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Surface properties of spider silk particles in solution
Abstract
Recombinant spider silk proteins, such as eADF4(C16), can be used for various applications. Colloidal particles of eADF4(C16) show potential as drug delivery systems. Tuning the colloidal properties of suspensions of eADF4(C16) particles represents a major prerequisite for their use in pharmaceutical formulations. In this study we determined the surface properties concerning inter-particle interactions by means of electrophoretic mobility and direct force measurements. The surface charge of eADF4(C16) spider silk particles was determined as a function of ionic strength and pH, respectively. The resulting electrophoretic mobility can be described using the O'Brien and White theory and is directly related to the amino acid sequence of the protein. We determined the extension of a fuzzy protein layer protruding into the solution by direct force measurements using a colloidal probe technique. This soft layer leads to deviations in the electrophoretic mobility and is responsible for additional repulsive forces at small separation distances. These steric forces lead to a stabilization of the particle suspension at high ionic strength.
... Electrophoretic methods are, in this respect, valuable techniques commonly used for the analytical characterization of spidroin particles. 7,13,14 A basic theory for electrophoresis of so-called hard particles, i.e., particles impermeable to ions and solvent, has been developed about a century ago and refined over the years to, e.g., account for surface ion-conduction processes and electric double layer polarization. 18 The numerical treatment of the standard electrokinetic model by O'Brien and White 19 is pivotal for analyzing situations where simplified analytical equations derived within the framework of the Debye−Huckel approximation are not applicable. ...
... 14,30,31 However, a recent study combining electrophoretic mobility with direct force measurements demonstrated that for such recombinant spidroin particles a pronounced diffuse interface with protruding protein segments exists, limiting the application of the classical O'Brien−White theory 19 (valid only for hard colloidal particles) to provide a comprehensive interpretation of the electrophoretic mobility as a function of pH and solution ionic strength. 13 Since the features of the spidroin particle surface depend critically on the production processing by salting-out, 32 a large number of parameters has to be taken into account. 12 Here, we address how far electrokinetic methods can be employed to characterize protein particles and to predict their overall colloidal stability. ...
... Beside both types of spidroin particles, silica particles (average diameter 6.8 μm, Bangs Laboratories Inc., Fishers, IN, U.S.A.) were immobilized on the functionalized dishes as "hard" internal standard. 13 The FluidFM setup enables the use of exchangeable colloidal probes. 35 First, the cantilever was approached near a silica particle, and an aspiration pressure of −800 mbar was applied. ...
Colloidal particles have been prepared from polyanionic and polycationic recombinant spider silk protein. The amino acid sequences of these spider silk proteins are identical except for 16 residues bearing either a cationic or an anionic ionizable group. Electrophoretic titration showed that protonation of the acidic and basic amino acids had significant impact on the electrophoretic mobility of the protein particles and, in particular, on their point of zero mobility (PZM). The experimentally determined PZMs are in good agreement with the theoretical values evaluated on the basis of the relevant amino acid sequences. A comprehensive description of the electrokinetic properties of the recombinant spider silk protein particles as a function of pH and solution ionic strength was provided from adequate application of electrokinetic theory for soft particles. Within the framework of this formalism, spider silk protein particles are viewed as porous colloids penetrable for ions and characterized by a finite penetration length for the electroosmotic flow. The differentiated electrokinetic properties of the particles were shown to be solely governed by the electrohydrodynamic features of their poorly charged outer peripheral layer with a thickness of about 10-20 nm. This finding was further corroborated experimentally by demonstrating that electrokinetics of particles bearing an additional outer layer consisting of oppositely charged spider silk proteins is entirely dominated thereby. The presence of a fuzzy, ion-permeable particle interface with an extension of several tenths of a nanometer was confirmed by direct measurement of the resulting steric forces using the colloidal probe atomic force microscopy (AFM) technique.
... Previously, it was shown that self-assembly of eADF4(C16) is triggered in the presence of phosphate ions (Pi). High concentrations of Pi (>400 mM) yield particles upon fast salting out and phase separation (Helfricht et al., 2013;Lammel et al., 2008;Slotta et al., 2008), while low Pi concentrations (<300 mM) trigger self-assembly into nano-fibrillar structures Slotta et al., 2007Slotta et al., , 2008 (Fig. 1 in Ref. (Humenik et al., 2015)). Assembly kinetics of eADF4(C16) fibrils could be explained by a nucleation dependent mechanism (Cohen et al., 2012;Eichner and Radford, 2011;Jarrett and Lansbury, 1993). ...
... In order to analyze the putative nucleation interface in more detail, b-sheet rich eADF4(C16) particles with a well-defined polymer-brush-like surface (Helfricht et al., 2013) were tested as seeds for fibril formation. eADF4(C16) was labeled either with the fluorophore N-hydroxysuccinimide-fluorescein or N-hydroxysuccinimide-rhodamine. ...
... eADF4(C16) monomers could also assemble on the surface of sub-micrometer eADF4(C16) particles (Fig. 6vi). Taking into account that eADF4(C16) particle surfaces reveal an interfacial layer of exposed brush-like protein chains (Helfricht et al., 2013), it is likely the sequence (polyalanine stretches) and its conformation on the interfacial layer surface that triggers fibril assembly on eADF4(C16) seeds and nuclei (represented as corresponding pale gray areas in Fig. 6). ...
Recombinant eADF4(C16) represents an engineered spider silk variant based on the sequence of the core domain of the natural dragline silk protein ADF4 of Araneus diadematus. Previously eADF4(C16) has been shown to self-assemble into cross- fibrils in a two-step process of nucleus formation and fibril growth. Here, it is shown that structurally converted low molecular weight oligomers can act as nuclei. Further, it could be determined that specifically potassium and phosphate ions strongly influence both nucleus formation as well as fibril growth. Nucleation of fibril assembly could be surpassed by seeding soluble protein with pre-assembled fibrils but also, unexpectedly, with eADF4(C16) sub-micrometer particles. The latter finding reveals that spider silk fibril assembly seems to be rather dependent on the protein sequence than on the structural features.
Copyright © 2015. Published by Elsevier Inc.
... Furthermore, silk materials are nontoxic and have low immunogenicity [7,[98][99][100][101]. Finally, silk materials have excellent mechanical stability and a controllable format and size, and they can be stored in a dried state because of their reversible swelling behavior [102], offering unlimited opportunities for the fabrication, functionalization, and processing of robust biomaterials. Figure 1. ...
