Human hazard potential of nanocellulose: quantitative insights from the literature.

This review aims to elucidate the current knowledge and future research needs regarding the hazard potential of nanocellulose to human health. Growing interest from research and industry alike has led to increasing likelihood of human contact to the material via various exposure routes. Although a number of comprehensive reviews on human health hazards of nanocellulose have been conducted, this paper brings new insights as it systematically analyzes and quantitatively assesses the results of in vivo and in vitro tests in terms of investigated endpoints, tested concentration ranges, physicochemical properties, surface modifications and source of the tested nanocellulose, exposure route, and cell lines used. The quality of the studies is further inspected based on various established criteria. Considering the rapid development of nanocellulose-based products and the novelty of the material, human health studies remain scarce. By assessing those that have been conducted, patterns and gaps were identified that will be helpful to guide future research. The results show that there are still significant uncertainties remaining, particularly regarding in vivo testing, with pulmonary exposure showing some cause for concern. Although a substantial number of in vitro studies have been undertaken, results are often conflicting. The detected effects could not be directly attributed to size of nanoparticles, cell lines, surface modifications or tested concentrations. This may also be linked to the varying quality of the studies. This review ends by identifying key gaps to help pave the way for future research and ensure the safe development and use of nanocellulose.

A Methodological Safe-by-Design Approach for the Development of Nanomedicines.

Safe-by-Design (SbD) concepts foresee the risk identification and reduction as well as uncertainties regarding human health and environmental safety in early stages of product development. The EU's NANoREG project and further on the H2020 ProSafe initiative, NanoReg2, and CALIBRATE projects have developed a general SbD approach for nanotechnologies (e.g., paints, textiles, etc.). Based on it, the GoNanoBioMat project elaborated a methodological SbD approach (GoNanoBioMat SbD approach) for nanomedicines with a focus on polymeric nanobiomaterials (NBMs) used for drug delivery. NBMs have various advantages such as the potential to increase drug efficacy and bioavailability. However, the nanoscale brings new challenges to product design, manufacturing, and handling. Nanomedicines are costly and require the combination of knowledge from several fields. In this paper, we present the GoNanoBioMat SbD approach, which allows identifying and addressing the relevant safety aspects to address when developing polymeric NBMs during design, characterization, assessment of human health and environmental risk, manufacturing and handling, and combines the nanoscale and medicine field under one approach. Furthermore, regulatory requirements are integrated into the innovation process.

How the Lack of Chitosan Characterization Precludes Implementation of the Safe-by-Design Concept.

Efficacy and safety of nanomedicines based on polymeric (bio)materials will benefit from a rational implementation of a Safe-by-Design (SbD) approach throughout their development. In order to achieve this goal, however, a standardization of preparation and characterization methods and their accurate reporting is needed. Focusing on the example of chitosan, a biopolymer derived from chitin and frequently used in drug and vaccine delivery vector preparation, this review discusses the challenges still to be met and overcome prior to a successful implementation of the SbD approach to the preparation of chitosan-based protein drug delivery systems.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 µg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

Hazard Assessment of Polymeric Nanobiomaterials for Drug Delivery: What Can We Learn From Literature So Far.

The physicochemical properties of nanobiomaterials, such as their small size and high surface area ratio, make them attractive, novel drug-carriers, with increased cellular interaction and increased permeation through several biological barriers. However, these same properties hinder any extrapolation of knowledge from the toxicity of their raw material. Though, as suggested by the Safe-by-Design (SbD) concept, the hazard assessment should be the starting point for the formulation development. This may enable us to select the most promising candidates of polymeric nanobiomaterials for safe drug-delivery in an early phase of innovation. Nowadays the majority of reports on polymeric nanomaterials are focused in optimizing the nanocarrier features, such as size, physical stability and drug loading efficacy, and in performing preliminary cytocompatibility testing and proving effectiveness of the drug loaded formulation, using the most diverse cell lines. Toxicological studies exploring the biological effects of the polymeric nanomaterials, particularly regarding immune system interaction are often disregarded. The objective of this review is to illustrate what is known about the biological effects of polymeric nanomaterials and to see if trends in toxicity and general links between physicochemical properties of nanobiomaterials and their effects may be derived. For that, data on chitosan, polylactic acid (PLA), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA) and policaprolactone (PCL) nanomaterials will be evaluated regarding acute and repeated dose toxicity, inflammation, oxidative stress, genotoxicity, toxicity on reproduction and hemocompatibility. We further intend to identify the analytical and biological tests described in the literature used to assess polymeric nanomaterials toxicity, to evaluate and interpret the available results and to expose the obstacles and challenges related to the nanomaterial testing. At the present time, considering all the information collected, the hazard assessment and thus also the SbD of polymeric nanomaterials is still dependent on a case-by-case evaluation. The identified obstacles prevent the identification of toxicity trends and the generation of an assertive toxicity database. In the future, in vitro and in vivo harmonized toxicity studies using unloaded polymeric nanomaterials, extensively characterized regarding their intrinsic and extrinsic properties should allow to generate such database. Such a database would enable us to apply the SbD approach more efficiently.

