Signal amplification of microarray-based immunoassay by optimization of nanoliposome formulations

The use of microarray-based immunoassay is often limited by its sensitivity. To increase the sensitivities of such an immunoassay, liposome encapsulation was explored. Two different liposome formations and several preparation methods were examined to optimize encapsulation and signal-enhancing efficacy for enzyme-linked immunosorbent assay (ELISA) and antibody array. The signal amplification by liposome encapsulation was demonstrated through a detection for foodborne pathogenic Listeria. In plate-trapped antigen (PTA) ELISA, horseradish peroxidase (HRP)-loaded liposome increased signal 9-fold more than the control. Limits of detection (LODs) of HRP-encapsulated liposome were 6.4×10(5) and 5.5×10(6)CFU/ml in sandwich ELISA and antibody array, respectively. Furthermore, when chromogenic 4-chloro-1-naphthol (4-CN) substrate was used for signal development in the antibody array, the signal could be detected with the naked eye. These results suggest that the liposome encapsulation technique can have great potential for signal amplification and, therefore, for increasing assay sensitivity for various formats of immunoassay, especially microarray-based format.     

Nanostructured Lipid Carriers (NLC) for Parenteral Delivery of an Anticancer Drug

The purpose of this research was to formulate nanostructured lipid carriers (NLC) for the parenteral delivery of an anticancer drug, all-trans retinoic acid (ATRA). The ATRA was incorporated into NLC by the de novo emulsification method. The effect of the formulation factor, i.e., type and oil ratio, initial ATRA concentration on physicochemical properties was determined. The anticancer efficacy of ATRA-loaded NLC on HL-60 and HepG2 cells was also studied. NLC was formulated using a blend of solid lipids (cetyl palmitate) and liquid lipids (soybean oil (S), medium-chain triglyceride (M), S/oleic acid (O; 3:1) and M/O (3:1)) at a weight ratio of 1:1. ATRA-loaded NLC had an average size of less than 200 nm (141.80 to 172.95 nm) with a narrow PDI and negative zeta potential that was within an acceptable range for intravenous injection. The results indicated that oleic acid enhanced the ATRA-loading capacity of NLC. In vitro ATRA release was only approximately 4.06% to 4.34% for 48 h, and no significant difference in ATRA release rate from all NLC formulations in accordance with the composition of the oil phase. Moreover, no burst release of the drug was observed, indicating that NLC could prolong the release of ATRA. The initial drug concentration affected the photodegradation rate but did not affect the release rate. All ATRA-loaded NLC formulations exhibited the photoprotective property. The cytotoxicity results showed that all ATRA-loaded NLC had higher cytotoxicity than the free drug and HL-60 cells were more sensitive to ATRA than HepG2 cells.     

Methylated N-(4-N,N-Dimethylaminobenzyl) Chitosan, a Novel Chitosan Derivative, Enhances Paracellular Permeability Across Intestinal Epithelial Cells (Caco-2)

The aim of this study was to investigate the effect of methylated N-(4-N,N-dimethylaminobenzyl) chitosan, TM-Bz-CS, on the paracellular permeability of Caco-2 cell monolayers and its toxicity towards the cell lines. The factors affecting epithelial permeability, e.g., degree of quaternization (DQ) and extent of dimethylaminobenzyl substitution (ES), were evaluated in intestinal cell monolayers of Caco-2 cells using the transepithelial electrical resistance and permeability of Caco-2 cell monolayers, with fluorescein isothiocyanate dextran 4,400 (FD-4) as a model compound for paracellular tight-junction transport. Cytotoxicity was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability assay. The results revealed that, at pH 7.4, TM-Bz-CS appeared to increase cell permeability in a concentration-dependent manner, and this effect was relatively reversible at lower doses of 0.05–0.5 mM. Higher DQ and the ES caused the permeability of FD-4 to be higher. The cytotoxicity of TM-Bz-CS depended on concentration, %DQ, and %ES. These studies demonstrated that this novel modified chitosan has potential as an absorption enhancer.     

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(?), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.     

Mucoadhesive property and biocompatibility of methylated N-aryl chitosan derivatives

The methylated N-aryl chitosan derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMCh) and methylated N-(4-pyridylmethyl) chitosan chloride (MPyMeCh), were synthesized by two steps, the reductive amination and the methylation. The physicochemical properties of chitosan derivatives were determined by ATR-FTIR, NMR, X-ray diffraction (XRD) and thermogravimetric (TG) techniques. The XRD analysis showed that the crystallinity and thermal stability of methylated chitosan derivatives were lower than those of chitosan. The effects of degree of quaternization (DQ), polymer structure and positive charge location on mucoadhesive property and cytotoxicity were investigated by using a mucin particle method and MTT assay compared to N,N,N-trimethylammonium chitosan chloride (TMChC). It was found that the mucoadhesive property and cytotoxicity increased with increasing DQ. At the DQ of 65%, the mucoadhesive property of the MDMCMCh was twofold lower than that of the TMChC. However, this phenomenon did not affect the mucoadhesive property when the DQ was higher than 65%. Surprisingly, the MPyMeCh showed the lowest toxicity even with the high DQ. These could be due to the resonance effect of the positive charge in the pyridine ring and the molecular weight after methylation. Finally, our result revealed that the mucoadhesive property was dependent on the DQ and polymer structure whereas the cytotoxicity was dependent on the combination of the polymer structure, positive charge location and molecular weight after methylation.     

