Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.     

Cationic starch nanoparticles based on polyelectrolyte complexes

Cationic starch nanoparticles were obtained by aqueous polyelectrolyte complex formation between cationic quaternary ammonium substituted starches and anionic sodium tripolyphosphate. The formation of nanosized starch particles of spherical shape was verified by dynamic light scattering and scanning electron microscopy measurements. The cationic starch nanoparticles of different constitution and containing various contents of free quaternary ammonium groups were produced and their zeta potential was modulated between +4 mV and +34 mV by varying polycation/polyanion ratio. Furthermore, the polyelectrolyte complex formation was confirmed by differential scanning calorimetry and FTIR analyses. The thermal stability of cationic starch nanoparticles increased with the introduction of polysalt into polyelectrolyte complex. The solubilization capacity of nanoparticles was varying with the concentration and composition as revealed by fluorescence probe experiments. The capability to accommodate hydrophobic pyrene quest molecule was decreasing with the increasing number of cationic groups in cationic starches and little depended on polyanion/polycation ratio in starch nanoparticles.     

Layer-by-layer engineering of biocompatible,decomposable core-shell structures

The objective of the present investigation was to fabricate composite colloidal particles consisting of a sacrificial, decomposable template of biodegradable nature covered with biocompatible polyelectrolyte multilayers using the layer-by-layer sequential adsorption technique. Poly-dl-lactic acid and poly(dl-lactic-co-glycolic acid) were chosen to design the microparticulate template, and a preliminary feasibility study was carried out with poly(styrene sulfonate sodium)-poly(allylamine hydrochloride) as shell components. The properties of both core-shell and hollow structures obtained by core dissolution were characterized by confocal laser scanning microscopy, microelectrophoresis, scanning force microscopy, and scanning electron microscopy. The concept was then extended to biocompatible polyelectrolytes as shell wall building blocks to deduce stable hollow capsules with tailored properties. Uniform, complete coating with oppositely charged polyelectrolyte pairs was achieved for all the combinations investigated. The results demonstrate that polyester microparticles could serve as viable alternative components to conventionally employed templates to derive hollow capsules with defined size, shape, and shell thickness. With all the components used for fabrication being biocompatible, these polyelectrolyte capsules may find interesting applications in the fields of biology, biochemistry, biotechnology, and drug delivery.     

Facile preparation of well-defined near-monodisperse chitosan/sodium alginate polyelectrolyte complex nanoparticles (CS/SAL NPs) via ionotropic gelification: A suitable technique for drug delivery systems

Polymeric nanoparticles have emerged as a promising approach for drug delivery systems. We prepared chitosan (CS)/sodium alginate (SAL) polyelectrolyte complex nanoparticles (CS/SAL NPs) via a simple and mild ionic gelation method by adding a CS solution to a SAL solution, and investigated the effects of molecular weight of the added CS, and the SAL:CS mass ratio on the formation of the polyelectrolyte complex nanoparticles. The well-defined CS/SAL NPs with near-monodisperse particle size of about 160 nm exhibited a pH stable structure, and pH responsive properties with a negatively or positively charged surface. The so-called “electrostatic sponge” structure of the polyelectrolyte complex nanoparticles enhanced their drug-loading capacity towards the differently charged model drug molecules, and favored controlled release. We also found that the drug-loading capacity was influenced by the nature of the drugs and the drug-loading media, while drug release was affected by the solubility of the drugs in the drug-releasing media. The biocompatibility and biodegradability of the polyelectrolytes in the polyelectrolyte complex nanoparticles were maintained by ionic interactions. These results indicate that CS/SAL NPs can represent a useful technique for pH-responsive drug delivery systems.     

CPMV-polyelectrolyte-templated gold nanoparticles

The use of polyelectrolyte surface-modified Cowpea mosaic virus (CPMV) for the templated synthesis of narrowly dispersed gold nanoparticles is described. The cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), is electrostatically bound to the external surface of the virus capsid; the polyelectrolyte promotes the adsorption of anionic gold complexes, which are then easily reduced, under mild conditions, to form a metallic gold coating. As expected, the templated gold nanoparticles can be further modified with thiol reagents. In contrast, reaction of polyelectrolyte-modified CPMV (CPMV-PA) with preformed gold nanoparticles results in the self-assembly of large, hexagonally packed, tessellated-spheres.     

