Interaction of polycations with cell-surface negative charges of epithelial cells.

The interaction between various polycations and cultured glomerular epithelial cells was studied by cell electrophoresis. It was shown that the glomerular epithelial cell presents a negatively charged surface which imparts a zeta potential of -29.0 +/- 1.5 mV at the peripheral layer of the plasma membrane. The pH at which the GEC charge became 50% reduced (pKa) was determined to be 3.0. A variety of polycations of various sizes and fixed and flexible geometries were tested for their capacity to neutralize the cell charge. All the polycations except cytochrome c and lysozyme were capable of completely neutralizing the cell. Cytochrome c could maximally neutralize only 50% of charge and lysozyme only 72% of charge. However, reduced and 'relaxed' molecules of cytochrome c and lysozyme efficiently neutralized the cell surface, as did larger sized 'flexible' polylysines. On the basis of these findings, we conclude that all polycations are not equal in their capacity to neutralize the cell surface. Flexible molecules in contrast to molecules with rigid structures were more effective in neutralizing the cell. This may likely be due to the exposure and availability of more cationic groups in a flexible molecule which results in stabilization of interaction with cells.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Physicochemical characterization of Escherichia coli. A comparison with gram-positive bacteria.

Eight Escherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r = 0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2-11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r = 0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH = 7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negative E. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS.     

XPS and ab initio calculation of surface states of sulfide minerals: pyrite,chalcopyrite and molybdenite

Sulfide minerals contain sulphur in a large variety of coordination environments. Consequently, the S 2p XPS of various mineral surface states undergo different shifts in binding energy (BE) relative to the bulk, depending on the charge distribution on the surface. This in turn depends on the number, type and position of the atoms on the fracture surface, which is determined by the fracture mechanism.

We have investigated three sulfide minerals: pyrite (tetrahedrally-coordinated S), chalcopyrite (tetrahedrally-coordinated S) and molybdenite (layered structure with trigonally-coordinated S). Comparison of conventional with surface sensitive synchrotron XPS shows that the S 2p spectrum displays two additional doublets at lower BE than the bulk signal for pyrite, and one doublet each at lower and at higher BE for chalcopyrite. Each of these signals is derived from surface states. Molybdenite shows no additional states. A BE shift to lower (higher) BE suggests a charge increase (decrease) on the S atoms relative to those in the bulk because of higher (lower) charge screening.

We have used ab initio density functional calculations to validate this interpretation of the experimental evidence, obtaining Mulliken population analyses for the possible fracture surfaces and comparing their charge distribution with the corresponding bulk charge distribution. Our calculations support the assignments of S 2p surface contributions as follows: the lower BE peak of chalcopyrite (160.84 eV) as under-coordinated surface S states, the higher BE peak of chalcopyrite (161.88 eV) as surface S polymers, the lowest BE peak of pyrite (161.3 eV) as surface S monomers, and the next lowest BE peak of pyrite (162.0 eV) as under-coordinated surface S dimers. The absence of any surface states in molybdenite is also confirmed by the models.     

Physicochemical characterization ofEscherichia coli

EightEscherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r=0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2–11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r=0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH=7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negativeE. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS.     

Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

Changes in physico-chemical properties of human large intestine tumour cells membrane

Tumour cells produce and excrete to blood many substances which are present in the cell itself in trace amounts only. Our work has been aimed at the determination of changes in electric charge and in phospholipid composition of large intestine normal mucosa and colorectal cancer cells.Surface charge density of tumour unaffected mucosa and of tissue sections from tumours, was measured by electrophoresis. The measurements were carried out at various pH of solution. Membrane isoelectric point was determined by measuring its electric charge in function of pH as well as total positive charge at low pH and total negative charge at high pH. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC. Four phospholipid classes were identified: PI, PS, PE and PC and their surface concentrations were determined.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.Tumour changes provoke an increase in surface charge density of large intestine membrane, whereas the content of individual phospholipids increased or decreased depending on a patient.     

