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.

Biodegradation-Resistant Multilayers Coated with Gold Nanoparticles. Toward a Tailor-made Artificial Extracellular Matrix.

Polymer multicomponent coatings such as multilayers mimic an extracellular matrix (ECM) that attracts significant attention for the use of the multilayers as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles were described. Nanoparticle coating acts as a semipermeable barrier that governs molecular transport into/from the multilayers and makes them biodegradation-resistant. Model protein lysozyme (mimics of ECM-soluble signals) diffuses into the multilayers as fast- and slow-diffusing populations existing in an equilibrium. Such a composite system may have high potential to be exploited as degradation-resistant drug-delivery platforms suitable for cell-based applications.

Controlling the Vaterite CaCO3 Crystal Pores. Design of Tailor-Made Polymer Based Microcapsules by Hard Templating.

The spherical vaterite CaCO3 microcrystals are nowadays widely used as sacrificial templates for fabrication of various microcarriers made of biopolymers (e.g., proteins, nucleic acids, enzymes) due to porous structure and mild template elimination conditions. Here, we demonstrated for the first time that polymer microcarriers with tuned internal nanoarchitecture can be designed by employing the CaCO3 crystals of controlled porosity. The layer-by-layer deposition has been utilized to assemble shell-like (hollow) and matrix-like (filled) polymer capsules due to restricted and free polymer diffusion through the crystal pores, respectively. The crystal pore size in the range of few tens of nanometers can be adjusted without any additives by variation of the crystal preparation temperature in the range 7-45 °C. The temperature-mediated growth mechanism is explained by the Ostwald ripening of nanocrystallites forming the crystal secondary structure. Various techniques including SEM, AFM, CLSM, Raman microscopy, nitrogen adsorption-desorption, and XRD have been employed for crystal and microcapsule analysis. A three-dimensional model is introduced to describe the crystal internal structure and predict the pore cutoff and available surface for the pore diffusing molecules. Inherent biocompatibility of CaCO3 and a possibility to scale the porosity in the size range of typical biomacromolecules make the CaCO3 crystals extremely attractive tools for template assisted designing tailor-made biopolymer-based architectures in 2D to 3D targeted at drug delivery and other bioapplications.

Temperature effect on the build-up of exponentially growing polyelectrolyte multilayers. An exponential-to-linear transition point.

In this study, the effect of temperature on the build-up of exponentially growing polyelectrolyte multilayer films was investigated. It aims at understanding the multilayer growth mechanism as crucially important for the fabrication of tailor-made multilayer films. Model poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers were assembled in the temperature range of 25-85 °C by layer-by-layer deposition using a dipping method. The film growth switches from the exponential to the linear regime at the transition point as a result of limited polymer diffusion into the film. With the increase of the build-up temperature the film growth rate is enhanced in both regimes; the position of the transition point shifts to a higher number of deposition steps confirming the diffusion-mediated growth mechanism. Not only the faster polymer diffusion into the film but also more porous/permeable film structure are responsible for faster film growth at higher preparation temperature. The latter mechanism is assumed from analysis of the film growth rate upon switching of the preparation temperature during the film growth. Interestingly, the as-prepared films are equilibrated and remain intact (no swelling or shrinking) during temperature variation in the range of 25-45 °C. The average activation energy for complexation between PLL and HA in the multilayers calculated from the Arrhenius plot has been found to be about 0.3 kJ mol(-1) for monomers of PLL. Finally, the following processes known to be dependent on temperature are discussed with respect to the multilayer growth: (i) polymer diffusion, (ii) polymer conformational changes, and (iii) inter-polymer interactions.

Hyaluronic Acid/Poly-l-Lysine Multilayers as Reservoirs for Storage and Release of Small Charged Molecules.

Polyelectrolyte multilayer films are nowadays very attractive for bioapplications due to their tunable properties and ability to control cellular response. Here we demonstrate that multilayers made of hyaluronic acid and poly-l-lysine act as high-capacity reservoirs for small charged molecules. Strong accumulation within the film is explained by electrostatically driven binding to free charges of polyelectrolytes. Binding and release mechanisms are discussed based on charge balance and polymer dynamics in the film. Our results show that transport of molecules through the film-solution interface limits the release rate. The multilayers might serve as an effective platform for drug delivery and tissue engineering due to high potential for drug loading and controlled release.

Isolation, biochemical characterization and anti-bacterial activity of BPIFA2 protein.

OBJECTIVE: Human BPIFA2 (parotid secretory protein) is a ubiquitous soluble salivary protein, which belongs to the PLUNC family of proteins. Having sequence similarity to bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein, PLUNC proteins are probably involved in local antibacterial response at mucosal sites, such as oral cavity. The aim of the study was to isolate and characterize human BPIFA2. DESIGN: In this paper, we report one-step affinity chromatography method for BPIFA2 purification from whole human saliva. The isolated BPIFA2 was identified by trypsin mass fingerprinting and characterized by electrophoretic methods. Antibacterial activity of BPIFA2 against model microorganism Pseudomonas aeruginosa was shown in minimum inhibitory concentration and time kill study assays. RESULTS: The protein showed microheterogeneity, both in molecular weight and pI value. BPIFA2 inhibited the growth of P. aeruginosa in microgram concentration range determined by minimum inhibitory concentration assay. In the time kill study, 32µg/mL BPIFA2 showed clear bactericidal activity and did not cause any aggregation of bacteria. CONCLUSION: Affinity chromatography is well suited for isolation of functional BPIFA2 with a potent bactericidal activity against P. aeruginosa.

Beyond H2S and NO interplay: hydrogen sulfide and nitroprusside react directly to give nitroxyl (HNO). A new pharmacological source of HNO.

Hydrogen sulfide (H2S) has been increasingly recognized as an important signaling molecule that regulates both blood pressure and neuronal activity. Attention has been drawn to its interactions with another gasotransmitter, nitric oxide (NO). Here, we provide evidence that the physiological effects observed upon the application of sodium nitroprusside (SNP) and H2S can be ascribed to the generation of nitroxyl (HNO), which is a direct product of the reaction between SNP and H2S, not a consequence of released NO subsequently reacting with H2S. Intracellular HNO formation has been confirmed, and the subsequent release of calcitonin gene-related peptide from a mouse heart has been demonstrated. Unlike with other thiols, SNP reacts with H2S in the same way as rhodanese, i.e., the cyanide transforms into a thiocyanate. These findings shed new light on how H2S is understood to interact with nitroprusside. Additionally, they offer a new and convenient pharmacological source of HNO for therapeutic purposes.