Regulated cell death modalities such as apoptosis and necroptosis play an important role in regulating different cellular processes. Currently, regulated cell death is identified using the golden ...standard techniques such as fluorescence microscopy and flow cytometry. However, they require fluorescent labels, which are potentially phototoxic. Therefore, there is a need for the development of new label-free methods. In this work, we apply Digital Holographic Microscopy (DHM) coupled with a deep learning algorithm to distinguish between alive, apoptotic and necroptotic cells in murine cancer cells. This method is solely based on label-free quantitative phase images, where the phase delay of light by cells is quantified and is used to calculate their topography. We show that a combination of label-free DHM in a high-throughput set-up (~10,000 cells per condition) can discriminate between apoptosis, necroptosis and alive cells in the L929sAhFas cell line with a precision of over 85%. To the best of our knowledge, this is the first time deep learning in the form of convolutional neural networks is applied to distinguish-with a high accuracy-apoptosis and necroptosis and alive cancer cells from each other in a label-free manner. It is expected that the approach described here will have a profound impact on research in regulated cell death, biomedicine and the field of (cancer) cell biology in general.
Cancer is a serious health problem mainly characterized by unregulated cell divisions. It is known that malign cells display cancer-specific glycans that can be recognized by lectins, proteins with ...capacity of specific binding to carbohydrates. One such protein is the
Dioclea violacea
lectin (DVL), a known antiproliferative lectin that has been applied in this work to create and optimize a methodology for a controlled-release system by adsorption of lectin into CaCO
3
particles. The system was tested by evaluating the cytotoxic effect of lectin particles against cervical cancer cells (HeLa) and compared to the non-conjugated lectin. DVL was adsorbed into CaCO
3
particles of two sizes (average of 3.6 μm and 0.7 μm), a low-cost, biocompatible and biodegradable material, and the effect of DVL/bioconjugates against HeLa cells has been investigated in vitro and in silico. The bioconjugates possessed adsorption and release compatible with the biological assays. Cells assays applying the bioconjugates demonstrated their cytotoxicity, with a stronger effect compared to the free lectin. In conclusion, the anticancer potential of DVL against HeLa cells has been increased by the construction and application of a controlled-release system based on the adsorption of the lectin on CaCO
3
particles with a relatively simple protocol. Also, the present data demonstrate the potential of lectin–particle bioconjugates as agents in cancer research and suggest an easy and low-cost methodology that can be extended to other lectins/proteins.
Graphical abstract
Regulated cell death (RCD) has a fundamental role in development, pathology, and tissue homeostasis. In order to understand the RCD mechanisms, it is essential to follow these processes in real time. ...Here, atomic force microscopy (AFM) is applied to morphologically and mechanically characterize four RCD modalities (intrinsic and extrinsic apoptosis, necroptosis, and ferroptosis) in murine tumor cell lines. The nano-topographical analysis revealed a distinct surface morphology in case of necroptosis, ∼ 200 nm membrane disruptions are observed. Using mechanical measurements, it is possible to detect the early onset of RCD. Combined elasticity and microrheology analysis allowed for a clear distinction between apoptotic and regulated necrotic cell death. Finally, immunofluorescence analysis of the cytoskeleton structure during the RCD processes confirm the measured mechanical changes. The results of this study not only demonstrate the possibility of early real-time cell death detection but also reveal important differences in the cytoskeletal dynamics between multiple RCD modalities.
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•AFM is a label-free method to distinguish apoptosis, necroptosis, and ferroptosis•Nanotopography and subtle morphologic changes are distinct for each RCD•Mechanobiology elasticity analysis reveals changes occurring at early stages of RCD•Microrheology data agree with mechanobiology Young's modulus analysis
Mechanobiology; Biotechnology; Cell Biology
Cells use glycans to encode information that modulates processes ranging from cell–cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is ...performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin’s specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.
•Cells are coated in a hybrid coating using polyelectrolytes and Au-nanoparticles using the LbL-method.•Au-nanoparticles were visualized by AFM and their concentration was determined using absorption ...spectrophotometry.•LBL-coating of the cells is verified using microscopy revealing a strong coverage.•Using a PrestoBlue assay, the viability of coated cells is confirmed.•Near-infrared laser is used to induce immediate drastic changes in the cellular membrane.
Encapsulation of cells has been an active area of research. Among various methods for encapsulation, Layer-by-Layer (LbL) offers extensive flexibility in the design of surfaces and their interfacial nanoarchitectonics. A diverse range of functionalities have been recently demonstrated for cell encapsulation including protection and improved circulation. Here, we present a new strategy of cell encapsulation in a hybrid coating containing LbL assembly functionalized with gold nanoparticle aggregates. The effect of this hybrid coating on cell viability was assessed. Subsequently, upon laser illumination the encapsulated cells undergo immediate necrosis caused by the localized heat generated by the laser beam on gold nanoparticle aggregates. Similarly to affecting polyelectrolyte multilayer capsules, one envisions controlling surface properties of cells remotely by a laser beam. Further applications of the proposed approach are expected to be in biomedicine.
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UiO-66-NH2 is a well-known Zr-MOF with a high surface area and good chemical stability, which is often employed for catalysis. The biological applications of Zr-MOFs are less often explored, however ...it has already been shown that they can be used for drug loading and release. In this work we show that UiO-66-NH2 (referred to as nano-MOF throughout the work) can be a host for accommodating β-NaYF4:Er,Yb@β-NaYF4 nanothermometers as well as gold nanoparticles (AuNPs) which can potentially take on the role of nanoheaters for plasmon-induced hyperthermia. Such a hybrid material can be made in an easy two step approach and yields nano-sized porous composites. At the same time, it could potentially still be used for drug delivery. A lipid bilayer is wrapped around the nano-MOF composites to further improve their biocompatibility with the human body. The hybrid composite shows good thermometry in the physiological range when dispersed in water.
