ABSTRACT
Lepidoptera comprise some of the most devastating herbivorous pest insects worldwide. One of the most promising novel pest control strategies is exploiting the RNA interference (RNAi) mechanism to target essential genes for knockdown and incite toxic effects in the target species without harming other organisms in the ecosystem. However, many insects are refractory to oral RNAi, often due to rapid degradation of ingested dsRNA in their digestive system. This is the case for many lepidopteran insects, including the beet armyworm Spodoptera exigua, which is characterized by a very alkaline gut environment (pH > 9.0) and a strong intestinal nucleolytic activity. In this research, guanidine-containing polymers were developed to protect dsRNA against nucleolytic degradation, specifically in high pH environments. First, their ability to protect dsRNA against nucleolytic degradation in gut juice of the beet armyworm S. exigua was investigated ex vivo. Polymers with high guanidine content provided a strong protection against nucleolytic degradation at pH 11, protecting the dsRNA for up to 30 h. Next, cellular uptake of the dsRNA and the polyplexes in lepidopteran CF203 midgut cells was investigated by confocal microscopy, showing that the polymer also enhanced cellular uptake of the dsRNA. Finally, in vivo feeding RNAi bioassays demonstrated that using these guanidine-containing polymer nanoparticles led to an increased RNAi efficiency in S. exigua. Targeting the essential gene chitin synthase B, we observed that the mortality increased to 53% in the polymer-protected dsRNA treatment compared to only 16% with the naked dsRNA and found that polymer-protected dsRNA completely halted the development of the caterpillars. These results show that using guanylated polymers as a formulation strategy can prevent degradation of dsRNA in the alkaline and strongly nucleolytic gut of lepidopteran insects. Furthermore, the polymer also enhances cellular uptake in lepidopteran midgut cells. This new delivery strategy could be of great use in further fundamental research in lepidopterans, using RNAi as a research tool, and could lead to future applications for RNAi-based pest control of lepidopteran insects.
ABSTRACT
Tissue engineering (TE) approaches often employ polymer-based scaffolds to provide support with a view to the improved regeneration of damaged tissues. The aim of this research was to develop a surface modification method for introducing chitosan as an antibacterial agent in both electrospun membranes and 3D printed poly-ε-caprolactone (PCL) scaffolds. The scaffolds were functionalized by grafting methacrylic acid N-hydroxysuccinimide ester (NHSMA) onto the surface after Ar-plasma/air activation. Subsequently, the newly-introduced NHS groups were used to couple with chitosan of various molecular weights (Mw). High Mw chitosan exhibited a better coverage of the surface as indicated by the higher N% detected by X-ray photoelectron spectroscopy (XPS) and the observations with either scanning electron microscopy (SEM)(for fibers) or Coomassie blue staining (for 3D-printed scaffolds). A lactate dehydrogenase assay (LDH) using L929 fibroblasts demonstrated the cell-adhesion and cell-viability capacity of the modified samples. The antibacterial properties against S. aureus ATCC 6538 and S. epidermidis ET13 revealed a slower bacterial growth rate on the surface of the chitosan modified scaffolds, regardless the chitosan Mw.
ABSTRACT
Pseudo-double network hydrogels based on vinylpyrrolidone and anionic methacrylic units were prepared, for the first-time, via a simple one step radical polymerization procedure using thermal or photoinitiation. These networks showed improved mechanical properties, in the hydrated state, compared with their single network cousins and were capable of hosting cells to confluence. Rapid cell detachment can be induced through simple mechanical agitation and the cell sheets can be transplanted easily without the need for a cell superstrate. The results reported in this work suggest that these hydrogels could be used as support systems for cell manipulation and are candidates to compete with the conventionally used thermoresponsive cell platforms based on poly-N-isopropylacrylamide (pNIPAm).
ABSTRACT
The differential reactivity of methylmethacrylate (MMA) and vinylpyrrolidone (VP) in free radical copolymerization, with stirring in methanol, renders an emulsified two phase system. The dispersed and continuous liquid phases contain copolymers rich in MMA and VP, respectively. When Fe(3)O(4) magnetic nanoparticles (mNPs) stabilized with tetramethylammonium hydroxide are added to this emulsion, the mNPs are located in the continuous phase. Very small chemical changes in the methacrylic or vinylic chains are able to guide the mNP toward the interface or to the inside of the dispersed phase since quite a selective functionalization of each phase may be achieved separately. Thus, a small addition of methacrylic acid as comonomer (0.5% molar) guides all of the mNPs to the interface while a 0.5% molar of sulfopropyl methacrylate induces the migration of all mNPs to the dispersed phase. When 0.5% molar of a VP derivative bearing sulfonate functionality is added, the mNPs are found both in the interface and in the continuous phase. The addition of water allows solid MMA-based microspheres to be obtained incorporating the mNPs selectively either at the surface or in the core.