Depth profiling of the elemental surface composition of the oral microorganismS. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass ofStreptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain,S. salivarius HBC12 and onS. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain,S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed forS. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

Changes in surface charge of mouse neuroblastoma cells during growth and morphological differentiation of the cell population

The surface charge of neuroblastoma cells (clone C1300-N18TG₂) was studied by microelectrophoresis. The surface charge of these cells was shown to be determined mainly by anionic groups of the membrane, located in a layer about 10 mm thick, with a density of 0.2 e/nm³, covering its outer surface. These groups interact with Ca ions with a binding constant of K_Ca=10–50 liters/mole and titrate corresponding to pK=3.8. The electrophoretic mobility of the neuroblastoma cells is reduced by trypsin, neuraminidase, and N-bromosuccinimide, which irreversibly neutralizes carboxyl groups, and is increased on treatment of the cells with tosyl chloride — a specific reagent for amino groups. The value of the surface charge also depends on the conditions of culture of the cell population. The process of morphological differentiation of the cells (termination of division, dendrite formation), induced by removal of serum from the medium, leads to an increase of about 30% in their electrophoretic mobility. If cells are cultured in medium containing 10 and 50% of blood serum, enabling them to multiply, variations are observed in the mean electrophoretic mobility, which are opposite in phase to the 24-hourly increase in the number of cells. It is suggested that these effects are determined by partial "self-synchronization" of the cell population. It is concluded that the surface charge of neuroblastoma cells, measured by the microelectrophoresis method, is determined mainly by carboxyl groups of peripheral proteins and gangliosides of the membrane, and that the content of these compounds in the membrane depends on the phase of cell development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 168–174, March–April, 1985.     

Spectroscopic study of extracellular polymeric substances from Bacillus subtilis: aqueous chemistry and adsorption effects

Reactions at ionizable functional groups in extracellular polymeric substances (EPS) from Bacillus subtilis are found to affect aqueous phase conformation and adsorption to mineral surfaces. Characterization by HPSEC, XPS, and FTIR indicates a wide range in apparent molecular mass (0.57-128 kDa), with functional group composition depending on cell growth phase (exponential vs stationary) and location in suspension (free vs cell-bound). ATR-FTIR spectroscopy shows complexation and dissociation of protons on acidic functional groups that result in alpha-helical protein conformation at pH < 2.6 and random coil (unordered) conformation at higher pH (>6). EPS exhibit higher affinity for adsorption to alpha-FeOOH than amorphous SiO(2) because of surface charge effects. Increased amide II band intensity and an amide I band shift to higher frequency indicate changes in protein structure upon adsorption. Goethite-EPS spectra show emergent vibrations consistent with P-O-Fe bonding, which suggests a role of phosphodiester groups in the adsorption reaction.     

Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa

Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, ¹³C and ²⁹Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68?nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix.     

Spatial distribution of mammalian cells dictated by material surface chemistry

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n-octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time-dependent process. Angle dependent x-ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was immobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.     

Amphiphilic self-assembled "polymeric drugs": morphology, properties, and biological behavior of nanoparticles

This article reports the fabrication and characterization of NPs based on the self-assembling of polymeric drugs with amphiphilic character synthetized from oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside methacrylate and vinyl pyrrolidone (OAGMA-VP). NPs were spherical, with an apparent hydrodynamic diameter between 91 and 226 nm and with zeta potential values that ensure stability. Atomic concentrations of C, O, and N, determined by X-ray photoelectron spectroscopy (XPS) of NPs, compared well with the corresponding theoretical values. High resolution XPS C1s spectra suggest that the carbons bound to heteroatoms or carbonyl groups are preferentially situated on the surface of the NP samples. ToF-SIMS spectra analyzed by principal component analysis (PCA) indicated that ions coming from acetyl and oleyl groups of OAGMA play important roles in the outer structure of NPs. Water contact angle and surface tension values of NPs were characteristic of hydrophilic surfaces, confirming the location of VP sequences on the surface. Cell culture assays showed that copolymeric NPs did not compromise biocompatibility of human fibroblasts according to ISO standard, but they were cytotoxic on a human glioblastoma cell line (A-172).     

Engineering surfaces for bioconjugation: developing strategies and quantifying the extent of the reactions

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.     

Changes in surface-charge density of blood cells after sudden unexpected death

The objective of the investigation was evaluation of postmortem changes of electric charge of human erythrocyte and thrombocyte membranes after sudden unexpected death. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells carried out at various pHs of electrolyte solution. The interactions between both erythrocyte and thrombocyte membranes and electrolyte ions were studied. Values of parameters characterizing the membrane--that is, the total surface concentrations of both acidic and basic groups and their association constants with solution ions--were calculated on the basis of a four-equilibria mathematical model. The model was validated by comparison of these values to experimental data. We established that examined electric properties of the cell membranes are affected by sudden unexpected death. Postmortem processes occurring in the cell membranes can lead to disorders of existing equilibria, which in turn result in changes in values of all the above-mentioned parameters.     

