Binding Mechanism of the Model Charged Dye Carboxyfluorescein to Hyaluronan/Polylysine Multilayers.

Biopolymer-based multilayers become more and more attractive due to the vast span of biological application they can be used for, e.g., implant coatings, cell culture supports, scaffolds. Multilayers have demonstrated superior capability to store enormous amounts of small charged molecules, such as drugs, and release them in a controlled manner; however, the binding mechanism for drug loading into the multilayers is still poorly understood. Here we focus on this mechanism using model hyaluronan/polylysine (HA/PLL) multilayers and a model charged dye, carboxyfluorescein (CF). We found that CF reaches a concentration of 13 mM in the multilayers that by far exceeds its solubility in water. The high loading is not related to the aggregation of CF in the multilayers. In the multilayers, CF molecules bind to free amino groups of PLL; however, intermolecular CF-CF interactions also play a role and (i) endow the binding with a cooperative nature and (ii) result in polyadsorption of CF molecules, as proven by fitting of the adsorption isotherm using the BET model. Analysis of CF mobility in the multilayers by fluorescence recovery after photobleaching has revealed that CF diffusion in the multilayers is likely a result of both jumping of CF molecules from one amino group to another and movement, together with a PLL chain being bound to it. We believe that this study may help in the design of tailor-made multilayers that act as advanced drug delivery platforms for a variety of bioapplications where high loading and controlled release are strongly desired.

In-situ assembly of Ca-alginate gels with controlled pore loading/release capability.

Development of tailor-made porous polymer scaffolds acting as a temporary tissue-construct for cellular organization is of primary importance for tissue engineering applications. Control over the gel porosity is a critical issue due to the need for cells to proliferate and migrate and to ensure the transport of nutrition and metabolites. Gel loading with bioactive molecules is desired for target release of soluble signals to guide cell function. Calcium-alginate hydrogels are one of the most popular gels successfully utilized as polymer scaffolds. Here we propose a benchtop approach to design porous alginate gels by dispersion of CaCO3 vaterite crystals in sodium alginate followed by the crystal elimination. CaCO3 crystals play a triple role being (i) cross-linkers (a source of calcium ions to cross-link gel network), (ii) pore-makers (leaching of crystals retains the empty pores), and (iii) reservoirs with (bio)molecules (by molecule preloading into the crystals). Pore dimensions, interconnectivity, and density can be adjusted by choosing the size, concentration, and packing of the sacrificial CaCO3 crystals. An opportunity to load the pores with biomolecules was demonstrated using FITC-labeled dextrans of different molecular masses from 10 to 500 kDa. The dextrans were preloaded into CaCO3 vaterite crystals, and the subsequent crystal removal resulted in encapsulation of dextrans inside the pores of the gel. The dextran release rate from the gel pores depends on the equilibration of the gel structure as concluded by comparing dextran release kinetics during gelation (fast) and dextran diffusion into the performed gel (slower). Macromolecule binding to the gel is electrostatically driven as found for lysozyme and insulin. The application of porous gels as scaffolds potentially offering biomacromolecule encapsulation/release performance might be useful for alginate gel-based applications such as tissue engineering.

Macromolecule loading into spherical, elliptical, star-like and cubic calcium carbonate carriers.

We fabricated calcium carbonate particles with spherical, elliptical, star-like and cubical morphologies by varying relative salt concentrations and adding ethylene glycol as a solvent to slow down the rate of particle formation. The loading capacity of particles of different isotropic (spherical and cubical) and anisotropic (elliptical and star-like) geometries is investigated, and the surface area of such carriers is analysed. Potential applications of such drug delivery carriers are highlighted.

Multifunctional polyelectrolyte microparticles for oral insulin delivery.

Multicomponent insulin-containing microparticles are prepared by layer-by-layer assembly of dextran sulfate and chitosan on the core of protein-polyanion complex with or without protease inhibitors. Oral bioavailability of the encapsulated insulin is improved due to the cumulative effect of each component. A physico-chemical study shows that the particle design allows adjustment of the pH-dependent profile of the insulin release, as well as mucoadhesive properties and Ca(2+) binding ability of the microparticles. Supplementing the microparticles with 2-3% protease inhibitors fully prevents proteolysis of human insulin. The pharmacological effect of microencapsulated insulin in doses 50-100 IU kg(-1) is demonstrated in chronic experiments after oral administration to diabetic rats fed ad libitum.

Magnetic porous sugar-functionalized PEG microgels for efficient isolation and removal of bacteria from solution.

Here, we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them, introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli . Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than nonporous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.

New surface-enhanced Raman scattering platforms: composite calcium carbonate microspheres coated with astralen and silver nanoparticles.

