Labeling nanoparticles: Dye leakage and altered cellular uptake (original) (raw)
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Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use
PLoS ONE, 2011
Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm.
Dual-Labelled Nanoparticles Inform on the Stability of Fluorescent Labels In Vivo
Pharmaceutics
Fluorescent labelling is commonly used to monitor the biodistribution of nanomedicines. However, meaningful interpretation of the results requires that the fluorescent label remains attached to the nanomedicine. In this work, we explore the stability of three fluorophores (BODIPY650, Cyanine 5 and AZ647) attached to polymeric hydrophobic biodegradable anchors. Using dual-labelled poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) nanoparticles that are both radioactive and fluorescent, we investigated how the properties of the fluorophores impact the stability of the labelling in vitro and in vivo. Results suggest that the more hydrophilic dye (AZ647) is released faster from nanoparticles, and that this instability results in misinterpretation of in vivo data. While hydrophobic dyes are likely more suitable to track nanoparticles in biological environments, quenching of the fluorescence inside the nanoparticles can also introduce artefacts. Altogether, this work raises awareness ab...
Comparative biodistribution in mice of cyanine dyes loaded in lipid nanoparticles
Two near infrared cyanine dyes, DiD (1,1 0-dioctadecyl-3,3,3 0 ,3 0-tetramethylindotricarbocyanine perchlo-rate) and ICG (Indocyanine Green) were loaded in lipid nanoparticles (LNP). DiD-LNP and ICG-LNP presented similar physicochemical characteristics (hydrodynamic diameter, polydispersity, zeta potential), encapsulation efficiency, and colloidal stability when stored in PBS buffer. However, whereas DiD had similar biodistribution than cholesteryl-1-14 C-oleate ([ 14 C]CHO, a constituent of the nanoparticle used as a reference radiotracer), ICG displayed a different biodistribution pattern, similar to that of the free dye, indicative of its immediate leakage from the nanovector after blood injection. NMR spectroscopy using Proton NOE (Nuclear Overhauser Effect) measurements showed that the localization of the dye in the lipid nanoparticles was slightly different: ICG, more amphiphilic than DiD, was found both inside the lipid core and at particle interface, whereas DiD, more hydrophobic, appeared exclusively located inside the particle core. The ICG release rate from the particles was 7% per 1 month under storage conditions (4 °C, dark, 10% of lipids), whereas no leakage could be detected for DiD. ICG leakage increased considerably in the presence of BSA 40 g/L (45% leakage in 24 h at 100 mg/mL of lipids), because of the high affinity of the fluorophore for plasma proteins. On the contrary, no DiD leakage was observed, until high dilution of the nanoparticles which triggered their dissociation (45% leakage in 24 h at 1 mg/mL of lipids). Altogether, the subtle difference in dye localization into the nanoparticles, the partial dissociation of the LNP in diluted media, and more importantly the high ICG affinity for plasma proteins, accounted for the differences observed in the fluorescence biodistribution after tail vein injection of the dye-loaded nanoparticles.
An Overview of Nanoparticles Commonly Used in Fluorescent Bioimaging
The article gives an overview on the various kinds of nanoparticles (NPs) that are widely used for purposes of fluorescent imaging, mainly of cells and tissue. Following an introduction and a discussion of merits of fluorescent NPs compared to molecular fluorophores, labels and probes, the article assesses the kinds and specific features of nanomaterials often used in bioimaging. These include fluorescently doped silicas and sol-gels, hydrophilic polymers (hydrogels), hydrophobic organic polymers, semiconducting polymer dots, quantum dots, carbon dots, other carbonaceous nanomaterials, upconversion NPs, noble metal NPs (mainly gold and silver), various others nanomaterials, and dendrimers. Another section covers coatings and methods for surface modification of NPs. Specific examples on the use of nanoparticles in (a) plain fluorescence imaging of cells, (b) targeted imaging, (c) imaging of chemical species, and (d) imaging of temperature are given next. A final section covers aspects of multimodal imaging (for example fluorescence/nmr), imaging combined with drug and gene delivery, or imaging combined with therapy or diagnosis. A Supporting Information gives specific examples for materials and methods used in imaging, sensing, multimodal imaging and theranostics such as imaging combined with drug delivery or photodynamic therapy. The article contains 270 references in the main part, and 157 references in the Supporting Information.
