Microrheology of novel cellulose stabilized oil-in-water emulsions.

Diffusing wave spectroscopy (DWS) is a powerful optical technique suitable to investigate turbid samples in a nondestructive and reproducible way, providing information on the static and dynamic properties of the system. This includes the relative displacement of emulsion droplets over time and changes in the viscoelastic properties. Here, novel and promising cellulose-based oil-in-water (O/W) emulsions were prepared and studied, for the first time, by DWS. Cellulose plays the role of a novel eco-friendly emulsifying agent. The hydrolysis time of cellulose was observed to affect the average size of the emulsion droplets and their stability; the longer the hydrolysis time, the more dispersed and stable the emulsions were found to be. Additionally, a good complementarity between the microrheology (DWS) and macrorheology (mechanical rheometer) data was found. Our work suggests that DWS is a highly attractive method to investigate the stability, aging and microrheology properties of cellulose-based emulsions, providing valuable insights on their microstructure. This technique is thus highly appealing for the characterization and design of novel emulsion formulations.

Elucidating the effect of additives on the alkyl chain packing of a double tail cationic surfactant.

HYPOTHESIS: Some low molecular weight additives can strongly influence the phase behavior of aqueous surfactant systems, and this offers an important handle to control the properties of surfactant solutions and thus to optimize the stability and performance of various formulations. EXPERIMENTS: The surfactant dioctadecyldimethylammonium chloride (DODAC) self-assembles into two lamellar phases in water, the gel phase (Lß) and the liquid crystalline phase (Lα). Here, we present approaches to tune the gel-to-liquid crystalline transition temperature (Tm) with the use of additives. The effects of urea, sodium butyrate and butyric acid on the packing behavior of DODAC were determined. The surfactant phases were characterized using polarized optical microscopy (POM), differential scanning calorimetry (DSC), and small/wide angle X-ray scattering (SWAXS). FINDINGS: All three additives - urea, sodium butyrate and butyric acid yield a single and stable lamellar phase. Urea and sodium butyrate have only minor effects on Tm, butyric acid gives a large decrease as it stabilizes the Lα phase with respect to the Lß phase. From the bilayer thickness of the gel phase an interdigitated or tilted packing of the surfactant molecules is suggested. The addition of sodium butyrate gives a highly interdigitated gel structure and resulted in the transition from lamellar liquid crystal to an isotropic L3 phase.

Development of carboxymethyl cellulose-chitosan hybrid micro- and macroparticles for encapsulation of probiotic bacteria.

Novel carboxymethyl cellulose-chitosan (CMC-Cht) hybrid micro- and macroparticles were successfully prepared in aqueous media either by drop-wise addition or via nozzle-spray methods. The systems were either physically or chemically crosslinked using genipin as the reticulation agent. The macroparticles (ca. 2mm) formed are found to be essentially of the core-shell type, while the microparticles (ca. 5µm) are apparently homogeneous. The crosslinked particles are robust, thermally resistant and less sensitive to pH changes. On the other hand, the physical systems are pH sensitive presenting a remarkable swelling at pH 7.4, while little swelling is observed at pH 2.4. Furthermore, model probiotic bacteria (Lactobacillus rhamnosus GG) was for the first time successfully encapsulated in the CMC-Cht based particles with acceptable viability count. Overall, the systems developed are highly promising for probiotic encapsulation and potential delivery in the intestinal tract with the purpose of modulating gut microbiota and improving human health.

Lipoprotein binding to anionic biopolyelectrolytes and the effect of glucose on nanoplaque formation in arteriosclerosis and Alzheimer's disease.

