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Sara La Manna,1 Alessia Cugudda,1 Flavia Anna Mercurio,2 Marilisa Leone,2 Sara Fortuna,3 Concetta Di Natale,4 Elena Lagreca,4 Paolo Antonio Netti,4 Valeria Panzetta,4 Daniela Marasco1 1Department of Pharmacy, CIRPEB: Research Center on Bioactive Peptides- University of Naples Federico II, Naples, 80131, Italy; 2Institute of Biostructures and Bioimaging (CNR), Naples, 80131, Italy; 3Italian Institute of Technology (IIT), Genova, 16152, Italy; 4Department of Ingegneria Chimica del Materiali e della Produzione Industriale (DICMAPI), University of Naples Federico II, Naples, 80125, ItalyCorrespondence: Daniela Marasco, Email daniela.marasco@unina.itIntroduction: SOCS3 (suppressor of cytokine signaling 3) protein is a crucial regulator of cytokine-induced inflammation, and its administration has been shown to have therapeutic effects. Recently, we designed a chimeric proteomimetic of SOCS3, mimicking the interfacing regions of a ternary complex composed of SOCS3, JAK2 (Janus kinase 2) and gp130 (glycoprotein 130) proteins. The derived chimeric peptide, KIRCONG chim, demonstrated limited mimetic function owing to its poor water solubility.Methods: We report investigations concerning a PEGylated variant of KIRCONG mimetic, named KIRCONG chim, bearing a PEG (Polyethylene glycol) moiety as a linker of noncontiguous SOCS3 regions. Its ability to bind to the catalytic domain of JAK2 was evaluated through MST (MicroScale Thermophoresis), as well as its stability in biological serum assays. The structural features of the cyclic compounds were investigated by CD (circular dichroism), nuclear magnetic resonance (NMR), and molecular dynamic (MD) studies. To evaluate the cellular effects, we employed a PLGA-nanoparticle as a delivery system after characterization using DLS and SEM techniques.Results: KIRCONG chim PEG-revealed selective penetration into triple-negative breast cancer (TNBC) MDA-MB-231 cells with respect to the human breast epithelial cell line (MCF10A), acting as a potent inhibitor of STAT3 phosphorylation.Discussion: Overall, the data indicated that miniaturization of the SOCS3 protein is a promising therapeutic approach for aberrant dysregulation of JAK/STAT during cancer progression.Keywords: SOCS3 protein, JAK/STAT pathway, mimetic chimeric peptides, nanoparticles

Alessio Smeraldo,1– 3,* Alfonso Maria Ponsiglione,1,* Andrea Soricelli,4 Paolo Antonio Netti,1– 3 Enza Torino1– 3 1Department of Chemical, Materials and Production Engineering, University of Naples “Federico II”, Naples, 80125, Italy; 2Interdisciplinary Research Center on Biomaterials, CRIB, Naples, 80125, Italy; 3Center for Advanced Biomaterials for Health Care, CABHC, Istituto Italiano di Tecnologia, IIT@CRIB, Naples, 80125, Italy; 4Department of Motor Sciences and Healthiness, University of Naples “Parthenope”, Naples, 80133, Italy*These authors contributed equally to this workCorrespondence: Enza Torino, Department of Chemical, Materials and Production Engineering, University of Naples “Federico II”, Piazzale Tecchio 80, Naples, 80125, Italy, Tel +39-328-955-8158, Email enza.torino@unina.itAbstract: The recent advancements in hybrid positron emission tomography–magnetic resonance imaging systems (PET/MRI) have brought massive value in the investigation of disease processes, in the development of novel treatments, in the monitoring of both therapy response and disease progression, and, not least, in the introduction of new multidisciplinary molecular imaging approaches. While offering potential advantages over PET/CT, the hybrid PET/MRI proved to improve both the image quality and lesion detectability. In particular, it showed to be an effective tool for the study of metabolic information about lesions and pathological conditions affecting the brain, from a better tumor characterization to the analysis of metabolic brain networks. Based on the PRISMA guidelines, this work presents a systematic review on PET/MRI in basic research and clinical differential diagnosis on brain oncology and neurodegenerative disorders. The analysis includes literature works and clinical case studies, with a specific focus on the use of PET tracers and MRI contrast agents, which are usually employed to perform hybrid PET/MRI studies of brain tumors. A systematic literature search for original diagnostic studies is performed using PubMed/MEDLINE, Scopus and Web of Science. Patients, study, and imaging characteristics were extracted from the selected articles. The analysis included acquired data pooling, heterogeneity testing, sensitivity analyses, used tracers, and reported patient outcomes. Our analysis shows that, while PET/MRI for the brain is a promising diagnostic method for early diagnosis, staging and recurrence in patients with brain diseases, a better definition of the role of tracers and imaging agents in both clinical and preclinical hybrid PET/MRI applications is needed and further efforts should be devoted to the standardization of the contrast imaging protocols, also considering the emerging agents and multimodal probes.Keywords: PET/MRI, contrast agents, radiotracers, brain oncology, medical imaging