For years, surgery, radiotherapy, and chemotherapy have been the gold standards to treat cancer, although continuing research has sought a more effective approach. While advances can be seen in the development of anticancer drugs, the tools that can improve their delivery remain a challenge. As anticancer drugs can affect the entire body, the control of their distribution is desirable to prevent systemic toxicity. The application of a suitable drug delivery platform may resolve this problem. Among other materials, silks offer many advantageous properties, including biodegradability, biocompatibility, and the possibility of obtaining a variety of morphological structures. These characteristics allow the exploration of silk for biomedical applications and as a platform for drug delivery. We have reviewed silk structures that can be used for local and systemic drug delivery for use in cancer therapy. After a short description of the most studied silks, we discuss the advantages of using silk for drug delivery. The tables summarize the descriptions of silk structures for the local and systemic transport of anticancer drugs. The most popular techniques for silk particle preparation are presented. Further prospects for using silk as a drug carrier are considered. The application of various silk biomaterials can improve cancer treatment by the controllable delivery of chemotherapeutics, immunotherapeutics, photosensitizers, hormones, nucleotherapeutics, targeted therapeutics (e.g., kinase inhibitors), and inorganic nanoparticles, among others.
... Measurement data were collected at the stationary planes for at least three times. The details on the procedure can be found elsewhere [57]. ...
Clays are not only ubiquitous in nature, but they are also used in huge quantities in a broad range of industrial applications, such as thixotropic drilling fluids, ore pelletizers, waste disposal sealants, or fillers in polymer nanocomposites. In order to model environmental processes or to design new materials on a rational base, it is of prime importance to determine and possibly modify the interfacial properties of clay platelets at the solid/electrolyte interface. In this context, the fundamental question rises how far the stoichiometric interlayer charges as determined by the composition of the silicate layer correlates with the diffuse double-layer properties. Here, this question is addressed by means of a series of purposely synthesized sodium 2:1 layered silicates with defined composition and hence interlayer charge densities, respectively. Platelets of layered silicates of large enough diameter to perform AFM colloidal probe measurements were produced by melt synthesis. For comparison also, a natural muscovite mica has been included in this study. The diffuse layer properties in electrolyte solution have been determined by direct force measurements using the colloidal probe AFM technique and by electrokinetic measurements, respectively. We find that the diffuse layer potential decreases with increasing interlayer charge of the 2:1 layered silicates. This counterintuitive finding is attributed to ion adsorption and was further corroborated by determining the quantitative adsorption of polyelectrolytes, namely poly(amidoamine) dendrimers.
... Measurement data were collected at the stationary planes for at least three times. The details on the procedure can be found elsewhere [57]. ...
Clays are not only ubiquitous in nature, but they are also used in huge quantities in a broad range of industrial applications, such as thixotropic drilling fluids, ore pelletizers, waste disposal sealants, or fillers in polymer nanocomposites. In order to model environmental processes or to design new materials on a rational base, it is of prime importance to determine and possibly modify the interfacial properties of clay platelets at the solid/electrolyte interface. In this context, the fundamental question rises how far the stoichiometric interlayer charges as determined by the composition of the silicate layer correlates with the diffuse double-layer properties. Here, this question is addressed by means of a series of purposely synthesized sodium 2:1 layered silicates with defined composition and hence interlayer charge densities, respectively. Platelets of layered silicates of large enough diameter to perform AFM colloidal probe measurements were produced by melt synthesis. For comparison also, a natural muscovite mica has been included in this study. The diffuse layer properties in electrolyte solution have been determined by direct force measurements using the colloidal probe AFM technique and by electrokinetic measurements, respectively. We find that the diffuse layer potential decreases with increasing interlayer charge of the 2:1 layered silicates. This counterintuitive finding is attributed to ion adsorption and was further corroborated by determining the quantitative adsorption of polyelectrolytes, namely poly(amidoamine) dendrimers.
Graphical abstract
... Spidroin particles substantially swell upon hydration (swelling factor of 2.3) inducing a drastic drop in elastic modulus from GPa in the dry state to MPa in the wet state [145], depending on the molecular weight of the respective spidroins. Direct force measurements using a colloidal probe technique revealed a polymer brush-like surface i.e., an ion-permeable particle interface protruding several tenths of nm into the solution [146,147]. Interestingly, it has been shown that particle surfaces can seed fibril growth (Fig. 6.4d) [102]. ...
The extraordinary mechanical properties of spider silk fibers result from the interplay of composition, structure and self-assembly of spider silk proteins (spidroins). Genetic approaches enabled the biotechnological production of recombinant spidroins which have been employed to unravel the self-assembly and spinning process. Various processing conditions allowed to explore non-natural morphologies including nanofibrils, particles, capsules, hydrogels, films or foams. Recombinant spider silk proteins and materials made thereof can be utilized for biomedical applications, such as drug delivery, tissue engineering or 3D-biomanufacturing.
... Using ionic liquids instead of aqueous buffers and high potassium phosphate concentrations to induce phase separation and nucleation in the protein solution or ultrasonication for particle production allowed enhanced size control and a reduced polydispersity index (Elsner et al. 2015;Lucke et al. 2015). eADF4(C16) (engineered Araneus diadematus fibroin 4) particles show a brush-like outer layer with protruding protein strands and a thickness of 30-50 nm covering a solid inner core (Helfricht et al. 2013). Importantly, no posttreatment with dehydrating agents is necessary to obtain water-insoluble particles, since the β-sheet content is high after the salting-out process . ...
Spider silk fibers have a sophisticated hierarchical structure composed of proteins with highly repetitive sequences. Their extraordinary mechanical properties, defined by a unique combination of strength and extensibility, are superior to most man-made fibers. Therefore, spider silk has fascinated mankind for thousands of years. However, due to their aggressive territorial behavior, farming of spiders is not feasible on a large scale. For this reason, biotechnological approaches were recently developed for the production of recombinant spider silk proteins. These recombinant proteins can be assembled into a variety of morphologies with a great range of properties for technical and medical applications. Here, the different approaches of biotechnological production and the advances in material processing toward various applications will be reviewed.
