Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles.

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks.

The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.

First record of Cotesia scotti () (Hymenoptera: Braconidae: Microgastrinae) comb. nov. parasitising Spodoptera cosmioides (Walk, 1858) and Spodoptera eridania (Stoll, 1782) (Lepidoptera: Noctuidae) in Brazil

ABSTRACT This is the first report of Cotesia scotti (Valerio and Whitfield) comb. nov. in Brazil, attacking larvae of the black armyworm, Spodoptera cosmioides, and the southern armyworm, S. eridania. The moth larvae were found respectively, infesting a protected cropping of organic tomato in Hidrolândia, Goiás, Brazil, and a transgenic soybean crop in São José dos Pinhais, Paraná, Brazil. Biological, molecular and morphological characters were used to confirm the identity of the specimens. Parasitoid identification presented a challenge since the species has most diagnostic characters of the genus Cotesia Cameron, but few in the poorly defined genus Parapanteles Ashmead. Based on morphological and molecular evidence, we transfer Parapanteles scotti to the genus Cotesia. The new combination is discussed by comparison with morphologically similar species and available molecular data.

Laponite nanoparticle-associated silated hydroxypropylmethyl cellulose as an injectable reinforced interpenetrating network hydrogel for cartilage tissue engineering.

Articular cartilage is a connective tissue which does not spontaneously heal. To address this issue, biomaterial-assisted cell therapy has been researched with promising advances. The lack of strong mechanical properties is still a concern despite significant progress in three-dimensional scaffolds. This article's objective was to develop a composite hydrogel using a small amount of nano-reinforcement clay known as laponites. These laponites were capable of self-setting within the gel structure of the silated hydroxypropylmethyl cellulose (Si-HPMC) hydrogel. Laponites (XLG) were mixed with Si-HPMC to prepare composite hydrogels leading to the development of a hybrid interpenetrating network. This interpenetrating network increases the mechanical properties of the hydrogel. The in vitro investigations showed no side effects from the XLG regarding cytocompatibility or oxygen diffusion within the composite after cross-linking. The ability of the hybrid scaffold containing the composite hydrogel and chondrogenic cells to form a cartilaginous tissue in vivo was investigated during a 6-week implantation in subcutaneous pockets of nude mice. Histological analysis of the composite constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and collagens. Overall, this new hybrid construct demonstrates an interpenetrating network which enhances the hydrogel mechanical properties without interfering with its cytocompatibility, oxygen diffusion, or the ability of chondrogenic cells to self-organize in the cluster and produce extracellular matrix components. This composite hydrogel may be of relevance for the treatment of cartilage defects in a large animal model of articular cartilage defects. STATEMENT OF SIGNIFICANCE: Articular cartilage is a tissue that fails to heal spontaneously. To address this clinically relevant issue, biomaterial-assisted cell therapy is considered promising but often lacks adequate mechanical properties. Our objective was to develop a composite hydrogel using a small amount of nano reinforcement (laponite) capable of gelling within polysaccharide based self-crosslinking hydrogel. This new hybrid construct demonstrates an interpenetrating network (IPN) which enhances the hydrogel mechanical properties without interfering with its cytocompatibility, O2 diffusion and the ability of chondrogenic cells to self-organize in cluster and produce extracellular matrix components. This composite hydrogel may be of relevance for the treatment of cartilage defects and will now be considered in a large animal model of articular cartilage defects.

BCG-loaded chitosan microparticles: interaction with macrophages and preliminary in vivo studies.

The aim of this study was to develop a novel BCG-loaded chitosan vaccine with high association efficiency which can afford efficient interaction with APC and elicit local and Th1-type-specific immune response after intranasal administration. Chitosan-suspended BCG and BCG-loaded chitosan-alginate microparticles were prepared by ionotropic gelation. Interaction with APC was evaluated by fluorescence microscopy using rBCG-GFP. Specific immune responses were evaluated following intranasal immunisation of mice. Cellular uptake was approximately two-fold higher for chitosan-suspended BCG. A single dose of BCG-loaded microparticles or chitosan-suspended BCG by intranasal route improved Th1-type response compared with subcutaneous BCG. Chitosan-suspended BCG originated the highest mucosal response in the lungs by intranasal route. These positive results indicate that the proposed approach of whole live BCG microencapsulation in chitosan-alginate for intranasal immunisation was successful in allowing efficient interaction with APC, while improving the cellular immune response, which is of interest for local immunisation against tuberculosis.

Development of a novel mucosal vaccine against strangles by supercritical enhanced atomization spray-drying of Streptococcus equi extracts and evaluation in a mouse model.

Strangles is an extremely contagious and sometimes deadly disease of the Equidae. The development of an effective vaccine should constitute an important asset to eradicate this worldwide infectious disease. In this work, we address the development of a mucosal vaccine by using a Supercritical Enhanced Atomization (SEA) spray-drying technique. Aqueous solutions containing the Streptococcus equi extracts and chitosan were converted into nanospheres with no use of organic solvents. The immune response in a mouse model showed that the nanospheres induced a well-balanced Th1 and Th2 response characterized by a unitary ratio between the concentrations of IgG2a and IgG1, together with IgA production. This strategy revealed to be an effective alternative for immunization against S. equi, and therefore, it may constitute a feasible option for production of a strangles vaccine.