PRESSURIZED SOLVENTS IN WHOLE-CELL BIOPROCESSING: METABOLIC AND STRUCTURAL PERTURBATIONS

Compressed and supercritical fluids, such as pressurized CO2, ethane, orpropane, provide a versatile and environmentally acceptable alternative to conventionalliquid organic solvents in bioprocessing applications – specifically in the areas ofproduct extraction, protein purification, microbial sterilization, and enzymatic and wholecellbiocatalysis. While their advantages have been well demonstrated, the effects ofcompressed and supercritical fluids on whole cells are largely unknown.Metabolic and structural perturbations of whole cells by compressed andsupercritical fluid solvents were examined. These perturbations exist as cell metabolismand membrane structure are influenced by pressure and the presence of a solventphase. Continuous cultures of Clostridium thermocellum (a model ethanol-producingthermophilic bacterium) were conducted under elevated hydrostatic and hyperbaricpressure to elucidate pressure- and solvent-effects on metabolism and growth.Fluorescence anisotropy was employed to study liposome fluidization due to thepresence of compressed and supercritical fluids and their partitioning/accumulation inthe phospholipid bilayer.Under elevated hydrostatic pressure (7.0 and 13.9 MPa; 333 K), significantchanges in product selectivity (towards ethanol) and growth were observed in C.thermocellum in conjunction with reduced maximum theoretical growth yields andincreased maintenance requirements. Similarly, metabolism and growth were greatlyinfluenced under hyperbaric pressure (1.8 and 7.0 MPa N2, ethane, and propane; 333K); however, severe inhibition was observed in the presence of supercritical ethane andliquid propane. These changes were attributed to mass-action effects on metabolicpathways, alterations in membrane fluidity, and the dominant role of phase toxicityassociated with compressed and supercritical fluids.Fluorescence anisotropy revealed fluidization and melting point depression ofdipalmitoylphosphatidylcholine liposomes in the presence of CO2, ethane, and propane(1.8 to 20.7 MPa; 295 to 333 K). The accumulation of these fluids within the bilayerupon pressurization and the ordering effects of pressure influenced liposome fluidity, themelting temperature, and the gel-fluid phase transition region. These resultsdemonstrate the disordering effects of compressed and supercritical fluids on biologicalmembranes and the ability to manipulate liposomes

Hydrophobic silver nanoparticles trapped in lipid bilayers: Size distribution, bilayer phase behavior, and optical properties

<p>Abstract</p> <p>Background</p> <p>Lipid-based dispersion of nanoparticles provides a biologically inspired route to designing therapeutic agents and a means of reducing nanoparticle toxicity. Little is currently known on how the presence of nanoparticles influences lipid vesicle stability and bilayer phase behavior. In this work, the formation of aqueous lipid/nanoparticle assemblies (LNAs) consisting of hydrophobic silver-decanethiol particles (5.7 ± 1.8 nm) embedded within 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers is demonstrated as a function of the DPPC/Ag nanoparticle (AgNP) ratio. The effect of nanoparticle loading on the size distribution, bilayer phase behavior, and bilayer fluidity is determined. Concomitantly, the effect of bilayer incorporation on the optical properties of the AgNPs is also examined.</p> <p>Results</p> <p>The dispersions were stable at 50°C where the bilayers existed in a liquid crystalline state, but phase separated at 25°C where the bilayers were in a gel state, consistent with vesicle aggregation below the lipid melting temperature. Formation of bilayer-embedded nanoparticles was confirmed by differential scanning calorimetry and fluorescence anisotropy, where increasing nanoparticle concentration suppressed the lipid pretransition temperature, reduced the melting temperature, and disrupted gel phase bilayers. The characteristic surface plasmon resonance (SPR) wavelength of the embedded nanoparticles was independent of the bilayer phase; however, the SPR absorbance was dependent on vesicle aggregation.</p> <p>Conclusion</p> <p>These results suggest that lipid bilayers can distort to accommodate large hydrophobic nanoparticles, relative to the thickness of the bilayer, and may provide insight into nanoparticle/biomembrane interactions and the design of multifunctional liposomal carriers.</p

