Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells.

Introduction: Triple negative breast cancer (TNBC), a highly aggressive subtype accounting for 15-20% of all breast cancer cases, faces limited treatment options often accompanied by severe side effects. In recent years, natural extracellular nanovesicles derived from plants have emerged as promising candidates for cancer therapy, given their safety profile marked by non-immunogenicity and absence of inflammatory responses. Nevertheless, the potential anti-cancer effects of Citrus limon L.-derived extracellular nanovesicles (CLENs) for breast cancer treatment is still unexplored. Methods: In this study, we investigated the anti-cancer effects of CLENs on two TNBC cell lines (4T1 and HCC-1806 cells) under growth conditions in 2D and 3D culture environments. The cellular uptake efficiency of CLENs and their internalization mechanism were evaluated in both cells using confocal microscopy. Thereafter, we assessed the effect of different concentrations of CLENs on cell viability over time using a dual approach of Calcein-AM PI live-dead assay and CellTiter-Glo bioluminescence assay. We also examined the influence of CLENs on the migratory and evasion abilities of TNBC cells through wound healing and 3D Matrigel drop evasion assays. Furthermore, Western blot analysis was employed to investigate the effects of CLENs on the phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal- regulated kinase (ERK) expression. Results: We found that CLENs were internalized by the cells via endocytosis, leading to decreased cell viability, in a dose- and time-dependent manner. Additionally, the migration and evasion abilities of TNBC cells were significantly inhibited under exposed to 40 and 80 µg/mL CLENs. Furthermore, down-regulated expression levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK), suggesting that the inhibition of cancer cell proliferation, migration, and evasion is driven by the inhibition of the PI3K/AKT and MAPK/ERK signaling pathways. Discussion: Overall, our results demonstrate the anti-tumor efficiency of CLENs against TNBC cells, highlighting their potential as promising natural anti-cancer agents for clinical applications in cancer treatment.

Unraveling the potential of graphene quantum dots against Mycobacterium tuberculosis infection.

Introduction: The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains has underscored the urgent need for novel therapeutic approaches. Carbon-based nanomaterials, such as graphene oxide (GO), have shown potential in anti-TB activities but suffer from significant toxicity issues. Methods: This study explores the anti-TB potential of differently functionalized graphene quantum dots (GQDs) - non-functionalized, L-GQDs, aminated (NH2-GQDs), and carboxylated (COOH-GQDs) - alone and in combination with standard TB drugs (isoniazid, amikacin, and linezolid). Their effects were assessed in both axenic cultures and in vitro infection models. Results: GQDs alone did not demonstrate direct mycobactericidal effects nor trapping activity. However, the combination of NH2-GQDs with amikacin significantly reduced CFUs in in vitro models. NH2-GQDs and COOH-GQDs also enhanced the antimicrobial activity of amikacin in infected macrophages, although L-GQDs and COOH-GQDs alone showed no significant activity. Discussion: The results suggest that specific types of GQDs, particularly NH2-GQDs, can enhance the efficacy of existing anti-TB drugs. These nanoparticles might serve as effective adjuvants in anti-TB therapy by boosting drug performance and reducing bacterial counts in host cells, highlighting their potential as part of advanced drug delivery systems in tuberculosis treatment. Further investigations are needed to better understand their mechanisms and optimize their use in clinical settings.

Slow and steady wins the race: Fractionated near-infrared treatment empowered by graphene-enhanced 3D scaffolds for precision oncology.

Surgically addressing tumors poses a challenge, requiring a tailored, multidisciplinary approach for each patient based on the unique aspects of their case. Innovative therapeutic regimens combined to reliable reconstructive methods can contribute to an extended patient's life expectancy. This study presents a detailed comparative investigation of near-infrared therapy protocols, examining the impact of non-fractionated and fractionated irradiation regimens on cancer treatment. The therapy is based on the implantation of graphene oxide/poly(lactic-co-glycolic acid) three-dimensional printed scaffolds, exploring their versatile applications in oncology by the examination of pro-inflammatory cytokine secretion, immune response, and in vitro and in vivo tumor therapy. The investigation into cell death patterns (apoptosis vs necrosis) underlines the pivotal role of protocol selection underscores the critical influence of treatment duration on cell fate, establishing a crucial parameter in therapeutic decision-making. In vivo experiments corroborated the profound impact of protocol selection on tumor response. The fractionated regimen emerged as the standout performer, achieving a substantial reduction in tumor size over time, surpassing the efficacy of the non-fractionated approach. Additionally, the fractionated regimen exhibited efficacy also in targeting tumors in proximity but not in direct contact to the scaffolds. Our results address a critical gap in current research, highlighting the absence of a standardized protocol for optimizing the outcome of photodynamic therapy. The findings underscore the importance of personalized treatment strategies in achieving optimal therapeutic efficacy for precision cancer therapy.