The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions?
Nanotechnology, Science and Applications, 2012
Nanoparticles (NPs) are materials with overall dimensions in the nanoscale range. They have unique physicochemical properties, and have emerged as important players in current research in modern medicine. In the last few decades, several types of NPs and microparticles have been synthesized and proposed for use as contrast agents for diagnostics and imaging and for drug delivery; for example, in cancer therapy. Yet specific targeting that will improve their delivery still represents an unsolved challenge. The mechanism by which NPs enter the cell has important implications not only for their fate but also for their impact on biological systems. Several papers in the literature discuss the potential risks related to NP exposure, and more recently the concept that even sublethal doses of NPs may elicit a cell response has been proposed. In this review, we intend to present an overall view of cell mechanisms that may be perturbed by cell-NP interaction. Published data, in fact, emphasize that NPs should no longer be viewed only as simple carriers for biomedical applications, but that they can also play an active role in mediating biological effects.
This article reviews the applications of nanotechnology in the fields of medical and life sciences. Nanoparticles have shown promising applications from diagnosis to treatment of various types of diseases including cancer. In this review, we discuss the applications of nanostructured materials such as nanoparticles, quantum dots, nanorods, nanowires, and carbon nanotubes in diagnostics, biomarkers, cell labeling, contrast agents for biological imaging, antimicrobial agents, drug delivery systems, and anticancer nanodrugs for treatment of cancer and other infectious diseases. The adverse affects of nanoparticles on human skin from daily use in cosmetics and general toxicology of nanoscale materials are also reviewed. KEYWORDS: Medical Applications, Nanoparticles, Contrast Agents for Biological Imaging, Cell Labeling, Antimicrobial Agents, Anticancer Nanodrugs, Cosmetics, Nanotoxicology.
Nanoparticles: Cellular Uptake and Cytotoxicity
Understanding the interactions of nanoparti-cles (NPs) with cells and how these interactions infl uence their cellular uptake is essential to exploring the biomedical applications of NPs, particularly for drug delivery. Various factors, whether differences in physical properties of NPs or variations in cell-membrane characteristics, infl uence NP-cell interactions and uptake processes. NP-cell membrane interactions may also infl uence intracellular traffi cking of NPs, their sorting into different intracellular compartments, cellular retention, and hence the effi cacy of encapsulated thera-peutics. A crucial consideration is whether such interactions might cause any toxicity,
Recent advances in the use of fluorescent nanoparticles for bioimaging
Nanomedicine, 2019
Rapid and recent progress in fluorescence microscopic techniques has allowed for routine discovery and viewing of biological structures and processes in unprecedented spatiotemporal resolution. In these imaging techniques, fluorescent nanoparticles (NPs) play important roles in the improvement of reporting systems. A short overview of recently developed fluorescent NPs used for advanced in vivo imaging will be discussed in this mini-review. The discussion begins with the contribution of fluorescence imaging in exploring the fate of NPs in biological systems. NP applications for in vivo imaging, including cell labeling, multimodal imaging and theranostic agents, are then discussed. Finally, despite all of the advancements in bioimaging, some unsolved challenges will be briefly discussed concerning future research directions.
Materials Today Communications, 2020
The utilization of nanomaterials in the biological and medical field is quickly progressing, particularly in areas where traditional diagnostics and treatment approaches have limited success. The success of nanomaterials in medical products such as biomedical implants, wound dressings and drug delivery systems rely upon their effective interaction between the extracellular matrix, cells, and intracellular components. Upon contact with mammalian cells, nanoparticles (NPs) begin to interact with the extracellular matrix, cell membrane, cytoplasmic proteins, nucleus, and other cellular organelles, which result in nanoparticle internalization and subsequent cellular responses. Such responses elicited by the mammalian cells as a result of the cell-nanomaterials interactions, both at the cellular and molecular level, are mainly determined by the morphological, chemical, and surface characteristics of the nanomaterials themselves. This review provides an overview of how such different attributes, such as chemical nature, size, shape, surface charge, topography, stiffness, and functional features of nanomaterials, influence the cell-nanomaterials interactions.
Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles
Biomacromolecules, 2010
Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.