Surface modification of poly(L-lactic acid) membrane via layer-by-layer assembly of silver nanoparticle-embedded polyelectrolyte multilayer

The improvement of hydrophilicity, antibacterial activity, hemocompatibility, and cytocompatibility of poly(L-lactic acid) (PLLA) membrane was developed via polyelectrolyte multilayer (PEM) immobilization. Colloidal silver nanoparticles were prepared by using dextran sulfate (DS) as a stabilizer to precede chemical reduction by dextrose. The polysaccharide PEMs, including chitosan (CH) and dextran sulfate (DS)-stabilized silver nanosized colloid (DSS), were successfully deposited on the aminolyzed PLLA membrane in a layer-by-layer (LBL) self-assembly manner. The obtained results showed that the contact angle of PLLA membranes decreased with PEMs grafting layers and reached a steady value after four bilayers of coating, hence suggesting that full coverage was achieved. The PLLA-PEM membranes with DSS as the outermost layer could resist platelet adhesion and human plasma fibrinogen (HPF) adsorption, while prolonging the blood coagulation time. The PLLA-PEM membranes could possess antibacterial activity against Methicilin-resistant Staphylococus aureus (MRSA). In addition, the proliferation and viability of human endothelial cells (ECs) on PLLA-PEM membranes could be significantly improved. Overall results demonstrated that such a fast, easy processing and shape-independent method for an antithrombogenic coating can be used for applications in hemodialysis devices.     

Stimuli-Responsive magnetic nanoparticles for monoclonal antibody purification

Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.     

Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.     

Core-shell Au nanoparticle formation with DNA-polymer hybrid coatings using aqueous ATRP

We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.     

In vitro studies with mammalian cell lines and gum arabic-coated magnetic nanoparticles

Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co-precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA-coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures.     

A new concept in polymeric thin-film composite nanofiltration membranes with antibacterial properties

A new, thin film, biofouling resistant, nanofiltration (NF) membrane was fabricated with two key characteristics, viz. a low rate of silver (Ag) release and long-lasting antibacterial properties. In the new approach, nanoparticles were embedded completely in a polymeric thin-film layer. A comparison was made between the new thin-film composite (TFC), NF membrane and thin-film nanocomposite (TFN), and antibacterial NF membranes. Both types of NF membrane were fabricated by interfacial polymerization on a polysulphone sublayer using m-phenylenediamine and trimesoyl chloride as an amine monomer and an acid chloride monomer, respectively. Energy dispersive X-ray (EDX) microanalysis demonstrated the presence of Ag nanoparticles. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to study the cross-sectional and surface morphological properties of the NF membranes. Permeability and salt rejection were tested using a dead-end filtration cell. Ag leaching from the membranes was measured using inductively coupled mass spectrometry (ICP–MS). Morphological studies showed that the TFC NF membranes had better thin-film formation (a more compact structure and a smoother surface) than TFN NF membranes. Performance experiments on TFC NF membranes revealed that permeability was good, without sacrificing salt rejection. The antibacterial properties of the fabricated membranes were tested using the disk diffusion method and viable plate counts. The antibiofouling properties of the membranes were examined by measuring the quantity of bacterial cells released from the biofilm formed (as a function of the amount of biofilm present). A more sensitive surface was observed compared to that of a typical antibacterial NF membrane. The Ag leaching rates were low, which will likely result in long-lasting antibacterial and biofouling resistant properties.     