Extreme transport of light in spheroids of tumor cells.

Extreme waves are intense and unexpected wavepackets ubiquitous in complex systems. In optics, these rogue waves are promising as robust and noise-resistant beams for probing and manipulating the underlying material. Localizing large optical power is crucial especially in biomedical systems, where, however, extremely intense beams have not yet been observed. We here discover that tumor-cell spheroids manifest optical rogue waves when illuminated by randomly modulated laser beams. The intensity of light transmitted through bio-printed three-dimensional tumor models follows a signature Weibull statistical distribution, where extreme events correspond to spatially-localized optical modes propagating within the cell network. Experiments varying the input beam power and size indicate that the rogue waves have a nonlinear origin. We show that these nonlinear optical filaments form high-transmission channels with enhanced transmission. They deliver large optical power through the tumor spheroid, and can be exploited to achieve a local temperature increase controlled by the input wave shape. Our findings shed light on optical propagation in biological aggregates and demonstrate how nonlinear extreme event formation allows light concentration in deep tissues, paving the way to using rogue waves in biomedical applications, such as light-activated therapies.

Curcumin-Functionalized Graphene Oxide Strongly Prevents Candida parapsilosis Adhesion and Biofilm Formation.

Candida parapsilosis is the major non-C. albicans species involved in the colonization of central venous catheters, causing bloodstream infections. Biofilm formation on medical devices is considered one of the main causes of healthcare-associated infections and represents a global public health problem. In this context, the development of new nanomaterials that exhibit anti-adhesive and anti-biofilm properties for the coating of medical devices is crucial. In this work, we aimed to characterize the antimicrobial activity of two different coated-surfaces, graphene oxide (GO) and curcumin-graphene oxide (GO/CU) for the first time, against C. parapsilosis. We report the capacity of GO to bind and stabilize CU molecules, realizing a homogenous coated surface. We tested the anti-planktonic activity of GO and GO/CU by growth curve analysis and quantification of Reactive Oxigen Species( ROS) production. Then, we tested the antibiofilm activity by adhesion assay, crystal violet assay, and live and dead assay; moreover, the inhibition of the formation of a mature biofilm was investigated by a viability test and the use of specific dyes for the visualization of the cells and the extra-polymeric substances. Our data report that GO/CU has anti-planktonic, anti-adhesive, and anti-biofilm properties, showing a 72% cell viability reduction and a decrease of 85% in the secretion of extra-cellular substances (EPS) after 72 h of incubation. In conclusion, we show that the GO/CU conjugate is a promising material for the development of medical devices that are refractory to microbial colonization, thus leading to a decrease in the impact of biofilm-related infections.

Nanotechnology in the COVID-19 era: Carbon-based nanomaterials as a promising solution.

The Coronavirus Disease 2019 (COVID-19) pandemic has led to collaboration between nanotechnology scientists, industry stakeholders, and clinicians to develop solutions for diagnostics, prevention, and treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections. Nanomaterials, including carbon-based materials (CBM) such as graphene and carbon nanotubes, have been studied for their potential in viral research. CBM unique effects on microorganisms, immune interaction, and sensitivity in diagnostics have made them a promising subject of SARS-CoV-2 research. This review discusses the interaction of CBM with SARS-CoV-2 and their applicability, including CBM physical and chemical properties, the known interactions between CBM and viral components, and the proposed prevention, treatment, and diagnostics uses.

Evaluation of the Toxic Activity of the Graphene Oxide in the Ex Vivo Model of Human PBMC Infection with Mycobacterium tuberculosis.

Graphene Oxide has been proposed as a potential adjuvant to develop improved anti-TB treatment, thanks to its activity in entrapping mycobacteria in the extracellular compartment limiting their entry in macrophages. Indeed, when administered together with linezolid, Graphene Oxide significantly enhanced bacterial killing due to the increased production of Reactive Oxygen Species. In this work, we evaluated Graphene Oxide toxicity and its anti-mycobacterial activity on human peripheral blood mononuclear cells. Our data show that Graphene Oxide, different to what is observed in macrophages, does not support the clearance of Mycobacterium tuberculosis in human immune primary cells, probably due to the toxic effects of the nano-material on monocytes and CD4+ lymphocytes, which we measured by cytometry. These findings highlight the need to test GO and other carbon-based nanomaterials in relevant in vitro models to assess the cytotoxic activity while measuring antimicrobial potential.

