RESUMO
The dynamic behavior of polyelectrolytes just after their encounter with the surface of bare colloidal particles is analyzed, using the flocculation properties of mono-dispersed polystyrene latex (PSL) particles. Applying a Standardized Colloid Mixing (SCM) approach, effects of ionic strength and charge density of polymer chain on the rate of flocculation, the electrophoretic mobility of particle coated with polyelectrolyte, and the thickness of adsorbed polymer layer were analyzed, focusing on distinguishing features of two modes of flocculation, namely bridging formation and charge neutralization. In the case of excess polymer dosage, the bridging flocculation clearly highlights the transient behavior of polymer conformation from random-coil-like in bulk solution to increasingly flatten on the surface. The adsorption of polymer chains leads to a stagnant layer of solvent near the solid wall, which is confirmed by electrokinetic data. In the regime near optimum dosage two cases emerge. For high charge density polymer, charge neutralization is dominant and advantageous for the continuous progress of flocculation by heterogeneous double layer interaction. As a function of elapsed time after the onset of mixing, crossover from bridging to charge neutralization is found. In the case of low charge density polymer, bridging flocculation is the mechanism. Fluid mixing is concluded to have an essential role in the formation of bridges.
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Soft microgel particles inherently possess qualities of both polymers as well as particles. We review the similarities and differences between soft microgel particles and stiff colloids at fluid-fluid interfaces. We compare two fundamental aspects of particle-laden interfaces namely the adsorption kinetics and the interactions between adsorbed particles. Although it is well established that the transport of both hard particles and microgels to the interface is driven by diffusion, the analysis of the adsorption kinetics needs reconsideration and a proper equation of state relating the surface pressure to the adsorbed mass should be used. We review the theoretical and experimental investigations into the interactions of particles at the interface. The rheology of the interfacial layers is intimately related to the interactions, and the differences between hard particles and microgels become pronounced. The assembly of particles into the layer is another distinguishing factor that separates hard particles from soft microgel particles. Microgels deform substantially upon adsorption and the stability of a microgel-stabilized emulsion depends on the conformational changes triggered by external stimuli.
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Viruses are among the simplest biological systems and are highly effective vehicles for the delivery of genetic material into susceptible host cells. Artificial viruses can be used as model systems for providing insights into natural viruses and can be considered a testing ground for developing artificial life. Moreover, they are used in biomedical and biotechnological applications, such as targeted delivery of nucleic acids for gene therapy and as scaffolds in material science. In a natural setting, survival of viruses requires that a significant fraction of the replicated genomes be completely protected by coat proteins. Complete protection of the genome is ensured by a highly cooperative supramolecular process between the coat proteins and the nucleic acids, which is based on reversible, weak and allosteric interactions only. However, incorporating this type of supramolecular cooperativity into artificial viruses remains challenging. Here, we report a rational design for a self-assembling minimal viral coat protein based on simple polypeptide domains. Our coat protein features precise control over the cooperativity of its self-assembly with single DNA molecules to finally form rod-shaped virus-like particles. We confirm the validity of our design principles by showing that the kinetics of self-assembly of our virus-like particles follows a previous model developed for tobacco mosaic virus. We show that our virus-like particles protect DNA against enzymatic degradation and transfect cells with considerable efficiency, making them promising delivery vehicles.
Assuntos
Proteínas do Capsídeo/química , DNA/administração & dosagem , Transfecção/métodos , Vírus/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , DNA/genética , Células HeLa , Humanos , Modelos Moleculares , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus/genética , Vírus/metabolismoRESUMO
Understanding the adsorption dynamics of soft microgel particles is a key step in designing such particles for potential applications as stimuli-responsive Pickering stabilizers for foams or emulsions. In this study we experimentally determine an equation of state (EOS) for poly (N-isopropylacrylamide) (PNIPAM) microgel particles adsorbed onto an air-water interface using a Langmuir film balance. We detect a finite surface pressure at very low surface concentration of particles, for which standard theories based on hard disk models predict negligible pressures, implying that the particles must deform strongly upon adsorption to the interface. Furthermore, we study the evolution of the surface pressure due to the adsorption of PNIPAM particles as a function of time using pendant drop tensiometry. The equation of state determined in the equilibrium measurements allows us to extract the adsorbed amount as a function of time. We find a mixed-kinetic adsorption that is initially controlled by the diffusion of particles towards the interface. At later stages, a slow exponential relaxation indicates the presence of a coverage-dependent adsorption barrier related to crowding of particles at the interface.
