Maria Luisa Dupuis

Nanoparticles were initially considered inert carriers for therapeutic payloads. This view has evolved substantially, as a growing body of experimental and clinical evidence indicates that engineered nanoparticles act as active immunomodulators, engaging pattern recognition receptors (PRRs), activating the complement cascade, reprogramming macrophage polarization, and inducing trained immunity in innate immune cells. This dual role of nanoparticles as both immunological agents and delivery vehicles represents a fundamental change that demands an integrative framework combining immunogenicity assessment, nanotoxicology, and therapeutic exploitation. This review provides an overview of the current state of the art at the nanomedicine–innate immunity interface across six nanoparticle classes, adopting a comparative framework that identifies both the immunological mechanisms shared among platforms and the specific features that distinguish individual nanoparticle systems. The analysis focuses on key innate immune populations, including dendritic cells (DCs), macrophages, natural killer (NK) cells, neutrophils and mast cells, as well as major innate immune signaling pathways, including the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS–STING) pathway, Toll-like receptor (TLR) signaling, and the NOD-like receptor protein 3 (NLRP3) inflammasome. The innate immune system represents a primary determinant of the biological outcome of nanomaterial exposure and provides a critical link to subsequent adaptive responses. Two methodological themes recur throughout the analysis. The first concerns attribution: distinguishing responses driven by the carrier itself from those driven by its cargo or by contaminants requires defined controls, and in their absence an observed response should not be ascribed to the nanomaterial. The second is inter-individual variability, which emerges as a determinant of outcome rather than as experimental noise, and which appears to arise from the combination of recipient and platform rather than from either alone, so that susceptibility established for one platform does not transfer to another. Our aim is to highlight unexplored connections and experimental gaps that currently limit clinical translation, offering an integrated interpretation of nanoparticle–innate immunity interactions that points to unresolved immunological challenges and oncology-relevant therapeutic opportunities for each class still awaiting validation.

Gold nanorods (AuNRs) were synthesized and optimized with the aim of obtaining strongly hydrophilic nanomaterials, suitable as a drug delivery system (DDS) for copper-based drugs. After careful purification, AuNRs were characterized by ultraviolet–visible–near-infrared spectroscopy (UV–Vis–NIR), showing two typical localized surface plasmon resonance (LSPR) bands in the range 550–750 nm. Fourier Transform Infrared (FT-IR) and high-resolution X-ray photoelectron (HR-XPS) spectroscopies verified the surface functionalization. Transmission electron microscopy (TEM) showed AuNRs with regular shape and size, with an aspect ratio (AR) of 2.6. Dynamic Light Scattering (DLS) measurements confirmed the size and the stability in water for up to 3 months. The AuNRs were conjugated with copper(I) drugs, i.e., [Cu(PTA)₄]BF₄ (PTA = 1,3,5-triaza-7-phosphadamantane). The drug loading procedures and efficiency were optimized, and the best loading was η (%) = 50 ± 7%. The non-covalent interactions of the Cu(I) complex with the AuNRs were studied by means of UV–Vis–NIR, ζ-potential, HR-TEM, FT-IR, synchrotron radiation-induced X-ray photoelectron (SR-XPS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy measurements. The MTT assay performed on Vero E6 cells showed that AuNRs and AuNR-Cu(I) conjugates had no significant effect on cell viability, being biocompatible, causing a reduction in cell viability only after prolonged exposure.

This paper reports the synthesis, structural characterization, and biological evaluation of a novel series of Cu^I and Cu^II complexes supported by an amantadine-functionalized bis(pyrazol-1-yl)acetate ligand (L^Ad) as potential anticancer agents for the treatment of glioblastoma (GBM). Comprehensive spectroscopic and structural investigations, including SR-XPS, XANES/EXAFS, and DFT modeling, confirmed the successful coordination of L^Ad to copper centers in both oxidation states, affording well-defined molecular architectures with distinct coordination geometries. Among the synthesized compounds, the Cu^I complexes bearing triphenylphosphine co-ligands (compounds 4 and 5) exhibited the strongest cytotoxicity against U87 MG and LN18 GBM cell lines, showing IC₅₀ values lower than those of cisplatin. These complexes induced a pronounced redox imbalance through reactive oxygen species (ROS) overproduction and glutathione (GSH) depletion, leading to G2/M cell cycle arrest and cell death. Flow cytometry and Western blot analyses demonstrated that cell death occurs via caspase-dependent apoptosis in LN18 cells, as evidenced by PARP cleavage, downregulation of Bcl-xL, release of cytochrome c, and mitochondrial translocation of Bax. Altogether, these findings highlight the potential of lipophilic amantadine-functionalized Cu^I complexes as promising anticancer candidates targeting glioma cells through mitochondrial dysfunction and redox-mediated pathways.