Vincenzo Quagliariello,1,* Raffaele Vecchione,2,* Alberta De Capua,2 Elena Lagreca,2 Rosario Vincenzo Iaffaioli,3 Gerardo Botti,4 Paolo A Netti,2 Nicola Maurea1 1Division of Cardiology, Istituto Nazionale Tumori- IRCCS- Fondazione G. Pascale, Napoli, Italia; 2Center for Advanced Biomaterial for Health Care (CABHC), Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, Naples, Italy; 3Multidisciplinary Studies in Oncology and Mediterranean Diet, Piazza Nicola Amore, Naples, Italy; 4Scientific Direction, Istituto Nazionale Tumori- IRCCS- Fondazione G. Pascale, Napoli, Italia*These authors contributed equally to this workCorrespondence: Vincenzo QuagliarielloDivision of Cardiology, Istituto Nazionale Tumori- IRCCS- Fondazione G. Pascale, Napoli, ItaliaTel +390815903349Email quagliariello.enzo@gmail.comIntroduction: CoenzymeQ10 (CoQ10) is a well-known antioxidant and anti-inflammatory agent with cardioprotective properties. However, clinical trials based on its oral administration have failed to provide significant effect on cardiac functionality. The main limitation of CoQ10 is based on its very low oral bioavailability and instability that limit dramatically its effects as a cardioprotective agent. Herein, we loaded CoQ10 in high bioavailable nano-emulsions (NEs) coated with chitosan or chitosan and hyaluronic acid in order to improve its performance.Methods: We tested cardioprotective and hepatoprotective effects of CoQ10-loaded nano-carriers against Doxorubicin and Trastuzumab toxicities in cardiomyocytes and liver cells through analysis of cell viability, lipid peroxidation, expression of leukotrienes, p65/NF-kB and pro-inflammatory cytokines involved in anticancer-induced cardio and hepatotoxicity.Results: Nano-carriers showed high stability and loading ability and increased cell viability both in hepatocytes and cardiomyocytes during anticancer treatments. We observed that these effects are mediated by the inhibition of lipid peroxidation and reduction of the inflammation. CoQ10-loaded nano-emulsions showed also strong anti-inflammatory effects reducing leukotriene B4 and p65/NF-κB expression and Interleukin 1β and 6 production during anticancer treatments.Discussion: Anthracyclines and Human epidermal growth factor receptor (HER2) inhibitors have shown significant anticancer effects in clinical practice but their use is characterized by cardiotoxicity and hepatotoxicity. Nano-carriers loaded with CoQ10 showed cardio and hepatoprotective properties mediated by reduction of oxidative damages and pro-inflammatory mediators. These results set the stage for preclinical studies of cardio and hepatoprotection in HER2+ breast cancer-bearing mice treated with Doxorubicin and Trastuzumab.Keywords: cardio-oncology, nano-medicine, Coenzyme Q10, doxorubicin, trastuzumab, inflammation

