RESUMEN
RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes' expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.
Asunto(s)
Genes Esenciales , Osteogénesis , Proteínas 14-3-3/genética , Perfilación de la Expresión Génica/métodos , Humanos , Osteogénesis/genética , Isomerasa de Peptidilprolil , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , Proteína de Unión a TATA-BoxRESUMEN
Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.
Asunto(s)
Nanopartículas , Corona de Proteínas , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nanopartículas/química , Nanopartículas/toxicidad , Corona de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Dióxido de Silicio/metabolismo , Dióxido de Silicio/toxicidad , Plata/metabolismo , Plata/toxicidadRESUMEN
The Wnt signalling pathway is one of the central signalling pathways in bone development, homeostasis and regulation of bone mineral density. It consists of numerous Wnt ligands, receptors and co-receptors, which ensure tight spatiotemporal regulation of Wnt signalling pathway activity and thus tight regulation of bone tissue homeostasis. This enables maintenance of optimal mineral density, tissue healing and adaptation to changes in bone loading. While the role of the canonical/ß-catenin Wnt signalling pathway in bone homeostasis is relatively well researched, Wnt ligands can also activate several non-canonical, ß-catenin independent signalling pathways with important effects on bone tissue. In this review, we will provide a thorough overview of the current knowledge on different non-canonical Wnt signalling pathways involved in bone biology, focusing especially on the pathways that affect bone cell differentiation, maturation and function, processes involved in bone tissue structure regulation. We will describe the role of the two most known non-canonical pathways (Wnt/planar cell polarity pathways and Wnt/Ca2+ pathway), as well as other signalling pathways with a strong role in bone biology that communicate with the Wnt signalling pathway through non-canonical Wnt signalling. Our goal is to bring additional attention to these still not well researched but important pathways in the regulation of bone biology in the hope of prompting additional research in the area of non-canonical Wnt signalling pathways.
Asunto(s)
Huesos/citología , Huesos/fisiología , Diferenciación Celular , Osteogénesis , Vía de Señalización Wnt , Animales , HumanosRESUMEN
Environmental or biomedical exposure to nanoparticles (NPs) can results in translocation and accumulation of NPs in the brain, which can lead to health-related problems. NPs have been shown to induce toxicity to neuronal cells through several direct mechanisms, but only a few studies have also explored the indirect effects of NPs, through consequences due to the exposure of neighboring cells to NPs. In this study, we analysed possible direct and indirect effects of NPs (polyacrylic acid (PAA) coated cobalt ferrite NP, TiO2 P25 and maghemite NPs) on immortalized mouse microglial cells and differentiated CAD mouse neuronal cells in monoculture (direct toxicity) or in transwell co-culture system (indirect toxicity). We showed that although the low NP concentrations (2-25 µg/mL) did not induce changes in cell viability, cytokine secretion or NF-κB activation of microglial cells, even low NP concentrations of 10 µg/mL can affect the cells and change their secretion of protein stress mediators. These can in turn influence neuronal cells in indirect exposure model. Indirect toxicity of NPs is an important and not adequately assessed mechanism of NP toxicity, since it not only affects cells on the exposure sites, but through secretion of signaling mediators, can also affect cells that do not come in direct contact with NPs.
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Supervivencia Celular/efectos de los fármacos , Microglía/efectos de los fármacos , Nanopartículas/toxicidad , Neuronas/efectos de los fármacos , Animales , Línea Celular , Citocinas/metabolismo , Ratones , Microglía/citología , Neuronas/citologíaRESUMEN
Many studies evaluated the short-term in vitro toxicity of nanoparticles (NPs); however, long-term effects are still not adequately understood. Here, we investigated the potential toxic effects of biomedical (polyacrylic acid and polyethylenimine coated magnetic NPs) and two industrial (SiO2 and TiO2) NPs following different short-term and long-term exposure protocols on two physiologically different in vitro models that are able to differentiate: L6 rat skeletal muscle cell line and biomimetic normal porcine urothelial (NPU) cells. We show that L6 cells are more sensitive to NP exposure then NPU cells. Transmission electron microscopy revealed an uptake of NPs into L6 cells but not NPU cells. In L6 cells, we obtained a dose-dependent reduction in cell viability and increased reactive oxygen species (ROS) formation after 24 h. Following continuous exposure, more stable TiO2 and polyacrylic acid (PAA) NPs increased levels of nuclear factor Nrf2 mRNA, suggesting an oxidative damage-associated response. Furthermore, internalized magnetic PAA and TiO2 NPs hindered the differentiation of L6 cells. We propose the use of L6 skeletal muscle cells and NPU cells as a novel approach for assessment of the potential long-term toxicity of relevant NPs that are found in the blood and/or can be secreted into the urine.
