Role of Surface Chemistry in the In Vitro Lung Response to Nanofibrillated Cellulose

Wood-derived nanofibrillated cellulose (NFC) has emerged as a sustainable material with a wide range of applications and increasing presence in the market. Surface charges are introduced during the preparation of NFC to facilitate the defibrillation process, which may also alter the toxicological properties of NFC. In the present study, we examined the in vitro toxicity of NFCs with five surface chemistries: nonfunctionalized, carboxymethylated, phosphorylated, sulfoethylated, and hydroxypropyltrimethylammonium-substituted. The NFC samples were characterized for surface functional group density, surface charge, and fiber morphology. Fibril aggregates predominated in the nonfunctionalized NFC, while individual nanofibrils were observed in the functionalized NFCs. Differences in surface group density among the functionalized NFCs were reflected in the fiber thickness of these samples. In human bronchial epithelial (BEAS-2B) cells, all NFCs showed low cytotoxicity (CellTiter-GloVR luminescent cell viability assay) which never exceeded 10% at any exposure time. None of the NFCs induced genotoxic effects, as evaluated by the alkaline comet assay and the cytokinesis-block micronucleus assay. The nonfunctionalized and carboxymethylated NFCs were able to increase intracellular reactive oxygen species (ROS) formation (chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate assay). However, ROS induction did not result in increased DNA or chromosome damage.

Download Full-text

Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽

10.3390/nano11081929 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1929

Author(s):

Tereza Cervena ◽

Andrea Rossnerova ◽

Tana Zavodna ◽

Jitka Sikorova ◽

Kristyna Vrbova ◽

...

Keyword(s):

Cytochalasin B ◽

Culture Media ◽

Methodological Approach ◽

Micronucleus Assay ◽

Toxicity Testing ◽

In Vitro Toxicity ◽

Testing Strategies ◽

Transmission Electron ◽

The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

Download Full-text

Potential of 2-Chloro-N-(4-fluoro-3-nitrophenyl)acetamide Against Klebsiella pneumoniae and In Vitro Toxicity Analysis

Molecules ◽

10.3390/molecules25173959 ◽

2020 ◽

Vol 25 (17) ◽

pp. 3959

Author(s):

Laísa Cordeiro ◽

Hermes Diniz-Neto ◽

Pedro Figueiredo ◽

Helivaldo Souza ◽

Aleson Sousa ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biological Activities ◽

High Capacity ◽

Pharmacokinetic Profile ◽

In Vitro Toxicity ◽

Antibacterial Drug ◽

Wide Range ◽

Good Potential

Klebsiella pneumoniae causes a wide range of community and nosocomial infections. The high capacity of this pathogen to acquire resistance drugs makes it necessary to develop therapeutic alternatives, discovering new antibacterial molecules. Acetamides are molecules that have several biological activities. However, there are no reports on the activity of 2-chloro-N-(4-fluoro-3-nitrophenyl)acetamide. Based on this, this study aimed to investigate the in vitro antibacterial activity of this molecule on K. pneumoniae, evaluating whether the presence of the chloro atom improves this effect. Then, analyzing its antibacterial action more thoroughly, as well as its cytotoxic and pharmacokinetic profile, in order to contribute to future studies for the viability of a new antibacterial drug. It was shown that the substance has good potential against K. pneumoniae and the chloro atom is responsible for improving this activity, stabilizing the molecule in the target enzyme at the site. The substance possibly acts on penicillin-binding protein, promoting cell lysis. The analysis of cytotoxicity and mutagenicity shows favorable results for future in vivo toxicological tests to be carried out, with the aim of investigating the potential of this molecule. In addition, the substance showed an excellent pharmacokinetic profile, indicating good parameters for oral use.

Download Full-text

Principles for assessing the genotoxicity of carbon nanomaterials in vitro (on the example of carbon nanotubes) (literature review)

Токсикологический вестник ◽

10.36946/0869-7922-2021-29-6-16-23 ◽

2021 ◽

Vol 29 (6) ◽

pp. 16-23

Author(s):

Gulnaz Faezovna Gabidinova ◽

Gyuzel Abdulkhalimovna Timerbulatova ◽

Liliya Minvagizovna Fatkhutdinova

Keyword(s):