Giovanni Smaldone,1,2 Annarita Falanga,3 Domenica Capasso,4 Daniela Guarnieri,5,6 Stefania Correale,1,7 Massimiliano Galdiero,8 Paolo A Netti,4 Massimo Zollo,9 Stefania Galdiero,1,2 Sonia Di Gaetano,1 Emilia Pedone1 1Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy; 2Department of Pharmacy and Interuniversity Research Center on Bioactive Peptides, Federico II University of Naples, Naples, Italy; 3Molecular Diagnostics and Pharmaceuticals Scarl, Naples, Italy; 4Special Center for Biotechnology, Federico II University of Naples, Naples, Italy; 5Center for Advanced Biomaterials for Health Care, Interdisciplinary Research Centre on Biomaterials, Italian Institute of Technology, Naples, Italy; 6Interdisciplinary Research Centre on Biomaterials, Federico II University of Naples, Naples, Italy; 7Kedrion S.p.A, Sant'antimo, Naples, Italy; 8Department of Experimental Medicine, Federico II University of Naples, Naples, Italy; 9CEINGE – Advanced Biotechnology Scarl, Naples, Italy Abstract: A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625–644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier. Keywords: delivery, IDP

A scattering snapshot hold an enormous potential for cell class and state classification, allowing to avoid costly fluorescence labelling. Beside convolutional neural networks show outstanding image classification performance compared to other state-of-the-art methods, regarding accuracy and speed. Therefore, we combined the two techniques (Light Scattering and Deep Learning) to identify living cells with high precision. Neural Networks show high prediction performance for known classes but struggles when unknown classes need to be identified. In such a scenario no prior knowledge of the unknown cell class can be used for the model training, which inevitably results in a misclassification. To overcome the hurdle, of identifying unknown cell classes, we must first define an in-distribution of known snapshots to afterwards detect out of distribution snapshots as unknowns. Ones, such a new cell class is identified, we can retrain our cell classifier with the obtained knowledge, so we dynamically update the cell class database. We applied this measurement approach to scattering pattern snapshots of different classes of living cells. Our outcome shows a precise cell classification, which can be applied to a wide range of single cell classification approaches.

Abstract This study aimed to find out rather higher-order thinking helped Junior High school students develop their reading comprehension or not. The research design that used in this research was quantitative and qualitative. The treatment group consists of 30 students, and the group of control also consists of 30 students. In response to an international educational problem, the Indonesian government has created a higher education system. To participate in global competitions in the twenty-first century, Students in Indonesian must think critically and creatively. Several research studies find how teachers use HOTS in many ways. The skill is integrated into the curriculum and application methods by the teacher. The study uses a T-test and interview to find out the effect of HOTS (higher-order thinking skill). The study was conducted at SMP NEGERI 7 in Medan. The test of the research instrument was split up into two parts: Pre-test and post-test, and variability of statistical measured based on the post-test result for two groups in terms of significance.

Carbon nanomaterials have been widely applied for cutting edge therapeutic applications as they offer tunable physio-chemical properties with economic scale-up options. Nuclear delivery of cancer drugs has been of prime focus since it controls important cellular signaling functions leading to greater anti-cancer drug efficacies. Better cellular drug uptake per unit drug injection drastically reduces severe side-effects of cancer therapies. Similarly, carbon dots (CDs) uptaken by the nucleus can also be used to set-up cutting edge nano delivery systems. In an earlier paper, we showed the cellular uptake and plasma membrane impact of combustion generated yellow luminescing CDs produced by our group from fuel rich combustion reactors in a one-step tunable production. In this paper, we aim to specifically study the nucleus by establishing the uptake kinetics of these combustion-generated yellow luminescing CDs. At sub-lethal doses, after crossing the plasma membrane, they impact the actin and microtubule mesh, affecting cell adhesion and migration; enter nucleus by diffusion processes; modify the overall appearance of the nucleus in terms of morphology; and alter chromatin condensation. We thus establish how this one-step produced, cost and bulk production friendly carbon dots from fuel rich combustion flames can be innovatively repurposed as potential nano delivery agents in cancer cells.