Arteriosclerosis with its clinical sequelae (cardiac infarction, stroke, peripheral arterial occlusive disease) and vascular/Alzheimer dementia not only result in far more than half of all deaths but also represent dramatic economic problems. The reason is, among others, that diabetes mellitus is an independent risk factor for both disorders, and the number of diabetics strongly increases worldwide. More than one-half of infants in the first 6months of life have already small collections of macrophages and macrophages filled with lipid droplets in susceptible segments of the coronary arteries. On the other hand, the authors of the Bogalusa Heart Study found a strong increase in the prevalence of obesity in childhood that is paralleled by an increase in blood pressure, blood lipid concentration, and type 2 diabetes mellitus. Thus, there is a clear linkage between arteriosclerosis/Alzheimer's disease on the one hand and diabetes mellitus on the other hand. Furthermore, it has been demonstrated that distinct apoE isoforms on the blood lipids further both arteriosclerotic and Alzheimer nanoplaque formation and therefore impair flow-mediated vascular reactivity as well. Nanoplaque build-up seems to be the starting point for arteriosclerosis and Alzheimer's disease in their later full clinical manifestation. In earlier work, we could portray the anionic biopolyelectrolytes syndecan/perlecan as blood flow sensors and lipoprotein receptors in cell membrane and vascular matrix. We described extensively molecular composition, conformation, form and function of the macromolecule heparan sulfate proteoglycan (HS-PG). In two supplementary experimental settings (ellipsometry, myography), we utilized isolated HS-PG for in vitro nanoplaque investigations and isolated human coronary artery segments for in vivo tension measurements. With the ellipsometry-based approach, we were successful in establishing a direct connection on a molecular level between diabetes mellitus on the one side and arteriosclerosis/Alzheimer's disease on the other side. Application of glucose at a concentration representative for diabetics and leading to glycation of proteins and lipids, entailed a significant increase in arteriosclerotic and Alzheimer nanoplaque formation. IDLapoE4/E4 was by far superior to IDLapoE3/E3 in plaque build-up, both in diabetic and non-diabetic patients. Recording vascular tension of flow-dependent reactivity in blood substitute solution and under application of different IDLapoE isoforms showed an impaired vasorelaxation for pooled IDL and IDLapoE4/E4, thus confirming the ellipsometric investigations. Incubation in IDLapoE0/E0 (apoE "knockout man"), however, resulted in a massive flow-mediated contraction, also complemented by strongly aggregated nanoplaques. In contrast, HDL was shown to present a powerful protection against nanoplaque formation on principle, both in the in vitro model and the in vivo scenario on the endothelial cell membrane. The competitive interplay with LDL is highlighted through the flow experiment, where flow-mediated, HDL-induced vasodilatation remains untouched by additional incubation with LDL. This is due to the four times higher affinity for the proteoglycan receptor of HDL as compared to LDL. Taken together, the studies demonstrate that while simplistic, the ellipsometry approach and the endothelial-mimicking proteoglycan-modified surfaces provide information on the initial steps of lipoprotein-related plaque formation, which correlates with findings on endothelial cells and blood vessels, and afford insight into the role of lipoprotein deposition and exchange phenomena at the onset of these pathophysiologies.

Mixed protein-DNA gel particles for DNA delivery: role of protein composition and preparation method on biocompatibility.

Mixtures of two cationic proteins were used to prepare protein-DNA gel particles, employing associative phase separation and interfacial diffusion (Morán et al., 2009a). By mixing the two proteins, we have obtained particles that displayed higher loading efficiency and loading capacity values than those obtained in single-protein systems. However, nothing is known about the adverse effects on haemocompatibility and cytotoxicity of these protein-DNA gel particles. Here, we examined the interaction of protein-DNA gel particles obtained by two different preparation methods, and their components, with red blood cells and established cells. From a haemolytic point of view, these protein-DNA gel particles were demonstrated to be promising long-term blood-contacting medical devices. Safety evaluation with the established cell lines revealed that, in comparison with proteins in solution, the cytotoxicity was reduced when administered in the protein-DNA systems. In comparison with large-sized particles, the cytotoxic responses of small-sized protein-DNA gel particles showed to be strongly dependent of both the protein composition and the cell line being the tumour cell line HeLa more sensitive to the deleterious effects of the mixed protein-based particles. The observed trends in haemolysis and cell viabilities were in agreement with the degree of complexation values obtained for the protein-DNA gel particles prepared by both preparation methods.

Cyclodextrin-grafted cellulose: physico-chemical characterization.

Cyclodextrins (CDs) can form inclusion complexes with a wide variety of molecules making them very attractive in different areas, such as pharmaceutics, biochemistry, food chemistry and textile. In this communication we will report on the physico-chemical characterization of cellulose modified with CDs by means of infra-red spectroscopy (FTIR), cross polarization magic angle spinning solid state nuclear magnetic resonance (CP-MAS NMR), polarized optical microscopy (POM) and thermal gravimetric analysis (TGA). Both CP-MAS NMR and FTIR indicate that CDs are chemically attached to cellulose backbone through the formation of ester bonds. Furthermore, the CD-grafted cellulose was dissolved in a "superphosphoric" acid solution but, despite the increase of hydrophilicity due to the modification, POM revealed that grafted cellulose was less soluble when compared to the unmodified polymer. The formation of a complex CD-cellulose network is suggested.