Surface-enhanced Raman scattering (SERS) microspectroscopy is a very promising label-free, noncontact, and nondestructive method for real-time monitoring of extracellular matrix (ECM) development and cell integration in scaffolds for tissue engineering. Here, we prepare a new type of micrometer-sized SERS substrate, core-shell microparticles composed of solid carbonate core coated with silver nanoparticles and polyhedral multishell fullerene-like structure, astralen. Astralen has been assembled with polyallylamine hydrochloride (PAH) by the layer-by-layer manner followed by Ag nanoparticle formation by means of a silver mirror reaction, giving the final structure of composite particles CaCO3(PAH/astralen)x/Ag, where x = 1-3. The components of the microparticle carry multiple functionalities: (i) an easy identification by Raman imaging (photostable astralen) and (ii) SERS due to a rough surface of Ag nanoparticles. A combination of Ag and astralen nanoparticles provides an enhancement of astralen Raman signal by more than 1 order of magnitude. Raman signals of commonly used scaffold components such as polylactide and polyvinyl alcohol as well as ECM component (hyaluronic acid) are significantly enhanced. Thus, we demonstrate that new mechanically robust and easily detectable (by astralen signal or optically) core-shell microspheres based on biocompatible CaCO3 can be used as SERS platform. Particle design opens many future perspectives for fabrication of SERS platforms with multiple functions for biomedical applications, for example, for theranostic.

Microfluidics as a tool to understand the build-up mechanism of exponential-like growing films.

Microfluidics is used here for the first time to efficiently tune the growth conditions for understanding the build-up mechanism of exponentially growing polyelectrolyte (PE) films. The velocity of PE supply and time of interaction can be successfully altered during the layer-by-layer assembly. Another advantage of this method is that the deposition of poly-L-lysine/hyaluronic acid (PLL/HA) films in microchannels can be monitored online by fluorescence microscopy. The study demonstrates that PE mass transport to the film surface and diffusion in the film are key parameters affecting PLL/HA film build-up. Increase of PE supply rate results in a change in the "transition" (exponential-to-linear growth) towards higher number of deposition steps, thus indicating a mass transport-mediated growth mechanism.

Control of cell adhesion by mechanical reinforcement of soft polyelectrolyte films with nanoparticles.

Chemical cross-linking is the standard approach to tune the mechanical properties of polymer coatings for cell culture applications. Here we show that the elastic modulus of highly swollen polyelectrolyte films composed of poly(L-lysine) (PLL) and hyaluronic acid (HA) can be changed by more than 1 order of magnitude by addition of gold nanoparticles (AuNPs) in a one-step procedure. This hydrogel-nanoparticle architecture has great potential as a platform for advanced cell engineering application, for example remote release of drugs. As a first step toward utilization of such films for biomedical applications we identify the most favorable polymer/nanoparticle composition for optimized cell adhesion on the films. Using atomic force microscopy (AFM) we determine the following surface parameters that are relevant for cell adhesion, i.e., stiffness, roughness, and protein interactions. Optimized cell adhesion is observed for films with an elastic modulus of about 1 MPa and a surface roughness on the order of 30 nm. The analysis further shows that AuNPs are not incorporated in the HA/PLL bulk but form clusters on the film surface. Combined studies of the elastic modulus and surface topography indicate a cluster percolation threshold at a critical surface coverage above which the film stiffness drastically increases. In this context we also discuss changes in film thickness, material density and swelling ratio due to nanoparticle treatment.

Synthesis of porous PEG microgels using CaCO3 microspheres as hard templates.

Porous poly(ethylene glycol) (PEG) microgels of both 17.6 and 8.3 µm in diameter are synthesized via hard templating with calcium carbonate (CaCO(3)) microparticles. The synthesis is performed in three steps: loading of PEG macromonomers into CaCO(3) microparticles, crosslinking via photopolymerization, and removal of the CaCO(3) template under acidic conditions. The resulting porous PEG microgels are inverse replicates of their templates as indicated by light microscopy, cryo-scanning electron microscopy (cryo-SEM), and permeability studies. Thus this process allows for the straightforward and highly reproducible synthesis of porous hydrogel particles of two different diameters and porosities that show great potential as carriers for drugs or nanomaterials.

Microfluidics meets soft layer-by-layer films: selective cell growth in 3D polymer architectures.

We present here the micropatterns of layer-by-layer (LbL) assembled soft films generated using microfluidic platform that can be exploited for selective cell growth. Using this method, the issue of cell adhesion and spreading on soft LbL-derived films, and simultaneous utilisation of such unmodified soft films to exploit their reservoir properties are addressed. This also paves the way for extending the culture of cells to soft films and other demanding applications like triggered release of biomolecules.

Anisotropic multicompartment micro- and nano-capsules produced via embedding into biocompatible PLL/HA films.