In situ SERS detection of dissolved nitrate on hydrated gold substrates

The accurate and fast measurement of nitrate in seawater is important for monitoring and controlling water quality to prevent ecologic and economic disasters. In this work we show that the in situ detection of nitrate in aqueous solution is feasible at nanomolar concentrations through surface enhanced Raman spectroscopy (SERS) using native nanostructured gold substrates without surface functionalization. Spectra were analyzed as collected or after standard normal variate (SNV) normalization, which was shown through Principal Component Analysis (PCA) to reduce spectral variations between sample sets and improve Langmuir adsorption model fits. An additional normalization approach based on the substrate silicon template showed that silicon provided an internal standard that accounted for the spectral variance without the need for SNV normalization. Nitrate adsorption was well-described by the Langmuir adsorption model, consistent with an adsorbed monolayer, and a limit of detection of 64 nM nitrate was obtained in ultrapure water, representing environmentally relevant concentrations. Free energy calculations based on the Langmuir adsorption constants, approximating equilibrium adsorption constants, and calculated self-energy arising from image charge, accounting for electrostatic interactions with a polarizable nanostructured substrate, suggest that nitrate adsorption was partially driven by an entropy gain presumably due to dehydration of the gold substrate and/or nitrate ion. This work is being extended to determine if similar statistical and normalization methods can be applied to nitrate detection in complex natural waters where non-target ions and molecules are expected to interfere

Albumin protein coronas render nanoparticles surface active: consonant interactions at air–water and at lipid monolayer interfaces

Protein coronas are known to alter the physicochemical properties, colloidal stability, and biological fate of nanoparticles. Using human serum albumin (HSA) and polystyrene nanoparticles (NPs) with anionic or cationic surface chemistries, we show that protein coronas also govern the surface activity of PS nanoparticles as well as their interactions with a model red blood cell (RBC) lipid monolayer. The adsorption kinetics of bare nanoparticles (no corona) and nanoparticles with a hard corona (HC) at an air–water interface were well-described theoretically, which revealed that the adsorption energy was greater with the corona due to hydrophobic interactions that were enhanced with protein restructuring. Corona complexation increased the concentration of nanoparticles at the interface and led to the formation of interfacial aggregates. Despite clear differences in monolayer structure, the compressibility of PS–HC monolayers was similar to free HSA, indicating that conformational changes associated with the protein were not restricted in a hard corona. The intrinsic behavior of the proteins driving the surface activity and compressibility of the complexes at an air–water interface was also observed at an air–lipid (RBC)–water interface. In this case the lipid monolayer acted as a barrier and reduced the interface concentration of bare nanoparticles. However, with a corona the nanoparticles penetrated into the monolayer and led to the formation of NP–HC–lipid ‘pillars’ that extended into air. Our results suggest that nanoparticle surface activity, and changes in surface activity due to corona formation, are insightful parameters to predicting nanoparticle–membrane interactions, complementing the conventional view that electrostatic forces are dominant

Partitioning of perfluorooctanoate into phosphatidylcholine bilayers is chain length-independent

The chain length dependence of the interaction of PFOA, a persistent environmental contaminant, with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated using steady-state fluorescence anisotropy spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). PFOA caused a linear depression of the main phase transition temperature Tm while increasing the width of the phase transition of all three phosphatidylcholines. Although PFOA\u27s effect on Tm and the transition width decreased in the order DMPC \u3e DPPC \u3e DSPC, its relative effect on the phase behavior was largely independent of the phosphatidylcholine. PFOA caused swelling of DMPC but not DPPC and DSPC liposomes at 37 °C in the DLS experiments, which suggests that PFOA partitions more readily into bilayers in the fluid phase. These findings suggest that PFOA\u27s effect on the phase behavior of phosphatidylcholines depends on the cooperativity and state (i.e., gel versus liquid phase) of the membrane. DLS experiments are also consistent with partial liposome solubilization at PFOA/lipid molar ratios \u3e 1, which suggests the formation of mixed PFOA–lipid micelles

