RESUMEN
Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.
Asunto(s)
Vesículas Extracelulares , Nanopartículas , Macrófagos/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas/metabolismo , Fagocitosis , Nanopartículas/toxicidadRESUMEN
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35−100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , ADN Tumoral Circulante , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Receptores ErbB/genética , Mutación , Inhibidores de Proteínas Quinasas , Oncogenes , Factor de Crecimiento Epidérmico/genética , Cromatografía en Gel , GenómicaRESUMEN
The design of targeted nanomedicines requires intracellular space- and time-resolved data of nanoparticle distribution following uptake. Current methods to study intracellular trafficking, such as dynamic colocalization by fluorescence microscopy in live cells, are usually low throughput and require extensive analysis of large datasets to quantify colocalization in several individual cells. Here a method based on flow cytometry to easily detect and characterize the organelles in which nanoparticles are internalized and trafficked over time is proposed. Conventional cell fractionation methods are combined with immunostaining and high-sensitivity organelle flow cytometry to get space-resolved data of nanoparticle intracellular distribution. By extracting the organelles at different times, time-resolved data of nanoparticle intracellular trafficking are obtained. The method is validated by determining how nanoparticle size affects the kinetics of arrival to the lysosomes. The results demonstrate that this method allows high-throughput analysis of nanoparticle uptake and intracellular trafficking by cells, therefore it can be used to determine how nanoparticle design affects their intracellular behavior.
Asunto(s)
Endocitosis , Nanopartículas , Citometría de Flujo , Nanomedicina , OrgánulosRESUMEN
Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.
Asunto(s)
Macrófagos/efectos de los fármacos , Nanopartículas/toxicidad , Poliestirenos/toxicidad , Dióxido de Silicio/toxicidad , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Células Cultivadas , Dispersión Dinámica de Luz , Estrés del Retículo Endoplásmico/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Lisosomas/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nanopartículas/química , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Poliestirenos/química , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Human hematopoietic progenitor cells (HPCs) are important for cell therapy in cancer and tissue regeneration. In vitro studies have shown a transient association of 40 nm polystyrene nanoparticles (PS NPs) with these cells, which is of interest for intelligent design and application of NPs in HPC-based regenerative protocols. In this study, we aimed to investigate the involvement of nanoparticles' size and membrane-attached glycan molecules in the interaction of HPCs with PS NPs, and compared it with monocytes. Human cord blood-derived HPCs and THP-1 cells were exposed to fluorescently labelled, carboxylated PS NPs of 40, 100 and 200 nm. Time-dependent nanoparticle membrane association and/or uptake was observed by measuring fluorescence intensity of exposed cells at short time intervals using flow cytometry. By pretreating the cells with neuraminidase, we studied the possible effect of membrane-associated sialic acids in the interaction with NPs. Confocal microscopy was used to visualize the cell-specific character of the NP association. RESULTS: Confocal images revealed that the majority of PS NPs was initially observed to be retained at the outer membrane of HPCs, while the same NPs showed immediate internalization by THP-1 monocytic cells. After prolonged exposure up to 4 h, PS NPs were also observed to enter the HPCs' intracellular compartment. Cell-specific time courses of NP association with HPCs and THP-1 cells remained persistent after cells were enzymatically treated with neuraminidase, but significantly increased levels of NP association could be observed, suggesting a role for membrane-associated sialic acids in this process. CONCLUSIONS: We conclude that the terminal membrane-associated sialic acids contribute to the NP retention at the outer cell membrane of HPCs. This retention behavior is a unique characteristic of the HPCs and is independent of NP size.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Monocitos/metabolismo , Nanopartículas/química , Ácidos Siálicos/química , Antígenos CD34/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/metabolismo , Endocitosis/efectos de los fármacos , Humanos , Tamaño de la Partícula , Poliestirenos , Propiedades de SuperficieRESUMEN
Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.