Glioblastoma (GBL) is one of the more malignant primary brain tumors; it is currently treated by a multimodality strategy including surgery, and radio- and chemotherapy, mainly consisting of temozolomide (TMZ)-based chemotherapy. Tumor relapse often occurs due to the establishment of TMZ resistance, with a patient median survival time of <2 years. The identification of natural molecules with strong anti-tumor activity led to the combination of these compounds with conventional chemotherapeutic agents, developing protocols for integrated anticancer therapies. Curcumin (CUR), resveratrol (RES), and its glucoside polydatin (PLD) are widely employed in the pharmaceutical and nutraceutical fields, and several studies have demonstrated that the combination of these natural products was more cytotoxic than the individual compounds alone against different cancers. Some of us recently demonstrated the synergistic efficacy of the sublingual administration of a new nutraceutical formulation of CUR+PLD in reducing tumor size and improving GBL patient survival. To provide some experimental evidence to reinforce these clinical results, we investigated if pretreatment with a combination of CUR+PLD can improve TMZ cytotoxicity on GBL cells by analyzing the effects on cell cycle, viability, morphology, expression of proteins related to cell proliferation, differentiation, apoptosis or autophagy, and the actin network. Cell viability was assessed using the MTT assay or a CytoSmart cell counter. CalcuSyn software was used to study the CUR+PLD synergism. The morphology was evaluated by optical and scanning electron microscopy, and protein expression was analyzed by Western blot. Flow cytometry was used for the cell cycle, autophagic flux, and apoptosis analyses. The results provide evidence that CUR and PLD, acting in synergy with each other, strongly improve the efficacy of alkylating anti-tumor agents such as TMZ on drug-resistant GBL cells through their ability to affect survival, differentiation, and tumor invasiveness.

Cancer is a complex disease that affects millions of people and remains a major public health problem worldwide. Conventional cancer treatments, including surgery, chemotherapy, immunotherapy, and radiotherapy, have limited achievements and multiple drawbacks, among which are healthy tissue damage and multidrug-resistant phenotype onset. Increasing evidence shows that many plants’ natural products, as well as their bioactive compounds, have promising anticancer activity and exhibit minimal toxicity compared to conventional anticancer drugs. However, their widespread use in cancer therapy is severely restricted by limitations in terms of their water solubility, absorption, lack of stability, bioavailability, and selective targeting. The use of nanoformulations for plants’ natural product transportation and delivery could be helpful in overcoming these limitations, thus enhancing their therapeutic efficacy and providing the basis for improved anticancer treatment strategies. The present review is aimed at providing an update on some phytocompounds (curcumin, resveratrol, quercetin, and cannabinoids, among others) and their main nanoformulations showing antitumor activities, both in vitro and in vivo, against such different human cancer types as breast and colorectal cancer, lymphomas, malignant melanoma, glioblastoma multiforme, and osteosarcoma. The intracellular pathways underlying phytocompound anticancer activity and the main advantages of nanoformulation employment are also examined. Finally, this review critically analyzes the research gaps and limitations causing the limited success of phytocompounds’ and nanoformulations’ clinical translation.

Glioblastoma (GBL) is one of the more malignant primary brain tumors, currently treated by a multimodality strategy including surgery, radio- and chemotherapy, mainly consisting of Temozolomide(TMZ)-based chemotherapy. Tumor relapse often occurs due to the establishment of TMZ resistance, with a patient median survival <2 years. The identification of natural molecules having strong anti-tumor activity, led to combine these compounds with conventional chemotherapeutic agents, developing protocols of integrated anticancer therapies. Curcumin (CUR), Resveratrol (RES), and its glucoside Polydatin (PLD) are widely employed in the pharmaceutical and nutraceutical fields, and several studies demonstrated that the combination of these natural products was more cytotoxic than the single compound alone in different cancers. Some of us recently demonstrated the synergistic efficacy of the sublingual administration of a new nutraceutical formulation of CUR+PLD in reducing tumor size and improving GBL patient survival. To deepen some mechanistic aspects of these results, we investigated if the pretreatment with a combination of CUR+PLD, can improve TMZ cytotoxicity on GBL cells, by analyzing the effects on cell cycle, viability and morphology, on proteins related to cell proliferation, differentiation, apoptosis or autophagy, and on actin network. Cell viability was assessed by MTT or by the cell counter CytoSmart. CalcuSyn software was used to study the CUR+PLD synergism. Morphology was evaluated by optical and scanning electron microscopy, and protein expression was analyzed by Western Blot. Flow cytometry was used for cell cycle, autophagic flux, and apoptosis analyses. The results obtained provide evidence that CUR and PLD, acting in synergy with each other, strongly improve the efficacy of alkylating antitumor agents such as TMZ, also in drug-resistant GBL cells, through their abilities to affect survival, differentiation, and tumor invasiveness.

