Colloidal force spectroscopy and cell biological investigations on biomimetic polyelectrolyte multilayer coatings composed of chondroitin sulfate and heparin

To promote osteoblast adhesion and proliferation on (bio)material surfaces, biomimetic coatings resembling the natural extracellular matrix (ECM) are desirable. The glycosamino glycans (GAGs) chondroitin sulfate (CS) and heparin (HEP) are promising candidates for a biomimetic coating since they are two of the most prevalent noncollagenous biomolecules constituting the ECM. Coatings containing CS and HEP were prepared employing the "layer by layer" technique yielding polyelectrolyte multilayers (PEMs). Physicochemical and mechanical characterization of the coatings were performed by means of streaming potential measurements and colloidal force spectroscopy. The capability of the coatings to support cell adhesion, spreading, proliferation, and maintenance of an osteoblastic phenotype was assessed with SaOS osteosarcoma cells. We demonstrate that PEMs constructed from CS as the polyanion display a low Young's modulus correlated with poorly supported cell adhesion and proliferation. When the CS was adsorbed onto a stiffer polypeptide PEM basis, the Young's modulus increased, and the cell response was significantly improved. For HEP coatings an intermediate Young's modulus and moderate cell adhesion and spreading were observed. No significant changes in stiffness or cell response were detected when HEP was adsorbed onto the polypeptide film.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

Polyelectrolyte multilayer microcapsules: Self-assembly and toward biomedical applications

Over the past few years, many studies have been performed involving the application of the Layer-by-Layer (LbL) deposition of oppositely charged polyelectrolytes onto charged colloidal particles, followed by the dissolution of the templates, ultimately resulting in polyelectrolyte multilayer microcapsules. The ease of preparation of polyelectrolyte multilayer microcapsules afforded by the LbL self-assembly technique, as well as the advantages of accurate control over size, composition, and the thickness of the multilayer shell make these capsules very promising for a number of applications in materials and biomedical science. In this review, we describe the assembly and stimuli-responsive properties (“smart” capsules) of polyelectrolyte multilayer microcapsules, and also discuss the potential of this technique in regard to biomedical applications. In addition, we illustrate two measurement techniques for determining the mechanical properties of polyelectrolyte multilayer microcapsules—(i) osmotic swelling and (ii) AFM compression experiments. These capsules are believed to have great potential for future applications, including biosensors, bioreactors, and carriers for targeted drug delivery.     

Low-fouling, biofunctionalized, and biodegradable click capsules

We report the synthesis of covalently stabilized hollow capsules from biodegradable materials using a combination of click chemistry and layer-by-layer (LbL) assembly. The biodegradable polymers poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) were modified with alkyne and azide moieties. Linear film buildup was observed for both materials on planar surfaces and colloidal silica templates. A variation of the assembly conditions, such as an increase in the salt concentration and variations in pH, was shown to increase the individual layer thickness by almost 200%. The biodegradable click capsules were analyzed with optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Capsules were uniform in size and had a regular, spherical shape. They were found to be stable between pH 2 and 11 and showed reversible, pH-responsive shrinking/swelling behavior. We also show that covalently stabilized PLL films can be postfunctionalized by depositing a monolayer of heterobifunctional poly(ethylene glycol) (PEG), which provides low-fouling properties and simultaneously enhances specific protein binding. The responsive, biodegradable click films reported herein are promising for a range of applications in the biomedical field.     

The reinforcement of gellan gel network by the immersion into salt solution

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.     

Ultrathin antibiotic walled microcapsules

Ultrathin microcapsules comprised of anionic polyelectrolytes (PE) and a polycationic aminoglycoside (AmG) antibiotic drug were prepared by depositing PE/AmG multilayers on zinc oxide (ZnO) colloid particles using the layer-by-layer self-assembly technique and subsequently dissolving the ZnO templated cores. The polyelectrolytes, dextran sulfate sodium (DxS) and poly(styrenesulfonate) (PSS), were selected owing to their different backbone structure. An aminoglycoside, tobramycin sulfate (TbS), was used for studying DxS/TbS or PSS/TbS multilayer films. The multilayer growth on ZnO cores was characterized by alternating zeta potential values that were different for the DxS/TbS and PSS/TbS multilayers due to the PE chemistry and its interaction with Zn(2+) ions. Transmission and scanning electron microscopy provide evidence of PE/TbS multilayer coating on ZnO core particles. The slow acid-decomposition of the ZnO cores using weak organic acids and the presence of sufficient quantity of Zn(2+) in the dispersion were required to produce antibiotic multilayer capsules. There was no difference in the morphological characteristics of the two types of capsules; although, the yield for [PSS/TbS](5) capsules was significantly higher than for [DxS/TbS](5) capsules which was related to the physicochemical properties of DxS/TbS/Zn(2+) and PSS/TbS/Zn(2+) complexes forming the capsule wall. The TbS quantity in the multilayer films was determined using a quartz crystal microbalance and high performance liquid chromatography techniques which showed less TbS loading in both, capsules and multilayers on planar gold substrate, than the theoretical DxS:TbS or PSS:TbS stoichiometric ratio. The decomposition of the [PE/TbS](6) multilayers was fastest in physiological buffer followed by mannitol and water. The decomposition rate of the [PSS/TbS](6) multilayers was slower than [DxS/TbS](6) monolayers. The incomplete decomposition of DxS/TbS under saline conditions suggests the major role of hydrogen bonding for stability of DxS/TbS multilayers. A combination of hydrogen bonding and hydrophobic interaction between phenyl rings in PSS was responsible for PSS/TbS multilayer stability. In vivo studies in rabbits highlight the safety and sustained drug delivery potential of the PE/AmG microcapsules. The antibiotic walled ultrathin capsules presented here are suitable for sustained ophthalmic antibiotic delivery.     