Asunto(s)
Nanopartículas/toxicidad , Pruebas de Toxicidad/métodos , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Células Musculares/metabolismo , Células Musculares/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Nanopartículas/química , Poliésteres/química , Ratas , Especies Reactivas de Oxígeno/metabolismo , Porcinos , Titanio/química , Urotelio/citologíaRESUMEN
The blood-urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of rhodamine B isothiocyanate-labelled polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.
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Endocitosis , Nanopartículas de Magnetita/química , Neoplasias de la Vejiga Urinaria/química , Urotelio/química , Resinas Acrílicas/química , Células Cultivadas , Cobalto/química , Compuestos Férricos/química , Humanos , Rodaminas/química , Neoplasias de la Vejiga Urinaria/patología , Urotelio/citologíaRESUMEN
Normal porcine urothelial cells have been shown to have a much lower rate of endocytosis than urothelial papillary neoplasm cells. This could be used as a mechanism for selective delivery of toxic compounds, such as polyethyleneimine coated nanoparticles (NPs). However, these NPs induce nonselective toxicity through direct membrane disruption. This toxicity can be reduced by functionalization of NPs with L-glutathione reduced or bovine serum albumin by reducing their surface charge. Functionalization was confirmed with Fourier Transform Infrared Spectroscopy, Dynamic Light Scattering and zeta potential measurements. Viability assays showed that bovine serum albumin coating reduced NPs cytotoxicity immediately after 3 h exposure and that such NPs were more toxic to urothelial papillary neoplasm cells compared to normal porcine urothelial cells at 50 µg/ml NPs concentration. However, 24 h after exposure, bovine serum albumin functionalized NPs had similar effect on viability of both cell lines. NPs showed some selective toxicity towards urothelial papillary neoplasm cells compared to normal cells after 3 h, however this was not confirmed after 24 h.
Asunto(s)
Nanopartículas de Magnetita/uso terapéutico , Polietileneimina , Neoplasias de la Vejiga Urinaria/terapia , Animales , Nanopartículas , Neoplasias , Porcinos , Células Tumorales Cultivadas , Vejiga UrinariaRESUMEN
Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.
Asunto(s)
Electroporación , Técnicas de Transferencia de Gen , Mioblastos/metabolismo , Transfección , Adolescente , Adulto , Supervivencia Celular , Células Cultivadas , Niño , Preescolar , Electroporación/métodos , Expresión Génica , Genes Reporteros , Humanos , Lactante , Lípidos , Cultivo Primario de Células , Transfección/métodos , Adulto JovenRESUMEN
Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.
Asunto(s)
Electroporación/métodos , Mioblastos/metabolismo , ARN Interferente Pequeño/genética , Transfección/métodos , Supervivencia Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mioblastos/citología , ARN Interferente Pequeño/metabolismoRESUMEN
Environmental exposure to nanoparticles (NPs) has significantly increased in the last decades, mostly due to increased environmental pollution and frequent use of NP containing consumer products. Such NPs may enter our body and cause various health-related problems. The brain is a particularly problematic accumulation site due to its physiological and anatomical restrictions. Several mechanisms of NP neurotoxicity have already been identified, however not enough is known especially regarding toxicity of engineered/industrial NPs. The focus of this in vitro study was on analysis of neurotoxicity of different engineered NPs, with which we come into contact in our daily lives; SiO2 NPs, food grade (FG) TiO2 NPs, TiO2 P25 and silver NPs as examples of industrial NPs, and polyacrylic acid (PAA) coated cobalt ferrite NPs as an example of biomedical NPs. All short term exposure experiments (24-72 h) were performed on SH-SY5Y human neuroblastoma cell line in vitro using higher (25-50 µg/ml) as well as lower (2-10 µg/ml), concentrations that are more relevant for in vivo NPs exposure. We show that NPs can cause neurotoxicity through different mechanisms, such as membrane damage, cell cycle interference, ROS formation and accumulation of autophagosomes, depending on their physico-chemical properties and stability in physiological media. Low, in vivo achievable concentrations of NPs induced only minor or no changes in vitro, however prolonged exposure and accumulation in vivo could negatively affect the cells. This was also shown in case of autophagy dysfunction for TiO2 P25 NPs and decrease of cell viability for TiO2 FG NPs, which were only evident after 72 h of incubation.