PFAS fluidize synthetic and bacterial lipid monolayers based on hydrophobicity and lipid charge

Poly- and Perfluoroalkyl substances (PFASs) are pollutants of emerging concern that persist in nature and pose environmental health and safety risks. PFAS disrupt biological membranes resulting in cellular inhibition, but the mechanism of disruption and the role of lipid composition remain unclear. We examine the role of phospholipid saturation and headgroup charge on the interactions between PFASs and phospholipid monolayers comprised of synthetic phosphocholine (PC) and phosphoglycerol (PG) lipids and prepared from bacteria membrane extracts rich in PG lipids from an environmentally relevant marine bacterium Alcanivorax borkumensis. When deposited on a buffered subphase containing PFAS, PFAS mixed within and fluidized zwitterionic and net-anionic monolayers leading to increases in monolayer compressibility that were driven by a combination of PFAS hydrophobicity and monolayer charge density. Differences in the monolayer response using saturated or unsaturated lipids are attributed to the ability of the unsaturated lipids to accommodate PFAS within ‘void space’ arising from the bent lipid tails. Similar fluidization and compressibility behavior were also observed in A. borkumensis lipid monolayers. This work provides new insight into PFAS partitioning into bacterial membranes and the effect PFAS have on the physicomechanical properties of zwitterionic and charged lipid monolayers

Critical new insights into the binding of poly- and perfluoroalkyl substances (PFAS) to albumin protein

With an increasing number of health-related impacts of per- and polyfluoroalkyl substances (PFAS) being reported, there is a pressing need to understand PFAS transport within both the human body and the environment. As proteins can serve as a primary transport mechanism for PFAS, understanding PFAS binding to proteins is essential for predictive physiological models where accurate values of protein binding constants are vital. In this work we present a critical analysis of three common models for analyzing PFAS binding to bovine serum albumin (BSA) based on fluorescence quenching: the Stern-Volmer model, the modified Stern-Volmer model, and the Hill equation. The PFAS examined include perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), and the replacement compound 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (HFPO-DA or GenX). While all three models capture the general effects of hydrophobicity and steric limitations to PFAS binding, the Hill equation highlighted a unique relationship between binding cooperativity and the number of fluorinated carbons, with PFOA exhibiting the greatest binding cooperativity. The significance of steric limitations was confirmed by comparing results obtained by fluorescence quenching, which is an indirect method based on specific binding, to those obtained by equilibrium dialysis where PFAS binding directly correlated with traditional measures of hydrophobicity. Finally, the binding constants were correlated with PFAS physicochemical properties where van der Waals volume best described the steric limitations observed by fluorescence quenching

Surface Activity of Poly(ethylene glycol)-Coated Silver Nanoparticles in the Presence of a Lipid Monolayer

We have investigated the surface activity of poly(ethylene glycol) (PEG)-coated silver nanoparticles (Ag-PEG) in the presence or absence of lipid monolayers comprised of monounsaturated dioleoylphosphocholine and dioleoylphosphoglycerol (DOPC/DOPG; 1:1 mol ratio). Dynamic measurements of surface pressure demonstrated that Ag-PEG were surface-active at the air/water interface. Surface excess concentrations suggested that at high Ag-PEG subphase concentrations, Ag-PEG assembled as densely packed monolayers in the presence and absence of a lipid monolayer. The presence of a lipid monolayer led to only a slight decrease in the excess surface concentration of Ag-PEG. Surface pressure–area isotherms showed that in the absence of lipids Ag-PEG increased the surface pressure up to 45 mN m–1 upon compression before the Ag-PEG surface layer collapsed. Our results suggest that surface activity of Ag-PEG was due to hydrophobic interactions imparted by a combination of the amphiphilic polymer coating and the hydrophobic dodecanethiol ligands bound to the Ag-PEG surface. With lipid present, Ag-PEG + lipid surface pressure–area (π–A) isotherms reflected Ag-PEG incorporation within the lipid monolayers. At high Ag-PEG concentrations, the π–A isotherms of the Ag-PEG + lipid films closely resembled that of Ag-PEG alone with a minimal contribution from the lipids present. Analysis of the subphase silver (Ag) and phosphorus (P) concentrations revealed that most of the adsorbed material remained at the air/lipid/water interface and was not forced into the aqueous subphase upon compression, confirming the presence of a composite Ag-PEG + lipid film. While interactions between “water-soluble” nanoparticles and lipids are often considered to be dominated by electrostatic interactions, these results provide further evidence that the amphiphilic character of a nanoparticle coating can also play a significant role

