Natural nanocontainer for the controlled delivery of glycerol as a moisturizing agent.

Natural halloysite nanotubes with a 15-nm internal lumen and a 50 nm outer diameter were investigated as a nanocontainer for the loading and extended release of glycerol for cosmetic applications. Cytotoxicity testing of the halloysite was conducted on 3T3 and MCF-7 cells, and the tubules showed no toxic effect on the cells for over 48 h. The capability of halloysite for loading glycerol was higher with the USA halloysite than with the New Zealand's, being approximately 20% and 2.3% by weight, respectively. The total elapsed time for releasing glycerol from the nanotubes exceeded 20 h. To further retard the glycerol release rate, the halloysite samples filled with glycerol were coated with several alternate layers of polyethyleneimine and polyacrylic acid. The release rate remained at the same level, however, probably due to the low molecular weight of the polyelectrolytes and the high solubility of glycerol in water.

Top-down and bottom-up approaches in production of aqueous nanocolloids of low solubility drug paclitaxel.

Nano-encapsulation of a poorly soluble anticancer drug was demonstrated with a sonication assisted layer-by-layer polyelectrolyte coating (SLbL). We changed the strategy of LbL-encapsulation from making microcapsules with many layers in the walls for encasing highly soluble materials to using a very thin polycation/polyanion coating on low solubility nanoparticles to provide them with good colloidal stability. SLbL encapsulation of paclitaxel resulted in stable 100-200 nm diameter colloids with a high electrical surface ξ-potential (of -45 mV) and drug content in the nanoparticles of 90 wt%. In the top-down approach, nanocolloids were prepared by rupturing a powder of paclitaxel using ultrasonication and simultaneous sequential adsorption of oppositely charged biocompatible polyelectrolytes. In the bottom-up approach paclitaxel was dissolved in organic solvent (ethanol or acetone), and drug nucleation was initiated by the addition of aqueous polyelectrolyte assisted by ultrasonication. Paclitaxel release rates from such nanocapsules were controlled by assembling multilayer shells with variable thicknesses and were in the range of 10-20 h.

Comparison of selective attachment and growth of smooth muscle cells on gelatin- and fibronectin-coated micropatterns.

Tissue engineering research has been on going for many years, people are making all the effort to explore the cell functions in cellular level and even in molecular level. Making the cells functional in an in vitro environment is a preliminary goal for the implantation and repair of complicated tissues/organs. Fabricating artificial ECM to mimic the in vivo environment is an essential approach in tissue engineering. The work in this paper is to study how rat aorta smooth muscle cells (RASMCs) behave in two engineered cell culture scaffolds: gelatin- and fibronectin (FN)-coated micropatterns. The investigation on the initial attachment and further growth of SMCs cultured on gelatin- and FN-coated micropatterns was addressed. This study focused on both the characterization of gelatin and fibronectin assembly properties and cell responses to these two protein-coated micropatterns. Thin film patterns with gelatin and fibronectin coatings were fabricated on microscope glass slides using photolithography, electrostatic layer-by-layer self-assembly and lift-off (LbL-LO) technologies. In this work, the scaffolds were built up by commonly used polyelectrolyte materials and proteins through LbL process, containing cationic poly(diallyldimethylammonium chloride) (PDDA), poly(allylamine hydrochloride) (PAH), anionic poly(sodium 4-styrenesulfonate) (PSS), gelatin and fibronectin. The resulting polyelectrolyte thin films were characterized by contact angle (CA), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and fluorescence microscopy. CA measurement shows the consistent hydrophylicity of gelatin surfaces in different number of layers with LbL deposition method. Different from our previous QCM measurement of gelatin, fibronectin does not show high electrostatic attraction to either positively or negatively charged polyelectrolytes, although it can be weakly assembled to both polyelectrolyte surfaces. AFM images show Gelatin- and FN-coated micropatterns are around 50-60 nm thick. RASMCs were cultured on these gelatin- and FN-coated micropatterns. It was observed that, for the cells cultured on gelatin-coated micropatterns, they initially landed on the gelatin-coated surface, not on the PDDA-coated surface in between. But further growth of the cells was affected by the shape of the patterns: strip pattern limited cell growth beyond the patterns, but square patterns could not. While, it was found interestingly, for the cells cultured on FN-coated micropatterns, SMCs initially landed on PDDA-coated surface, and then migrated to FN-coated both square and strip patterns. These findings indicate that both gelatin and fibronectin are adhesive proteins, but they have different effects on the initial attachment and later growth for SMCs.