Carbon Nanomaterials ◽

Physicochemical Characterization ◽

Gene Mutations ◽

Micronucleus Assay ◽

Bibliographic Databases ◽

Chromosomal Damage ◽

Colorimetric Test ◽

Wide Range ◽

Dna Strand

Introduction. Genotoxicity of nanomaterials (NM) is becoming a major concern when investigating new NM for their safety. Each mutagen is considered to be potentially carcinogenic, therefore a genotoxicity assessment is necessary. However, a clear strategy for assessing the genotoxic effect of NM has not yet been developed. Material and methods. The material for the analysis have included literature sources from the bibliographic databases PubMed, Scopus, RSCI. Results. Physicochemical characterization of NM is carried out using high-resolution microscopic and light scattering methods. Before testing for genotoxicity, it is necessary to know the cytotoxicity of the tested NM in order to select the appropriate concentration range. The most important and significant tests are based on the cell viability. MTT assay is a colorimetric test that evaluates the metabolic activity of cells. In addition, viability can be determined using microscopy, flow cytometry, determination of lactate dehydrogenase. Genotoxicity evaluation can be carried out only after the preliminary steps. The strategy should include genotoxicity endpoints: DNA damage, gene mutations, chromosomal damage. The in vitro mammalian gene mutation test, usually performed using mouse lymphoma cells, detects a wide range of genetic damage, including gene deletions. The most common test for detecting chromosomal damage is an in vitro micronucleus assay. DNA strand breaks are most often assessed using the comet DNA assay. Conclusion. Compulsory stages in the study of the genotoxicity of nanomaterials should be preliminary studies, including physicochemical characterization and assessment of cytotoxicity, as well as the study of the endpoints of genotoxicity and potential mechanisms.

Download Full-text

In Vitro Biological Impact of Nanocellulose Fibers on Human Gut Bacteria and Gastrointestinal Cells

Nanomaterials ◽

10.3390/nano10061159 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1159 ◽

Cited By ~ 2

Author(s):

Viviana R. Lopes ◽

Maria Strømme ◽

Natalia Ferraz

Keyword(s):

Cell Response ◽

Lactobacillus Reuteri ◽

Membrane Integrity ◽

Gut Bacteria ◽

Degree Of Crystallinity ◽

Surface Functional Group ◽

Fiber Morphology ◽

Oral Toxicity ◽

Cell Membrane Integrity

Wood-derived nanofibrillated cellulose (NFC) has long been recognized as a valuable nanomaterial for food-related applications. However, the safety of NFC cannot be predicted just from the chemical nature of cellulose, and there is a need to establish the effect of the nanofibers on the gastrointestinal tract, to reassure the safe use of NFC in food-related products. The present work selected the intestinal cells Caco-2 and the gut bacteria Escherichia coli and Lactobacillus reuteri to evaluate the in vitro biological response to NFC. NFC materials with different surface modifications (carboxymethylation, hydroxypropyltrimethylammonium substitution, phosphorylation and sulfoethylation) and unmodified NFC were investigated. The materials were characterized in terms of surface functional group content, fiber morphology, zeta potential and degree of crystallinity. The Caco-2 cell response to the materials was evaluated by assessing metabolic activity and cell membrane integrity. The effects of the NFC materials on the model bacteria were evaluated by measuring bacterial growth (optical density at 600 nm) and by determining colony forming units counts after NFC exposure. Results showed no sign of cytotoxicity in Caco-2 cells exposed to the NFC materials, and NFC surface functionalization did not impact the cell response. Interestingly, a bacteriostatic effect on E. coli was observed while the materials did not affect the growth of L. reuteri. The present findings are foreseen to contribute to increase the knowledge about the potential oral toxicity of NFC and, in turn, add to the development of safe NFC-based food products.

Download Full-text

In Vitro Toxicity: Mechanisms, Alternatives and Validation — A Report from the 19th Annual Scientific Meeting of the Scandinavian Society for Cell Toxicology

Alternatives to Laboratory Animals ◽

10.1177/026119290203000307 ◽

2002 ◽

Vol 30 (3) ◽

pp. 307-308

Author(s):

Anna Forsby

Keyword(s):

Animal Experiments ◽

Scientific Meeting ◽

In Vitro Cytotoxicity ◽

Annual Scientific ◽

In Vitro Toxicity ◽

In Vitro Tests ◽

Mechanisms Of Toxicity ◽

Scandinavian Society ◽

Wide Range

The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held in 2001 at Sørup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.

Download Full-text

Modulation of Inflammatory Mediators by Polymeric Nanoparticles Loaded with Anti-Inflammatory Drugs

Pharmaceutics ◽

10.3390/pharmaceutics13020290 ◽

2021 ◽

Vol 13 (2) ◽

pp. 290

Author(s):

Gloria María Pontes-Quero ◽

Lorena Benito-Garzón ◽

Juan Pérez Cano ◽

María Rosa Aguilar ◽

Blanca Vázquez-Lasa

Keyword(s):

