Polylactic acid (PLA), a biodegradable and biocompatible polymer produced from renewable resources, has been widely used as a nanoparticulate platform for antigen and drug delivery. Despite generally regarded as safe, its immunotoxicological profile, when used as a polymeric nanoparticle (NP), is not well-documented. Thus, this study intends to address this gap, by evaluating the toxicity of two different sized PLA NPs (PLA A NPs and PLA B NPs), produced by two nanoprecipitation methods and extensively characterized regarding their physicochemical properties in in vitro experimental conditions. After production, PLA A NPs mean diameter (187.9 ± 36.9 nm) was superior to PLA B NPs (109.1 ± 10.4 nm). Interestingly, when in RPMI medium, both presented similar mean size (around 100 nm) and neutral zeta potential, possibly explaining the similarity between their cytotoxicity profile in PBMCs. On the other hand, in DMEM medium, PLA A NPs presented smaller mean diameter (75.3 ± 9.8 nm) when compared to PLA B NPs (161.9 ± 8.2 nm), which may explain its higher toxicity in RAW 264.7. Likewise, PLA A NPs induced a higher dose-dependent ROS production. Irrespective of size differences, none of the PLA NPs presented an inflammatory potential (NO production) or a hemolytic activity in human blood. The results herein presented suggest the hypothesis, to be tested in the future, that PLA NPs presenting a smaller sized population possess increased cytotoxicity. Furthermore, this study emphasizes the importance of interpreting results based on adequate physicochemical characterization of nanoformulations in biological medium. As observed, small differences in size triggered by the dispersion in cell culture medium can have repercussions on toxicity, and if not correctly evaluated can lead to misinterpretations, and subsequent ambiguous conclusions.

A Gram-positive, endospore-forming, alkalitolerant bacterial strain, designated CGII, was isolated from the wastewater of a cassava flour mill in the state of São Paulo, Brazil and submitted to phylogenetic studies and biochemical tests. The 16S rRNA gene sequence indicated the highest degree of genomic similarity with MLB2 strains of Bacillus lehensis (100%). A two-level central composite rotatable design was then employed to optimize the medium composition and culture conditions for the production of the enzyme cyclodextrin glycosyltransferase (CGTase) in shake-flask and bioreactors. CCTase activity was measured under different production conditions, such as culture medium, agitation and aeration. Highest enzyme production by B. lehensis was achieved in 72 h with a maximal activity of 134.05 U mL −1 . The response surface method demonstrated that the proposed model achieved a good level of agreement with experimental data, with a correlation coefficient of 0.910, thereby confirming the adequate reliability of the model.

Blanco K.C., de Moraes F.F., Bernardi N.S., Vettori M.H.P.B., Monti R., Contiero J. (2014): Cyclodextrin production by Bacillus lehensis isolated from cassava starch: Characterisation of a novel enzyme. Czech J. Food Sci., 32: 48-53.The properties of a previously unknown enzyme, denominated cyclodextrin glycosyltransferase, produced from Bacillus lehensis, were evaluated using affinity chromatography for protein purification. Enzyme characteristics (optimum pH and temperature; pH and temperature stability), the influence of substances on the enzyme activity, enzyme kinetics, and cyclodextrin production were analysed. Cyclodextrin glycosyltransferase was purified up to 320.74-fold by affinity chromatography using β-cyclodextrin as the binder and it exhibited 8.71% activity recovery. This enzyme is a monomer with a molecular weight of 81.27 kDa, as estimated by SDS-PAGE. Optimum temperature and pH for cyclodextrin glycosyltransferase were 55°C and 8.0, respectively. The Michaelis-Menten constant was 8.62 g/l during maximum velocity of 0.858 g/l·h.

Poly-ε-caprolactone (PCL) is a biodegradable polyester that has FDA and CE approval as a medical device. Nonetheless, the lack of toxicity exhibited by the polymer cannot be extrapolated to its nanomaterial conformation. Despite PCL-based NPs being widely studied in the biomedical field for their advantages as controlled drug delivery systems, little data describe PCL NPs’ toxicity, particularly immunotoxicity. This work assessed different PCL-based delivery systems intended for protein delivery regarding their immunotoxicity and hemocompatibility. Two different molecular weight PCL polymers were used, as well as blends with chitosan and glucan. Results showed that the presence of NaOH during the production of PCL2 NPs and PCL2/glucan NPs induced PCL alkali hydrolysis, generating more reactive groups (carboxyl and hydroxyl) that contributed to an increased toxicity of the NPs (higher reduction in peripheral blood mononuclear cell viability and lower hemocompatibility). PCL2/glucan NPs showed an anti-inflammatory activity characterized by the inhibition of LPS stimulated nitric oxide (NO) and TNF-α. In conclusion, generalizations among different PCL NP delivery systems must be avoided, and immunotoxicity assessments should be performed in the early stage of product development to increase the clinical success of the nanomedicine.

No abstract