Hamiltonian formalism and the Garrett-Munk spectrum of internal waves in the ocean.

Wave turbulence formalism for long internal waves in a stratified fluid is developed, based on a natural Hamiltonian description. A kinetic equation appropriate for the description of spectral energy transfer is derived, and its anisotropic self-similar stationary solution corresponding to a direct cascade of energy toward the short scales is found. This solution is very close to the high wave-number limit of the Garrett-Munk spectrum of long internal waves in the ocean. In fact, a small modification of the Garrett-Munk formalism includes a spectrum consistent with the one predicted by wave turbulence.

In-vitro release characteristics of tetracycline HCl, khellin and nicotinamide adenine dineculeotide from halloysite; a cylindrical mineral.

The use of halloysite clay as a low cost alternative to more traditional microencapsulation systems is reported. Halloysite is an alumino-silicate clay which demonstrates a predominately cylindrical geometry, uniquely characterized by a hollow core or series of voids with diameters ranging from 16-50 nm. These nanoscale-to-mesoscale microcylinders are capable of entrapping active agents within the core lumen as well as within any void spaces contained in the multilayered walls of the cylinder. Some of the active agents associated with the clay are also bound to the external surfaces of the clay. Delivery of the active agent is first by desorption of the active agent from the exterior surfaces and exposed ends of the microcylinders, and is followed by a second more prolonged phase dominated by pore diffusion from the ends of the cylinders. Halloysite is capable of retaining and releasing a range of active ingredients. Both hydrophilic and hydrophobic agents may be entrapped following appropriate pre-treatment of the clay to render it lipophilic. Here, a unique low cost alternative microcylindrical delivery system: the clay mineral halloysite, is investigated.

Biocolloids with ordered urease multilayer shells as enzymatic reactors.

The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

Influence of bromide on electrochemistry of photosynthetic reaction center films on gold electrodes.

A strong influence of bromide ion was found on voltammetry of layered films of photosynthetic reaction center (RC) protein and polyions on gold electrodes. Similar, but not identical, cyclic voltammetry peaks were observed for polyion films on gold with and without RC when the buffer solutions contained bromide ion. CVs of RC films were quite different in the absence of bromide. These new findings suggest that previously published results were biased by significant background peaks involving bromide ion adsorption/desorption.

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy.

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.

Electroactive Films of Alternately Layered Polycations and Iron-Sulfur Protein Putidaredoxin on Gold.

Layered, electrochemically active films of bacterial iron-sulfur protein putidaredoxin (Pdx) and poly(dimethyldiallyammonium) (PDDA) polycations were constructed on gold electrodes coated with mercaptopropane sulfonate (MPS) and on quartz slides. Second-derivative UV-vis spectra suggested similar structures of Pdx in films and solutions at pH 7. Direct electrochemistry was achieved between Pdx and gold electrodes in these films, with significantly better electrochemical reversibility than in cast Nafion-lipid-Pdx films. A formal potential dispersion model gave a good fit to square wave voltammograms by regression analysis and was used to estimate an average apparent rate constant of 4.5 s(-1). Reduced Pdx in the polyion films did not react with its natural redox partner cytochrome P450(cam) because of unfavorable thermodynamics in the film environment. Copyright 2000 Academic Press.

Fast reversible electron transfer for photosynthetic reaction center from wild type Rhodobacter sphaeroides re-constituted in polycation sandwiched monolayer film.