Place-Based Attributes Predict Community Membership in a Mobile Phone Communication Network

Social networks can be organized into communities of closely connected nodes, a property known as modularity. Because diseases, information, and behaviors spread faster within communities than between communities, understanding modularity has broad implications for public policy, epidemiology and the social sciences. Explanations for community formation in social networks often incorporate the attributes of individual people, such as gender, ethnicity or shared activities. High modularity is also a property of large-scale social networks, where each node represents a population of individuals at a location, such as call flow between mobile phone towers. However, whether or not place-based attributes, including land cover and economic activity, can predict community membership for network nodes in large-scale networks remains unknown. We describe the pattern of modularity in a mobile phone communication network in the Dominican Republic, and use a linear discriminant analysis (LDA) to determine whether geographic context can explain community membership. Our results demonstrate that place-based attributes, including sugar cane production, urbanization, distance to the nearest airport, and wealth, correctly predicted community membership for over 70% of mobile phone towers. We observed a strongly positive correlation (r = 0.97) between the modularity score and the predictive ability of the LDA, suggesting that place-based attributes can accurately represent the processes driving modularity. In the absence of social network data, the methods we present can be used to predict community membership over large scales using solely place-based attributes.     

Chitosan-Mediated siRNA Delivery <Emphasis Type="Italic">In Vitro</Emphasis>: Effect of Polymer Molecular Weight,Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.     

Spatial Heterogeneity,Host Movement and Mosquito-Borne Disease Transmission

Mosquito-borne diseases are a global health priority disproportionately affecting low-income populations in tropical and sub-tropical countries. These pathogens live in mosquitoes and hosts that interact in spatially heterogeneous environments where hosts move between regions of varying transmission intensity. Although there is increasing interest in the implications of spatial processes for mosquito-borne disease dynamics, most of our understanding derives from models that assume spatially homogeneous transmission. Spatial variation in contact rates can influence transmission and the risk of epidemics, yet the interaction between spatial heterogeneity and movement of hosts remains relatively unexplored. Here we explore, analytically and through numerical simulations, how human mobility connects spatially heterogeneous mosquito populations, thereby influencing disease persistence (determined by the basic reproduction number R ₀), prevalence and their relationship. We show that, when local transmission rates are highly heterogeneous, R ₀ declines asymptotically as human mobility increases, but infection prevalence peaks at low to intermediate rates of movement and decreases asymptotically after this peak. Movement can reduce heterogeneity in exposure to mosquito biting. As a result, if biting intensity is high but uneven, infection prevalence increases with mobility despite reductions in R ₀. This increase in prevalence decreases with further increase in mobility because individuals do not spend enough time in high transmission patches, hence decreasing the number of new infections and overall prevalence. These results provide a better basis for understanding the interplay between spatial transmission heterogeneity and human mobility, and their combined influence on prevalence and R ₀.     

<Emphasis Type="Italic">In vitro</Emphasis> Permeability Enhancement in Intestinal Epithelial Cells (Caco-2) Monolayer of Water Soluble Quaternary Ammonium Chitosan Derivatives

The aim of this study was to investigate the effects of a type of hydrophobic moiety, extent of N-substitution (ES), and degree of quaternization (DQ) of chitosan (CS) on the transepithelial electrical resistance and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4,400 (FD-4) as the model compound for paracellular tight junction transport. CS was substituted with hydrophobic moiety, an aliphatic aldehyde (n-octyl) or aromatic aldehyde (benzyl), for the improved hydrophobic interaction with cell membrane, and they were quaternized with Quat-188 to render CS soluble. The factors affecting the epithelial permeability have been evaluated in the intestinal cell monolayers, Caco-2 cells. Cytotoxicity was evaluated by using the trypan blue and MTT viability assay. The results revealed that at pH 7.4 CSQ appeared to increase cell permeability in dose-dependent manner, and this effect was relatively reversible at the lower doses of 0.05–1.25 mM. The higher DQ and ES caused the higher permeability of FD-4. Cytotoxicity of CSQ was concentration, %DQ, and %ES dependent. Substitution with hydrophobic moiety caused decreasing in permeability of FD-4 and cytotoxicity by benzyl group had more effect than octyl group. These studies demonstrated that these novel modified chitosan derivatives had potential for using as absorption enhancers.