Ecotoxicology of nanomaterials: the role of invertebrate testing

Engineered nanomaterials represent a new and expanding class of chemicals whose environmental hazard is actually poorly determined. The peculiar behavior of nanomaterials makes them much more similar to new chemicals than to the corresponding bulk materials; this feature imposes reliable and standardized evaluation protocols for toxicity and ecotoxicity assessments. General rules for assessing nanotoxicity and the state of the art are periodically published in reports by control agencies. This review highlights the role of invertebrates as valuable and validated test organisms for assessing ecotoxicity of new and/or untested chemicals. The general scarcity of experimental data, their unequal distribution among the different nanomaterials and environmental conditions, the difficulties in manipulating nanomaterials and obtaining stable and homogeneous suspensions, the confusion arising from a not well defined metrics are discussed

Upgrading Monocytes Therapy for Critical Limb Ischemia Patient Treatment: Pre-Clinical and GMP-Validation Aspects

Advanced cell therapy medicinal products (ATMP) are at the forefront of a new range of biopharmaceuticals. The use of ATMP has evolved and increased in the last decades, representing a new approach to treating diseases that are not effectively managed with conventional treatments. The standard worldwide recognized for drug production is the Good Manufacturing Practices (GMP), widely used in the pharma production of synthesized drugs but applying also to ATMP. GMP guidelines are worldwide recognized standards to manufacture medicinal products to guarantee high quality, safety, and efficacy. In this report, we describe the pre-clinical and the GMP upgrade of peripheral blood mononuclear cell (PBMC) preparation, starting from peripheral blood and ending up with a GMP-grade clinical product ready to be used in patients with critical limb ischemia (CLI). We also evaluated production in hypoxic conditions to increase PBMC functional activity and angiogenic potential. Furthermore, we extensively analyzed the storage and transport conditions of the final product as required by the regulatory body for ATMPs. Altogether, results suggest that the whole manufacturing process can be performed for clinical application. Peripheral blood collected by a physician should be transported at room temperature, and PBMCs should be isolated in a clean room within 8 h of venipuncture. Frozen cells can be stored in nitrogen vapors and thawed for up to 12 months. PBMCs resuspended in 5% human albumin solution should be stored and transported at 4 degrees C before injection in patients within 24 h to thawing. Hypoxic conditioning of PBMCs should be implemented for clinical application, as it showed a significant enhancement of PBMC functional activity, in particular with increased adhesion, migration, and oxidative stress resistance. We demonstrated the feasibility and the quality of a GMP-enriched suspension of monocytes as an ATMP, tested in a clean room facility for all aspects related to production in respect of all the GMP criteria that allow its use as an ATMP. We think that these results could ease the way to the clinical application of ATMPs

Human Adipose-Derived Stem Cell-Conditioned Medium Promotes Vascularization of Nanostructured Scaffold Transplanted into Nude Mice

Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration

Antimicrobial activity of nanoconjugated glycopeptide antibiotics and their effect on Staphylococcus Aureus biofilm

In the era of antimicrobial resistance, the use of nanoconjugated antibiotics is regarded as a promising approach for preventing and fighting infections caused by resistant bacteria, including those exacerbated by the formation of difficult-to-treat bacterial biofilms. Thanks to their biocompatibility and magnetic properties, iron oxide nanoparticles (IONPs) are particularly attractive as antibiotic carriers for the targeting therapy. IONPs can direct conjugated antibiotics to infection sites by the use of an external magnet, facilitating tissue penetration and disturbing biofilm formation. As a consequence of antibiotic localization, a decrease in its administration dosage might be possible, reducing the side effects to non-targeted organs and the risk of antibiotic resistance spread in the commensal microbiota. Here, we prepared nanoformulations of the ‘last-resort’ glycopeptides teicoplanin and vancomycin by conjugating them to IONPs via surface functionalization with (3-aminopropyl) triethoxysilane (APTES). These superparamagnetic NP-TEICO and NP-VANCO were chemically stable and NP-TEICO (better than NP-VANCO) conserved the typical spectrum of antimicrobial activity of glycopeptide antibiotics, being effective against a panel of staphylococci and enterococci, including clinical isolates and resistant strains. By a combination of different methodological approaches, we proved that NP-TEICO and, although to a lesser extent, NP-VANCO were effective in reducing biofilm formation by three methicillin-sensitive or resistant Staphylococcus aureus strains. Moreover, when attracted and concentrated by the action of an external magnet, NP-TEICO exerted a localized inhibitory effect on S. aureus biofilm formation at low antibiotic concentration. Finally, we proved that the conjugation of glycopeptide antibiotics to IONPs reduced their intrinsic cytotoxicity toward a human cell line. Copyright © 2021 Berini, Orlandi, Gamberoni, Martegani, Armenia, Gornati, Bernardini and Marinelli

Enzyme activation by alternating magnetic field: Importance of the bioconjugation methodology

Iron oxide nanoparticles (NPs) are attractive materials for enzyme immobilization and, thanks to their superparamagnetism, can be accessed by remote stimuli. This can be exploited to activate molecules that are not remotely actuable. Here, we demonstrate that thermophilic enzymes chemically linked to NPs can be activated in a \u201cwireless\u201d fashion by an external alternate magnetic field (AMF). To this aim, we have conjugated, with different binding strategies, the thermophilic enzymes \u3b1-amylase and L-aspartate oxidase to iron oxide NPs obtaining NP-enzyme systems with activities depending on the different orientations and stretching of the enzymes. Since enzyme activation occurs without a significant rise of the \u201coverall\u201d temperature of the systems, we have speculated a local NP-enzyme heating that does not immediately interest the rest of the solution that remains at relatively low temperature, low enough to allow non-thermophilic enzymes to work together with the NP-conjugated thermophilic enzymes. Nanoactuation of thermophilic enzymes by AMF has potential applications in different fields. Indeed, multi-enzymatic processes with enzymes with different temperature optima could be carried out in the same reaction pot and thermolabile products could be efficiently produced by thermophilic enzymes without suffering for the high temperatures. Moreover, our findings represent a proof of concept of the possibility to achieve a fine-tuning of the enzyme-NP system with the aim to intervene in cell metabolism

Retromer stabilization results in neuroprotection in a model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS

Efficacy of N-acetylcysteine and all-trans retinoic acid in restoring in vitro effective hemopoiesis in myelodysplastic syndromes

We evaluated the in vitro effect on clonogenic potential (CFU-GM) and apoptosis in myelodysplastic syndromes (MDS) progenitors of an anti-oxidant (N-acetylcysteine, NAC) and/or a differentiating (all-trans retinoic acid, ATRA) agent. NAC significantly reduced apoptosis, both NAC and ATRA induced an increase in CFU-GM, but NAC seemed to be particularly effective in the high risk (HR) MDS. NAC + ATRA conferred a significant advantage in terms of CFU-GM with respect to NAC and ATRA alone. Tumor Necrosis Factor-alpha (TNF-alpha) levels decreased after incubation with NAC in the MDS samples. This study shows that ineffective hemopoiesis in MDS could benefit from both NAC and ATRA, suggesting that anti-oxidant treatment may play a role in guaranteeing MDS cell survival, predisposing them towards differentiatio