Sensing red blood cell nano-mechanics: Toward a novel blood biomarker for Alzheimer's disease.

Red blood cells (RBCs) are characterized by a remarkable elasticity, which allows them to undergo very large deformation when passing through small vessels and capillaries. This extreme deformability is altered in various clinical conditions, suggesting that the analysis of red blood cell (RBC) mechanics has potential applications in the search for non-invasive and cost-effective blood biomarkers. Here, we provide a comparative study of the mechanical response of RBCs in patients with Alzheimer's disease (AD) and healthy subjects. For this purpose, RBC viscoelastic response was investigated using atomic force microscopy (AFM) in the force spectroscopy mode. Two types of analyses were performed: (i) a conventional analysis of AFM force-distance (FD) curves, which allowed us to retrieve the apparent Young's modulus, E; and (ii) a more in-depth analysis of time-dependent relaxation curves in the framework of the standard linear solid (SLS) model, which allowed us to estimate cell viscosity and elasticity, independently. Our data demonstrate that, while conventional analysis of AFM FD curves fails in distinguishing the two groups, the mechanical parameters obtained with the SLS model show a very good classification ability. The diagnostic performance of mechanical parameters was assessed using receiving operator characteristic (ROC) curves, showing very large areas under the curves (AUC) for selected biomarkers (AUC > 0.9). Taken all together, the data presented here demonstrate that RBC mechanics are significantly altered in AD, also highlighting the key role played by viscous forces. These RBC abnormalities in AD, which include both a modified elasticity and viscosity, could be considered a potential source of plasmatic biomarkers in the field of liquid biopsy to be used in combination with more established indicators of the pathology.

Advanced usage of Ti3C2Tx MXenes for photothermal therapy on different 3D breast cancer models.

Globally, breast cancer is the most diagnosed invasive cancer among women. Current therapies (e.g., chemotherapy) show numerous limitations due to the lack of selectivity and involved side effects, which urgently asks for novel approaches with enhanced tumor-killing efficacy. We previously demonstrated that MXenes, new bioactive nanomaterials with promising photophysical properties, are capable to increase the efficiency of the targeted breast cancer photothermal therapy (PTT). In this work, we investigated the effect of few- and multi-layer Ti3C2Tx MXenes mediated-PTT on two different 3D reliable breast cancer models such as conventional and bio-printed spheroids. We performed PTT on both cancer models using a non-toxic MXene concentration of 50 µg/mL. After PTT, a significant reduction in the cell viability along with a notable increase in reactive oxygen species (ROS) was observed. Moreover, we studied the effect of PTT on the migration of macrophages and endothelial cells toward cancer regions in both 3D models. Our results indicate that PTT mediated by both few- and multi-layer MXenes significantly modulates the tumor progression through cells' death by increasing the temperature, which holds particularly true for the bio-printed model.

Tuning the immune system by nanoparticle-biomolecular corona.

Nanotechnology has a great potential to revolutionize the landscape of medicine, but an inadequate understanding of the nanomaterial-biological (nano-bio) interface hampers its ultimate clinical translation. Surface attachment of biomolecules provides a new biological identity of nanoparticles that plays a crucial role in vivo as it can activate the immune system triggering inflammatory responses, clearance from the body, and cellular toxicity. In this review, we summarize and critically analyze progress in understanding the relationship between the biological identity of nanoparticles and immune system activation. Accordingly, we discuss the implications of biomolecular corona on nanotoxicity, immune safety, and biocompatibility. We also highlight a perspective on engineering the biological identity of nanoparticles for modulating immunological responses.

Evaluation of Novel Guanidino-Containing Isonipecotamide Inhibitors of Blood Coagulation Factors against SARS-CoV-2 Virus Infection.