This review will present the latest research related to the production and application of spider silk and silk-based materials in reconstructive and regenerative medicine and tissue engineering, with a focus on musculoskeletal tissues, and including skin regeneration and tissue repair of bone and cartilage, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Natural spider silk synthesis is reviewed, and the further recombinant production of spider silk proteins. Research insights into possible spider silk structures, like fibers (1D), coatings (2D), and 3D constructs, including porous structures, hydrogels, and organ-on-chip designs, have been reviewed considering a design of bioactive materials for smart medical implants and drug delivery systems. Silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Novel biomaterials with silk fibroin can mimic the tissue structure and promote regeneration and new tissue growth. Silk proteins are important in designing tissue-on-chip or organ-on-chip technologies and micro devices for the precise engineering of artificial tissues and organs, disease modeling, and the further selection of adequate medical treatments. Recent research indicates that silk (films, hydrogels, capsules, or liposomes coated with silk proteins) has the potential to provide controlled drug release at the target destination. However, even with clear advantages, there are still challenges that need further research, including clinical trials.
During past few decades, the demand for the replacement of damaged organs is increasing consistently. This is due to the advancement in tissue engineering, which opens the possibility of regeneration of damaged organs or tissues into functional parts with the help of 3D bioprinting. Bioprinting technology presents an excellent potential to develop complex structures with precise control over cell suspension and structure. A brief description of different types of 3D bioprinting techniques, including inkjet‐based, laser‐based, and extrusion‐based bioprinting is presented here. Due to innate advantageous features like tunable biodegradability, biocompatibility, elasticity and mechanical robustness, silk has carved a niche in the realm of tissue engineering. In this review article, the focus is to highlight the possible approach of exploring silk as bioink for fabrication of bioprinted implants using 3D bioprinting. This review discusses different type of degumming, dissolution techniques for extraction of proteins from different sources of silk. Different recently reported 3D bioprinting techniques suitable for silk‐based bioink are further elaborated. Postprinting characterization of resultant scaffolds are also describe here. However, there is an astounding progress in 3D bioprinting technology, still there is a need to develop further the current bioprinting technology to make it suitable for generation of heterogeneous tissue construct. The possibility of utilizing the adhesive property of sericin to consider it as bioink is elaborated.
Robust, biocompatible, and facile coatings are promising for improving the in vivo performance of medical implants and devices. Here, we demonstrate the formation of nanothin silk coatings by leveraging the biomimetic self-assembly of eADF(C16), an amphiphilic recombinant protein based on the Araneus diadematus dragline spidroin ADF4. These coatings result from concurrent adsorption and supramolecular assembly of eADF4(C16)induced by KH2PO4, thereby providing a mild one-pot coating strategy in which coating rate can be controlled by protein and KH2PO4 concentration. The thickness of coatings range from 2 – 30 nm depending on time immersed in the aqueous coating solution. Coatings can be formed on hydrophobic and hydrophilic substrates regardless of surface chemistry and without requiring specialized surface activation. Moreover, coatings appear to be stable through vigorous rinsing and prolonged agitation in water. Grazing incidence wide angle X-ray scattering, single-molecule force spectroscopy, and Congo red staining techniques confirm the formation of β-sheet nanocrystals within the eADF4(C16)coating, which contributes to the cohesive and adhesive stability of the material. Coatings are exceptionally smooth in the dry state and are hydrophilic regardless of substrate hydrophobicity. In aqueous conditions, coatings exhibit the properties of a hydrogel material.
Silk is a source of marvel for centuries as one of nature's high-performance materials. More recently, chemical and structural analysis techniques have helped explore the relationship between silk's properties and its hierarchical structure. Furthermore, recombinant protein engineering as well as polymer and organic synthesis techniques have enabled the production of silk-like materials. It has become apparent that silk is a supramolecular polymer with many of the properties exhibited by well-known synthetic supramolecular materials, such as block copolymers, liquid crystals, thermoplastic elastomers, and self-assembling peptides. In this review, the hierarchical structure and supramolecular assembly of silk are discussed in comparison to these synthetic supramolecular systems. By focusing on the connections between chemical structure, nanoscale molecular organization, and material properties, the aim is to provide perspectives on the rational design of advanced soft matter to supramolecular chemists and molecular engineers who look to nature for inspiration.
Silk is mostly known as a luxurious textile, which originates from silkworms first cultivated in China. A deeper look into the variety of silk reveals that it can be used for much more, in nature and by humanity. For medical purposes, natural silks were recognized early as a potential biomaterial for surgical threads or wound dressings; however, as biomedical engineering advances, the demand for high-performance, naturally derived biomaterials becomes more pressing and stringent. A common problem of natural materials is their large batch-to-batch variation, the quantity available, their potentially high immunogenicity, and their fast biodegradation. Some of these common problems also apply to silk; therefore, recombinant approaches for producing silk proteins have been developed. There are several research groups which study and utilize various recombinantly produced silk proteins, and many of these have also investigated their products for biomedical applications. This review gives a critical overview over of the results for applications of recombinant silk proteins in biomedical engineering.
The adhesion of alginate hydrogels to solid surfaces was probed by atomic force microscopy (AFM) in the sphere/plane geometry. For this purpose a novel approach has been developed for the immobilization of soft colloidal probes onto AFM-cantilevers, which is inspired by techniques originating from cell biology. The aspiration and consecutive manipulation of hydrogel beads by micropipettes allows the entire manipulation sequence to be carried-out in situ. Hence, any alteration of the hydrogel beads upon drying can be excluded. The adhesive behaviour of alginate hydrogels was first evaluated by determining the distribution of pull-off forces on self-assembled monolayers (SAMs) terminating in different functional groups (-CH3, -OH, -NH2, -COOH). It was demonstrated that solvent exclusion plays practically no role in the adhesion process, in clear difference to solid colloidal probes. The adhesion of alginate beads is dominated by chemical interactions rather than solvent exclusion, in particular in the case of amino-terminated SAMs. The data set acquired on the SAMs provided the framework to relate the adhesion of alginate beads on recombinant spider silk protein films to specific functional groups. The preparation of soft colloidal probes and the presented approach in analysing the adhesive behaviour is not limited to alginate hydrogel beads but can be generally applied for probing and understanding the adhesion behaviour of hydrogels on a wide range of substrates, which would be relevant for various applications such as biomedical surface modification or tissue engineering.