Inflammatory Mediators ◽

Water Solubility ◽

Articular Chondrocytes ◽

Surface Charges ◽

Raw264.7 Macrophages ◽

Drug Concentrations ◽

Wide Range ◽

Inflammatory Drugs ◽

Anti Inflammatory

The first-line treatment of osteoarthritis is based on anti-inflammatory drugs, the most currently used being nonsteroidal anti-inflammatory drugs, selective cyclooxygenase 2 (COX-2) inhibitors and corticoids. Most of them present cytotoxicity and low bioavailability in physiological conditions, making necessary the administration of high drug concentrations causing several side effects. The goal of this work was to encapsulate three hydrophobic anti-inflammatory drugs of different natures (celecoxib, tenoxicam and dexamethasone) into core-shell terpolymer nanoparticles with potential applications in osteoarthritis. Nanoparticles presented hydrodynamic diameters between 110 and 130 nm and almost neutral surface charges (between −1 and −5 mV). Encapsulation efficiencies were highly dependent on the loaded drug and its water solubility, having higher values for celecoxib (39–72%) followed by tenoxicam (20–24%) and dexamethasone (14–26%). Nanoencapsulation reduced celecoxib and dexamethasone cytotoxicity in human articular chondrocytes and murine RAW264.7 macrophages. Moreover, the three loaded systems did not show cytotoxic effects in a wide range of concentrations. Celecoxib and dexamethasone-loaded nanoparticles reduced the release of different inflammatory mediators (NO, TNF-α, IL-1β, IL-6, PGE2 and IL-10) by lipopolysaccharide (LPS)-stimulated RAW264.7. Tenoxicam-loaded nanoparticles reduced NO and PGE2 production, although an overexpression of IL-1β, IL-6 and IL-10 was observed. Finally, all nanoparticles proved to be biocompatible in a subcutaneous injection model in rats. These findings suggest that these loaded nanoparticles could be suitable candidates for the treatment of inflammatory processes associated with osteoarthritis due to their demonstrated in vitro activity as regulators of inflammatory mediator production.

Download Full-text

Can the In Vivo Maximum Tolerated Dose be Predicted Using In Vitro Techniques? A Working Hypothesis

Alternatives to Laboratory Animals ◽

10.1177/026119299101900403 ◽

1991 ◽

Vol 19 (4) ◽

pp. 393-402

Author(s):

Ravi Shrivastava ◽

Gareth W. John ◽

Ginette Rispat ◽

Annick Chevalier ◽

Roy Massingham

Keyword(s):

Cell Death ◽

Research Effort ◽

Maximum Tolerated Dose ◽

In Vitro Cytotoxicity ◽

Toxicity Tests ◽

In Vitro Toxicity ◽

Working Hypothesis ◽

Wide Range

All new chemical entities synthesised in our laboratories have routinely been subjected to in vitro toxicity tests. Out of curiosity, we established a working hypothesis in which the in vitro data could be empirically transformed to predict the in vivo four-week standard maximum tolerated dose (MTD) studies in rats and dogs. As a first step to verifying this hypothesis, we report here the findings of an in vitro cytotoxicity study of 25 compounds randomly selected from our files, possessing a wide range of pharmacological activities and for which data from standard four-week MTD studies were available. Single blind in vitro toxicity studies in three carefully selected types of primary and cell line cultures were carried out. In vitro CT50 (concentration inducing 50% cell death) and CT100 (concentration inducing 100% cell death) values were obtained for each of the three cell types and, using empirical assumptions, these results were used to predict the MTD in vivo in the rat and dog. The actual in vivo threshold and toxic doses were obtained from the MTD study reports. The in vivo toxicity values predicted from the in vitro toxicity results with this series of 25 compounds showed a better than 80% correlation with the actual in vivo results obtained in the MTD studies. Whether or not in vitro cytotoxicity predictions are ultimately found to be directly and consistently related to the MTD in vivo for all pharmacological classes of compounds will require many additional studies, but it is hoped that these results will stimulate the necessary research effort required to answer this question.

Download Full-text

Biodegradable nanoparticles loaded with levodopa and/or curcumin for treatment of Parkinson’s disease

European Journal of Public Health ◽

10.1093/eurpub/ckab120.070 ◽

2021 ◽

Vol 31 (Supplement_2) ◽

Author(s):

Marco André Cardoso ◽

Bassam Felipe Mogharbel ◽

Ana Carolina Irioda ◽

Priscila Elias Ferreira Stricker ◽

Robson Camilotti Slompo ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Pc12 Cells ◽

Neurodegenerative Disorder ◽

Toxicity Testing ◽

In Vitro Toxicity ◽

Age Related ◽

Wide Range ◽

Low Cytotoxicity

Abstract Background Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Levodopa (L-DOPA) remains the standard gold drug available for the treatment of PD. Curcumin has a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, anti-amyloid, antitumor properties. Copolymers composed of poly(ethylene oxide) (PEO) and biodegradable polyesters like poly(ε-caprolactone) (PCL) that can self-assemble into nanoparticles (NP). This study describes the development of NH2-PEO-PCL diblock copolymer positively charged and modified by the addition of glutathione (GSH) on the outer surface, resulting in a synergistic delivery of L-DOPA and curcumin that would be able to pass the blood-brain barrier. Methods The NH2-PEO-PCL nanoparticles suspensions were prepared using a nanoprecipitation and solvent displacement method and were coated with GSH. NP was submitted to various characterizations assays, and to ensure the bioavailability, Vero and PC12 cells were treated with various concentrations of the loaded and unloaded NP to observe cytotoxicity. Results NP has successfully loaded L-DOPA and curcumin was stable after freeze-drying, capable of advancing into in vitro toxicity testing. After being treated up to 72 hours of various concentrations of L-DOPA and curcumin loaded NP Vero and PC12 cells, the viability of the treated cells maintained a high percentage indicating that the NPs are biocompatible. Conclusions NP consisting of NH2-PEO-PCL have been characterized as potential formulations for brain delivery of L-DOPA and curcumin, and obtained results also indicate that the developed biodegradable nanomicelles were blood compatible, presented low cytotoxicity even in longer exposure times.

Download Full-text