Molecular Modeling for Nanomaterial-Biology Interactions: Opportunities, Challenges, and Perspectives.

Injection of nanoparticles (NP) into the bloodstream leads to the formation of a so-called "nano-bio" interface where dynamic interactions between nanoparticle surfaces and blood components take place. A common consequence is the formation of the protein corona, that is, a network of adsorbed proteins that can strongly alter the surface properties of the nanoparticle. The protein corona and the resulting structural changes experienced by adsorbed proteins can lead to substantial deviations from the expected cellular uptake as well as biological responses such as NP aggregation and NP-induced protein fibrillation, NP interference with enzymatic activity, or the exposure of new antigenic epitopes. Achieving a detailed understanding of the nano-bio interface is still challenging due to the synergistic effects of several influencing factors like pH, ionic strength, and hydrophobic effects, to name just a few. Because of the multiscale complexity of the system, modeling approaches at a molecular level represent the ideal choice for a detailed understanding of the driving forces and, in particular, the early events at the nano-bio interface. This review aims at exploring and discussing the opportunities and perspectives offered by molecular modeling in this field through selected examples from literature.

Computational Assessment of the Pharmacological Profiles of Degradation Products of Chitosan.

Chitosan is a natural polymer revealing an increased potential to be used in different biomedical applications, including drug delivery systems, and tissue engineering. It implies the evaluation of the organism response to the biomaterial implantation. Low-molecular degradation products, the chito-oligomers, are resulting mainly from the influence of enzymes, which are found in the organism fluids. Within this study, we have performed the computational assessment of pharmacological profiles and toxicological effects on human health of small chito-oligomers with distinct molecular weights, deacetylation degrees, and acetylation patterns. Our approach is based on the fact that regulatory agencies and researchers in the drug development field rely on the use of modeling to predict biological effects and to guide decision making. To be considered as valid for regulatory purposes, every model that is used for predictions should be associated with a defined toxicological endpoint and has appropriate robustness and predictivity. Within this context, we have used FAF-Drugs4, SwissADME, and PreADMET tools to predict the oral bioavailability of chito-oligomers and SwissADME, PreADMET, and admetSAR2.0 tools to predict their pharmacokinetic profiles. The organs and genomic toxicities have been assessed using admetSAR2.0 and PreADMET tools but specific computational facilities have been also used for predicting different toxicological endpoints: Pred-Skin for skin sensitization, CarcinoPred-EL for carcinogenicity, Pred-hERG for cardiotoxicity, ENDOCRINE DISRUPTOME for endocrine disruption potential and Toxtree for carcinogenicity and mutagenicity. Our computational assessment showed that investigated chito-oligomers reflect promising pharmacological profiles and limited toxicological effects on humans, regardless of molecular weight, deacetylation degree, and acetylation pattern. According to our results, there is a possible inhibition of the organic anion transporting peptides OATP1B1 and/or OATP1B3, a weak potential of cardiotoxicity, a minor probability of affecting the androgen receptor, and phospholipidosis. Consequently, these results may be used to guide or to complement the existing in vitro and in vivo toxicity tests, to optimize biomaterials properties and to contribute to the selection of prototypes for nanocarriers.