In this work, for the first time, different parts of the Finger Lime (Citrus australasica F. Muell.), such as pulp, peel and seeds, were analyzed by HS-SPME-GC/MS, and NMR techniques in order to describe its volatile and non-volatile chemical profile. The results highlighted the presence of a high number of terpenes with limonene as principal component in all investigated parts (ranging from 40.4% to 62.6%) and molecules belonging to the classes of amino acids, organic acids, carbohydrates, fatty acids, phenols and miscellaneous compounds that followed a different trend between the investigated different parts. In this study, the inhibition of ChEs (AChE and BChE) was evaluated using the spectrophotometric method of Ellman. The results showed that only peel extract weakly inhibited AChE (14%). Based on these data, this extract was further investigated by GC/MS after derivatization. Furthermore, peel extract was chosen to evaluate the in vitro effects on two human glioblastoma cells lines (U87 and LN18). Flow cytometry results showed that citrus extract was more effective in down-regulating the expression of the adhesion molecule CD44. In fact, after 72 h with 400 µg/mL of citrus extract, CD44 expression levels were reduced in both U87 and LN18 glioblastoma cell lines. This was confirmed by immunofluorescence analysis, which also showed a modification of CD44 antigen localization in both U87 and LN18 cell lines. Moreover, wound assay data supported its ability to reduce glioblastoma cell’s motility. The migration ability of U87 cells decreased (85% control vs. 50% at 400 μg/mL), while it was even more pronounced in resistant LN18 cells (93% control vs. 15% at 400 μg/mL). The findings highlighted that citrus peel extract could have an anti-invasive activity for glioma management.

Coagulation disorders are described in COVID-19 and long COVID patients. In particular, SARS-CoV-2 infection in megakaryocytes, which are precursors of platelets involved in thrombotic events in COVID-19, long COVID and, in rare cases, in vaccinated individuals, requires further investigation, particularly with the emergence of new SARS-CoV-2 variants. CD147, involved in the regulation of inflammation and required to fight virus infection, can facilitate SARS-CoV-2 entry into megakaryocytes. MCT4, a co-binding protein of CD147 and a key player in the glycolytic metabolism, could also play a role in SARS-CoV-2 infection. Here, we investigated the susceptibility of megakaryocytes to SARS-CoV-2 infection via CD147 and MCT4. We performed infection of Dami cells and human CD34⁺ hematopoietic progenitor cells induced to megakaryocytic differentiation with SARS-CoV-2 pseudovirus in the presence of AC-73 and syrosingopine, respective inhibitors of CD147 and MCT4 and inducers of autophagy, a process essential in megakaryocyte differentiation. Both AC-73 and syrosingopine enhance autophagy during differentiation but only AC-73 enhances megakaryocytic maturation. Importantly, we found that AC-73 or syrosingopine significantly inhibits SARS-CoV-2 infection of megakaryocytes. Altogether, our data indicate AC-73 and syrosingopine as inhibitors of SARS-CoV-2 infection via CD147 and MCT4 that can be used to prevent SARS-CoV-2 binding and entry into megakaryocytes, which are precursors of platelets involved in COVID-19-associated coagulopathy.

Hypovitaminosis D is involved in various inflammatory, infectious and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Moreover, the active form of vitamin D, calcitriol, has been shown to modulate the immune response, playing an anti-inflammatory effect. However little is known about the mechanisms underlying this anti-inflammatory effect and the potential sex differences of calcitriol immune regulation. Hence, the aim of this study was to investigate whether calcitriol could act differently in modulating T cell immunity of age-matched male and female healthy donors. We analyzed the effects of calcitriol in T lymphocytes from healthy women and men on the expression levels of the vitamin D receptor (VDR) and pro- and anti-inflammatory cytokine production. We showed that a treatment with calcitriol induced a significant increase in the VDR expression levels of activated T lymphocytes from male and female healthy subjects. Moreover, we found that calcitriol significantly reduced the expression level of pro-inflammatory cytokines IL-17, INF-γ and TNF-α in the T lymphocytes of both sexes. Notably, we observed that calcitriol induced a significant increase in the expression level of anti-inflammatory cytokine IL-10 only in the T lymphocytes from female healthy donors. In conclusion, our study provides new insights regarding the sex-specific anti-inflammatory role of calcitriol in T cell immunity.

Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs’ cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.