Two-dimensional transition metal dichalcogenides, particularly MoS 2 , are interesting materials for many applications in aerospace research, radiation therapy and bioscience more in general. Since in many of these applications MoS 2 -based nanomaterials can be placed in an aqueous environment while exposed to ionizing radiation, both experimental and theoretical studies of their behaviour under these conditions is particularly interesting. Here, we study the effects of tiny imparted doses of 511 keV photons to MoS 2 nanoflakes in water solution. To the best of our knowledge, this is the first study in which ionizing radiation on 2D-MoS 2 occurs in water. Interestingly, we find that, in addition to the direct interaction between high-energy photons and nanoflakes, reactive chemical species, generated by γ-photons induced radiolysis of water, come into play a relevant role. A radiation transport Monte Carlo simulation allowed determining the elements driving the morphological and spectroscopical changes of 2D-MoS 2 , experimentally monitored by SEM microscopy, DLS, Raman and UV-vis spectroscopy, AFM, and X-ray photoelectron techniques. Our study demonstrates that radiolysis products affect the Molybdenum oxidation state, which is massively changed from the stable + 4 and + 6 states into the rarer and more unstable + 5. These findings will be relevant for radiation-based therapies and diagnostics in patients that are assuming drugs or contrast agents containing 2D-MoS 2 and for aerospace biomedical applications of 2DMs investigating their actions into living organisms on space station or satellites., (© 2024. The Author(s).)

Recently, a variety of microenvironmental biophysical stimuli have been proved to play a crucial role in regulating cell functions. Among them, morpho-physical cues, like curvature, are emerging as key regulators of cellular behavior. Changes in substrate curvature have been shown to impact the arrangement of Focal Adhesions (FAs), influencing the direction and intensity of cytoskeleton generated forces and resulting in an overall alteration of cell mechanical identity. In their native environment, cells encounter varying degrees of substrate curvature, and in specific organs, they are exposed to dynamic changes of curvature due to periodic tissue deformation. However, the mechanism by which cells perceive substrate curvature remains poorly understood. To this aim, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. Employing a combined experimental and simulative approach, human adipose-derived stem cells were exposed to controlled curvature intensity and frequency. During this exposure, measurements were taken on FAs extension and orientation, cytoskeleton organization and cellular/nuclear alignment. The data clearly indicated a significant influence of the substrate curvature on cell adhesion processes. These findings contribute to a better understanding of the mechanisms through which cells perceive and respond to substrate curvature signals. STATEMENT OF SIGNIFICANCE: This work is our contribution to the comprehension of substrate curvature's function as a crucial regulator of cell adhesion at the scale of focal adhesions and cell mechanical identity. In recent years, a large body of knowledge is continuously growing providing comprehension of the role of various microenvironmental biophysical stimuli in regulating cell functions. Nevertheless, little is known about the role of substrate curvature, in particular, when cells are exposed to this stimulus in a dynamic manner. To address the role of substrate curvature on cellular behavior, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. The experiment data made it abundantly evident that the substrate curvature had a major impact on the mechanisms involved in cell adhesion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2 μL) at 25 °C with a higher sensitivity (LOD 1.3 fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30 min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N = 7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inorganic medicinal compounds represent a unique and versatile source of potential therapeutics in many diseases and, more recently, in neurodegeneration. Herein we investigated the effects of two η 6 -arene Ru(II) complexes on the self-aggregation processes of several amyloidogenic peptides endowed with different kinetics and primary sequences. The Ru(II) complexes exhibit, around the metal ion, two chlorides, one NHC = N -heterocyclic carbene, with a glucosyl and a methyl substituent and separately a hexamethylbenzene, which is named Ru1 , and one benzene, named Ru2 . Both complexes were demonstrated to bind monomeric amyloids suppressing aggregation as evidenced in thioflavin T (ThT) binding assays and autofluorescence experiments. Electrospray ionization mass spectrometry (ESI-MS) indicated the formation of direct adducts between amyloid and metal complexes, which determined the marked conformational variation of peptides and a rescue of cellular viability in SH-SY5Y cells. The complex Ru2 was demonstrated to be a more potent inhibitor of amyloid aggregation compared to Ru1 likely because of the less hindrance of the arene moiety. The presented data strongly support the in vitro ability of η 6 -arene Ru(II) complexes to suppress amyloid aggregation, providing insights into their potential application as novel therapeutics in neurodegenerative diseases.