Mechanical properties of DNA films

The Young's dynamical modulus (E) and the DNA film logarithmic decrement (theta) at frequencies from 50 Hz to 20 kHz are measured. These values are investigated as functions of the degree of hydration and temperature. Isotherms of DNA film hydration at 25 degrees C are measured. The process of film hydration changing with temperature is studied. It is shown that the Young's modulus for wet DNA films (E = 0.02-0.025 GN m-2) strongly increases with decreasing hydration and makes E = 0.5-0.7 GN m-2. Dependence of E on hydration is of a complex character. Young's modulus of denatured DNA films is larger than that of native ones. All peculiarities of changing of E and theta of native DNA films (observed at variation of hydration) vanish in the case of denatured ones. The native and denatured DNA films isotherms are different and depend on the technique of denaturation. The Young's modulus of DNA films containing greater than 1 g H2O/g dry DNA is found to decrease with increasing temperature, undergoing a number of step-like changes accompanied by changes in the film hydration. At low water content (less than 0.3 g H2O/g dry DNA), changing of E with increasing temperature takes place smoothly. The denaturation temperature is a function of the water content.     

Encapsulating live cells with water-soluble chitosan in physiological conditions

A new class of microcapsules was prepared under physiological conditions by polyelectrolyte complexation between two oppositely-charged, water-soluble polymers. The microcapsules consisted of an inner core of half N-acetylated chitosan and an outer shell of methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA) (52.2%) (MAA-HEMA-MMA) terpolymer. Both 400 and 150 kDa half N-acetylated chitosans maintained good water solubility and supplied enough protonated amino groups to coacervate with terpolymer at pH 7.0-7.4, in contrast to other chitosan-based microcapsules which must be prepared at pH <6.5. The viscosity of half N-acetylated chitosan solutions between 80 and 3000 cPas allowed the formation of microcapsules with spherical shape. Molar mass, pH and concentration of half N-acetylated chitosan, and reaction time, influenced the morphology, thickness and porosity of the microcapsules. Microcapsules formed with high concentration of half N-acetylated chitosan exhibited improved mechanical stability, whereas microcapsules formed with low concentration of half N-acetylated chitosan exhibited good permeability. This 3D microenvironment has been configured to cultivate sensitive anchorage-dependent cells such as hepatocytes to maintain high level of functions.     

Elasticity of native and cross-linked polyelectrolyte multilayer films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.     

Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine)

In general, microcapsules prepared from alginate and polycations lack mechanical strength because the interaction between alginate and polycations is ionic instead of covalent, which represents a much stronger bond. To increase the mechanical strength of the capsule, we prepared photosensitive microcapsules that could form covalent bonds between polymers in the capsular membrane by light irradiation. Two types of photosensitive poly(allylamine), with 5% and 10% of amino groups modified by alpha-phenoxycinnamylidene acetylchloride, were synthesized. Both photopolymers exhibited an absorption maximum at 325 nm and were capable of crosslinking upon light exposure. These photosensitive polymers were used for the preparation of microcapsules. The capsules formed from this photosensitive poly(allylamine) and alginate were strengthened significantly by light irradiation. Only 28% of the microcapsules prepared from the 5%-modified photopolymer fractured after 48 h of shaking at 150 rpm. This fracture percentage is much lower when compared with the 60% of capsules fractured when prepared from the untreated poly(allylamine). By using poly(allylamine) at 10% modification, the mechanical strength was improved only slightly, with 26% of capsules fractured. Analysis of the permeability test indicated that the photo-crosslinked capsular membrane was freely permeable to cytochrome c and myoglobin, but less permeable to serum albumin. The encapsulation method was used to entrap and culture IW32 mouse leukemia cells. The cells proliferated to a density of about 1.1 x 10(7) cells/mL in the capsules after 7 days of cultivation. Concurrently, the concentration of erythropoietin in the microcapsules increased to 800 mU/mL. This new encapsulation technique has great potential in the application of a bioindustrial cell-culturing process.     

