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Ochratoxin A-induced cytotoxicity, genotoxicity and reactive oxygen species in kidney cells: an integrative approach of complementary endpoints

Publication . Costa, J.G.; Saraiva, N.; Guerreiro, P.S.; Louro, Henriqueta; Silva, Maria João; Miranda, J.P.; Castro, M.; Batinic-Haberle, I.; Fernandes, A.S.; Oliveira, N.G.

Ochratoxin A (OTA) is a well-known nephrotoxic and potential carcinogenic agent but no consensus about the molecular mechanisms underlying its deleterious effects has been reached yet. The aim of this study is to integrate several endpoints concerning OTA-induced toxicological effects in Vero kidney cells in order to obtain additional mechanistic data, especially regarding the influence of reactive oxygen species (ROS). One innovative aspect of this work is the use of the superoxide dismutase mimic (SODm) MnTnHex-2-PyP as a mechanistic tool to clarify the involvement of oxidative stress in OTA toxicity. The results showed concentration and time-dependent cytotoxic effects of OTA (crystal violet, neutral red and LDH leakage assays). While the SODm mildly increased cell viability, trolox and ascorbic acid had no effect with regards to this endpoint. OTA induced micronuclei formation. Using the FPG modified comet assay, OTA modestly increased the % of DNA in tail, revealing the presence of oxidative DNA lesions. This mycotoxin increased apoptosis, which was attenuated by SODm. In addition, the SODm decreased the ROS accumulation observed in DHE assay. Taken together, our data suggest that ROS partially contribute to the cytotoxicity and genotoxicity of OTA, although other mechanisms may be relevant in OTA-induced deleterious effects.

2015-12-02Journal article

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Safety evaluation of novel polymeric nanocarriers for drug delivery using human osteoblasts

Publication . Dias, Kamila; Louro, Henriqueta; Gonçalves, Lídia; Bettencourt, Ana; Silva, Maria João

The development of novel nanocarriers has been recognized as a promising approach to improve drug release profiles on targeted sites, being the assessment of their biocompatibility and safety a critical point of the process. The objective of this work was to characterize the cellular interactions and the potential toxicity of the novel developed polymeric nanoparticles (NPs), using human osteoblasts. For this purpose, Poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (PMMA-Eud, 50:50) NPs were produced and their physicochemical properties were characterized. The safety evaluation of both NPs was conducted through the characterization of cellular uptake (fluorescence microscopy), cyto- and genotoxicity (MTT, Comet and Micronucleus assays) using both normal and differentiated osteoblast cell line (MG-63-ATCC®CRL-1427™). The results confirmed the successful uptake of PMMA and PMMA-Eud by the cells. Both NPs were not cytotoxic in differentiated cells, although a moderate toxicity was detected in undifferentiated cells. As to their genotoxic potential, NPs induced primary DNA damage in osteoblasts, especially under a short-term exposure. Noteworthy, none of the NPs caused chromosome breaks, indicating that the primary DNA lesions were not converted into permanent genetic damage. On the other hand, an increased cell proliferative capacity was noted for PMMA-NPs, which deserves further investigation. In conclusion, the safety assessment of the two NPs indicated that both are biocompatible but display a weak genotoxicity that should be explored in other in vitro endpoints, e.g., gene mutation, and in vivo studies. Moreover, understanding how physicochemical features relate to toxicity will support the design of safer formulations for biomedical purposes.

2017-05-08Conference object

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Levofloxacin-loaded bone cement delivery system: highly effective against intracellular bacteria and Staphylococcus aureus biofilms

Publication . Ferreira, Magda; Rzhepishevska, Olena; Grenho, Liliana; Malheiros, Danila; Gonçalves, Lídia; Almeida, António J.; Jordao, Luisa; Ribeiro, Isabel A.; Ramstedt, Madeleine; Gomes, Pedro; Bettencourt, Ana