Reconstructing the microscale villous organisation and functionality of the small intestine is essential for developing in vitro platforms tailored for absorption studies as well as for investigating intestinal morphogenesis in development and disease. However, the current fabrication techniques able to mimic the villus-crypt axis poses significant challenges in terms of reconstruction of the complex 3D microarchitecture. These challenges extend beyond mere structural intricacies to encompass the incorporation of diverse cell types and the management of intricate fluid dynamics within the system. Here, we introduce a novel microfluidic device called In-Crypts , which integrates a cell-instructive membrane aimed at inducing and guiding Caco-2 cells morphogenesis. Patterned topographical cues embossed onto the porous membrane induce the formation of a well-organized intestinal epithelium, characterized by proliferating crypt-like domains and differentiated villus-like regions. Notably, our cell-instructive porous membrane effectively sustains stem cells development, faithfully replicating the niche environment of in vivo intestinal crypts thus mirroring the cell biogeography observed in vivo . Moreover, by introducing dynamic fluid flow, we provide a faithful recapitulation of the native microenvironmental shear stress experienced by the intestinal epithelium. This stress plays a crucial role in influencing cell behaviour, differentiation, and overall functionality, thus offering a highly realistic model for studying intestinal physiology and pathology. The resulting intestinal epithelium exhibits significantly denser regions of mucus and microvilli, characteristic typically absent in static cultures, upregulating more than 1.5 of the amount expressed in the classical flattened configuration, enhanced epithelial cell differentiation and increased adsorptive surface area. Hence, the innovative design of In-Crypts proves the critical role of employing a cell-instructive membrane in argument the physiological relevance of organs-on-chips. This aspect, among others, will contribute to a more comprehensive understanding of organism function, directly impacting drug discovery and development., (Creative Commons Attribution license.)

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression., (© 2024 Wiley‐VCH GmbH.)

Currently, the biomimetic approach of drawing inspiration from nature has frequently been employed in designing drug nanocarriers (NCs) of actively target various diseases, ranging from cancer to neuronal and inflammation pathologies. The cell-membrane coating can confer upon the inner nanomaterials a biological identity and the functions exhibited by the cells from which the membrane is derived. Monocyte- and macrophage-membrane-coated nanomaterials have emerged as an ideal delivery system to target inflamed vasculature. Herein, we developed two biomimetic NCs using a human-derived leukaemia monocytic cell line (THP-1), either undifferentiated or differentiated by phorbol 12-myristate 13-acetate (PMA) into adherent macrophage-like cells as membrane sources for NC coating. We employed a secondary oil-in-water nano-emulsion (SNE) as the inner core, which served as an optimal NC for high payloads of lipophilic compounds. Two different biomimetic systems were produced, combining the biomimetic features of biological membranes with the physicochemical and nano-sized characteristics of SNEs. These systems were named Monocyte NEsoSome (M-NEsoSome) and Macrophage NEsoSome (M0-NEsoSome). Their uptake ability was investigated in tumour necrosis factor alfa (TNFα)-treated human umbilical vein endothelial cells (HUVECs), selected as a model of inflamed endothelial cells. The M0 membrane coating demonstrated accelerated internalisation compared with the monocyte coating and notably surpassed the uptake rate of bare NCs. In conclusion, M0-NEsoSome NCs could be a therapeutic system for targeting inflamed endothelial cells and potentially delivering anti-inflammatory drugs in vascular inflammation.

