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A better understanding of the pathophysiology of cardiac fat depots is crucial to describe their role in the development of cardiovascular diseases. To this end, we have developed a method to isolate mature fat cells from the pericardial adipose tissue (PAT), the most accessible cardiac fat depot during cardiac surgery. Using enzymatic isolation, we were able to successfully obtain mature fat cells together with the corresponding cells of the stromal vascular fraction (SVF). We subjected the PAT adipocytes to thorough morphological and molecular characterization, including detailed fatty acid profiling, and simultaneously investigated their reactivity to external stimuli. Our approach resulted in highly purified fat cells with sustained viability for up to 72â¯h after explantation. Remarkably, these adipocytes responded to multiple challenges, including pro-inflammatory and metabolic stimuli, indicating their potential to trigger a pro-inflammatory response and modulate endothelial cell behavior. Furthermore, we have created conditions to maintain whole PAT in culture and preserve their viability and reactivity to external stimuli. The efficiency of cell recovery combined with minimal dedifferentiation underscores the promise for future applications as a personalized tool for screening and assessing individual patient responses to drugs and supplements or nutraceuticals.
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The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of ß-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3ß (glycogen synthase kinase 3ß). Herein, we show that GSK3ß is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3ß activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3ß complex formation. Specifically, GSK3ß activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the ß-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.
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Apoptose/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antígenos CD34/metabolismo , Benzamidas , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Ciclina D1/metabolismo , Citocinas/farmacologia , Dasatinibe , Sinergismo Farmacológico , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesilato de Imatinib , Indóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Maleimidas/farmacologia , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , beta Catenina/metabolismoRESUMO
The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.
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Lipídeos , Teste de Materiais , Nanopartículas , Tamanho da Partícula , Transfecção , Humanos , Transfecção/métodos , Nanopartículas/química , Lipídeos/química , Células Jurkat , Materiais Biocompatíveis/química , Sobrevivência Celular/efeitos dos fármacos , DNA/administração & dosagem , DNA/química , Linfócitos T/metabolismo , Linfócitos T/citologia , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro-patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi-nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer-by-layer (LbL) polyelectrolyte multilayer deposition was combined with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self-assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4-styrene-sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly-L-arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)5-coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro.
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Técnicas de Cultura de Células/instrumentação , Fibras Musculares Esqueléticas/citologia , Nanoestruturas/química , Polímeros/química , Ácidos Sulfônicos/química , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Fenômenos Fisiológicos Celulares/fisiologia , Meios de Cultura , Camundongos , Microscopia de Fluorescência , Poliaminas/química , Propriedades de Superfície , Engenharia TecidualRESUMO
Currently, cancer represents a major cause of death in the world, despite all the progress made in developing new therapies [...].
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In the last decade, hyaluronic acid (HA) has attracted an ever-growing interest in the biomedical engineering field as a biocompatible, biodegradable, and chemically versatile molecule. In fact, HA is a major component of the extracellular matrix (ECM) and is essential for the maintenance of cellular homeostasis and crosstalk. Innovative experimental strategies in vitro and in vivo using three-dimensional (3D) HA systems have been increasingly reported in studies of diseases, replacement of tissue and organ damage, repairing wounds, and encapsulating stem cells for tissue regeneration. The present work aims to give an overview and comparison of recent work carried out on HA systems showing advantages, limitations, and their complementarity, for a comprehensive characterization of their use. A special attention is paid to the use of HA in three important areas: cancer, diseases of the central nervous system (CNS), and tissue regeneration, discussing the most innovative experimental strategies. Finally, perspectives within and beyond these research fields are discussed.