Characterisation of ionogenic groups and estimation of the net negative electric charge on the surface of cells using natural pH gradients

The technique of isoelectric focusing has been extended to the study of the cell surface. A few tumour cell types and normal liver cells have been examined and are found to have characteristic isoelectric points. The isoelectric point of a cell, it is shown, provides information about the ionogenic groups present on its surface. The net electric charge borne by cells at their isoelectric points can be used to predict their electrophoretic mobilities in buffers at physiological pH and ionic strengths.     

X-ray photoelectron spectroscopy analysis of whole cells and isolated cell walls of gram-positive bacteria: comparison with biochemical analysis.

The surface chemical composition of whole cells and isolated cell walls of four coryneform bacteria and of a Bacillus brevis strain has been determined by X-ray photoelectron spectroscopy (XPS). The XPS data were converted into concentrations of model compounds: peptides, polysaccharides, and hydrocarbonlike compounds. The composition of the surface of B. brevis differed markedly from that of coryneforms: the peptide concentration was about twice higher in the former case, which is attributed to the presence of an S-layer at the cell surface; in contrast, the surface of coryneforms was rich in hydrocarbonlike compounds (about 40%), which was concomitant with a high water contact angle. The peptide surface concentration of the isolated cell walls of the five strains deduced from XPS data fitted well with the total peptide content determined by biochemical analysis, which supports the validity of XPS to determine the overall macromolecular composition of the bacterial cell surface. Compared to biochemical analysis of isolated cell walls, XPS analysis of whole cells provides information which concerns directly the cell surface (2- to 5-nm-thick layer) and is less subject to alteration via losses of cell wall constituents or contamination by intracellular compounds.     

Changes in cell surface anionogenic groups during differentiation of Herpetomonas samuelpessoai mediated by dimethylsulfoxide

The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treated Herpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation of H. samuelpessoai induced by DMSO treatment caused a significant increase in the net negative surface charge. In flagellates exposed to DMSO, more cationized ferritin, colloidal iron hydroxide, and sendai virus particles bound to the cell surface. Treatment of both untreated and DMSO-treated flagellates with neuraminidase decreased markedly the EPM of cells to the cathodic pole. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. samuelpessoai. Thin-layer chromatography showed that N-acetyl and N,O-diacylneuraminic acids, in equal proportions, were present in H. samuelpessoai. However, N-acetylneuraminic acid predominates in DMSO-treated cells.     

Interactions of erythrocytes with an artificial wall: influence of the electrical surface charge

Electrical charge on any biological surface plays a crucial role in its interaction with other molecules or surfaces. Here, we study, under flow conditions, the interactions of erythrocytes with an artificial surface: a platinum microelectrode whose charge density ranges from –15 to +27 μC/cm². This artificial surface could be similar in surface charge to an endothelium or a biomaterial. In this model, interactions are measured as a transient relative increase of the electrolyte resistance obtained by impedance measurement of a microelectrode. A maximal interaction of erythrocytes with the charged surface is calculated in the 0 to +10 μC/cm² charge density range. At negative surface charge, a less efficient contact was obtained because of electrostatic repulsion forces. High positive surface charge (charge density >10 μC/cm²) does not improve the contact but induces a progressive decrease in the contact efficiency, which could be explained by a rearrangement of macromolecules on the erythrocyte surface or an effect of positive groups on the cell membrane. This work suggests that a greater surface area of contact is obtained in the 0 to +10 μC/cm² charge density range and that this is provided by more molecular bridges. Received: 23 February 1996 / Accepted: 26 April 1996     

Electrophoretic Studies on Plant Protoplasts II. Relative Amounts of Various Charged Groups on the Surface of Barley Mesophyll Protoplasts

Electrophoretic mobilities of barley mesophyll cell protoplastsmodified by chemical and enzymatic treatments were measuredin media at various pH values to elucidate the contributionof phosphate, carboxylate and amino groups to the surface chargedensity. Existence of these charged groups was confirmed byresults of treatment of protoplasts with glutaraldehyde (foramino groups), acid phosphatase (for phosphate groups) and l-ethyl-3-(3-dimethylaminopropyl)carbodi-imidetogether with glycine methyl ester (for carboxylate groups).The relative amounts of these groups were estimated from thecurves of surface charge density () vs. surface pH (pH_s) ofthe treated protoplasts, in terms of simplified acid-base dissociationcurves. The estimated ratio of the amounts of phosphate, carboxylateand amino groups was approximate 0.5 :0.5 : 0.7 for the native(unmodified) barley mesophyll cell protoplasts, when the totalnegative charge on the native protoplasts was assumed to be1. (Received January 9, 1989; Accepted April 28, 1989)