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Nanopartículas/toxicidad , Células-Madre Neurales/efectos de los fármacos , Resinas Acrílicas , Autofagosomas/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/patología , Supervivencia Celular , Cobalto , Compuestos Férricos , Alimentos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Síndromes de Neurotoxicidad/patología , Especies Reactivas de Oxígeno/metabolismo , Titanio/toxicidadRESUMEN
Stem cell-based therapeutics is a rapidly developing field associated with a number of clinical challenges. One such challenge lies in the implementation of methods to track stem cells and stem cell-derived cells in experimental animal models and in the living patient. Here, we provide an overview of cell tracking in the context of cardiac and neurological disease, focusing on the use of iron oxide-based particles (IOPs) visualized in vivo using magnetic resonance imaging (MRI). We discuss the types of IOPs available for such tracking, their advantages and limitations, approaches for labeling cells with IOPs, biological interactions and effects of IOPs at the molecular and cellular levels, and MRI-based and associated approaches for in vivo and histological visualization. We conclude with reviews of the literature on IOP-based cell tracking in cardiac and neurological disease, covering both preclinical and clinical studies.
Asunto(s)
Rastreo Celular , Compuestos Férricos/química , Cardiopatías/terapia , Imagen Molecular , Enfermedades del Sistema Nervioso/terapia , Células Madre/citología , Animales , HumanosRESUMEN
Polyethyleneimine (PEI) is a polycationic compound frequently used as a transfection agent. However, cytotoxicity of PEI and PEI-coated nanoparticles (PEI NPs) is still a major obstacle in its use. In this study we report a method for reducing cytotoxicity of PEI NPs by addition of glutathione in NPs synthesis. Glutathione reduced cytotoxic effects for at least 30% and decreased observed oxidative stress response compared to standard formulation. Results showed that the effect was partially due to reduced zeta potential and partially due to protective antioxidant properties of glutathione. Addition of glutathione to cell culture media with concurrent exposure to PEI NPs proved to be insufficient for cytotoxicity reduction. Additionally, we compared internalization pathways of both PEI NPs and GSH NPs. NPs were only found in endosomes and no NPs were found free in the cytosol, as would be expected according to so called proton sponge hypothesis.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Cobalto/toxicidad , Compuestos Férricos/toxicidad , Glutatión/metabolismo , Nanopartículas del Metal/toxicidad , Polietileneimina/toxicidad , Animales , Células CHO , Cricetulus , Fenómenos Magnéticos , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Increased environmental pollution has been suggested as one of the possible causes for increased incidence of neurodegenerative and developmental disorders. Through the environmental pollution, everyday consumer products and nanomedical applications, we are also exposed to various nanoparticles (NPs). Specific types of NPs have been shown to be able to cause neural damage in vivo through processes such as disruption of the blood-brain barrier, induction of neuroinflammation, increase in oxidative stress and protein aggregation. In this study, we analysed the influence of PEI-coated magnetic NPs designed for biotechnological applications and industrial SiO2, TiO2 N and TiO2 P25 NPs on intracellular localization and solubility of fused in farcoma (FUS) and TAR-DNA binding protein 43 (TDP-43) that are important pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). SH-SY5Y neuroblastoma cells and B16 mouse melanoma cells were exposed to NPs for 24 h and analysed using confocal microscopy and Western blot. Exposure to 50 µg/ml TiO2 N and 4 µg/ml PEI NPs in SH-SY5Y cells caused cell toxicity-induced changes in expression in different biochemical/cellular fractions for both FUS and TDP-43 proteins. TiO2 N induced a drop in nuclear levels of TDP-43 and increase in cytoplasmic levels of FUS, while PEI NPs increased nuclear levels of FUS. Furthermore, TiO2 N and PEI induced a reduction of FUS and TDP-43 quantity in the less soluble urea fraction. No formation of stress granules was observed. These results demonstrate that TiO2 N and PEI NPs can affect the behaviour of FUS and TDP-43 proteins; however, the changes were relatively minor compared to pathological changes even for the high NP concentrations (50 µg/ml) used in this study.