New strategies for enhancing drug delivery to the blood-brain barrier (BBB) represent a major challenge in treating cerebral diseases. Nanoemulsion-based nanocarriers represent an ideal candidate to improve drug delivery thanks to their versatility in functionalization and cargo protection. In this work, a paclitaxel-loaded nano-emulsion has been firstly functionalized and stabilized with two layers constituted of chitosan and hyaluronic acid, and, secondly, the latter has been conjugated to the CRT peptide. CRT is a bioactive peptide that selectively recognizes bEnd.3 cells, a model of the BBB, thanks to its interactions with transferrin (Tf) and its receptor (TfR). Cytotoxic results showed a 41.5% higher uptake of CRT functionalized nano-emulsion than the negative control, demonstrating the ability of this novel tool to be accumulated in brain endothelium tissue. Based upon these results, our approach can be fully generalizable to the design of multifunctional nanocarriers for delivery of therapeutic agents to the central nervous systems.

Background: Among mechanoporation techniques for intracellular delivery, microfluidic approaches succeed in high delivery efficiency and throughput. However, especially the entry of large cargoes (e.g. DNA origami, mRNAs, organic/inorganic nanoparticles) is currently impaired since it requires large cell membrane pores with the need to apply multi-step processes and high forces, dramatically reducing cell viability., Results: Here, HiViPore presents as a microfluidic viscoelastic contactless compression for one-step cell mechanoporation to produce large pores while preserving high cell viability. Inducing an increase of curvature at the equatorial region of cells, formation of a pore with a size of ~ 1 μm is obtained. The poration is coupled to an increase of membrane tension, measured as a raised fluorescence lifetime of 12% of a planarizable push-pull fluorescent probe (Flipper-TR) labelling the cell plasma membrane. Importantly, the local disruptions of cell membrane are transient and non-invasive, with a complete recovery of cell integrity and functions in ~ 10 min. As result, HiViPore guarantees cell viability as high as ~ 90%. In such conditions, an endocytic-free diffusion of large nanoparticles is obtained with typical size up to 500 nm and with a delivery efficiency up to 12 times higher than not-treated cells., Conclusions: The proposed one-step contactless mechanoporation results in an efficient and safe approach for advancing intracellular delivery strategies. In detail, HiViPore solves the issues of low cell viability when multiple steps of poration are required to obtain large pores across the cell plasma membrane. Moreover, the compression uses a versatile, low-cost, biocompatible viscoelastic fluid, thus also optimizing the operational costs. With HiViPore, we aim to propose an easy-to-use microfluidic device to a wide range of users, involved in biomedical research, imaging techniques and nanotechnology for intracellular delivery applications in cell engineering., (© 2024. The Author(s).)

Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodeling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodeling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to fully replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodeling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed gland-like structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Potent synthetic drugs, as well as biomolecules extracted from plants, have been investigated for their selectivity toward cancer cells. The main limitation in cancer treatment is the ability to bring such molecules within each single cancer cell, which requires accumulation in the peritumoral region followed by homogeneous spreading within the entire tissue. In the last decades, nanotechnology has emerged as a powerful tool due to its ability to protect the drug during blood circulation and allow enhanced accumulation around the leaky regions of the tumor vasculature. However, the ideal size for accumulation of around 100 nm is too large for effective penetration into the dense collagen matrix. Therefore, we propose a multistage system based on graphene oxide nanosheet-based quantum dots (GOQDs) with dimensions that are 12 nm, functionalized with hyaluronic acid (GOQDs-HA), and deposited using the layer-by-layer technique onto an oil-in-water nanoemulsion (O/W NE) template that is around 100 nm in size, previously stabilized by a biodegradable polymer, chitosan. The choice of a biodegradable core for the nanocarrier is to degrade once inside the tumor, thus promoting the release of smaller compounds, GOQDs-HA, carrying the adsorbed anticancer compound, which in this work is represented by curcumin as a model bioactive anticancer molecule. Additionally, modification with HA aims to promote active targeting of stromal and cancer cells. Cell uptake experiments and preliminary penetration experiments in three-dimensional microtissues were performed to assess the proposed multistage nanocarrier.

Azopolymers are light-responsive materials that hold promise to transform in vitro cell culture systems. Through precise light illumination, they facilitate substrate pattern formation and erasure, allowing for the dynamic control and creation of active interfaces between cells and materials. However, these materials exhibit a tendency to locally detach from the supporting glass in the presence of aqueous solutions, such as cell culture media, due to the formation of blisters, which are liquid-filled cavities generated at the azopolymer film-glass interface. These blisters impede precise structurization of the surface of the azomaterial, limiting their usage for surface photoactivation in the presence of cells. In this study, we present a cost-effective and easily implementable method to improve the azopolymer-glass interface stability through silane functionalization of the glass substrate. This method proved to be efficient in preventing blister formation, thereby enabling the dynamic modulation of the azopolymer surface in situ for live-cell experiments. Furthermore, we proved that the light-illumination conditions used to induce azopolymer surface variations do not induce phototoxic effects. Consequently, this approach facilitates the development of a photoswitchable azopolymer cell culture platform for studying the impact of multiple in situ inscription and erasure cycles on cell functions while maintaining a physiological wet microenvironment.

The stiffness of the extracellular matrix plays a crucial role in cell motility and spreading, influencing cell morphology through cytoskeleton organization and transmembrane proteins' expression. In this context, mechanical characterization of both cells and the extracellular matrix gains prominence for enhanced diagnostics and clinical decision-making. Here, we investigate the combined effect of mechanotransduction and ionizing radiations on altering cells' mechanical properties, analysing mammary cell lines (MCF10A and MDA-MB-231) after X-ray radiotherapy (2 and 10 Gy). We found that ionizing radiations sensitively affect adenocarcinoma cells cultured on substrates mimicking cancerous tissue stiffness (15 kPa), inducing an increased structuration of paxillin-rich focal adhesions and cytoskeleton: this process translates in the augmentation of tension at the actin filaments level, causing cellular stiffness and consequently affecting cytoplasmatic/nuclear morphologies. Deeper exploration of the intricate interplay between mechanical factors and radiation should provide novel strategies to orient clinical outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mottareale, Frascogna, La Verde, Arrichiello, Muto, Netti, Fusco, Panzetta and Pugliese.)

There is a growing interest in the search for metal-based therapeutics for protein misfolding disorders such as Alzheimer's disease (AD). A novel and largely unexplored class of metallodrugs is constituted by paddlewheel diruthenium complexes, which exhibit unusual water solubility and stability and unique coordination modes to proteins. Here, we investigate the ability of the complexes [Ru 2 Cl(DPhF)(O 2 CCH 3 ) 3 ]·H 2 O ( 1 ), [Ru 2 Cl(DPhF) 2 (O 2 CCH 3 ) 2 ]·H 2 O ( 2 ), and K 2 [Ru 2 (DPhF)(CO 3 ) 3 ]·3H 2 O ( 3 ) (DPhF - = N , N '-diphenylformamidinate) to interfere with the amyloid aggregation of the Aβ 1-42 peptide. These compounds differ in charge and steric hindrance due to the coordination of a different number of bulky ligands. The mechanisms of action of the three complexes were studied by employing a plethora of physicochemical and biophysical techniques as well as cellular assays. All these studies converge on different mechanisms of inhibition of amyloid fibrillation: complexes 1 and 2 show a clear inhibitory effect due to an exchange ligand process in the Ru 2 unit aided by aromatic interactions. Complex 3 shows no inhibition of aggregation, probably due to its negative charge in solution. This study demonstrates that slight variations in the ligands surrounding the bimetallic core can modulate the amyloid aggregation inhibition and supports the use of paddlewheel diruthenium complexes as promising therapeutics for Alzheimer's disease.