Asunto(s)
Células Epiteliales/metabolismo , Pulmón/metabolismo , Nanopartículas , Poliestirenos , Línea Celular Tumoral , Células Epiteliales/citología , Humanos , Pulmón/citología , Análisis EspectralRESUMEN
PVP-Hypericin (PVP: polyvinylpyrrolidone) is a potent anti-cancer photosensitizer for photodynamic diagnosis (PDD) and therapy (PDT). However, cellular targets and mechanisms involved in the cancer-selectivity of the photosensitizer are not yet fully understood. This paper gives new insights into the differential transport and localization of PVP-Hypericin in cancer and normal cells which are essential to unravel the mechanisms of action and cancer-selectivity. Temporal (TICS) and spatiotemporal (STICS) image correlation spectroscopy are used for the assessment of PVP-Hypericin diffusion and/or velocity in the case of concerted flow in human cervical epithelial HeLa and human lung carcinoma A549 cells, as well as in human primary dendritic cells (DC) and human peripheral blood mononuclear cells (PBMC). Spatiotemporal image cross-correlation spectroscopy (STICCS) based on organelle specific fluorescent labeling is employed to study the accumulation of the photosensitizer in nucleus, mitochondria, early-endosomes and lysosomes of the cells and to assess the dynamics of co-migrating molecules. Whereas STICS and TICS did not show a remarkable difference between the dynamics of PVP-Hypericin in HeLa, A549 and DC cells, a significantly different diffusion rate of the photosensitizer was measured in PBMC. STICCS detected a stationary accumulation of PVP-Hypericin within the nucleus, mitochondria, early endosomes and lysosomes of HeLa and A549 cells. However, significant flow due to the directed motion of the organelles was detected. In contrast, no accumulation in the nucleus and mitochondria of DC and PBMC could be monitored.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/farmacocinética , Povidona/farmacocinética , Análisis Espectral/métodos , Antracenos , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Perileno/farmacocinéticaRESUMEN
The identification of the molecular composition of extracellular vesicles (EV) by omics approaches, including proteomics, requires the separation of EV from non-EV confounding factors present in the source biofluid. In this protocol, we present the sequential implementation of density gradient ultracentrifugation and size-exclusion chromatography to prepare EV from cell-conditioned medium with high specificity and repeatability. This approach enables the recovery of intact purified EV suited for downstream functional assays and biomarker discovery by omics approaches.
Asunto(s)
Técnicas Citológicas , Vesículas Extracelulares , Vesículas Extracelulares/química , Fraccionamiento Celular , Medios de Cultivo Condicionados , Humanos , Técnicas Citológicas/métodos , Proteómica , Centrifugación por Gradiente de Densidad , Cromatografía en GelRESUMEN
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.
Asunto(s)
Vesículas Extracelulares/metabolismo , Citometría de Flujo/métodos , Línea Celular , Humanos , Manejo de Especímenes , UltracentrifugaciónRESUMEN
Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.
Asunto(s)
Pulpa Dental/citología , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Adolescente , Inductores de la Angiogénesis/metabolismo , Animales , Factores Quimiotácticos/farmacología , Pollos , Endocitosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/ultraestructura , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/ultraestructura , Neovascularización Fisiológica/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Factores de Tiempo , Adulto JovenRESUMEN
Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.
Asunto(s)
Vesículas Extracelulares/metabolismo , Nanopartículas del Metal/química , Animales , Humanos , Ratones , RatasRESUMEN
OBJECTIVES: In this study, we investigate in human cervical epithelial HeLa cells the intracellular dynamics and the mutual interaction with the organelles of the poly-l-lactic acid nanoparticles (PLLA NPs) carrying the naturally occurring hydrophobic photosensitizer hypericin. METHODS: Temporal and spatiotemporal image correlation spectroscopy was used for the assessment of the intracellular diffusion and directed motion of the nanocarriers by tracking the hypericin fluorescence. Using image cross-correlation spectroscopy and specific fluorescent labelling of endosomes, lysosomes and mitochondria, the NPs dynamics in association with the cell organelles was studied. Static colocalization experiments were interpreted according to the Manders' overlap coefficient. KEY FINDINGS: Nanoparticles associate with a small fraction of the whole-organelle population. The organelles moving with NPs exhibit higher directed motion compared to those moving without them. The rate of the directed motion drops substantially after the application of nocodazole. The random component of the organelle motions is not influenced by the NPs. CONCLUSIONS: Image correlation and cross-correlation spectroscopy are most appropriate to unravel the motion of the PLLA nanocarrier and to demonstrate that the rate of the directed motion of organelles is influenced by their interaction with the nanocarriers. Not all PLLA-hypericin NPs are associated with organelles.