Medical therapies are often accompanied by un-wanted side-effects or, even worse, targeted cells can develop drug resistance leading to an ineffective treatment. Therefore, drug delivery systems are under investigation to lower the risk thereof. Drug carriers should be biocompatible, biodegradable, nontoxic, non-immunogenic, and should show controllable drug loading and release properties. Previous studies qualified spider silk particles as drug delivery carriers, however, cellular uptake was only tested with unloaded spider silk particles. Here, the effect of drug loading on cellular uptake of previously established spider silk-based particles made of eADF4(C16), eADF4(C16)RGD, eADF4(C16)R8G and eADF4(κ16) was investigated. Fluorescently labelled polyethylenimine was used as a model substance for loading eADF4(C16), eADF4(C16)RGD or eADF4(C16)R8G particles, and fluorescently labelled ssDNA was used for loading eADF4(κ16) particles. Upon loading polyanionic eADF4(C16) and eADF4(C16)RGD particles with polycationic polyethylenimine the cellular uptake efficiency was increased, while the uptake of eADF4(C16)R8G and polycationic eADF4(κ16) particles was decreased upon substance loading. The latter could be circumvented by coating substance-loaded eADF4(κ16) particles with an additional layer of eADF4(κ16) (layer-by-layer coating). Further, it could be shown that eADF4(C16)RGD and eADF4(κ16) uptake was based on clathrin-mediated endocytosis, whereas macropinocytosis was more important in case of eADF4(C16) and eADF4(C16)R8G particle uptake. Finally, it was confirmed that drugs, such as doxorubicin, can be efficiently delivered into and released within cells when spider silk particles were used as a carrier.
We report on direct force measurements between single melt-electrospun 1,3,5-cyclohexanetrisamide (CTA) fibers in crossed-cylinder geometry. The two CTA compounds selected for this study differ in their peripheral substituents: aliphatic (-C10H21) and fluorinated chains (-CH2C6F13), respectively. Melt-electrospinning of the CTAs results in smooth and circular fibers with diameters of about 5 μm. Individual segments of these fibers were attached to tipless atomic force microscope (AFM) cantilevers and used to measure long-range interaction forces versus a second fiber from the same compound in crossed-cylinder geometry. This geometry is well-known from the surface force apparatus and allows for the normalization of forces according to the Derjaguin approximation. From symmetrical measurements, i.e. measurements between fibers from the same type of CTA, we quantify the diffuse layer properties in aqueous electrolyte solutions within the framework of the Derjaguin, Landau, Verwey and Overbeek (DLVO) theory. Apparent diffuse layer potentials resulting from the fits to the full solutions of the Poisson–Boltzmann equation show that the fiber surfaces bear a negative surface charge. Most likely, the origin of charging is the adsorption of hydroxyl ions as residual charges from the electrospining process would be compensated upon the immersion in the electrolyte solutions. Such ion adsorption processes are well-known for other hydrophobic surfaces such as aliphatic and fluorinated self-assembled monolayers (SAMs) (terminating with CH3 and CF3 groups). The apparent diffuse layer potentials for CTA fibers are comparable to the values reported for these SAMs.
Spider silk fibers are well known for their mechanical properties, and they are therefore in the focus of materials scientists. Additionally, silks display biocompatibility making them interesting materials for applications in medicine or cosmetics. Due to the low abundance of natural spider silk proteins because of the spider's cannibalism, the recombinant spider silk protein eADF4 has been established for material science applications. Once processed into micron-sized particles by controlled salting-out, these particles can be used as drug delivery vehicles. For any application of the silk particles it is important to know their mechanical characteristics for processing and storage reasons. Here, we examine the swelling behavior and mechanics of these particles. Upon hydration, a drastic drop in elastic modulus occurs by orders of magnitude, from 0.8 GPa in the dry state to 2.99 MPa in the wet state. Importantly, the elastic modulus of recombinant silk particles can be tuned by varying the molecular weight of the used proteins, as well as chemical crosslinking thereof.
Drug delivery systems allow tissue/cell specific targeting of drugs in order to reduce total drug amounts administered to an organism and potential side effects upon systemic drug delivery. Most drug delivery systems are polymer-based, but the number of possible materials is limited since many commercially available polymers induce allergic or inflammatory responses or lack either biodegradability or the necessary stability in vivo. Spider silk proteins represent a new class of (bio)polymers that can be used as drug depots or drug delivery systems. The recombinant spider silk protein eADF4(C16), which can be processed into different morphologies such as particles, films, or hydrogels, has been shown to fulfil most criteria necessary for its use as biomaterial. Further, eADF4(C16) particles have been shown to be well-suited for drug delivery. Here, a new method was established for particle production to reduce particle size and size distribution. Importantly, cellular uptake of these particles was shown to be poor in HeLa cells. Therefore, variants of eADF4(C16) with inversed net charge or incorporated cell penetrating peptides and receptor interacting motifs were tested, showing much better cellular uptake. Interestingly, uptake of all silk variant particles was mainly achieved by clathrin-mediated endocytosis.
Drug delivery carriers stabilize drugs and control their release, expanding the therapeutic window, and avoiding side effects of otherwise freely diffusing drugs in the human body. Materials used as carrier vehicles have to be biocompatible, biodegradable, nontoxic, and nonimmunogenic. Previously, particles made of the recombinant spider silk protein eADF4(C16) could be effectively loaded with positively and neutrally charged model substances. Here, a new positively charged variant thereof, named eADF4(κ16), has been engineered. Its particle formation is indistinguishable to that of polyanionic eADF4(C16), but in contrast polycationic eADF4(κ16) allows incorporation of negatively charged substances. Both high-molecular-weight substances, such as nucleic acids, and low-molecular-weight substances could be efficiently loaded onto eADF4(κ16) particles, and release of nucleic acids was shown to be well controlled. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.