Hydration Repulsion Effects of the Formation of Supported Lipid Bylayers

When zwitterionic lipids fuse onto substrates such as silica (SiO2), the water of hydration between the two approaching surfaces must be removed, giving rise to an effective hydration repulsion. Removal of water around the polar headgroups of the lipid and the silanols (SiOH) of SiO2 allows supported lipid bilayer (SLB) formation, although an interstitial water layer remains between the lipid and surface. The importance of hydration repulsion in SLB formation is demonstrated by monitoring fusion of zwitterionic lipids onto silica (SiO2) nanoparticles heat treated to control the silanol group (SiOH) density and thus the amount of bound water. SLB formation, observed by cryo-TEM and nanodifferential scanning calorimetry, was found to be slower for the more hydrated surfaces. Although the SiOH density decreased with increasing heat treatment temperature, z-potentials were the same for all the SiO2. This arose since at the pH ¼ 8 of the experiments, only isolated silanols, with a pKa ¼ 4.9, and not hydrogen bonded silanols, with a pKa ¼ 8.5, were dissociated/charged.1 Since there were no differences in double layer forces between the SUVs and SiO2, which are the largest and most important interactions determining lipid fusion onto surfaces,2,3 the slower rate of SLB formation of DMPC onto SiO2 nanoparticles with higher silanol densities and more bound water was therefore attributed to greater hydration repulsion of the more hydrated nanoparticles. For SiO2 heated to 1000 °C, with only a few isolated silanols, little adsorbed water and many hydrophobic Si–O–Si groups, particle aggregation occurred and lipid sheaths formed around the nanoparticle aggregates

Low-dose chemotherapy of hepatocellular carcinoma through triggered-release from bilayer-decorated magnetoliposomes

Low-dose (LD) chemotherapy is a promising treatment strategy that may be improved by controlled delivery. Polyethylene glycol-stabilized bilayer-decorated magnetoliposomes (dMLs) have been designed as a stimuli-responsive LD chemotherapy drug delivery system and tested in vitro using Huh-7 hepatocellular carcinoma cell line. The dMLs contained hydrophobic superparamagnetic iron oxide nanoparticles within the lipid bilayer and doxorubicin hydrochloride (DOX, 2 μM) within the aqueous core. Structural analysis by cryogenic transmission electron microscopy and dynamic light scattering showed that the assemblies were approximately 120 nm in diameter. Furthermore, the samples consisted of a mixture of dMLs and bare liposomes (no nanoparticles), which provided dual burst and spontaneous DOX release profiles, respectively. Cell viability results show that the cytotoxicity of DOX-loaded dMLs was similar to that of bare dMLs (∼10%), which indicates that spontaneous DOX leakage had little cytotoxic effect. However, when subjected to a physiologically acceptable radiofrequency (RF) electromagnetic field, cell viability was reduced up to 40% after 8 h and significant cell death (\u3e90%) was observed after 24 h. The therapeutic mechanism was intracellular RF-triggered DOX release from the dMLs and not intracellular hyperthermia due to nanoparticle heating via magnetic losses. [Refer to PDF for graphical abstract