Development of biodegradable scaffolds based on patient-specific arterial configuration

Biodegradable scaffolds are of great value in tissue engineering. We have developed a method for fabricating patient-specific vascular scaffolds from a biocompatible and biodegradable polymer, poly(L-lactide-co-epsilon-caprolactone). This method's usefulness is due to flexibility in the choice of materials and vascular configurations. Here, we present a way to fabricate scaffolds of human carotid artery by combining processes of rapid prototyping, lost wax, dip coating, selective dissolution, and salt leaching. The result was the successful development of porous biodegradable scaffolds, with mechanical strength covering the range of human blood vessels (1-3 MPa). Human umbilical vein endothelial cells were also cultured on the scaffolds and their biocompatibility was confirmed by cell growth. The Young's modulus of scaffolds could be controlled by changing polymer concentration and porosity. The wall thickness of the tubular scaffold was also controllable by adjusting polymer concentration and pull-up velocity during dip coating. We believe that this fabrication technique can be applied to patient-specific regeneration of blood vessels.     

Spontaneous loading of positively charged macromolecules into alginate-templated polyelectrolyte multilayer microcapsules

A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca(2+)-cross-linked alginate hydrogel within {poly(allylamine hydrochloride)/poly (styrene sulfonate)}(4) ({PAH/PSS}(4)) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}(4) microcapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated microcapsules, which showed the loading occurred in <2 min for dextran-amino and <10 min for peroxidase, respectively. A high loading efficiency of 25 mug peroxidase in approximately 1.0 x 10(7) microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 mug/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.     

Protamine assembled in multilayers on colloidal particles can be exchanged and released

Biocomposite thin films assembled on colloidal particles by means of layer-by-layer adsorption have been suggested as drug carriers and diagnostic devices. Protamine (PRM)/dextransulfate (DXS) and protamine/bovine serum albumine (BSA) multilayers were fabricated on colloidal silica and subsequently investigated by means of fluorescence activated cell sorting (FACS) and microelectrophoresis. Fluorescein labeled polyelectrolytes were embedded at different positions in the multilayers as a marker for layer growth. FACS showed that PRM and DXS formed regular growing stable multilayers, yet adsorbed PRM can be nevertheless exchanged with PRM in solution during layer formation and also after the multilayer formation has been completed. Up to 90% of the PRM pool was available for exchange. PRM together with BSA as demonstrated by SFM did not form multilayers under the applied conditions although the zeta-potential, commonly used as an indicator for stepwise adsorption, observed characteristic alternations. The capability of bound PRM to exchange with PRM in solution is attributed to its relatively small size. The demonstrated exchange may have importance in designing multilayers with smart release features. Furthermore, FACS proved to be a rather suitable means to quantify the aggregation behavior during coating and washing. Singulets, doublets, triplets, and aggregates of higher order could be clearly resolved. The aggregation of particles coated with PRM/DXS layers was higher than that of silica particles coated with PAH/PSS layers. In the first case about 50% of all recorded events are attributed to aggregats, while the PAH/PSS coating produced only about 10% aggregates.     

Protein-calcium carbonate coprecipitation: a tool for protein encapsulation

A new approach of encapsulation of proteins in polyelectrolyte microcapsules has been developed using porous calcium carbonate microparticles as microsupports for layer-by-layer (LbL) polyelectrolyte assembling. Two different ways were used to prepare protein-loaded CaCO3 microparticles: (i) physical adsorption--adsorption of proteins from the solutions onto preformed CaCO3 microparticles, and (ii) coprecipitation--protein capture by CaCO3 microparticles in the process of growth from the mixture of aqueous solutions of CaCl2 and Na2CO3. The latter was found to be about five times more effective than the former (approximately 100 vs approximately 20 mug of captured protein per 1 mg of CaCO3). The procedure is rather mild; the revealed enzymatic activity of alpha-chymotrypsin captured initially by CaCO3 particles during their growth and then recovered after particle dissolution in EDTA was found to be about 85% compared to the native enzyme. Core decomposition and removal after assembly of the required number of polyelectrolyte layers resulted in release of protein into the interior of polyelectrolyte microcapsules (PAH/PSS)5 thus excluding the encapsulated material from direct contact with the surrounding. The advantage of the suggested approach is the possibility to control easily the concentration of protein inside the microcapsules and to minimize the protein immobilization within the capsule walls. Moreover, it is rather universal and may be used for encapsulation of a wide range of macromolecular compounds and bioactive species.     