Nanoparticles: From Diagnosis to Therapy
International Journal of Medical Nano Research, 2016
Cancer is a leading cause of death, the second most common cause, exceeded only by heart disease. Still, the current clinical imaging methods and treatments are in many situations unable to provide timely detection and curative therapy. The field of drug delivery stands to be significant advances in nanotechnology and benefits of novel nanotechnology in oncology already starts. New strategies are being designed to deliver chemotherapeutic drugs or imaging agents to the tumor at higher concentrations with minimal damage to normal tissues. This review will focus on how nanoparticles are able to function as carriers for chemotherapeutic drugs to increase their therapeutic index; how can be used as imaging agents to detect and monitor cancer progression.
Nanomaterials: Impact on Cells and Cell Organelles
Nanomaterial, 2014
Colloidal nanoparticles designed for the interactions with cells are very small, nanoscale objects usually consisting of inorganic cores and organic shells that are dispersed in a buffer or biological medium. Tuning the material properties the nanoparticles are made from, a number of different properties that can be exploited in specific biological applications are possible, i.e. targeting, labelling, drug delivery, use as diagnostic tools or therapy. For all biological applications of nanoparticles, it is important to understand their interactions with the surrounding biological environment in order to predict their biological impact, in particular when designing the nanoparticles for diagnostic and therapeutic purpose. Due to the high surface-to-volume ratio, the surface of nanomaterials is very reactive. When exposed to biological fluids, the proteins and biomolecules present therein tend to associate with the nanoparticles' surfaces. This phenomenon is defined as biomolecular corona formation. The biomolecular corona plays a key role in the interaction between nanoparticles and biological systems, impacting on how these particles interact with biological systems on a cellular and molecular level. This book chapter describes the nature of the interactions at the bio-nano interface, shows the design strategy of nanoparticles for nanomedicine, and defines the concepts of biomolecular corona and biological identity of nanoparticles. Moreover, it describes the interaction of functionalised nanomaterials with cell organelles and intracellular fate of nanoparticles and it shows therapeutic application of gold nanoparticles as dose enhancers in radiotherapy.
Insights into Cellular Uptake of Nanoparticles
Current Drug Delivery, 2015
Nanomaterials promise to improve disease diagnosis and treatment by enhancing the delivery of drugs, genes, biomolecules and imaging agents to specific subcellular targets. In order to optimize nanomaterial design for this purpose, a comprehensive understanding of how these materials are taken up and transported within the cell is required. In this review, we discuss the endocytic pathways employed by different types of nanoparticles with emphasis on the influence of nanoparticle surface modification. The use of pharmacological inhibition to probe internalization and intracellular trafficking pathways of nanoparticles is critically evaluated. Finally, approaches to target-specific delivery of therapeutics via nanoparticles into the cytoplasm and nucleus are addressed.
Toxicology, 2017
At many portals of entry the relative uptake by phagocytes and non-phagocytic cells has a prominent effect on availability and biological action of nanoparticles (NPs). Cellular uptake can be determined for fluorescence-labeled NPs. The present study compares three methods (plate reader, flow cytometry and image analysis) in order to investigate the influence of particle size and functionalization and medium content on cellular uptake of fluorescence-labeled polystyrene particles and to study the respective method's suitability for uptake studies. For comparison between the techniques, ratios of macrophage to alveolar epithelial cell uptakes were used. Presence of serum protein in the exposure solution decreased uptake of carboxyl-functionalized and non-functionalized particles; there was no clear effect for the amine-functionalized particles. The 200 nm non-or carboxyl-functionalized NPs were taken up preferentially by phagocytes while for amine-functionalized particles preference was lowest. The presence of the serum slightly increased the preference for these particles. In conclusion, due to the possibility of calibration, plate reader measurements might present a better option than the other techniques to (semi)quantify differences between phagocytes and nonphagocytic cells for particles with different fluorescence. In order to obtain unbiased data the fluorescent labeling has to fulfill certain requirements.