Staphylococcus aureus is a major pathogen in bone associated infections due to its ability to adhere and form biofilms on bone and/or implants. Moreover, recrudescent and chronic infections have been associated with S. aureus capacity to invade and persist within osteoblast cells. With the growing need of novel therapeutic tools, this research aimed to evaluate some important key biological properties of a novel carrier system composed of acrylic bone cement (polymethylmethacrylate - PMMA), loaded with a release modulator (lactose) and an antibiotic (levofloxacin). Levofloxacin-loaded bone cement (BC) exhibited antimicrobial effects against planktonic and biofilm forms of S. aureus (evaluated by a flow chamber system). Moreover, novel BC formulation showed high anti-bacterial intraosteoblast activity. This fact led to the conclusion that levofloxacin released from BC matrices could penetrate the cell membrane of osteoblasts and be active against S. aureus strains in the intracellular environment. Furthermore, levofloxacin-BC formulations showed no significant in vitro cytotoxicity and no allergic potential (measured by the in vivo chorioallantoic membrane assay). Our results indicate that levofloxacin-loaded BC has potential as a local antibiotic delivery system for treating S. aureus associated bone infections.

2017-10-30Journal article

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Toxicity screening of a novel poly(methylmethacrylate)-Eudragit nanocarrier on L929 fibroblasts

Publication . Graça, Diogo; Louro, Henriqueta; Santos, Joana; Dias, Kamila; Almeida, Antonio J; Gonçalves, Lídia; Silva, Maria João; Bettencourt, Ana

Translation of innovative drug delivery nanosystems into the market involves an early toxicity screening in the development phase. Previously, we showed that inclusion of the polymer Eudragit (EUD) into poly(methylmethacrylate) (PMMA) nanoparticles (NP) resulted in a novel nanocarrier (PMMA-EUD) with an improved biomedical performance. The safety of this novel nanoparticulate system (PMMA-EUDNPs) was assessed in this work and compared to that of the original PMMANPs by using an integrated approach, comprising in vitro toxicity assessment and NPs physicochemical characterization in water and cell medium. For toxicity assessment several endpoints were analysed, including cell death, oxidative stress, and genotoxicity using L929 fibroblasts. PMMA-EUDNPs proved to be more hydrophobic than the original PMMANPs. Also, charge of both NPs was strongly affected by cell medium. On the other hand, the novel nanosystem was easily uptaken by L929 cells and did not display relevant in vitro cytotoxic or genotoxic effects. On the contrary, PMMANPs were less internalized in cells and proved to be genotoxic, as measured by the micronucleus assay. To conclude, our results provide initial evidence about the safety of the novel and promising PMMA-EUD nanoparticulate system, enabling its further development towards applications for drug delivery.

2017-07-05Journal article

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Biocompatibility Assessment of Polymeric Nanoparticle Carriers for Drug Delivery in Human Osteoblasts

Publication . Dias, Kamila; Louro, Henriqueta; Gonçalves, Lídia; Silva, Maria João; Bettencourt, Ana

Bone infections after prosthetic surgery represent a painful and costly process that should be prevented. The use of nanocarriers as efficient drug-delivery systems represents an innovative strategy to reduce systemic toxicity and avoid antimicrobial resistance. Still, great efforts are required to ensure the biocompatibility and safety of new nanocarriers before their medical application. In this work, the FDA-approved polymers poly(methyl methacrylate) (PMMA), and Eudragit RL 100 (Eud) were used to obtain two sets of nanoparticles, plain PMMA and PMMA-Eud (50:50). Their biocompatibility was assessed in a normal and differentiated human osteoblast cell line. The nanoparticles synthesis was conducted by a previously optimized methodology and their physicochemical properties, including size distribution, surface charge, morphology and aggregation/agglomeration states, were analysed during different preparation steps. Furthermore, the cell uptake and cytotoxicity as well as and their potential to produce reactive oxygen species (ROS) were also studied. Cell assays were performed following exposure of MG63 (ATCC®CRL-1427™) under normal and differentiation culture conditions, to a concentration range of both polymeric nanoparticles. The results of the uptake studies confirmed that both nanoparticles are internalized by osteoblasts. For both formulations, neither a significant decrease in cell viability nor oxidative stress induction was observed. The comparative analysis of data obtained in undifferentiated osteoblast and in those under differentiation conditions showed similar effects being the latter slightly less sensitive to the nanoparticles toxicity. Based on the reported data, PMMA and PMMA-Eud can be described as biocompatible nanomaterials and be further explored in a perspective of potential carriers for drug delivery, reducing adverse effects of conventional treatments. However, another key property i.e., genotoxicity, needs to be assessed before ensuring their safety for human health. This approach isn’t only relevant for understanding the nano-bio interface by disclosing how physicochemical features relate to toxicological events, but also to support the safe-by-design concept.

2017-05Conference object

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