Coagulation factor Xa (fXa) and thrombin (thr) are widely expressed in pulmonary tissues, where they may catalyze, together with the transmembrane serine protease 2 (TMPRSS2), the coronaviruses spike protein (SP) cleavage and activation, thus enhancing the SP binding to ACE2 and cell infection. In this study, we evaluate in vitro the ability of approved (i.e., dabigatran and rivaroxaban) and newly synthesized isonipecotamide-based reversible inhibitors of fXa/thr (cmpds 1-3) to hinder the SARS-CoV-2 infectivity of VERO cells. Nafamostat, which is a guanidine/amidine antithrombin and antiplasmin agent, disclosed as a covalent inhibitor of TMPRSS2, was also evaluated. While dabigatran and rivaroxaban at 100 µM concentration did not show any effect on SARS-CoV-2 infection, the virus preincubation with new guanidino-containing fXa-selective inhibitors 1 and 3 did decrease viral infectivity of VERO cells at subtoxic doses. When the cells were pre-incubated with 3, a reversible nanomolar inhibitor of fXa (Ki = 15 nM) showing the best in silico docking score toward TMPRSS2 (pdb 7MEQ), the SARS-CoV-2 infectivity was completely inhibited at 100 µM (p < 0.0001), where the cytopathic effect was just about 10%. The inhibitory effects of 3 on SARS-CoV-2 infection was evident (ca. 30%) at lower concentrations (3-50 µM). The covalent TMPRSS2 and the selective inhibitor nafamostat mesylate, although showing some effect (15-20% inhibition), did not achieve statistically significant activity against SARS-CoV-2 infection in the whole range of test concentrations (3-100 µM). These findings suggest that direct inhibitors of the main serine proteases of the blood coagulation cascade may have potential in SARS-CoV-2 drug discovery. Furthermore, they prove that basic amidino-containing fXa inhibitors with a higher docking score towards TMPRSS2 may be considered hits for optimizing novel small molecules protecting guest cells from SARS-CoV-2 infection.

miRNome and Proteome Profiling of Small Extracellular Vesicles Secreted by Human Glioblastoma Cell Lines and Primary Cancer Stem Cells.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite available therapeutic interventions, it is very difficult to treat, and a cure is not yet available. The intra-tumoral GBM heterogeneity is a crucial factor contributing to poor clinical outcomes. GBM derives from a small heterogeneous population of cancer stem cells (CSCs). In cancer tissue, CSCs are concentrated within the so-called niches, where they progress from a slowly proliferating phase. CSCs, as most tumor cells, release extracellular vesicles (EVs) into the surrounding microenvironment. To explore the role of EVs in CSCs and GBM tumor cells, we investigated the miRNA and protein content of the small EVs (sEVs) secreted by two GBM-established cell lines and by GBM primary CSCs using omics analysis. Our data indicate that GBM-sEVs are selectively enriched for miRNAs that are known to display tumor suppressor activity, while their protein cargo is enriched for oncoproteins and tumor-associated proteins. Conversely, among the most up-regulated miRNAs in CSC-sEVs, we also found pro-tumor miRNAs and proteins related to stemness, cell proliferation, and apoptosis. Collectively, our findings support the hypothesis that sEVs selectively incorporate different miRNAs and proteins belonging both to fundamental processes (e.g., cell proliferation, cell death, stemness) as well as to more specialized ones (e.g., EMT, membrane docking, cell junction organization, ncRNA processing).

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols.

In the last 20 years, bone regenerative research has experienced exponential growth thanks to the discovery of new nanomaterials and improved manufacturing technologies that have emerged in the biomedical field. This revolution demands standardization of methods employed for biomaterials characterization in order to achieve comparable, interoperable, and reproducible results. The exploited methods for characterization span from biophysics and biochemical techniques, including microscopy and spectroscopy, functional assays for biological properties, and molecular profiling. This review aims to provide scholars with a rapid handbook collecting multidisciplinary methods for bone substitute R&D and validation, getting sources from an up-to-date and comprehensive examination of the scientific landscape.

INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids' death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

3D-printed graphene polylactic acid devices resistant to SARS-CoV-2: Sunlight-mediated sterilization of additive manufactured objects.

Additive manufacturing has played a crucial role in the COVID-19 global emergency allowing for rapid production of medical devices, indispensable tools for hospitals, or personal protection equipment. However, medical devices, especially in nosocomial environments, represent high touch surfaces prone to viral infection and currently used filaments for 3D printing can't inhibit transmission of virus [1]. Graphene-family materials are capable of reinforcing mechanical, optical and thermal properties of 3D printed constructs. In particular, graphene can adsorb near-infrared light with high efficiency. Here we demonstrate that the addition of graphene nanoplatelets to PLA filaments (PLA-G) allows the creation of 3D-printed devices that can be sterilized by near-infrared light exposure at power density analog to sunlight. This method has been used to kill SARS-CoV-2 viral particles on the surface of 3D printed PLA-G by 3 min of exposure. 3D-printed PLA-G is highly biocompatible and can represent the ideal material for the production of sterilizable personal protective equipment and daily life objects intended for multiple users.

