Not Only Redox: The Multifaceted Activity of Cerium Oxide Nanoparticles in Cancer Prevention and Therapy

Much information is accumulating on the effect of cerium oxide nanoparticles (CNPs) as cell-protective agents, reducing oxidative stress through their unique ability of scavenging noxious reactive oxygen species via an energy-free, auto-regenerative redox cycle, where superoxides and peroxides are sequentially reduced exploiting the double valence (Ce3+/Ce4+) on nanoparticle surface. In vitro and in vivo studies consistently report that CNPs are responsible for attenuating and preventing almost any oxidative damage and pathology. Particularly, CNPs were found to exert strong anticancer activities, helping correcting the aberrant homeostasis of cancer microenvironment, normalizing stroma-epithelial communication, contrasting angiogenesis, and strengthening the immune response, leading to reduction of tumor mass in vivo. Since these homeostatic alterations are of an oxidative nature, their relief is generally attributed to CNPs redox activity. Other studies however reported that CNPs exert selective cytotoxic activity against cancer cells and sensitize cancer cells to chemotherapy- and radiotherapy-induced apoptosis: such effects are hardly the result of antioxidant activity, suggesting that CNPs exert such important anticancer effects through additional, non-redox mechanisms. Indeed, using Sm-doped CNPs devoid of redox activity, we could recently demonstrate that the radio-sensitizing effect of CNPs on human keratinocytes is independent from the redox switch. Mechanisms involving particle dissolution with release of toxic Ce4+ atoms, or differential inhibition of the catalase vs. SOD-mimetic activity with accumulation of H2O2 have been proposed, explaining such intriguing findings only partially. Much effort is urgently required to address the unconventional mechanisms of the non-redox bioactivity of CNPs, which may provide unexpected medicinal tools against cancer

Improved multidetector asymmetrical-flow field-flow fractionation method for particle sizing and concentration measurements of lipid-based nanocarriers for RNA delivery

Lipid-based nanoparticles for RNA delivery (LNP-RNA) are revolutionizing the nanomedicine field, with one approved gene therapy formulation and two approved vaccines against COVID-19, as well as multiple ongoing clinical trials. As for other innovative nanopharmaceuticals (NPhs), the advancement of robust methods to assess their quality and safety profiles—in line with regulatory needs—is critical for facilitating their development and clinical translation. Asymmetric-flow field-flow fractionation coupled to multiple online optical detectors (MD-AF4) is considered a very versatile and robust approach for the physical characterisation of nanocarriers, and has been used successfully for measuring particle size, polydispersity and physical stability of lipid-based systems, including liposomes and solid lipid nanoparticles. However, the unique core structure of LNP-RNA, composed of ionizable lipids electrostatically complexed with RNA, and the relatively labile lipid-monolayer coating, is more prone to destabilization during focusing in MD-AF4 than previously characterised nanoparticles, resulting in particle aggregation and sample loss. Hence characterisation of LNP-RNA by MD-AF4 needs significant adaptation of the methods developed for liposomes. To improve the performance of MD-AF4 applied to LNP-RNA in a systematic and comprehensive manner, we have explored the use of the frit-inlet channel where, differently from the standard AF4 channel, the particles are relaxed hydrodynamically as they are injected. The absence of a focusing step minimizes contact between the particle and the membrane, reducing artefacts (e.g. sample loss, particle aggregation). Separation in a frit-inlet channel enables satisfactory reproducibility and acceptable sample recovery in the commercially available MD-AF4 instruments. In addition to slice-by-slice measurements of particle size, MD-AF4 also allows to determine particle concentration and the particle size distribution, demonstrating enhanced versatility beyond standard sizing measurements.publishedVersio

A novel synthetic approach of cerium oxide nanoparticles with improved biomedical activity