We present a novel strategy to fabricate anisotropic multicompartment Janus capsules by embedding larger containers into a soft poly-L-lysine/hyaluronic acid (PLL/HA) polymeric film, followed by adsorption of smaller containers on top of their unmasked surface. This research is also attractive for developing substrates for cell cultures.

Bio-interfaces--interaction of PLL/HA thick films with nanoparticles and microcapsules.

The interaction of biocompatible, exponentially grown films composed of poly-L-lysine (PLL) and hyaluronic acid (HA) polymers with gold nanoparticles and microcapsules is studied. Both aggregated and non-aggregated nanoparticle states are achieved; desorption of PLL accounts for aggregation of nanoparticles. The presence of aggregates of gold nanoparticles on films enables remote activation by near-infrared irradiation due to local, nanometer confined heating. Thermally shrunk microcapsules, which are remarkably monodisperse upon preparation but gain polydispersity after months of storage, are also adsorbed onto films. PLL polymers desorbed from films interact with microcapsules introducing a charge imbalance which leads to an increase of the microcapsule size, thus films amplify this effect. Multifunctional, biocompatible, thick gel films with remote activation and release capabilities are targeted for cell cultures in biology and tissue engineering in medicine.

Near-IR remote release from assemblies of liposomes and nanoparticles.

Set free by light: Near-IR (NIR) laser-initiated remote release of fluorescent dye from complexes of liposome-gold-nanoparticle aggregates is demonstrated (see fluorescence images). Complexes of the desired size are shown to be a viable approach to the construction of vesicle-based drug-delivery systems with light-triggered remote release characteristics. This opens up a new method to manipulate liposome-based drug-delivery systems in a biocompatible way by using the near-IR spectral range.

Real-time assessment of spatial and temporal coupled catalysis within polyelectrolyte microcapsules containing coimmobilized glucose oxidase and peroxidase.

The encapsulation of biological enzymes within polyelectrolyte microcapsules is an important step toward microscale devices for processing and analytical applications, one which could be applied to the realization of minimally invasive sensing technology. In this work, the encapsulation and functional characterization of a bienzymatic coupled catalytic system within polyelectrolyte microcapsules is described. The two components, glucose oxidase (GOx) and horseradish peroxidase (HRP), were coprecipitated with calcium carbonate microspheres, followed by layer-by-layer assembly to form ultrathin polymer film coatings that act as capsule walls after removal of the sacrificial carbonate cores. Encapsulated concentrations of GOx and HRP were determined to be 19.7 +/- 1.0 and 29.4 +/- 3.6 mg/mL, respectively. An 85% decrease in the rate of glucose consumption relative to GOx and HRP in free solution was observed, which is attributed to substrate diffusion limitations. To further understand the temporal and spatial dynamics of the two-step reaction, a technique for monitoring microscale glucose consumption was developed using confocal imaging techniques. Time-based acquisition of capsule/Amplex Red suspensions was performed, from which it was observed that the high concentration of enzyme immobilized within the capsule walls resulted in a greater rate and quantity of glucose consumption at the capsule periphery when compared to glucose consumption within the capsule interior. These findings demonstrate the function of a bienzymatic catalytic system within the controlled environment of polyelectrolyte microspheres and a novel approach to analysis of the internal reactions using confocal imaging that will allow direct comparison with reaction-diffusion modeling and further explorations to optimize the distribution and activity of the encapsulated species.

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.

Protein encapsulation via porous CaCO3 microparticles templating.

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

Matrix polyelectrolyte microcapsules: new system for macromolecule encapsulation.

A new approach to fabricate polyelectrolyte microcapsules is based on exploiting porous inorganic microparticles of calcium carbonate. Porous CaCO3 microparticles (4.5-5.0 microns) were synthesized and characterized by scanning electron microscopy and the Brunauer-Emmett-Teller method of nitrogen adsorption/desorption to get a surface area of 8.8 m2/g and an average pore size of 35 nm. These particles were used as templates for polyelectrolyte layer-by-layer assembly of two oppositely charged polyelectrolytes, poly(styrene sulfonate) and poly(allylamine hydrochloride). Calcium carbonate core dissolution resulted in formation ofpolyelectrolyte microcapsules with an internal matrix consisting of a polyelectrolyte complex. Microcapsules with an internal matrix were analyzed by confocal Raman spectroscopy, scanning electron microscopy, force microscopy, and confocal laser-scanning fluorescence microscopy. The structure was found to be dependent on a number of polyelectrolyte adsorption treatments. Capsules have a very high loading capacity for macromolecules, which can be incorporated into the capsules by capturing them from the surrounding medium into the capsules. In this paper, we investigated the loading by dextran and bovine serum albumin as macromolecules. The amount of entrapped macromolecules was determined by two independent methods and found to be up to 15 pg per microcapsule.

Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase.

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.