Modulating the nitric oxide - cyclic GMP pathway in the pressure-overloaded left ventricle and group II pulmonary hypertension.

Group II pulmonary hypertension (PH) commonly occurs in the setting of a pressure-overloaded left ventricle (LV) which is also conducive to the development of heart failure with preserved ejection fraction. Population trends and a high prevalence of underlying causative conditions, such as essential hypertension or aortic stenosis, have increased the awareness of the pressure-overloaded LV and associated group II pulmonary hypertension. Patients often exhibit poor exercise tolerance and signs of heart failure indistinguishable from systolic heart failure; but effective medical treatments in this area have been lacking. Recent preclinical work has shed light on how the down-regulated nitric oxide - cyclic GMP pathway (within the myocardium and pulmonary vasculature) contributes to the pathophysiology of these associated conditions. This article will discuss the impact of the nitric oxide - cyclic GMP pathway on the pathogenesis of the pressure-overloaded LV and group II pulmonary hypertension, and will also introduce the potential therapeutic value of modulating this pathway.

The antimicrobial reagent role on the degradation of model cellulose film.

The effect of the antimicrobial agent TMPAC (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) on the cellulase activity on model cellulose substrate was investigated by in situ-null ellipsometry. The cellulases used were extracted from Trichoderma viride and Aspergillus niger, and the model cellulose film was prepared by spin-coating silicon oxide wafers with cellulose solubilized in N-methylmorpholine-N-oxide/dimethyl sulfoxide solution. Upon enzyme addition to the previously equilibrated cellulose film, the initial enzyme adsorption on the substrate was followed by an overall decrease in film mass owing to enzymatic digestion of the cellulose. The loss of cellulose film mass was associated with a non-monotonously behavior of the cellulose film thickness. The activities of the two enzymes were different, a much higher degradation rate being observed for the Trichoderma viride cellulase. The degradation rate with this cellulase decreased significantly when the cellulose film was treated with the antimicrobial agent. The antimicrobial agent did not affect the cellulose degradation catalyzed by the Aspergillus niger cellulase. It was, hence, demonstrated for the first time that, depending on the cellulase type, the antimicrobial agent can inhibit enzymatic activity at the solid-liquid interface.

The effect of chain length on the melting temperature and size of dialkyldimethylammonium bromide vesicles.

Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) were used to obtain the gel to liquid-crystalline phase transition temperature (Tm) and the apparent hydrodynamic radius (Rh) of spontaneously formed cationic vesicles of dialkyldimethylammonium bromide salts (CnH2n+1)2(CH3)2N+.Br-, with varying chain lengths. The preparation of cationic vesicles from aqueous solution of these surfactants, for n=12, 14, 16 and 18 (DDAB, DTDAB, DHDAB and DODAB, respectively), requires the knowledge of the surfactant gel to liquid-crystalline phase transition temperature, or melting temperature (Tm) since below this temperature these surfactants are poorly or not soluble in water. That series of cationic surfactants has been widely investigated as vesicle-forming surfactants, although C12 and C18, DDAB and DODAB are by far the most investigated from this series. The dependence of Tm of these surfactants on the number n of carbons in the surfactant tails is reported. The Tm obtained by DSC increases non-linearly with n, and the vesicle apparent radius Rh is about the same for DHDAB and DODAB, but much smaller for DDAB.

Surface complexation of DNA with insoluble monolayers. Influence of divalent counterions.

DNA interacts with insoluble monolayers made of cationic amphiphiles as well as with monolayers of zwitterionic lipids in the presence of divalent ions. Binding to dioctadecyldimethylammonium bromide (DODAB) or distearoyl-sn-glycero-3-phosphocholine (DSPC) monolayers in the presence of calcium is accompanied by monolayer expansion. For the positively charged DODAB monolayer, this causes a decrease of surface potential, while an increase is observed for the DSPC monolayers. Binding to dipalmitoyl-sn-glycero-3-phosphocholine preserves most of the liquid expanded-liquid condensed coexistence region. The liquid condensed domains adopt an elongated morphology in the presence of DNA, especially in the presence of calcium. The interaction of DNA with phospholipid monolayers is ion specific: the presence of calcium leads to a stronger interaction than magnesium and barium. These results were confirmed by bulk complexation studies.