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Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by five mutations in the RAB7A gene. Autophagy and late endocytic trafficking were already characterized in CMT2B. Indeed, impairment of autophagy and an increase in lysosomal degradative activity were found in cells expressing the mutant proteins. Recently, we described a novel RAB7 mutation associated with predominantly motor CMT2 and impaired EGFR trafficking. With the aim to analyze the autophagy process and lysosomal activity in CMT2B fibroblasts carrying the p.K126R RAB7 novel mutation and to investigate further the causes of the different phenotype, we have performed Western blot, immunofluorescence and cytometric analyses monitoring autophagic markers and endocytic proteins. Moreover, we investigated lipophagy by analyzing accumulation of lipid droplets and their co-localization with endolysosomal degradative compartments. We found that cells expressing the RAB7K126R mutant protein were characterized by impairment of autophagy and lipophagy processes and by a moderate increase in lysosomal activity compared to the previously studied cells carrying the RAB7V162M mutation. Thus, we concluded that EGFR trafficking alterations and a moderate increase in lysosomal activity with concomitant impairment of autophagy could induce the specific predominantly motor phenotype observed in K126R patients.
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Proteínas rab de Ligação ao GTP , proteínas de unión al GTP Rab7 , Autofagia/genética , Doença de Charcot-Marie-Tooth , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Humanos , Laminopatias , Lisossomos/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Protein-based microfibers are biomaterials of paramount importance in materials science, nanotechnology, and medicine. Here we describe the spontaneous in situ formation and secretion of nanostructured protein microfibers in 2D and 3D cell cultures of 3T3 fibroblasts and B104 neuroblastoma cells upon treatment with a micromolar solution of either unmodified terthiophene or terthiophene modified by mono-oxygenation (thiophene â thiophene S-oxide) or dioxygenation (thiophene â thiophene S,S-dioxide) of the inner ring. We demonstrate via metabolic cytotoxicity tests that modification to the S-oxide leads to a severe drop in cell viability. By contrast, unmodified terthiophene and the respective S,S-dioxide cause no harm to the cells and lead to the formation and secretion of fluorescent and electroactive protein-fluorophore coassembled microfibers with a large aspect ratio, a micrometer-sized length and width, and a nanometer-sized thickness, as monitored in real-time by laser scanning confocal microscopy (LSCM). With respect to the microfibers formed by unmodified terthiophene, those formed by the S,S-dioxide display markedly red-shifted fluorescence and an increased n-type character of the material, as shown by macroscopic Kelvin probe in agreement with cyclovoltammetry data. Electrophoretic analyses and Q-TOF mass spectrometry of the isolated microfibers indicate that in all cases the prevalent proteins present are vimentin and histone H4, thus revealing the capability of these fluorophores to selectively coassemble with these proteins. Finally, DFT calculations help to illuminate the fluorophore-fluorophore intermolecular interactions contributing to the formation of the microfibers.
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The development of nanovectors for precise gene therapy is increasingly focusing on avoiding uncontrolled inflammation while still being able to effectively act on the target sites. Herein, we explore the use of non-viral hybrid polyelectrolyte nanocomplexes (hPECs) for gene delivery, which display good transfection efficacy coupled with non-inflammatory properties. Monodisperse hPECs were produced through a layer-by-layer self-assembling of biocompatible and biodegradable polymers. The resulting nanocomplexes had an inner core characterized by an EGFP-encoding plasmid DNA (pDNA) complexed with linear polyethyleneimine or protamine (PEI or PRM) stabilized with lecithin and poly(vinyl alcohol) (PVA) and an outer layer consisting of medium-molecular-weight chitosan (CH) combined with tripolyphosphate (TPP). PEI- and PRM-hPECs were able to efficiently protect the genetic cargo from nucleases and to perform a stimuli-responsive release of pDNA overtime, thus guaranteeing optimal transfection efficiency. Importantly, hPECs revealed a highly cytocompatible and a non-inflammatory profile in vitro. These results were further supported by evidence of the weak and unspecific interactions of serum proteins with both hPECs, thus confirming the antifouling properties of their outer shell. Therefore, these hPECs represent promising candidates for the development of effective, safe nanotools for gene delivery.
