L_{o} coexistence regime was imaged using the technique of Brewster angle microscopy (BAM) as a function of T and the corresponding power spectral density function (PSDF) obtained. Monolayer morphology passed from stable circular domains and a sharp peak in PSDF to stable dendritic domains and a divergence of the correlation length as the critical point was approached from below. The critical point was found to be consistent at ≈38^{∘}C from both isotherm and BAM results. In the critical regime the scaling behavior of the transition followed the two-dimensional Ising model. Additionally, we obtained a precritical regime, over a temperature range of ≈8^{∘}C below T_{c}, characterized by fluctuations in the order parameter at the macroscopic scale and at the mesoscopic scale characterized by unstable domains of fingering or dendritic morphology as well as proliferation of a large number of small sized domains, multiple peaks in the power spectra, and a corresponding fluctuation in the peak q values with T. Further, while comparing temperature studies on an ensemble of MyA monolayers with those on a single monolayer, the system was found to be not strictly ergodic in that the ensemble development did not strictly match with the time development in the system. In particular, the critical temperature was found to be lowered in the latter. These results clearly show that the critical behavior in fatty acid monolayer phase transitions have features of both complex and nonequilibrium systems.

Counterion effects on nano-confined metal-drug-DNA complexes.

We have explored morphology of DNA molecules bound with Cu complexes of piroxicam (a non-steroidal anti-inflammatory drug) molecules under one-dimensional confinement of thin films and have studied the effect of counterions present in a buffer. X-ray reflectivity at and away from the Cu K absorption edge and atomic force microscopy studies reveal that confinement segregates the drug molecules preferentially in a top layer of the DNA film, and counterions enhance this segregation.

Dynamics driven by lipophilic force in Langmuir monolayers: In-plane and out-of-plane growth.

While monolayer area fraction versus time (A(n)-t) curves obtained from surface pressure-area (π-A) isotherms for desorption-dominated (DD) processes in Langmuir monolayers of fatty acids represent continuous loss, those from Brewster angle microscopy (BAM) also show a two-dimensional (2D) coalescence. For nucleation-dominated (ND) processes both techniques suggest competing processes, with BAM showing 2D coalescence alongside multilayer formation. π enhances both DD and ND processes with a lower cutoff for ND processes, while temperature has a lower cutoff for DD but negligible effect on ND processes. Hydrocarbon chain length has the strongest effect, causing a crossover from DD to ND dynamics. Imaging ellipsometry of horizontally transferred films onto Si(100) shows Stranski-Krastanov-like growth for ND process in an arachidic acid monolayer resulting in successive stages of monolayer, trilayer, and multilayer islands, ridges from lateral island coalescence, and shallow wavelike structures from ridge coalescence on the film surface. These studies show that lipophilic attraction between hydrocarbon chains is the driving force at all stages of long-term monolayer dynamics.

Stability and softening of a lipid monolayer in the presence of a pain-killer drug.

The aim of this study is to investigate the interaction of a drug (Piroxicam, 4-hydroxy-2-methyl-N-(2-pyridinyl)-2H-1,2-benzothiazine-3-carboxamide 1,1 dioxide) with a lipid (DMPC) monolayer used as a membrane-mime in terms of drug-induced changes in stability and compressibility with variation in temperature, surface-pressure, drug-dose and ionic states of the monolayers. Drug-induced fluidization is noticed in the π-A isotherms through increase in phase-transition pressure at constant temperature. The long-term dynamics of the lipid-monolayer is characterized by algebraic decays in surface-energy E with time t, E∼t(-p1,2,3), with an initial decay exponent p1 that changes to p2 after ∼1000 s, and, at high surface pressures and/or drug-dose, to a third exponent p3 after ∼3500 s, suggesting structural reorganizations in the monolayer. With increasing drug-lipid ratio (D/L), p1 shows a decrease ending at an almost constant value after 0.05, p2 shows an almost negligible lowering while p3 shows a monotonic and considerable increase. The reorganization is summarized by proposing two mechanisms: (a) 'charging-discharging' where drug-molecules sitting parallel to the interface increase headgroup separations and (b) 'discharging-charging' where drug-molecules sitting roughly perpendicular to the interface bring headgroups closer. Drug-induced softening of lipid-monolayers is characterized by the compressibilites of pure and mixed lipid monolayers. Compressibility-change (i.e., compressibility difference between drug/lipid and pure lipid monolayer) with pressure is maximum in the LE-LC transition zone and compressibility-change with drug-dose reveals an optimum dose of drug for maximum increase in compressibility. Molecular dynamics simulation shows that the ordering in the different parts of the lipid chains is changed to different extents in the presence of drugs with maximum change near the headgroups and again points to an optimum dose for maximum disorder.

Evolution of self-organized two-dimensional patterns of nanoclusters through demixing.

A mixture of dodecanethiol-capped Au nanoparticles (AuNPs) and the amphiphilic fatty acid, stearic acid, spread as a monomolecular layer on water surface, is observed with Brewster angle microscopy (BAM) to form a two-dimensional network of AuNP clusters through demixing, at concentration of AuNPs by weight (ρ[over ¯])>10% and the surface pressure (π)≥10mNm^{-1}. For π=15mNm^{-1}, the number of nodes (n) remains unchanged till ∼2 hours and then changes over to a lower n state, where the pattern consists of almost perfect circles with greater in-plane thickness of the AuNP lamellae. For the higher n state the mean-square fluctuation of BAM intensity remains flat and then decays as f(ξ)=ξ^{2α} with α∼0.6 (correlated fluctuations) over the length scales of 400µm-6µm and below 6µm, respectively. For the lower n state the fluctuation decays almost over the entire length scale with α=0.3, indicating emergence of aperiodicity from quasiperiodicity and a changeover to anticorrelated fluctuations. These patterns can be looked at as two distinct chaotic trajectories in the I-I^{'} phase space of the system (I being the scattered light intensity at any position of the pattern and I^{'} its gradient) with characteristic Lyapunov exponents.

Atomic force microscopy in biofilm study.

Biofilms have been classically visualized by Scanning Electron Microscopy (SEM). The complex operating procedure of SEM restricts its use in routine practice. There is a need of newer visualizing techniques for examining surfaces of biofilms, in particular under ambient conditions. We have presented the unique advantages of atomic force microscopy (AFM) in studying surfaces of biofilms through analyses of the height images obtained on biofilms of two gram positive and one gram negative bacteria, namely Staphylococcus aureus, Nocardia brasiliensis and Pseudomonas aeruginosa, respectively. Biofilm quality of the three different bacteria, ageing effects on Nocardia spp. biofilm surface and effects of the antibiotic ciprofloxacin at different doses on Staphylococcus and Pseudomonas biofilm surfaces have been investigated under ambient conditions and distinctive features have been observed.

Evolution of nanoparticle-induced distortion on viral polyhedra.

Morphological changes in the polyhedra of the Bombyx mori L. nuclear polyhedrosis virus (BmNPV), a baculovirus causing the deadly grasserie disease in silkworms, brought about by mixing with lipophilically capped amorphous silica nanoparticles (LASN, average size 10 ± 2 nm) were studied with scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. SEM shows that the regular octagonal polyhedra facets are replaced by a larger number of newly formed irregular ones. The average number of facets reveals a nonlinear growth pattern with nanoparticle (NP) concentration, where an initial linear region ends in a plateau. IR bands corresponding to vibration modes of the capping show (a) a saturation of the area under the band with NP concentration, indicating a correlation with attachment to viral polyhedra and (b) a narrowing of the band per NP from the linear to the plateau portions of the distortion curve, suggesting non-equilibrium and equilibrium situations, respectively.