Spider silk fibers are well known for their mechanical properties, and they are therefore in the focus of materials scientists. Additionally, silks display biocompatibility making them interesting materials for applications in medicine or cosmetics. Due to the low abundance of natural spider silk proteins because of the spider's cannibalism, the recombinant spider silk protein eADF4 has been established for material science applications. Once processed into micron-sized particles by controlled salting-out, these particles can be used as drug delivery vehicles. For any application of the silk particles it is important to know their mechanical characteristics for processing and storage reasons. Here, we examine the swelling behavior and mechanics of these particles. Upon hydration, a drastic drop in elastic modulus occurs by orders of magnitude, from 0.8 GPa in the dry state to 2.99 MPa in the wet state. Importantly, the elastic modulus of recombinant silk particles can be tuned by varying the molecular weight of the used proteins, as well as chemical crosslinking thereof.
In this note we present a novel approach to prepare colloidal probes for atomic force microscopy by sintering. A central element of this procedure is the introduction of an inorganic "fixation neck" between the cantilever and a micrometer-sized silica particle that is acting as probe. This procedure overcomes previous restrictions for the probe particles, which had to be low melting point materials, such as borosilicate glass or latex particles. The here-presented colloidal probes from silica can withstand large mechanical forces. Additionally, they have high chemical resistivity due to the absence of adhesives and the well-studied surface chemistry of colloidal silica.
Images and force measurements taken by an atomic‐force microscope (AFM) depend greatly on the properties of the spring and tip used to probe the sample’s surface. In this article, we describe a simple, nondestructive procedure for measuring the force constant, resonant frequency, and quality factor of an AFM cantilever spring and the effective radius of curvature of an AFM tip. Our procedure uses the AFM itself and does not require additional equipment.
Nature has evolved a range of materials that compete with man-made materials in physical properties; one of these is spider silk. Silk is a fibrous material that exhibits extremely high strength and toughness with regard to its low density. In this review we discuss the molecular structure of spider silk and how this understanding has allowed the development of recombinant silk proteins that mimic the properties of natural spider silks. Additionally, we will explore the material morphologies and the applications of these proteins. Finally, we will look at attempts to combine the silk structure with chemical polymers and how the structure of silk has inspired the engineering of novel polymers.
Direct force measurements between oppositely charged latex particles in aqueous electrolyte solutions were carried out with a multiparticle colloidal probe technique based on atomic force microscopy. Force profiles between two dissimilarly charged surfaces can be only described when charge regulation effects are taken into account, while constant charge or constant potential boundary conditions are inappropriate. Surface potentials and regulation parameters are determined from force data obtained in symmetric systems with the Poisson-Boltzmann theory and constant regulation approximation. The resulting quantities are used to predict the force profiles in asymmetric systems, and good agreement between theory and experiment is found. These findings show that charge regulation is important to quantify double-layer forces in asymmetric systems.
In a significant fraction of the Escherichia coli cytosolic proteins, the N-terminal methionine residue incorporated during the translation initiation step is excised. The N-terminal methionine excision is catalyzed by methionyl-aminopeptidase (MAP). Previous studies have suggested that the action of this enzyme could depend mainly on the nature of the second amino acid residue in the polypeptide chain. In this study, to achieve a systematic analysis of the specificity of MAP action, each of the 20 amino acids was introduced at the penultimate position of methionyl-tRNA synthetase of E. coli and the extent of in vivo methionine excision was measured. To facilitate variant protein purification and N-terminal sequence determination, an expression shuttle vector based on protein fusion with beta-galactosidase was used. From our results, methionine excision catalyzed by MAP is shown to obey the following rule: the catalytic efficiency of MAP, and therefore the extent of cleavage, decreases in parallel with the increasing of the maximal side-chain length of the amino acid in the penultimate position. This molecular model accounts for the rate of N-terminal methionine excision in E. coli, as deduced from the analysis of 100 protein N-terminal sequences.
The Derjaguin approximation states that the interaction force between two curved surfaces is proportional to their effective radius, whereby the inverse effective radius is the arithmetic mean of the inverse curvature radii of the surfaces involved. The present study investigates the validity of this approximation with an atomic force microscope (AFM) by measuring interaction forces between colloidal particles of different sizes, but of identical composition. Forces were measured between silica particles of 2.0, 4.8 and 6.8 microm in diameter in KCl electrolyte solution with and without adsorbed poly(amido amine) (PAMAM) dendrimers. The Derjaguin approximation could be confirmed at all distances investigated, including those comparable with the characteristic length scales of the surface roughness or the surface charge heterogeneities. For the conditions investigated, the Derjaguin approximation turns out to be surprisingly robust.
The controlled delivery of water-soluble substances is one important issue in pharmaceutical and medical applications. Biocompatible polymers which can easily be processed in an all aqueous process with controllable and adjustable properties have been thoroughly investigated in the past for their use as drug delivery vehicles. Recently, we established sub-microparticles produced from the engineered spider silk protein eADF4(C16) as potential carriers for highly water-soluble drugs. Here, we investigate the influence of crosslinking on the structural integrity of the sub-microparticles and the effect on drug loading and release. To analyze the order-of-addition influences of processing of sub-microparticles on drug loading and release, we tested five different preparation routes. We showed that the preparation route largely influences the loading capacity of the eADF4(C16) sub-microparticles. In the preferred preparation route, rhodamine B and the protein are co-precipitated by salting-out, yielding the highest loading. Further, crosslinking the proteins with APS (ammonium persulfate) and Rubpy (Tris(2,2′- bipyridyl)dichlororuthenium(II)) has an impact on loading as well as on the release behavior of drug molecules as shown exemplarily with rhodamine B.
The equations which govern the ion distributions and velocities, the electrostatic potential and the hydrodynamic flow field around a solid colloidal particle in an applied electric field are reexamined. By using the linearity of the equations which determine the electrophoretic mobility, we show that for a colloidal particle of any shape the mobility is independent of the dielectric properties of the particle and the electrostatic boundary conditions on the particle surface. The mobility depends only on the particle size and shape, the properties of the electrolyte solution in which it is suspended, and the charge inside, or electrostatic potential on, the hydrodynamic shear plane in the absence of an applied field or any macroscopic motion.New expressions for the forces acting in the particle are derived and a novel substitution is developed which leads to a significant decoupling of the governing equations. These analytic developments allow for the construction of a rapid, robust numerical scheme for the solution of the governing equations which we have applied to the case of a spherical colloidal particle in a general electrolyte solution. We describe a computer program for the conversion of mobility measurements to zeta potential for a spherical colloidal particle which is far more flexible than the Wiersema graphs which have traditionally been used for the interpretation of mobility data. Furthermore it is free of the high zeta potential convergence difficulties which limited Wiersema's calculations to moderate values of ζ. Some sample computations in typical 1:1 and 2:1 electrolytes are exhibited which illustrate the existence of a maximum in the mobility at high zeta potentials. The physical explanation of this effect is given. The importance of the mobility maximum in testing the validity of the governing equations of electrophoresis and its implications for the colloid chemist's picture of the Stern layer are briefly discussed.