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The distribution of ions and charge at solid-water interfaces plays an essential role in a wide range of processes in biology, geology and technology. While theoretical models of the solid-electrolyte interface date back to the early 20th century, a detailed picture of the structure of the electric double layer has remained elusive, largely because of experimental techniques have not allowed direct observation of the behaviour of ions, i.e. with subnanometer resolution. We have made use of recent advances in high-resolution Atomic Force Microscopy to reveal, with atomic level precision, the ordered adsorption of the mono- and divalent ions that are common in natural environments to heterogeneous gibbsite/silica surfaces in contact with aqueous electrolytes. Complemented by density functional theory, our experiments produce a detailed picture of the formation of surface phases by templated adsorption of cations, anions and water, stabilized by hydrogen bonding.
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The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0-80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration-induced inactivation that takes place during the first activity measurement, (2) a phase of first-order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well.
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Biocatálise , Dipeptídeos/biossíntese , Furanos/metabolismo , Subtilisinas/metabolismo , Desidratação , Furanos/química , CinéticaRESUMO
Coating DNA is an effective way to modulate its physical properties and interactions. Current chemosynthetic polymers form DNA aggregates with random size and shape. In this study, monodisperse protein diblock copolymers are produced at high yield in recombinant yeast. They carry a large hydrophilic colloidal block (≈400 amino acids) linked to a short binding block (≈12 basic amino acids). It is demonstrated that these protein polymers complex single DNA molecules as highly stable nanorods, reminiscent of cylindrical viruses. It is proposed that inter- and intramolecular bridging of DNA molecules are prevented completely by the small size of the binding block attached to the large colloidal stability block. These protein diblocks serve as a scaffold that can be tuned for application in DNA-based nanotechnology.
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DNA/química , Polímeros/química , Engenharia de Proteínas/métodos , Sistema Livre de Células , Coloides/química , Eletroforese em Gel de Poliacrilamida , Técnicas de Transferência de Genes , Vetores Genéticos , Interações Hidrofóbicas e Hidrofílicas , Luz , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Peptídeos/química , Pichia , Plasmídeos/metabolismo , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Propriedades de Superfície , Água/químicaRESUMO
Estimating the environmental exposure to manufactured nanomaterials is part of risk assessment. Because nanoparticles aggregate with each other (homoaggregation) and with other particles (heteroaggregation), the main route of the removal of most nanoparticles from water is aggregation, followed by sedimentation. The authors used water samples from two rivers in Europe, the Rhine and the Meuse. To distinguish between small (mainly natural organic matter [NOM]) particles and the remainder of the natural colloids present, both filtered and unfiltered river water was used to prepare the particle suspensions. The results show that the removal of nanoparticles from natural river water follows first-order kinetics toward a residual concentration. This was measured in river water with less than 1 mg L(-1) CeO(2) nanoparticles. The authors inferred that the heteroaggregation with or deposition onto the solid fraction of natural colloids was the main mechanism causing sedimentation in relation to homoaggregation. In contrast, the NOM fraction in filtered river water stabilized the residual nanoparticles against further sedimentation for up to 12 d. In 10 mg L(-1) and 100 mg L(-1) CeO(2) nanoparticle suspensions, homoaggregation is likely the main mechanism leading to sedimentation. The proposed model could form the basis for improved exposure assessment for nanomaterials.
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Coloides/química , Modelos Químicos , Nanopartículas/química , Rios/química , Fenômenos Químicos , Cinética , Nanoestruturas/química , Países Baixos , Suspensões/química , Poluentes Químicos da Água/químicaRESUMO
With the aim of determining suitable conditions for uptake and release of globular proteins on microgels, we studied the interaction between phosphated, highly cross-linked, negatively charged oxidized potato starch polymer (OPSP) microgel particles and lysozyme from hen eggs. Our microgel shows a typical protein-induced deswelling behavior for charged microgels. The protein distributes rather homogenously through the microgel. We found that at low salt concentration the saturation protein uptake Gammasat increases with increasing pH. This is because the binding capacity is mainly determined by charge compensation: with increasing pH, the (positive) charge on the lysozyme molecules decreases, while the (negative) charge of the microgel particles increases. Therefore, more protein molecules are needed to compensate for the charge on the gel and the binding capacity increases. The protein binding affinity, however, decreases sharply with increasing pH, presumably because this affinity is mainly sensitive to the lysozyme charge density. At high pH the binding affinity is relatively low, and by adding salt, the protein can easily be released from the gel. This leads to a maximum in the curves of Gammasat versus pH, and this maximum shifts to lower pH values with increasing ionic strength. We conclude that, for protein uptake and release applications, the present system works best around pH 5 due to a sufficiently high binding affinity and a sufficiently high binding capacity.