Natural polymers have found increased use in a wider range of applications due to their less harmful effects. Notably, bacterial cellulose has gained significant consideration due to its exceptional physical and chemical properties and its substantial biocompatibility, which makes it an attractive candidate for several biomedical applications. This study attempts to thoroughly unravel the microstructure of bacterial cellulose precursors, known as bioflocculants, which to date have been poorly characterised, by employing both electron and optical microscopy techniques. Here, starting from bioflocculants from Symbiotic Culture of Bacteria and Yeast (SCOBY), we proved that their microstructural features, such as porosity percentage, cellulose assembly degree, fibres' density and fraction, change in a spatio-temporal manner during their rising toward the liquid-air interface. Furthermore, our research identified a correlation between electron and optical microscopy parameters, enabling the assessment of bioflocculants' microstructure without necessitating offline sample preparation procedures. The ultimate goal was to determine their potential suitability as a novel cellulose-based building block material with tuneable structural properties. Our investigations substantiate the capability of SCOBY bioflocculants, characterized by distinct microstructures, to successfully assemble within a microfluidic device, thereby generating a cellulose sheet endowed with specific and purposefully designed structural features., (© 2024. The Author(s).)

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricate in vitro tissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors ( μ TPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly of μ TPs into predefined shapes. Compared to cell-aggregated bioink, our μ TPs represent a functional unit where cells are embedded in their specific ECM. μ TPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days, μ TPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) ( µ TP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter. µ TP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel at T > 30 °C), ensuring µ TPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, until µ TPs fused into a continuous connective tissue. µ TPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues., (Creative Commons Attribution license.)

Mammalian sperm motility is getting more relevant due to rising infertility rates worldwide, generating the need to improve conventional analysis and diagnostic approaches. Nowadays, computer assisted sperm analysis (CASA) technologies represent a popular alternative to manual examination which is generally performed by observing sperm motility in very confined geometries. However, under physiological conditions, sperm describe three-dimensional motility patterns which are not well reconstructed by the limited depth of standard acquisition chambers. Therefore, affordable and more versatile alternatives are needed. Here, a motility analysis in unconfined conditions is proposed. In details, the analysis is characterized by a significant longer duration -with respect to conventional systems- with the aim to observe eventually altered motility patterns. Brightfield acquisition in rectangular glass capillaries captured frozen-thawed bovine spermatozoa which were analyzed by means of a self-written tracking routine and classified in sub-populations, based on their curvilinear velocity. To test the versatility of our approach, cypermethrin -a commonly used pesticides- known to be responsible for changes in sperm motility was employed, assessing its effect at three different time-steps. Experimental results showed that such drug induces an increase in sperm velocity and progressiveness as well as circular pattern formation, likely independent of wall interactions. Moreover, this resulted in a redistribution of sperm with the rapid class declining in number with time, but still showing an overall velocity increase. The flexibility of the approach permits parameter modifications with the experimental needs, allowing us to conduct a comprehensive examination of sperm motility. This adaptability facilitated data acquisition which can be computed at different frame rates, extended time periods, and within deeper observation chambers. The suggested approach for sperm analysis exhibits potential as a valuable augmentation to current diagnostic instruments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Canonico, De Clemente, Fardilha, Ferreira, Maremonti, Dannhauser, Causa and Netti.)