Cerium oxide nanoparticles (CNPs) are novel synthetic antioxidant agents proposed for treating oxidative stress-related diseases. The synthesis of high-quality CNPs for biomedical applications remains a challenging task. A major concern for a safe use of CNPs as pharmacological agents is their tendency to agglomerate. Herein we present a simple direct precipitation approach, exploiting ethylene glycol as synthesis co-factor, to synthesize at room temperature nanocrystalline sub-10 nm CNPs, followed by a surface silanization approach to improve nanoparticle dispersibility in biological fluids. CNPs were characterized using transmission electron microscopy (TEM) observations, X-ray diffraction (XRD) analysis, thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (H-1-NMR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurements. CNP redox activity was studied in abiotic systems using electron spin resonance (ESR) measurements, and in vitro on human cell models. In-situ silanization improved CNP colloidal stability, in comparison with non-functionalized particles, and allowed at the same time improving their original biological activity, yielding thus functionalized CNPs suitable for biomedical applications

The Use of Baclofen as a Treatment for Alcohol Use Disorder: A Clinical Practice Perspective

Alcohol use disorder (AUD) is a brain disorder associated with high rates of mortality and morbidity worldwide. Baclofen, a selective gamma-aminobutyric acid-B (GABA-B) receptor agonist, has emerged as a promising drug for AUD. The use of this drug remains controversial, in part due to uncertainty regarding dosing and efficacy, alongside concerns about safety. To date there have been 15 randomized controlled trials (RCTs) investigating the use of baclofen in AUD; three using doses over 100 mg/day. Two additional RCTs have been completed but have not yet been published. Most trials used fixed dosing of 30–80 mg/day. The other approach involved titration until the desired clinical effect was achieved, or unwanted effects emerged. The maintenance dose varies widely from 30 to more than 300 mg/day. Baclofen may be particularly advantageous in those with liver disease, due to its limited hepatic metabolism and safe profile in this population. Patients should be informed that the use of baclofen for AUD is as an “off-label” prescription, that no optimal fixed dose has been established, and that existing clinical evidence on efficacy is inconsistent. Baclofen therapy requires careful medical monitoring due to safety considerations, particularly at higher doses and in those with comorbid physical and/or psychiatric conditions. Baclofen is mostly used in some European countries and Australia, and in particular, for patients who have not benefitted from the currently used and approved medications for AUD

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

On the issue of transparency and reproducibility in nanomedicine.

Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio-nano papers, the community responds

A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers

Abstract: Breast cancer (BC) risk for BRCA1 and BRCA2 mutation carriers varies by genetic and familial factors. About 50 common variants have been shown to modify BC risk for mutation carriers. All but three, were identified in general population studies. Other mutation carrier-specific susceptibility variants may exist but studies of mutation carriers have so far been underpowered. We conduct a novel case-only genome-wide association study comparing genotype frequencies between 60,212 general population BC cases and 13,007 cases with BRCA1 or BRCA2 mutations. We identify robust novel associations for 2 variants with BC for BRCA1 and 3 for BRCA2 mutation carriers, P < 10−8, at 5 loci, which are not associated with risk in the general population. They include rs60882887 at 11p11.2 where MADD, SP11 and EIF1, genes previously implicated in BC biology, are predicted as potential targets. These findings will contribute towards customising BC polygenic risk scores for BRCA1 and BRCA2 mutation carriers

A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers

Breast cancer (BC) risk for BRCA1 and BRCA2 mutation carriers varies by genetic and familial factors. About 50 common variants have been shown to modify BC risk for mutation carriers. All but three, were identified in general population studies. Other mutation carrier-specific susceptibility variants may exist but studies of mutation carriers have so far been underpowered. We conduct a novel case-only genome-wide association study comparing genotype frequencies between 60,212 general population BC cases and 13,007 cases with BRCA1 or BRCA2 mutations. We identify robust novel associations for 2 variants with BC for BRCA1 and 3 for BRCA2 mutation carriers, P < 10−8, at 5 loci, which are not associated with risk in the general population. They include rs60882887 at 11p11.2 where MADD, SP11 and EIF1, genes previously implicated in BC biology, are predicted as potential targets. These findings will contribute towards customising BC polygenic risk scores for BRCA1 and BRCA2 mutation carriers

Cerium oxide nanoparticles as novel radio-protective and radio-sensitizing agents