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Pediatric brain tumors represent the most common types of childhood cancer and novel diagnostic and therapeutic solutions are urgently needed. The gold standard treatment option for brain cancers in children, as in adults, is tumor resection followed by radio- and chemotherapy, but with discouraging therapeutic results. In particular, the last two treatments are often associated to significant neurotoxicity in the developing brain of a child, with resulting disabilities such as cognitive problems, neuroendocrine, and neurosensory dysfunctions/deficits. Nanoparticles have been increasingly and thoroughly investigated as they show great promises as diagnostic tools and vectors for gene/drug therapy for pediatric brain cancer due to their ability to cross the blood-brain barrier. In this review we will discuss the developments of nanoparticle-based strategies as novel precision nanomedicine tools for diagnosis and therapy in pediatric brain cancers, with a particular focus on targeting strategies to overcome the main physiological obstacles that are represented by blood-brain barrier.
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The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.
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For centuries, macroalgae, or seaweeds, have been a significant part of East Asian diets. In Europe, seaweeds are not considered traditional foods, even though they are increasingly popular in Western diets in human food applications. In this study, a biological processing method based on semi-solid fermentation was optimized for the treatment of the seaweed Gracilaria gracilis. For the first time, selected lactic acid bacteria and non-conventional coagulase-negative staphylococci were used as starter preparations for driving a bio-processing and bio-stabilization of raw macroalga material to obtain new seaweed-based food prototypes for human consumption. Definite food safety and process hygiene criteria were identified and successfully applied. The obtained fermented products did not show any presence of pathogenic or spoilage microorganisms, thereby indicating safety and good shelf life. Lactobacillus acidophilus-treated seaweeds revealed higher α-amylase, protease, lipase, endo-cellulase, and endo-xylanase activity than in the untreated sample. This fermented sample showed a balanced n-6/n-3 fatty acid ratio. SBM-11 (Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus) and PROMIX 1 (Staphylococcus xylosus) treated samples showed fatty acid compositions that were considered of good nutritional quality and contained relevant amounts of isoprenoids (vitamin E and A). All the starters improved the nutritional value of the seaweeds by significantly reducing the insoluble indigestible fractions. Preliminary data were obtained on the cytocompatibility of G. gracilis fermented products by in vitro tests. This approach served as a valid strategy for the easy bio-stabilization of this valuable but perishable food resource and could boost its employment for newly designed seaweed-based food products.
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In our search for thiophene fluorophores that can overcome the limits of currently available organic dyes in live-cell staining, we synthesized biocompatible dithienothiophene-S,S-dioxide derivatives (DTTOs) that were spontaneously taken up by live mouse embryonic fibroblasts and HeLa cells. Upon treatment with DTTOs, the cells secreted nanostructured fluorescent fibrils, while cell viability remained unaltered. Comparison with the behavior of other cell-permeant, newly synthesized thiophene fluorophores showed that the formation of fluorescent fibrils was peculiar to DTTO dyes. Laser scanning confocal microscopy of the fluorescent fibrils showed that most of them were characterized by helical supramolecular organization. Electrophoretic analysis and theoretical calculations suggested that the DTTOs were selectively recognized by the HyPro component of procollagen polypeptide chains and incorporated through the formation of multiple H-bondings.
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Fibroblastos/química , Fluorescência , Corantes Fluorescentes/química , Tiofenos/química , Animais , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Tiofenos/síntese químicaRESUMO
In this work, the feasibility of sterilizing a water suspension of poly-3-hexylthiophene nanoparticles (P3HT-NPs) is investigated using ionizing radiation, either γ-rays or high-energy electrons (e-beam). It is found that regardless of the irradiation source, the size, polydispersity, aggregation stability, and morphology of the NPs are not affected by the treatment. Furthermore, the impact of ionizing radiation on the physicochemical properties of NPs at different absorbed radiation doses (10-25 kGy) and dose rates (kGy time-1 ) is evaluated through different spectroscopic techniques. The results indicate that delivering a high dose of radiations (25 kGy) at a high dose rate, that is, kGy s-1 , as achieved by e-beam irradiation, preserves the characteristics of the polymeric NPs. Differently, the same radiation dose but delivered at a lower dose rate, that is, kGy h-1 , as attained by using a γ-source, can modify the physicochemical properties of the polymer. Sterility tests indicate that an absorbed dose of 10 kGy, delivered either with γ-rays or e-beam, is already sufficient for effective sterilization of the colloidal suspension and for reducing the endotoxin content. Finally, NPs irradiated at different doses, exhibit the same cytocompatibility and cell internalization characteristics in human neuroblastoma SH-SY5Y cells of NPs prepared under aseptic conditions.