Polyion adsorption onto catanionic surfaces. A Monte Carlo study.

The adsorption of a single and negatively charged polyion with varying flexibility onto a surface carrying both negative and positive charges representing a charged membrane surface has been investigated by using a simple model employing Monte Carlo simulations. The polyion was represented by a sequence of negatively charged hard spheres connected with harmonic bonds. The charged surface groups were also represented by charged hard spheres, and they were positioned on a hard surface slightly protruding into the solution. The surface charges were either frozen in a liquidlike structure or laterally mobile. With a large excess of positive surface charges, the classical picture of a strongly adsorbed polyion with an extended and flat configuration emerged. However, adsorption also appeared at a net neutral surface or at a weakly negatively charged surface, and at these conditions the adsorption was stronger with a flexible polyion as compared to a semiflexible one, two features not appearing in simpler models containing homogeneously charged surfaces. The presence of charged surface patches (frozen surface charges) and the ability of polarization of the surface charges (mobile surface charges) are the main reasons for the enhanced adsorption. The stronger adsorption with the flexible chain is caused by its greater ability to spatially correlate with the surface charges.

Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo.

BACKGROUND: Chitosans of high molecular weights have emerged as efficient nonviral gene delivery systems, but the properties and efficiency of well-defined low molecular weight chitosans (<5 kDa) have not been studied. We therefore characterized DNA complexes of such low molecular weight chitosans and related their physical shape and stability to their efficiency as gene delivery systems in vitro and in vivo. METHODS: Individual complexes between six different chitosan oligomers (6-, 8-, 10-, 12-, 14- and 24-mers) and fluorescence-labeled T4 DNA were visualized and classified into six physical shapes using video-enhanced fluorescence microscopy. The effects of chitosan chain length, charge ratio (+/-) and solvent properties (pH and ionic strength) on the stability and structure of the complexes were studied. Gene expression in vitro and in vivo were studied using a luciferase reporter gene. RESULTS: Free DNA appeared as extended coils. Chitosan complexes had a variety of physical shapes depending on the experimental conditions. In general, the fraction of complexes that had nonaggregated, globular structures increased with increasing chain length of the chitosan oligomer, increasing charge ratio and reduction of pH (from 6.5 to 3.5). A further increase in charge ratio for globular complexes or a further reduction in pH (to 2.5) increased the fraction of aggregates, indicating a window where pharmaceutically desirable globules are obtained. Gene transfection efficiencies in vitro and in vivo were related to the physical shape and stability of the complexes. Only the 24-mer formed stable complexes that gave a high level of gene expression comparable to that of high molecular weight ultrapure chitosan (UPC) in vitro and in vivo. CONCLUSIONS: Chitosan oligomers form complexes with DNA in a structure-dependent manner. We conclude that the 24-mer, which has more desirable physical properties than UPC, is more attractive as a gene delivery system than the conventional high molecular weight chitosans.

DNA-lipid systems. A physical chemistry study.

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

DNA-lipid systems. A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection

Sodium polyacrylate potentiates the anti-adhesion effect of a cellulose-derived polymer.

Available methods for postoperative adhesion prevention are insufficient. A previous study demonstrated that LM-200, a bioadhesive cellulose derivative was effective in reducing adhesions. Increasing the viscosity of a polymer solution enhances the tissue separating properties. Theoretically, a combination of sodium polyacrylate (PA) and LM-200 would give more viscous solutions than LM-200 alone, and thus be more efficacious. Therefore the efficacy of various combinations of LM-200 and PA was investigated. A lesion was created in the peritoneum of mice. The solutions to be tested, or saline, were given intraperitoneally. One week post-operatively, adhesion formation was quantified and expressed as a percentage of the original lesion covered with adhesions. PA (0.01 and 0.03 wt%) given separately did not differ in adhesion reducing effect from LM-200 (p = 0.3710 and 0.3481) but PA (0.1 wt%) resulted in significantly less adhesion formation (p = 0.0004). The effect of LM-200 increased significantly when adding PA (0.01 wt%) (p = 0.0007) or PA (0.03 wt%) (p < 0.0001). When adding PA (0.1 wt%) the effect was even more pronounced (p < 0.0001). The combination of a bioadhesive cellulose derivative and the polymer PA, was effective in reducing postoperative adhesion formation and a dose-dependent increase in efficacy was obtained compared to using the two components separately.