The adhesion between colloidal silica particles and modified electrodes has been studied by direct force measurements with the colloidal probe technique based on the atomic force microscope (AFM). The combination of potentiostatic control of gold electrodes and their chemical surface modification by self-assembled monolayers (SAMs) allows for the decoupling of forces due to the electrical double layers and chemical functionality of the solid/liquid interface. Adhesion on such electrodes can be tuned over a large range in dependence of the externally applied potential and the aqueous solution's ionic strength. By utilizing cantilevers with a high force constant, it is possible to separate the various contributions to the adhesion in an unambiguous manner. These contributions comprise diffuse layer overlap, van-der Waals forces, solvent exclusion, and electrocapillarity. A quantitative description of the observed adhesion forces is obtained by taking into account the surface roughness of the silica particle. The main component in the adhesion, which is tuned by the external potential, originates from the overlap of the electrical double layers. Instead, effects due to electrocapillarity are only of minor importance. Based on our quantitative analysis a new approach is proposed that allows tuning the adhesion force as a function of the externally applied potential. This approach is supposed to have important implications for the design of microelectromechanical systems (MEMS), the development of electrochemical sensors as well as for micro- and nanomanipulation.
The interaction energy between microscopic bodies is almost exclusively determined assuming perfectly smooth and geometrically regular surfaces. Quite often, such interactions fail to explain several colloidal phenomena. These inexplicable behaviors of colloidal systems are generally ascribed to surface chemical and morphological heterogeneities. Here, we employ the surface element integration technique to determine the interaction energy between surfaces containing morphological heterogeneity. Random asperities are generated to represent surface morphological heterogeneity (roughness), and their influence on the DLVO interaction potential is investigated. Incorporation of surface roughness causes a significant reduction in the repulsive interaction energy, the extent of which depends on the size of the asperities and their densities on the surface. Predictions of interaction energy indicate that the DLVO interaction energy profiles for rough surfaces deviate significantly from those derived assuming perfectly smooth surfaces, particularly at very short separation distances.
An atomic force microscope has been used to study the forces between a silica sphere in the colloidal size range and silica or mica flat surfaces as a function of distance of separation. At low ionic strength, independent electrokinetic measurements (ζ potentials) of both the spheres (by electrophoresis) and flat surfaces (by streaming potential) under the same conditions show excellent agreement with the diffuse double layer potentials derived from the force data using conventional DLVO theory. At higher ionic strength, the electrokinetically derived potentials were found to deviate from those derived from the fitted atomic force microscopy data, and a short range steric type repulsion was observed between the surfaces, the magnitude of which increased with decreasing pH.
The forces between hydrophilic and hydrophobic silica particles and an air bubble were measured in pure water and in NaCl solutions using an atomic force microscope. In addition to the expected double-layer and van der Waals forces, strong long-range attractive forces were also observed. A long-range attraction was also measured between a hydrophilic silica particle and a hydrophobic silica plate. A gas bubble thus behaves like a hydrophobic surface. The long-ranged attractive component of the force disappeared when the anionic surfactant sodium dodecylsulfate (SDS) was added to the solution. This effect is explicable in terms of surfactant adsorption at the hydrophobic interfaces which renders them hydrophilic. A ''thermodynamic'' model is proposed that appears to be consistent with these and previous force and wetting experiments on hydrophobic surfaces. It is also demonstrated that a nonzero water contact angle on clean hydrophilic silica and similar hydrophilic surfaces can arise from DLVO forces alone and is not necessarily an indication of surface contamination or some hydrophobic component in the force.
A general expression is derived for the electrophoretic mobility of a soft particle, i.e., a spherical hard colloidal particle of radius a coated with a layer of polyelectrolytes of thickness d in an electrolyte solution, In the limit of d → 0, the mobility expression tends to that for a spherical hard particle (Ohshima et al., J. Chem. Soc. Faraday Trans.2 79, 1613 (1983)), whereas in the limit of a → ∞, it tends to that for a plate-like soft particle derived previously (J. Colloid Interface Sci.130, 281 (1989)). Also in the limit of a → 0 and low potentials, the obtained mobility expression tends to a mobility formula for a spherical polyelectrolyte (a charged porous sphere with no particle core) derived by Hermans and Fujita (K. Ned. Akad. Wet. Proc. Ser. B58, 182 (1955)). A simple approximate analytic mobility expression is presented.
Electrophoretic mobilities (EPM) of negatively charged latex spheres were measured as a function of salt type and salt concentration.
The measured values of EPM were analyzed using a standard electrokinetic model that includes double layer relaxation and the
Poisson–Boltzmann model of diffuse double layer. Calculated values of EPM were in good agreement with experimental data taken
in simple 1:1 (KCl) and 1:2 (Na2SO4) electrolyte solutions without using any fit parameters. For 2:1 electrolytes (CaCl2 and MgCl2), however, the magnitude of EPM calculated by the model was higher than the measured values of EPM at higher electrolyte
concentrations. The difference between measured and calculated EPM was reduced by assuming the distance of slipping plane
x
s = 0.25nm or by assuming the decrease of the magnitude of surface charge density from −0.07 to −0.025C/m2. These are probably due to the accumulation of divalent counterions in the vicinity of a particle’s surface.
Polystyrene microspheres having roughly the same size but different negative surface charge densities were prepared by emulsion
polymerization. The amount of sulfate groups on the surface of the particles was controlled by variation of the amount and
the decomposition rate of the initiator used, potassium, persulfate. After the cleaning process involving dialysis and extensive
ultrafiltration the surface-charge density of the samples was determined and their electrokinetic behavior was studied.
A simple model based on the Gouy-Chapman theory and the O’Brian-White approach allows the calculation of the dependence of
the electrophoretic mobility on salt concentration. Comparison of the theoretical and experimental curves showed that they
were in good agreement in a number of qualitative features. Moreover, the model revealed that a monotonously increasing zeta
potential with falling electrolyte concentration results in a mobility maximum, and that this so-called atypical behavior
is in accordance with the standard electrokinetic theory. No ion adsorption mechanism or the existence of a charged hairy
layer, current standard explanations for this anomality, had to be invoked.