Protein encapsulation via porous CaCO3 microparticles templating

Porous microparticles of calcium carbonate with an average diameter of 4.75 microm were prepared and used for protein encapsulation in polymer-filled microcapsules by means of electrostatic layer-by-layer assembly (ELbL). Loading of macromolecules in porous CaCO3 particles is affected by their molecular weight due to diffusion-limited permeation inside the particles and also by the affinity to the carbonate surface. Adsorption of various proteins and dextran was examined as a function of pH and was found to be dependent both on the charge of the microparticles and macromolecules. The electrostatic effect was shown to govern this interaction. This paper discusses the factors which can influence the adsorption capacity of proteins. A new way of protein encapsulation in polyelectrolyte microcapsules is proposed exploiting the porous, biocompatible, and decomposable microparticles from CaCO3. It consists of protein adsorption in the pores of the microparticles followed by ELbL of oppositely charged polyelectrolytes and further core dissolution. This resulted in formation of polyelectrolyte-filled capsules with protein incorporated in interpenetrating polyelectrolyte network. The properties of CaCO3 microparticles and capsules prepared were characterized by scanning electron microscopy, microelectrophoresis, and confocal laser scanning microscopy. Lactalbumin was encapsulated by means of the proposed technique yielding a content of 0.6 pg protein per microcapsule. Horseradish peroxidase saves 37% of activity after encapsulation. However, the thermostability of the enzyme was improved by encapsulation. The results demonstrate that porous CaCO3 microparticles can be applied as microtemplates for encapsulation of proteins into polyelectrolyte capsules at neutral pH as an optimal medium for a variety of bioactive material, which can also be encapsulated by the proposed method. Microcapsules filled with encapsulated material may find applications in the field of biotechnology, biochemistry, and medicine.     

Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.     

Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4–5 nm x-y size for the smallest to 40–70 nm for the largest, and of channel-like structures 25–30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric range.Abbreviations AFM atomic-force microscopy - EM electron microscopy - TMAFM tapping-mode atomic-force microscopy     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein -chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.     

Preparation and antibacterial activity of chitosan nanoparticles

Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.     

细胞膜仿生修饰纳米粒肿瘤治疗的研究进展

大量研究证明,细胞膜仿生修饰通过将不同细胞膜包被于纳米粒表面,赋予纳米粒新的生物学功能.纳米粒被细胞膜仿生修饰后,获得了细胞膜表面丰富的蛋白质并保留了纳米粒的高载药能力,延长体内循环时间,使纳米粒具有逃避免疫系统,跨越各种生理屏障的能力.本文总结了近年来细胞膜仿生修饰纳米粒用于肿瘤治疗的最新进展,讨论了细胞膜仿生修饰纳...     

Study on the Effect of Poly(Styrene 4-Sulfonic Acid-co-Maleic Acid) Toward Metallization and Plasmonic Tuning of Silver Nanoparticle Thin Films

Thin films with tunable optical properties from yellow to metallic were prepared from a monolayer coating of silver nanoparticles (AgNP) onto a polyelectrolyte multilayer (PEM) thin film. The AgNP were synthesized using various concentrations of stabilizing polyelectrolytes leading to a competitive adsorption concept in which AgNP compete with excess polyelectrolytes to coat the cationic PEM top layer. The AgNP were synthesized by chemical reduction of Ag salts using poly(styrene 4-sulfonic acid-co-maleic acid) (PSS-co-MA) as stabilizing agent to produce nanoparticles coated with both a strong acid (sulfonic) and a weak acid (carboxylic) moiety. Although all the nanoparticle solutions displayed a characteristic bright yellow due to the localized surface plasmon band around 420 nm, the monolayer films of nanoparticles obtained after dipping displayed striking different optical properties. When using a high PSS-co-MA content in the solution, a pale-yellow film was obtained which color shifted to orange and metallic when the capping concentration was decreased from 0.25 to 0.001 mM. The optical properties of the AgNP film could be further changed by galvanic replacement of the Ag with gold ions to produce a gold monolayer. These results are interesting to produce surface with tunable catalytic properties, tunable optical properties, or to be used as primer for the metallization of polymeric surfaces.

     

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high-throughput analysis of mumps virus and a mumps virus-specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11-mercaptoundecanoic acid (MUA). Poly-L-lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly-L-lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 x 10(5) to 14 x 10(5) pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose-dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high-throughput analysis of intact virus and serodiagnosis of infectious diseases.