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Nanopartículas , Água , Raios gama , Humanos , Doses de Radiação , EsterilizaçãoRESUMO
Nanotechnology offers innovative tools for the design of biomimetic nanocarriers for targeted cancer therapy. These nano-systems present several advantages such as cargo's protection and modulation of its release, inclusion of stimuli-responsive elements, and enhanced tumoral accumulation. All together, these nano-systems suffer low therapeutic efficacy in vivo because organisms can recognize and remove foreign nanomaterials. To overcome this important issue, different modifications on nanoparticle surfaces were exploited in order to reach the desired therapeutic efficacy eliciting, also, the response of immune system against cancer cells. For this reason, more recently, a new strategy involving cell membrane-covered nanoparticles for biomedical application has been attracting increasing attention. Membranes from red blood cells, platelets, leukocytes, tumor, and stem cells, have been exploited as biomimetic coatings of nanoparticles for evading clearance or stimulated immune system by maintaining in the same way their targeting capability. In this review, the use of different cell sources as coating of biomimetic nanocarriers for cancer therapy is discussed.
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Background: A great number of therapeutic limitations, such as chemoresistance, high dosage, and long treatments, are still present in cancer therapy, and are often followed by side effects such as infections, which represent the primary cause of death among patients. Methods: We report pH- and enzymatic-responsive hybrid clustered nanoparticles (HC-NPs), composed of a PCL polymeric core loaded with an anticancer drug, such as Imatinib Mesylate (IM), and coated with biodegradable multilayers embedded with antibacterial and anticancer baby-ship silver NPs, as well as a monoclonal antibody for specific targeting of cancer cells conjugated on the surface. Results: The HC-NPs presented an onion-like structure that serially responded to endogenous stimuli. After internalization into targeted cancer cells, the clustered nanoparticles were able to break up, thanks to intracellular proteases which degraded the biodegradable multilayers and allowed the release of the baby-ship NPs and the IM loaded within the pH-sensible polymer present inside the mothership core. In vitro studies validated the efficiency of HC-NPs in human chronic leukemic cells. This cellular model allowed us to demonstrate specificity and molecular targeting sensitivity, achieved by using a combinatorial approach inside a single nano-platform, instead of free administrations. The combinatory effect of chemotherapic drug and AgNPs in one single nanosystem showed an improved cell death efficacy. In addition, HC-NPs showed a good antibacterial capacity on Gram-negative and Gram-positive bacteria. Conclusions: This study shows an important combinatorial anticancer and antimicrobial effect in vitro.
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We show the influence of two functional ions (Cu2+ and La3+ ), incorporated into a quaternary (Si, Ca, Na, P) sol-gel derived bioactive glass system, on its particle size, cytotoxicity, and bioactivity. By doping the parent glass with the two ions in singular or combined forms, 15 doped glasses were prepared by a rapid sol-gel technique. The influence of the combined doping on the particle size and cell viability was successfully evaluated by the aid of signal-to-noise-ratio (S/N), using Taguchi analysis. This allowed us to analyze the complex interplay of effects between these ions, and the marked differences in biocompatibility between the three cell types studied. Cu addition had a significant effect on reducing the glass particle size, while both increased density. Cell viability was significantly improved for some doping combinations, demonstrating that while combined Cu-La doping was beneficial for biocompatibility with lymphoblasts, individual high-Cu or low-La doping was better with fibroblasts, and either high-Cu or low-La doping, or certain combined Cu-La combinations, were the optimum for osteoblasts. However, the bioactivity of doped samples was generally similar to that of the parent glass, although both La, and particularly Cu, did appear to aid dissolution of ions when immersed in SBF, act as glass modifiers, and encourage HAp crystallization. The results reveal that potential synergistic benefits can be obtained by combining the effects on the mean particle size, density, cytotoxicity, and bioactivity of the glasses. The greatly improved biocompatibility of some of the doped glasses makes them promising candidates for biomedical applications.