Electrophoretic mobility studies of highly charged amidine latex particles were carried out to validate the standard electrokinetic model. The electrophoretic mobilities were measured on a standard commercial laser Doppler velocimetry electrophoresis apparatus. In a plot against the salt concentration, a pronounced mobility maximum was observed. This observation, which is a direct consequence of the standard electrokinetic model, showed good agreement with theory.
The engineered and recombinant spider silk protein eADF4(C16) has been shown to be a promising biomaterial for the use as drug delivery system. In previous studies, eADF4(C16) particles were loaded with low molecular weight drugs exhibiting a positive net-charge and sufficient hydrophobicity. Here, we demonstrate that also macromolecular drugs like proteins can be loaded on eADF4(C16) particles. Using lysozyme as a model protein, remarkably high loading of up to 30% [w/w] was feasible and high loading efficiencies of almost 100% were obtained. Furthermore, using confocal laser scanning microscopy, it is demonstrated that fluorescently labeled lysozyme is not only adsorbed to the negatively charged particles' surface, but also diffusing into the matrix of eADF4(C16) particles. The release of lysozyme is shown to be dependent on the ionic strength and pH of the release medium. To improve the long-term stability of eADF4(C16) containing dispersions, lyophilization is shown as a suitable tool. Disaccharides (sucrose, trehalose) and mannitol served as stabilizers to prevent aggregation and/or particle degradation during freeze-drying. The slowly biodegradable eADF4(C16) particles are a promising new particulate drug carrier system for the delivery of susceptible drugs like therapeutic proteins.
Spider silk has been in the focus of research mainly due to the superior mechanical characteristics of silk fibers. However, it has been previously shown that spider silk proteins can also adopt other morphologies such as submicroparticles. This study examines the applicability of such particles as drug carriers. Particle characterization revealed that particles made of the engineered spider silk protein eADF4(C16) are colloidally stable in solution. Here, it is shown that small molecules with positive net-charge can diffuse into the negatively charged spider silk protein matrix driven by electrostatic interactions. The loading efficiencies correlate with the distribution coefficient (logD) of small molecules of weak alkaline nature. Interestingly, constant release rates can be realized for a period of two weeks at physiological conditions in vitro, with accelerated release rates within acidic environments. Enzymatic degradation studies of eADF4(C16) particles indicated that the silk proteins degrade slowly and the particles decrease in size. Along with their all-aqueous and easy preparation, drug loaded eADF4(C16) particles provide a high potential for diverse applications in which controlled release from biodegradable carriers is desired.
Steric forces between polymer brushes and atomic force microscope tips were investigated. We studied two systems: polystyrene(PS) grafted to silicon in cyclohexane and poly(ethylene oxide)/poly(methacrylic acid) (PEO/PMAA) diblock copolymer adsorbed with the PMAA block to aluminum oxide in aqueous medium. On approach exponentially decaying repulsive forces were observed in both systems. With a homemade heat stage we could adjust the temperature. Increasing the temperature between 19 and 53 degrees C led to a linear increase of the decay length for PS in cyclohexane. Also the work required to bring the tip to a certain distance increased roughly linearly with temperature. This supports the view that the repulsion is of entropic origin. At the same time this demonstrates that the temperature dependence of surface forces could be routinely measured. For PEO in water the repulsive force was not significantly affected by a change in temperature. Approaching and retracting parts of force curves measured with PS in cyclohexane were in most cases indistinguishable. In contrast, for PEO in water a significant hysteresis was observed. This might be caused by an escape of polymers underneath the tip of the atomic force microscope. When retracting the tip in some cases the stretching of individual polymers was observed in both systems. Stretching force vs distance curves could be described by a wormlike chain model with typical persistence lengths of 1 nm.
Due to their extraordinary mechanical and biochemical properties, silks have long been in focus of research. In vivo, fibers are formed from silk proteins, in vitro, however, a variety of materials can be produced in addition to fibers including capsules, particles, films, foams, and gels. The versatility of silk proteins, along with their biocompatibility, biodegradability, and potential for processing in aqueous solution under ambient conditions make silk‐based materials good candidates for biomedical applications such as drug delivery systems and scaffolds for tissue engineering. Here, we summarize recent progress in research employing recombinantly produced engineered spider silk proteins with a focus on the fundamentals of silk protein processing. We highlight recombinant spider silk films and particles as morphologies that represent model systems with adjustable material properties controlled by process parameters.
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Interaction forces between ionizable surfaces across an electrolyte solution on the Poisson-Boltzmann level are discussed within the constant regulation approximation. The chemical response of each surface is expressed in terms of two parameters, namely, the diffuse layer potential and the regulation parameter p. Both parameters are easily available because they arise naturally within classical equilibrium models for a single noninteracting surface. This approximation, thus, eliminates the need to treat the more intricate problem of two chemical adsorption equilibria coupled to the overlapping double layers between the surfaces. The ensuing simplicity makes this approach extremely versatile for the analysis of experimental data. The classical boundary condition of constant potential corresponds to p = 0, and that of constant charge corresponds to p = 1. While this approximation is rigorously correct at large separations, we find that it remains excellent down to contact in many realistic situations, such as in symmetric or asymmetric systems involving metal oxides or silica described by the 1-pK basic Stern model.
Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable. This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.
The van der Waals and electrostatic interaction energies between a single particle and a flat plate were measured using the optical technique of total internal reflection microscopy (TIRM). The particles used were 15-µm-diameter polystyrene latex spheres and the flat plate was a polished BK-7 glass slide. The measurements were performed in aqueous solutions of ionic strength ranging from 3 to 7 mM, and the particle-plate separation distances ranged from approximately 25 to 300 nm. During measurement, the particle was trapped in a secondary energy well formed by the repulsive electrostatic and attractive van der Waals forces; however, the particle was free to undergo Brownian motion at all times. These measurements, which capitalize on the extreme sensitivity of the TIRM technique, are believed to be the first direct measurement of the van der Waals interaction energy in aqueous solutions at separation distances where retardation effects are substantial. Comparison of the measured energy wells with predictions made with traditional energy equations produced only fair agreement; specifically, the measured well depths were consistently lower than predicted. However, when the measured results were compared with predictions made using the recent model of L. Suresh and J. Y. Walz ([J. Colloid Interface Sci. 183, 199 (1996)] for rough surfaces, very good agreement was obtained. The asperity heights yielding the best agreement ranged between 14 and 33 nm, with an average height of 26 nm. This value is consistent with previous estimates of the roughness height obtained by measuring the particle sedimentation velocity [J. Y. Walz and L. Suresh, J. Chem. Phys. 103, 10714 (1995)]. Copyright 1997 Academic Press.