Asunto(s)
Nanopartículas , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/administración & dosificación , Poliésteres/química , Antracenos , Difusión , Portadores de Fármacos/química , Endosomas/metabolismo , Fluorescencia , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lisosomas/metabolismo , Mitocondrias/metabolismo , Orgánulos/metabolismo , Perileno/administración & dosificación , Perileno/química , Perileno/metabolismo , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Análisis EspectralRESUMEN
Nanosized objects, such as nanoparticles and other drug carriers used in nanomedicine, once in contact with biological environments are modified by adsorption of biomolecules on their surface. The presence of this corona strongly affects the following interactions at cell and organism levels. It has been shown that corona proteins can be recognized by cell receptors. However, it is not known whether the composition of this acquired layer can also affect the mechanisms nanoparticles use to enter cells. This is of particular importance when considering that the same nanoparticles can form different coronas for instance in vitro when exposed to cells in different serum amounts or in vivo depending on the exposure or administration route. Thus, in this work, different coronas were formed on 50 nm silica by exposing them to different serum concentrations. The uptake efficiency in HeLa cells was compared, and the uptake mechanisms were characterized using transport inhibitors and RNA interference. The results showed that the nanoparticles were internalized by cells via different mechanisms when different coronas were formed, and only for one corona condition was uptake mediated by the LDL receptor. This suggested that coronas of different composition can be recognized differently by cell receptors, and this in turn leads to internalization via different mechanisms. Similar studies were performed using other cells, including A549 cells and primary HUVEC, and different nanoparticles, namely 100 nm liposomes and 200 nm silica. Overall, the results confirmed that the corona composition can affect the mechanisms of nanoparticle uptake by cells.
Asunto(s)
Portadores de Fármacos/farmacología , Nanomedicina , Nanopartículas/química , Corona de Proteínas/química , Adsorción/efectos de los fármacos , Vías de Administración de Medicamentos , Portadores de Fármacos/química , Células HeLa , Humanos , Liposomas/química , Liposomas/farmacología , Nanopartículas/uso terapéutico , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
CD34+ hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon.
Asunto(s)
Antígenos CD34/metabolismo , Células Madre Hematopoyéticas/citología , Nanopartículas/química , Poliestirenos/química , Proteínas Bacterianas/química , Muerte Celular , Proliferación Celular , Células Cultivadas , Proteínas Fluorescentes Verdes/química , Células Madre Hematopoyéticas/metabolismo , Humanos , Recién Nacido , Cinética , Proteínas Luminiscentes/química , Modelos BiológicosRESUMEN
Despite many investigations have focused on the pristine toxicity of gold nanoparticles (GNPs), little is known about the outcome of co-exposure and interaction of GNPs with heavy metals which can possibly detoxify or potentiate them. Here, the combined exposure of nickel (II) sulfate (NiSO4) and GNPs on the maturation response of dendritic cells (DCs) was explored. Exposure to GNPs or NiSO4 separately induced cell activation. When cells were exposed to a mixture of both, however, the observed cell activation pattern indicated a competitive rather than an additive effect of both inducers with levels similar to those induced by NiSO4 alone. Quantification of the GNP uptake by DCs demonstrated a significant decrease in intracellular gold content during co-incubation with NiSO4. An extensive physiochemical characterization was performed to determine the interaction between GNPs and NiSO4 in the complex physiological media using nanoparticle tracking analyses, disc centrifugation, UV-visible spectroscopy, ICP-MS analyses, zeta potential measurements, electron microscopy, and proteomics. Although GNPs and NiSO4 did not directly interact with each other, the presence of NiSO4 in the physiological media resulted in changes in GNPs' charge and their associated protein corona (content and composition), which may contribute to a decreased cellular uptake of GNPs and sustaining the nickel-induced DC maturation. The presented results provide new insights in the interaction of heavy metals and NPs in complex physiological media. Moreover, this study highlights the necessity of mixture toxicology, since these combined exposures are highly relevant for human subjection to NPs and risk assessment of nanomaterials.