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Materiais Biocompatíveis/toxicidade , Cerâmica/toxicidade , Cobre/toxicidade , Lantânio/toxicidade , Dióxido de Silício/toxicidade , Materiais Biocompatíveis/química , Cátions/química , Cátions/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cerâmica/química , Cobre/química , Humanos , Lantânio/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Transição de Fase , Dióxido de Silício/químicaRESUMO
Background: A hallmark of glioblastoma is represented by their ability to widely disperse throughout the brain parenchyma. The importance of developing new anti-migratory targets is critical to reduce recurrence and improve therapeutic efficacy. Methods: Polydimethylsiloxane substrates, either mechanically uniform or presenting durotactic cues, were fabricated to assess GBM cell morphological and dynamical response with and without pharmacological inhibition of NNMII contractility, of its upstream regulator ROCK and actin polymerization. Results: Glioma cells mechanotactic efficiency varied depending on the rigidity compliance of substrates. Morphologically, glioma cells on highly rigid and soft bulk substrates displayed bigger and elongated aggregates whereas on durotactic substrates the same cells were homogeneously dispersed with a less elongated morphology. The durotactic cues also induced a motility change, cell phenotype dependent, and with cells being more invasive on stiffer substrates. Pharmacological inhibition of myosin or ROCK revealed a rigidity-insensitivity, unlike inhibition of microfilament contraction and polymerization of F-actin, suggesting that alternative signalling is used to respond to durotactic cues. Conclusions: The presence of a distinct mechanical cue is an important factor in cell migration. Together, our results provide support for a durotactic role of glioma cells that acts through actomyosin contractility to regulate the aggressive properties of GBM cells.
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One of the key challenges in materials science is to control the properties of a material by directing its supramolecular arrangement. Here we show that iridium complexes, such as FIrpic, Ir-PPY, and Ir-MDQ, can be organized into crystalline and phosphorescent nanoparticles through the nanoprecipitation method, which allows thorough modification of their functional properties. Moreover, we found that it is possible to combine different iridium complexes into a single multicomponent nanostructure, thus creating nanoparticles whose photonic properties derive from the close spatial proximity of the electronic excited states of the different Ir complexes. The morphology of all nanoparticles was fully characterized by microscopic and spectroscopic techniques, and their ordered arrangement was assessed by X-ray diffraction (XRD) and high-resolution transmission electron microscopy (TEM) measurements. We demonstrate that the nanostructuring of the complexes influences their optical and redox properties-by promoting a fine-tuning of emission, photoluminescence quantum yield, excited state lifetime, HOMO/LUMO energy levels, and energy-transfer processes-as well as their interaction with living cells. Investigations on glioblastoma U-251 MG cells demonstrate that nanostructuring represents an effective tool to regulate the efficiency of cell loading, cell viability, colocalization, and penetration in 3D spheroids.
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The use of intrinsic chiral molecules opens the door to bio-imaging specific tools and to the development of target-therapy. In this work the synthesis and characterization of polythiophenes with alkyl side chains containing one R or S chiral carbon is reported. Enantiopure chiral nanoparticles (R or S NPs) were prepared from the polymers by a reprecipitation method. UV-vis, photoluminescence and circular dichroism spectroscopy of the NPs are described. In vitro analysis and metabolic assays show that both R and S NPs are efficiently taken-up by fibroblast cells without signs of toxicity. SDS-PAGE experiments show that formation of hard protein 'corona' enhances the chirality difference between nanoparticles. Co-localization experiments demonstrate that the cells are able to discriminate between the enantiomeric R and S nanoparticles. Finally, experiments carried out on Gram negative and Gram positive bacteria show that the enantiomeric NPs display different antibacterial activity.