Prevention of adhesions by surfactants and cellulose derivatives in mice.

OBJECTIVE: To evaluate the efficacy of hydrophobically modified ethyl (hydroxyethyl) cellulose (cellulose), sodium hyaluronate (hyaluronate) and phosphatidylglycerol, in the reduction of adhesion formation. DESIGN: Controlled study. SETTING: Experimental academic unit, Sweden. MATERIAL: NMRI mice. Solutions: (1) cellulose, (2) hyaluronate, (3) phosphatidylglycerol, (4) phosphatidylglycerol and cellulose, and (5) phosphatidylglycerol, cellulose and hyaluronate. INTERVENTIONS: A standard lesion was created in the parietal peritoneum in mice. One of the viscous solutions to be tested, or saline, was given intraperitoneally. MAIN OUTCOME MEASURES: Amount of adhesions found one week postoperatively. RESULTS: Cellulose; phosphatidylglycerol and cellulose; and phosphatidylglycerol, cellulose and hyaluronate all significantly reduced the amount of adhesions (p=0.0002, p=0.002, p < 0.0001), as did the hyaluronate alone (p < 0.05). Phosphatidylglycerol alone did not reduce the amount of adhesions. Combining cellulose with phosphatidylglycerol, or with hyaluronate, did not improve efficacy. CONCLUSION: Cellulose and hyaluronate were effective in reducing the formation of adhesions. Combining cellulose with hyaluronate or phosphatidylglycerol or both did not improve efficacy.

Modification of the lyotropic liquid crystalline microstructure of amphiphilic block copolymers in the presence of cosolvents.

This article reviews the results of recent investigations on the macroscopic (phase behavior) and microscopic (microstructure) aspects of the role of cosolvents on the self-assembly of amphiphilic copolymers. A comprehensive account of the systematic studies performed in ternary isothermal systems consisting of a representative poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer (Pluronic P105, EO37PO58EO37), water and a polar cosolvent (such as glycerol, propylene glycol or ethanol) is presented. The effect of cosolvents on the copolymer phase behavior is quantified in terms of the highest cosolvent/water ratio able to maintain the liquid crystalline structures. The effect of cosolvents on the microstructure of the lyotropic liquid crystals is quantified in terms of the degree of relative swelling per cosolvent content per copolymer content, a parameter that characterizes the given cosolvent and copolymer. The set of correlations on the cosolvent effects on the phase behavior or microstructure to the cosolvent physicochemical characteristics (such as octanol/water partition coefficient or solubility parameter) have led to the development of a hypothesis that accounts for the cosolvent effects on the self-assembly of PEO-PPO-PEO block copolymers and can be used to predict them. The rich structural diversity and the potential for a precise an convenient modification of the lyotropic liquid crystalline microstructure of the PEO-PPO-PEO block copolymers is discussed in comparison to the phase behavior of the low-molecular nonionic surfactants.

Clinical significance of defective dendritic cell differentiation in cancer.

Defective dendritic cell (DC) function has been described previously in cancer patients and tumor-bearing mice. It can be an important factor in the escape of tumors from immune system control. However, the mechanism and clinical significance of this phenomenon remain unclear. Here, 93 patients with breast, head and neck, and lung cancer were investigated. The function of peripheral blood and tumor draining lymph node DCs was equally impaired in cancer patients, consistent with a systemic rather than a local effect of tumor on DCs. The number of DCs was dramatically reduced in the peripheral blood of cancer patients. This decrease was associated with the accumulation of cells lacking markers of mature hematopoietic cells. The presence of these immature cells was closely associated with the stage and duration of the disease. Surgical removal of tumor resulted in partial reversal of the observed effects. The presence of immature cells in the peripheral blood of cancer patients was closely associated with an increased plasma level of vascular endothelial growth factor but not interleukin 6, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, interleukin 10, or transforming growth factor-beta and was decreased in lung cancer patients receiving therapy with antivascular endothelial growth factor antibodies. These data indicate that defective DC function in cancer patients is the result of decreased numbers of competent DCs and the accumulation of immature cells. This effect may have significant clinical implications.

Production and characterization of monoclonal antibodies to ARVCF.

We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.