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Oro/toxicidad , Nanopartículas del Metal/toxicidad , Níquel/toxicidad , Antígenos de Superficie/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interacciones Farmacológicas , Endocitosis/efectos de los fármacos , Citometría de Flujo , Oro/química , Oro/metabolismo , Humanos , Nanopartículas del Metal/química , Níquel/química , Corona de Proteínas/metabolismo , Propiedades de SuperficieRESUMEN
The study of cell-nanoparticle interactions is an important aspect for understanding drug delivery using nanocarriers. In this regard, advances in fluorescence based microscopy are useful for the investigation of temporal and spatial behavior of nanoparticles (NPs) within the intracellular environment. In this work, we focus on the delivery of the naturally-occurring hydrophobic photosensitizer Hypericin in human lung carcinoma A549 cells by using biodegradable poly L-lactic acid NPs. For the first time, Hypericin containing NPs are prepared by combining the miniemulsion technique with the solvent evaporation method. This approach yields an efficient loading of the NPs with Hypericin and allows for additional cargo molecules. To monitor the release of Hypercin from the NPs, an additional fluorescent lipophilic dye Coumarin-6 is incorporated in the NPs. Temporal and spatiotemporal image correlation spectroscopy is used to determine the fate of the NPs carrying the potential cargo. Both directed and non-directed motions are detected. By using image cross-correlation spectroscopy and specific fluorescent labeling of endosomes, lysosomes and mitochondria, the dynamics of the cargo loaded NPs in association with the organelles is studied.
Asunto(s)
Cumarinas/administración & dosificación , Portadores de Fármacos/administración & dosificación , Colorantes Fluorescentes/administración & dosificación , Nanopartículas/administración & dosificación , Perileno/análogos & derivados , Tiazoles/administración & dosificación , Antracenos , Transporte Biológico , Línea Celular Tumoral , Cumarinas/química , Portadores de Fármacos/química , Colorantes Fluorescentes/química , Humanos , Ácido Láctico/química , Nanopartículas/química , Perileno/administración & dosificación , Perileno/química , Poliésteres , Polímeros/química , Análisis Espectral , Tiazoles/químicaRESUMEN
In a rat model of global fetal and perinatal asphyxia, we investigated if asphyxia and long-lasting brain tolerance to asphyxia (preconditioning) are mediated by modifications in inflammatory cytokines and ceramide metabolism genes in prefrontal cortex, hippocampus and caudate-putamen at the age of 8months. Most significant changes were found in prefrontal cortex, with reduced LAG1 homolog ceramide synthase 1 expression after both types of asphyxia. Additionally, sphingosine kinase 1 was upregulated in those animals that experienced the combination of fetal and perinatal asphyxia (preconditioning), suggesting increased cell proliferation. While cytokine levels are normal, levels of ceramide genes were modulated both after fetal and perinatal asphyxia in the adult prefrontal cortex. Moreover, the combination of two subsequent asphyctic insults provides long-lasting neuroprotection in the prefrontal cortex probably by maintaining normal apoptosis and promoting cell proliferation. Better understanding of the effects of asphyxia on ceramide metabolism will help to understand the changes leading to brain tolerance and will open opportunities for the development of new neuroprotective therapies.