•Nano Zr-MOFs are suitable hosts for accommodating NaYF4:Er,Yb@NaYF4 nanothermometers and gold nanoparticle nanoheaters.•Such hybrid materials are non-toxic to human cells, however incorporating AuNPs increases the toxicity.•These hybrid composites show good thermometric performance in the physiological range.
Employing luminescence thermometry in the biomedical field is undeniably appealing as many health conditions are accompanied by temperature changes. In this work, we show our ongoing efforts and ...results at designing novel vehicles for dual-mode thermometry and pH-dependent drug release based on hollow spheres. Hereby for that purpose, we exploit the hollow Y 2 O 3 and Y 2 O 2 SO 4 host materials. These two inorganic hollow phosphors were investigated and showed to have excellent upconversion Er 3+ –Yb 3+ luminescence properties and could be effectively used as optical temperature sensors in the physiological temperature range when induced by near-infrared CW light (975 nm). Further, doxorubicin was exploited as a model anti-cancer drug to monitor the pH-dependent drug release of these materials showing that they can be used for simultaneous thermometry and drug delivery applications.
Cancer is a serious health problem mainly characterized by unregulated cell divisions. It is known that malign cells display cancer-specific glycans that can be recognized by lectins, proteins with ...capacity of specific binding to carbohydrates. One such protein is the Dioclea violacea lectin (DVL), a known antiproliferative lectin that has been applied in this work to create and optimize a methodology for a controlled-release system by adsorption of lectin into CaCO.sub.3 particles. The system was tested by evaluating the cytotoxic effect of lectin particles against cervical cancer cells (HeLa) and compared to the non-conjugated lectin. DVL was adsorbed into CaCO.sub.3 particles of two sizes (average of 3.6 mum and 0.7 mum), a low-cost, biocompatible and biodegradable material, and the effect of DVL/bioconjugates against HeLa cells has been investigated in vitro and in silico. The bioconjugates possessed adsorption and release compatible with the biological assays. Cells assays applying the bioconjugates demonstrated their cytotoxicity, with a stronger effect compared to the free lectin. In conclusion, the anticancer potential of DVL against HeLa cells has been increased by the construction and application of a controlled-release system based on the adsorption of the lectin on CaCO.sub.3 particles with a relatively simple protocol. Also, the present data demonstrate the potential of lectin-particle bioconjugates as agents in cancer research and suggest an easy and low-cost methodology that can be extended to other lectins/proteins. Graphical abstract
In recent years, the importance of the investigation of regulated cell death (RCD) has significantly increased and different methods are proposed for the detection of RCD including biochemical as ...well as fluorescence assays. Researchers have shown that early stages of cell death could be detected by using AFM. Although AFM offers a high single‐cell resolution and sensitivity, the throughput (<100 cells/h) limits a broad range of biomedical applications of this technique. Here, a microfluidics‐based mechanobiology technique, named shear flow deformability cytometry (sDC), is used to investigate and distinguish dying cells from viable cells purely based on their mechanical properties. Three different RCD modalities (i.e., apoptosis, necroptosis, and ferroptosis) are induced in L929sAhFas cells and analysed using sDC. Using machine learning on the extracted parameters, it was possible to predict the dead or viable state with 92% validation accuracy. A significant decrease in elasticity can be noticed for each of these RCD modalities by analysing the deformation of the dying cells. Analysis of morphological characteristics such as cell size and membrane irregularities also indicated significant differences in the RCD induced cells versus control cells. These results highlight the importance of mechanical properties during RCD and the significance of label‐free techniques, such as sDC, which can be used to detect regulated cell death and can be further linked with sorting of live and dead cells.
In this work, we investigate the viability to use a microfluidics‐based approach, shear flow deformability cytometry (sDC), to measure mechanical properties of cells undergoing regulated cell death. Three different regulated cell death modalities were observed. Using machine learning prediction on the parameters extracted from sDC, it was possible to predict with 92% accuracy the live/dead state of cells. The main difference between live and dead cells showed to be the deformation of the cells which is strongly linked to the mechanical state of cells.
Biomaterials composed of food polysaccharides are of great interest for future biomedical applications due to their great biocompatibility, tunable mechanical properties, and complex architectural ...designs that play a crucial role in the modulation of cell adhesion and proliferation. In this work, a facile approach was designed to obtain novel 3D alginate-CaCO3 hybrid hydrogel particles in situ. Controlling the gel concentration from 3 to 20 mg·mL−1 allows us to control the alginate-CaCO3 hydrogel particles' size and density (size variation from 1.86 to 2.34 mm and density from 1.22 to 1.29 mg/mm3). This variable also has a considerable influence on the mineralization process resulting in CaCO3 particles with varied sizes and amounts within the hydrogel beads. The measurements of Young's modulus showed that the inclusion of CaCO3 particles into the alginate hydrogel improved its mechanical properties, and Young's modulus of these hybrid hydrogel particles had a linear relationship with alginate content and hydrogel particle size. Cell experiments indicated that alginate-CaCO3 hybrid hydrogel particles can support osteoblastic cell proliferation and growth. In particular, the amount of hydroxyapatite deposition on the cell membrane significantly increased after the treatment of cells with hybrid hydrogel particles, up to 20-fold. This work offers a strategy for constructing inorganic particle-doped polysaccharide hybrid hydrogel scaffolds that provide the potential to support cell growth.
A crystal ball model of cells growing on hybrid colloidal hydrogel beads. Display omitted
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