Nanomedicine is the novel frontier of the prevention and treatment of an impressive series of severe human diseases. The search for therapeutic nanomaterials is fervent. In particular, finding a reliable and effective nano-antioxidant (as opposed to molecular anti-oxidants) is a main focus of current pharmacological research, since many serious diseases, including tumors, chronic inflammation, diabetes, neurodegenerative diseases, etc., imply oxidative stress. Among intrinsically active nano anti-oxidants, special attention has been given to cerium oxide nanoparticles (CNPs), showing promising anti-inflammatory and anti-degenerative properties. CNP biological properties may be associated to the presence of two different surface defects on their surface: the co-existence of Ce3+ and Ce4+ oxidation states and the consequent formation of charge-compensating oxygen vacancies. The pharmacological potential of CNPs as nanoantioxidant has been always attributed to the redox changes in the Ce oxidation state (Ce4+/Ce3+ redox switch), which trigger the abatement of the noxious intracellular reactive oxygen species (ROS), thereby protecting from the cytotoxic effects induced by oxidative stress. Very recently CNPs have been shown to possess a peculiar anti-cancer activity. CNPs seem able to selectively sensitize cancer cells to apoptosis, while protecting healthy tissues from exogenous carcinogenic sources. Therefore a promising use of CNPs in cancer therapy is emerging. However the mechanisms behind their selective anti-cancer activity are still unclear. Here we present a mechanistic analysis of the effects of CNPs against two different carcinogenic radiation sources, UV-rays and X-rays, on in vitro cell models. First, we demonstrate that CNPs are able to strongly counteract the pro-oxidant and pro-mutagenic effects of UV-rays and X-rays, although with different mechanisms. Second, we show that CNPs are able to radio-sensitize resistant cells to X-ray-induced apoptosis by enhancing the stringency of their defective DNA damage response. By Sm doping, which decreases the number of Ce3+ sites while keeping the same content of oxygen vacancies, is possible to perform a mechanistic analysis of the role of CNPs surface defects (Ce3+/Ce4+ redox couple vs. oxygen vacancies) in their anti-cancer activity. First, we correlate the protective effects of CNPs against UV-rays cytotoxicity to the activity of their Ce3+/Ce4+ redox couple. Second, we show that in cells irradiated with X-rays CNPs act as direct anti-oxidants, thanks to the activity of the Ce3+/Ce4+ redox couple, as well as by a peculiar nonredox mechanism, probably involving oxygen vacancies. Investigating this peculiar non-redox activity of CNPs, we show that CNPs are able to inhibit the activity of lipoxygenases (LOX) and cyclooxygenases (COX), strongly reducing their pro-oxidant, pro-survival and pro-mutagenic effects. A specific cause-effect relationship is found between the inhibition of 5-LOX enzymes and the protection against X-ray-induced damage to cellular membranes and to DNA. Interestingly, the inhibition of 5-LOX by CNPs increases the X-ray-induced apoptosis by increasing selectively the sensitivity of radio-resistant, e.g. transformed, cells to radiation treatments. The finding that CNP inhibit LOX and COX metabolism will help to better focus their pharmacological potential in radiation therapy as well as in pathologies involving the hyperactivation of these enzymes, including cancer, neurodegenerations and chronic inflammation. CNPs seem promising nanomaterials for the treatment of many diseases, including cancer. However the synthesis of CNPs suitable for biomedical applications is still a challenging task. One of the main challenges for a safe and efficient use of CNPs as pharmacological agents is their tendency to agglomerate in biological media. The formation of precipitates is expected to decrease the active surface area of CNPs, thereby reducing their biological activity. Importantly, nanoparticle agglomeration could also cause toxicity and deleterious side effects by accumulating on target organs (e.g. spleen and kidney) and by depressing the inflammatory response. Therefore, to reduce CNP agglomeration, we develop a base-catalyzed precipitation approach exploiting ethylene glycol as synthesis co-factor, followed by a post-synthesis surface functionalization with a poly (ethylene glycol)-terminated silane. We demonstrate that in situ silanization improves CNP colloidal stability with respect to the non-functionalized nanoparticles, at the same time allowing maintaining their pristine biological activity, yielding thus functionalized CNPs suitable for in vivo applications