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Espacio Intracelular/metabolismo , Nanopartículas , Proteína FUS de Unión a ARN/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Proteínas de Unión al ADN/química , Humanos , Espacio Intracelular/química , Ratones , Nanopartículas/química , Nanopartículas/toxicidad , Proteína FUS de Unión a ARN/química , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
Potential nanoparticle (NP) toxicity is one of crucial problems that limit the applicability of NPs. When designing NPs for biomedical and biotechnological applications it is thus important to understand the mechanisms of their toxicity. In this study, we analysed the stress responses of previously designed polyacrylic acid (PAA) and polyethylenimine (PEI) coated NPs on primary human myoblasts (MYO) and B16 mouse melanoma cell line. Negatively charged PAA did not induce cell toxicity, reactive oxygen species (ROS) or activate the transcription factor NF-κB in either cell line even at high concentrations (100µg/ml). On the other hand, positively charged PEI NPs induced a concentration dependent necrotic cell death and an increase in ROS following 24h incubation already at low concentrations (>4µg/ml). Moreover, PEI NPs induced NF-κB activation 15-30min after incubation in MYO cells, most probably through activation of TLR4 receptor. Interestingly, there was no NF-κB response to PEI NPs in B16 cells.
Asunto(s)
Cobalto/química , Cobalto/toxicidad , Compuestos Férricos/química , Compuestos Férricos/toxicidad , Mioblastos/efectos de los fármacos , Nanopartículas/química , Nanopartículas/toxicidad , Resinas Acrílicas/química , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Mioblastos/citología , Mioblastos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Polietileneimina/química , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo.
Asunto(s)
Resinas Acrílicas , Endocitosis/efectos de los fármacos , Nanopartículas de Magnetita , Resinas Acrílicas/química , Resinas Acrílicas/toxicidad , Animales , Células CHO , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cobalto/química , Cobalto/toxicidad , Cricetinae , Cricetulus , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidad , RatonesRESUMEN
In recent years, nanoparticles (NPs) and related applications have become an intensive area of research, especially in the biotechnological and biomedical fields, with magnetic NPs being one of the promising tools for tumor treatment and as MRI-contrast enhancers. Several internalization and cytotoxicity studies have been performed, but there are still many unanswered questions concerning NP interactions with cells and NP stability. In this study, we prepared functionalized magnetic NPs coated with polyacrylic acid, which were stable in physiological conditions and which were also nontoxic short-term. Using fluorescence, scanning, and transmission electron microscopy, we were able to observe and determine the internalization pathways of polyacrylic acid-coated NPs in Chinese hamster ovary cells. With scanning electron microscopy we captured what might be the first step of NPs internalization - an endocytic vesicle in the process of formation enclosing NPs bound to the membrane. With fluorescence microscopy we observed that NP aggregates were rapidly internalized, in a time-dependent manner, via macropinocytosis and clathrin-mediated endocytosis. Inside the cytoplasm, aggregated NPs were found enclosed in acidified vesicles accumulated in the perinuclear region 1 hour after exposure, where they stayed for up to 24 hours. High intracellular loading of NPs in the Chinese hamster ovary cells was obtained after 24 hours, with no observable toxic effects. Thus polyacrylic acid-coated NPs have potential for use in biotechnological and biomedical applications.