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.     

Preparation and characterization of polyaniline/chitosan blend film

Films consisting of a blend of a chitosan hydrogel and a conductive polymer, polyaniline (PANI), were prepared and characterized for their electrical and mechanical properties. Polyaniline in emeraldine base (EB) form was dispersed in chitosan solution and blend films were obtained by solution casting. The PANI particles in the blend films were then doped with HCl where we observed reductions in the film tensile strength and Young's modulus by about 30%, but the films electrical conductivity increased by 6 orders of magnitude. The highest electrical conductivity of the blend films was of the order 10⁻⁴ S/cm. The electrical and mechanical properties of the films varied with polyaniline content, acid dopant type, acid dopant concentration, and doping time.     

Mechanical properties of the lateral cortex of mammalian auditory outer hair cells.

Mammalian auditory outer hair cells generate high-frequency mechanical forces that enhance sound-induced displacements of the basilar membrane within the inner ear. It has been proposed that the resulting cell deformation is directed along the longitudinal axis of the cell by the cortical cytoskeleton. We have tested this proposal by making direct mechanical measurements on outer hair cells. The resultant stiffness modulus along the axis of whole dissociated cells was 3 x 10(-3) N/m, consistent with previously published values. The resultant axial and circumferential stiffness moduli for the cortical lattice were 5 x 10(-4) N/m and 3 x 10(-3) N/m, respectively. Thus the cortical lattice is a highly orthotropic structure. Its axial stiffness is small compared with that of the intact cell, but its circumferential stiffness is within the same order of magnitude. These measurements support the theory that the cortical cytoskeleton directs electrically driven length changes along the longitudinal axis of the cell. The Young's modulus of the circumferential filamentous components of the lattice were calculated to be 1 x 10(7) N/m2. The axial cross-links, believed to be a form of spectrin, were calculated to have a Young's modulus of 3 x 10(6) N/m2. Based on the measured values for the lattice and intact cell cortex, an estimate for the resultant stiffness modulus of the plasma membrane was estimated to be on the order of 10(-3) N/m. Thus, the plasma membrane appears to be relatively stiff and may be the dominant contributor to the axial stiffness of the intact cell.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

IL-1beta decreases the elastic modulus of human tenocytes.

Cellular responses to mechanical stimuli are regulated by interactions with the extracellular matrix, which, in turn, are strongly influenced by the degree of cell stiffness (Young's modulus). It was hypothesized that a more elastic cell could better withstand the rigors of remodeling and mechanical loading. It was further hypothesized that interleukin-1beta (IL-1beta) would modulate intracellular cytoskeleton polymerization and regulate cell stiffness. The purpose of this study was to investigate the utility of IL-1beta to alter the Young's modulus of human tenocytes. Young's modulus is the ratio of the stress to the strain, E = stress/strain = (F/A)/(deltaL/L0), where L0 is the equilibrium length, deltaL is the length change under the applied stress, F is the force applied, and A is the area over which the force is applied. Human tenocytes were incubated with 100 pM recombinant human IL-1beta for 5 days. The Young's modulus was reduced by 27-63%. Actin filaments were disrupted in >75% of IL-1beta-treated cells, resulting in a stellate shape. In contrast, immunostaining of alpha-tubulin showed increased intensity in IL-1beta-treated tenocytes. Human tenocytes in IL-1beta-treated bioartificial tendons were more tolerant to mechanical loading than were untreated counterparts. These results indicate that IL-1beta reduced the Young's modulus of human tenocytes by disrupting the cytoskeleton and/or downregulating the expression of actin and upregulating the expression of tubulins. The reduction in cell modulus may help cells to survive excessive mechanical loading that may occur in damaged or healing tendons.     

Donnan membrane equilibrium,sedimentation equilibrium,and coil expansion of DNA in salt solutions

This is a review of applications of the McMillan-Mayer-Hill virial theory and the ionic double-layer theory to dilute colloidal solutions, in particular, solutions of DNA. Interactions of highly charged colloidal rods are developed in terms of the second virial coefficients between two rods, and between one rod and one small co-ion. The relevant cluster integrals are evaluated with interaction potentials based on the Poisson-Boltzmann equation. The treatment is extended to the intrachain repulsion responsible for the statistical swelling of coiled DNA (excluded volume effect). The theory is compared with three sets of experimental data: The salt distribution in Donnan membrane equilibria of DNA-salt solutions, sedimentation equilibria of short DNA fragments at different ionic strengths, and the intrinsic viscosity of T7 DNA in NaCl solutions. In all cases the theory agrees well with the experiments. The agreement is not convincing for the sedimentation equilibrium at low ionic strength, because here the experimental DNA concentration is too high for the truncated dilute solution expansion of the DNA-salt repulsion.