Principles for optimization and validation of mRNA lipid nanoparticle vaccines against COVID-19 using 3D bioprinting.

BioNTech/Pfizer's Comirnaty and Moderna's SpikeVax vaccines consist in mRNA encapsulated in lipid nanoparticles (LNPs). The modularity of the delivery platform and the manufacturing possibilities provided by microfluidics let them look like an instant success, but they are the product of decades of intense research. There is a multitude of considerations to be made when designing an optimal mRNA-LNPs vaccine. Herein, we provide a brief overview of what is presently known and what still requires investigation to optimize mRNA LNPs vaccines. Lastly, we give our perspective on the engineering of 3D bioprinted validation systems that will allow faster, cheaper, and more predictive vaccine testing in the future compared with animal models.

Graphene nanoplatelet and graphene oxide functionalization of face mask materials inhibits infectivity of trapped SARS-CoV-2.

Recent advancements in bidimensional nanoparticles production such as graphene (G) and graphene oxide (GO) have the potential to meet the need for highly functional personal protective equipment (PPE) against SARS-CoV-2 infection. The ability of G and GO to interact with microorganisms provides an opportunity to develop engineered textiles for use in PPE and limit the spread of COVID-19. PPE in current use in high-risk settings for COVID transmission provides only a physical barrier that decreases infection likelihood and does not inactivate the virus. Here, we show that virus pre-incubation with soluble GO inhibits SARS-CoV-2 infection of VERO cells. Furthermore, when G/GO-functionalized polyurethane or cotton was in contact SARS-CoV-2, the infectivity of the fabric was nearly completely inhibited. The findings presented here constitute an important innovative nanomaterial-based strategy to significantly increase PPE efficacy in protection against the SARS-CoV-2 virus that may implement water filtration, air purification, and diagnostics methods.

Biosynthesis and physico-chemical characterization of high performing peptide hydrogels@graphene oxide composites.

Hydrogels based on short peptide molecules are interesting biomaterials with wide present and prospective use in biotechnologies. A well-known possible drawback of these materials can be their limited mechanical performance. In order to overcome this problem, we prepared Fmoc-Phe3self-assembling peptides by a biocatalytic approach, and we reinforced the hydrogel with graphene oxide nanosheets. The formulation here proposed confers to the hydrogel additional physicochemical properties without hampering peptide self-assembly. We investigated in depth the effect of nanocarbon morphology on hydrogel properties (i.e. morphology, viscoelastic properties, stiffness, resistance to an applied stress). In view of further developments towards possible clinical applications, we have preliminarily tested the biocompatibility of the composites. Our results showed that the innovative hydrogel composite formulation based on FmocPhe3 and GO is a biomaterial with improved mechanical properties that appears suitable for the development of biotechnological applications.

Inhibiting the Growth of 3D Brain Cancer Models with Bio-Coronated Liposomal Temozolomide.

Nanoparticles (NPs) have emerged as an effective means to deliver anticancer drugs into the brain. Among various forms of NPs, liposomal temozolomide (TMZ) is the drug-of-choice for the treatment and management of brain tumours, but its therapeutic benefit is suboptimal. Although many possible reasons may account for the compromised therapeutic efficacy, the inefficient tumour penetration of liposomal TMZ can be a vital obstacle. Recently, the protein corona, i.e., the layer of plasma proteins that surround NPs after exposure to human plasma, has emerged as an endogenous trigger that mostly controls their anticancer efficacy. Exposition of particular biomolecules from the corona referred to as protein corona fingerprints (PCFs) may facilitate interactions with specific receptors of target cells, thus, promoting efficient internalization. In this work, we have synthesized a set of four TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical-chemical properties. We have demonstrated that precoating liposomal TMZ with a protein corona made of human plasma proteins can increase drug penetration in a 3D brain cancer model derived from U87 human glioblastoma multiforme cell line leading to marked inhibition of tumour growth. On the other side, by fine-tuning corona composition we have also provided experimental evidence of a non-unique effect of the corona on the tumour growth for all the complexes investigated, thus, clarifying that certain PCFs (i.e., APO-B and APO-E) enable favoured interactions with specific receptors of brain cancer cells. Reported results open new perspectives into the development of corona-coated liposomal drugs with enhanced tumour penetration and antitumour efficacy.