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Portadores de Fármacos/química , Géis/química , Muramidase/química , Animais , Galinhas , Feminino , Concentração de Íons de Hidrogênio , Concentração Osmolar , Oxirredução , Polímeros/química , Amido/químicaRESUMO
Vaccine efficacy is strongly enhanced by antibody-mediated targeting of vaccine components to dendritic cells (DCs), which are professional antigen presenting cells. However, the options to link antigens or immune modulators to a single antibody are limited. Here, we engineered versatile nano- and micrometer-sized slow-release vaccine delivery vehicles that specifically target human DCs to overcome this limitation. The nano- (NPs) and microparticles (MPs), with diameters of approximately 200nm and 2microm, consist of a PLGA core coated with a polyethylene glycol-lipid layer carrying the humanized targeting antibody hD1, which does not interact with complement or Fc receptors and recognizes the human C-type lectin receptor DC-SIGN on DCs. We studied how these particles interact with human DCs and blood cells, as well as the kinetics of PLGA-encapsulated antigen degradation within DCs. Encapsulation of antigen resulted in almost 38% degradation for both NPs and MPs 6days after particle ingestion by DCs, compared to 94% when nonencapsulated, soluble antigen was used. In contrast to the MPs, which were taken up rather nonspecifically, the NPs effectively targeted human DCs. Consequently, targeted delivery only improved antigen presentation of NPs and induced antigen-dependent T cell responses at 10-100 fold lower concentrations than nontargeted NPs.
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Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos , Lectinas Tipo C/imunologia , Anticorpos/imunologia , Anticorpos/metabolismo , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos/imunologia , Antígenos/metabolismo , Moléculas de Adesão Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Ácido Láctico , Lectinas Tipo C/metabolismo , Polietilenoglicóis/metabolismo , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptores de Superfície Celular , Vacinas/imunologia , Vacinas/metabolismoRESUMO
The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.
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Lactalbumina/farmacocinética , Muramidase/farmacocinética , Adsorção , Animais , Galinhas , Feminino , Cinética , Dióxido de SilícioRESUMO
The influence of succinylation on lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability of succinylated lysozyme is lower than that of native lysozyme. For succinylated lysozyme, the denaturation temperature (Td) varies in the range of 325-333 K (52-60 degrees C) and the associated denaturation enthalpy (DeltadenH) varies between 225 and 410 kJ/mol. For lysozyme, Td is 342-349 K (69-76 degrees C) and DeltadenH is 440-500 kJ/mol. From these data, the change in the heat capacity (DeltadenCp) upon thermal denaturation is derived. For lysozyme, DeltadenCp is 7.5 kJ/mol/K, and for succinylated lysozyme, it is 16.7 kJ/mol/K. The value of DeltadenCp for lysozyme is comparable to previously reported values. The high value of DeltadenCp for succinylated lysozyme is explained in terms of an extended degree of unfolding of the secondary structure and exposure of the apolar parts of the succinyl groups. Furthermore, the Gibbs energy of denaturation, as a function of temperature, derived from the thermodynamic analysis of the calorimetric data, indicates a cold-denaturated state of succinylated lysozyme below 20 degrees C. However, because a denatured state at low temperatures could not be detected by CD or fluorescence measurements, the native state may be considered to be metastable at those conditions.
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Temperatura Alta , Muramidase/química , Ácido Succínico/química , Varredura Diferencial de Calorimetria , Estabilidade Enzimática , Estrutura Molecular , Desnaturação Proteica , Espectrometria de Fluorescência , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The influence of electrostatic interactions on protein adsorption was studied by comparing the adsorption of lysozyme and succinylated lysozyme at silica surfaces. The succinylation affects the charge of the protein, but also the stability. Although changes in stability can have an influence on adsorption, our data show that the primary effect can be entirely understood in terms of electrostatic interactions. The adsorbed amount as a function of pH has a maximum for both proteins. This maximum coincides with the isoelectric point for succinylated lysozyme, and is close to the isoelectric point for lysozyme. At pH values where the protein is electrostatically repelled by the sorbent, higher ionic strengths increase adsorption, and for electrostatic attraction higher ionic strengths decrease adsorption.