Direct reversible electron transfer for photosynthetic reaction center from wild type Rhodobacter sphaeroides re-constituted in polycation sandwiched monolayer film was observed in this work. The redox potential E0' = 0.46 V vs. NHE for first primary donor redox couple P/P+ was accurately measured from reversible CV or SWV peaks, which were quite close to those obtained from optic redox titration method. Reaction center (RC) in film was found re-constituted in such an ordered way that the orientation of RC favored the electron transfer in film. Thus, the protein electroactivity seems to be turned on in this artificial biomimic thin film. Furthermore, RC in the film features a photo-induced redox-peak fluctuation, suggesting an intact and functional state for RC in such film. Redox peaks were also found dependent of pH, implying a proton-coupled electron transfer occurring in film. Charge recombination was observed accompanied with change of electrochemical driving force. Electrochemical model assuming several classes of electroactive sites in the films on the electrode with a dispersion of standard potentials successfully fits SWV experimental data at different pulse height and frequency.

Ultrathin films of charged polysaccharides assembled alternately with linear polyions.

As a means of preparation of biocompatible molecular surfaces, an alternate assembly of charged polysaccharides and oppositely-charged synthetic polymers was conducted. Cationic chitosan was assembled alternately with anionic poly(sodium styrenesulfonate) (PSS) at pH 4. Regular film growth and its dependence on ionic strength were detected by the quartz crystal microbalance (QCM) method. Averaged film thicknesses for the chitosan + PSS layer were 15, 31, 46, and 69 A, respectively, when 0, 0.25, 0.5, and 1 M of NaCl was contained in aqueous chitosan. Adsorption of chitosan did not reach saturation in 20 min at 0 M NaCl, while the adsorption became saturated within 6 min with 0.25 M NaCl. Anionic sodium chondroitin sulfate was also assembled in alternation with cationic poly(dimethyldiallylammonium chloride) (PDDA) at pH 6.5. The adsorption of chondroitin sulfate was less sensitive to ionic strength. Surface morphology of chitosan-PSS films was investigated by non-contact atomic force microscopy (AFM) observation. Maximum height difference and Ra value for a 1000 x 1000 nm area were 11 and 0.69 nm, respectively, indicating the formation of a molecularly flat surface by alternate layer-by-layer adsorption.

Sequential actions of glucose oxidase and peroxidase in molecular films assembled by layer-by-layer alternate adsorption.

Molecular films of protein/polyion layers were assembled by means of alternate adsorption through electrostatic interaction. Glucose oxidase (GOD) and peroxidase (POD) were assembled in combination with sodium poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI), respectively. Enzyme activities of those films on specific substrates (glucose and H(2)O(2)) were examined by coloring reaction of dye DA67. A multienzyme film containing GOD layer and POD layer was prepared by alternate adsorption of POD/PSS followed by PEI/GOD. Sequential redox reaction of glucose/H(2)O(2)/DA67 was demonstrated successfully with this supramolecular system.

Packaging of DNA in cauliflower mosaic virus and bacteriophage SD studied with magnetic birefringence.

Magnetic birefringence measurements are reported on solutions of two double-stranded DNA containing viruses: icosahedral cauliflower mosaic virus (CaMV) and SD, an isometric bacteriophage with a short tail. Assuming the signal comes predominantly from the DNA, constraints can be put on its packing geometry. The anisotropy of CaMV is consistent with the DNA having a near orthogonally wound spool-like structure. The DNA anisotropy is smaller in SD but is compatible with a spool-like structure if the windings are tilted about the spool axis or if a significant proportion of the DNA is effectively isotropically packed. Intermediate structures are also possible.

Structure of bacteriophage T7. Small-angle X-ray and neutron scattering study.

Small-angle x-ray and neutron scattering techniques were applied to bacteriophage T7 solutions at different scattering densities. Scattering curves determined under a variety of experimental conditions were used to derive a set of parameters characterizing the shape, size, and weight of the whole phage particle and of its DNA and protein components. The T7 head has an icosahedral shape with an edge of 37.7 +/- 0.5 nm, a volume of (12.0 +/- 1.0) x 10(4) nm3, and a small tail amounting to 6--7% of the head volume. The intraphage DNA region is most probably a hollow sphere. The best fit to the data was obtained with a model in which the hollow sphere filled with a protein core with a diameter of 24 nm. The average degree of swelling (i.e., the ratio of the hydrated to the dry volume) of the particle is 2.3; the degree of swelling of the DNA component is higher, 3.2, and that of the protein part is lower, 1.2.