Spiders produce a variety of silks, and the cloning of genes for silk fibroins reveals a clear link between protein sequence and structure-property relationships. The fibroins produced in the spider's major ampullate (MA) gland, which forms the dragline and web frame, contain multiple repeats of motifs that include an 8-10 residue long poly-alanine block and a 24-35 residue long glycine-rich block. When fibroins are spun into fibres, the poly-alanine blocks form (&bgr;)-sheet crystals that crosslink the fibroins into a polymer network with great stiffness, strength and toughness. As illustrated by a comparison of MA silks from Araneus diadematus and Nephila clavipes, variation in fibroin sequence and properties between spider species provides the opportunity to investigate the design of these remarkable biomaterials.
Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.
The current theoretical approaches to electrokinetics of gels or polyelectrolyte layers are based on the assumption that the position of the very interface between the aqueous medium and the gel phase is well defined. Within this assumption, spatial profiles for the volume fraction of polymer segments (phi), the density of fixed charges in the porous layer (rho fix), and the coefficient modeling the friction to hydrodynamic flow (k) follow a step-function. In reality, the "fuzzy" nature of the charged soft layer is intrinsically incompatible with the concept of a sharp interface and therefore necessarily calls for more detailed spatial representations for phi, rho fix, and k. In this paper, the notion of diffuse interface is introduced. For the sake of illustration, linear spatial distributions for phi and rho fix are considered in the interfacial zone between the bulk of the porous charged layer and the bulk electrolyte solution. The corresponding distribution for k is inferred from the Brinkman equation, which for low phi reduces to Stokes' equation. Linear electrostatics, hydrodynamics, and electroosmosis issues are analytically solved within the context of streaming current and streaming potential of charged surface layers in a thin-layer cell. The hydrodynamic analysis clearly demonstrates the physical incorrectness of the concept of a discrete slip plane for diffuse interfaces. For moderate to low electrolyte concentrations and nanoscale spatial transition of phi from zero (bulk electrolyte) to phi o (bulk gel), the electrokinetic properties of the soft layer as predicted by the theory considerably deviate from those calculated on the basis of the discontinuous approximation by Ohshima.
Colloidal silica is known to be stable at high salt concentrations and low pH, where silica is basically uncharged. This observation is in qualitative disagreement with the theory of Derjaguin, Landau, Verwey, and Overbeek (DLVO), which predicts rapid aggregation (or coagulation) under these conditions. This study reports a very different behaviour for Stöber-type silica heated at 800 degrees C, as these particles follow DLVO theory quantitatively. Unheated samples behave approximatively according to DLVO theory, but they show systematic deviations, in particular, featuring higher stability at low pH. The heat treatment also substantially modifies the charging properties, as heated particles show titratable surface charge densities in the range expected for the water-silica interface, while much higher charge densities are observed for the unheated samples. The electrophoretic mobilities, on the other hand, are hardly influenced by the heat treatment. We suspect that the suspension stability of the unheated particles is influenced by the presence of a hairy-layer of polysilicilic acid chains on the surface.
In this report, the status quo and recent progress in electrokinetics are reviewed. Practical rules are recommended for performing electrokinetic measurements and interpreting their results in terms of well-defined quantities, the most familiar being the zeta-potential or electrokinetic potential. This potential is a property of charged interfaces and it should be independent of the technique used for its determination. However, often the zeta-potential is not the only property electrokinetically characterizing the electrical state of the interfacial region; the excess conductivity of the stagnant layer is an additional parameter. The requirement to obtain the zeta-potential is that electrokinetic theories be correctly used and applied within their range of validity. Basic theories and their application ranges are discussed. A thorough description of the main electrokinetic methods is given; special attention is paid to their ranges of applicability as well as to the validity of the underlying theoretical models. Electrokinetic consistency tests are proposed in order to assess the validity of the zeta-potentials obtained. The recommendations given in the report apply mainly to smooth and homogeneous solid particles and plugs in aqueous systems; some attention is paid to nonaqueous media and less ideal surfaces.
The electrostatic, hydrodynamic, and swelling properties of a well-defined, functionalized carboxymethyldextran (CMD) polysaccharide are investigated in aqueous NaNO3 solution over a broad ionic strength range. The impact of the polycarboxylate charge and molar mass of the CMD macromolecules on their electrohydrodynamic features is thoroughly examined by combined protolytic titration, dynamic light scattering, and electrokinetic analyses. Electrophoretic mobility data obtained for sufficiently high electrolyte concentrations reveal a typical soft particle behavior. Upon decrease of the ionic strength, mobilities strongly increase in magnitude while significant electrostatic swelling takes place, as reflected in a decrease in the diffusion coefficients. CMD entities undergo conformational transitions from compact random coil at large ionic strengths to swollen coil and possibly a wormlike structure at lower NaNO3 concentrations. The magnitude of the variations in size and mobility with electrolyte concentration strongly depends on the overall charge of the CMD entity as well as on its molar mass. These factors control the stiffness of the constituent polymer chains and thus the degree of macromolecular permeability ("softness"). Using the soft-diffuse interface formalism previously developed for the electrohydrodynamics of charged permeable macromolecules, a quantitative analysis of the electrophoretic mobility data is presented. The measured values of the diffusion coefficient and space charge density Gamma degrees, as evaluated independently from the modeling of potentiometric titration curves, are taken into account in a self-consistent manner. It is found that large CMD entities of low charge densities are the most permeable to flow penetration with a limited heterogeneous electrostatic stiffening of the chains, whereas small CMD entities of larger Gamma degrees significantly expand upon lowering the ionic strength, giving rise to a strong anisotropy for the spatial distribution of polymer chain density.