Cellular interactions of therapeutically delivered nanoparticles
Expert Opinion on Drug Delivery, 2011
Introduction: Nanoparticles (NPs) are used extensively in drug delivery. They are administered through various routes in the host, and their uptake by the cellular environment has been observed in several pathways. After uptake, NPs interact with cells to different extents, depending on their size, shape, surface properties, ligands tagged to the surface and tumor architecture. Complete understanding of such cellular uptake mechanisms and interactions of NPs is important for their effective use in drug delivery. Areas covered: This article describes the various cellular pathways for NP uptake, and the factors affecting NP uptake and interactions with cells. Understanding these two important aspects will help in the future design of NPs for effective and targeted drug delivery. Expert opinion: Surface charge and ligands tagged on the surface of NPs play a critical role in their uptake and interaction with cells; so surface modifications of NPs can offer increased drug delivery effectiveness, for example, the coupling of ligands on the surface of NPs can increase cellular binding, and NPs in biological fluids can be coated with proteins and as such can exert biological effects. All of the factors affecting NP uptake need to be investigated thoroughly before interpreting any NP-cellular interactions.
Pharmaceutics, 2021
Fluorescently labeled nanoparticles are widely used for evaluating their distribution in the biological environment. However, dye leakage can lead to misinterpretations of the nanoparticles’ biodistribution. To better understand the interactions of dyes and nanoparticles and their biological environment, we explored PLGA nanoparticles labeled with four widely used dyes encapsulated (coumarin 6, rhodamine 123, DiI) or bound covalently to the polymer (Cy5.5.). The DiI label was stable in both aqueous and lipophilic environments, whereas the quick release of coumarin 6 was observed in model media containing albumin (42%) or liposomes (62%), which could be explained by the different affinity of these dyes to the polymer and lipophilic structures and which we also confirmed by computationalmodeling (log PDPPC/PLGA: DiI—2.3, Cou6—0.7). The importance of these factors was demonstrated by in vivo neuroimaging (ICON) of the rat retina using double-labeled Cy5.5/Cou6-nanoparticles: encapsulated Cou6 quickly leaked into the tissue, whereas the stably bound Cy.5.5 label remained associated with the vessels. This observation is a good example of the possiblemisinterpretation of imaging results because the coumarin 6 distribution creates the impression that nanoparticles effectively crossed the blood–retina barrier, whereas in fact no signal fromthe corematerial was found beyond the blood vessels.
The impact of nanoparticles on cellular functions
epub.oeaw.ac.at
Nanotoxicology deals with the effects of nanomaterials on cells and living organ-isms. Few toxicological data, however, are available for most nano-objects. There is general agreement that the surface struc-ture of nanoparticles plays a key role in the interaction with cells and therefore ...
Cellular uptake of nanoparticles: journey inside the cell
Chemical Society reviews, 2017
Nanoscale materials are increasingly found in consumer goods, electronics, and pharmaceuticals. While these particles interact with the body in myriad ways, their beneficial and/or deleterious effects ultimately arise from interactions at the cellular and subcellular level. Nanoparticles (NPs) can modulate cell fate, induce or prevent mutations, initiate cell-cell communication, and modulate cell structure in a manner dictated largely by phenomena at the nano-bio interface. Recent advances in chemical synthesis have yielded new nanoscale materials with precisely defined biochemical features, and emerging analytical techniques have shed light on nuanced and context-dependent nano-bio interactions within cells. In this review, we provide an objective and comprehensive account of our current understanding of the cellular uptake of NPs and the underlying parameters controlling the nano-cellular interactions, along with the available analytical techniques to follow and track these proces...
Journal of Nanobiotechnology, 2012
Background: Nanoparticles (NPs) are currently used in a wide variety of fields such as technology, medicine and industry. Due to the novelty of these applications and to ensure their success, a precise characterization of the interactions between NPs and cells is essential. Findings: The current study explores the uptake of polystyrene NPs by 1321N1 human astrocytoma and A549 human lung carcinoma cell lines. In this work we show for the first time a comparison of the uptake rates of fluorescently labeled carboxylated polystyrene (PS) NPs of different sizes (20, 40 and 100 nm) in two different cell types, keeping the number of NPs per unit volume constant for all sizes. We propose a reliable methodology to control the dose of fluorescently labeled NPs, by counting individual NPs using automated particle detection from 3D confocal microscopy images. The possibility of detecting individual NPs also allowed us to calculate the size of each nanoparticle and compare the fluorescence of single NPs across different sizes, thereby providing a robust platform for normalization of NP internalization experiments as measured by flow cytometry. Conclusions: Our findings show that 40 nm NPs are internalized faster than 20 nm or 100 nm particles in both cell lines studied, suggesting that there is a privileged size gap in which the internalization of NPs is higher.
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Following the Fate of Dye-Containing Liposomes In Vitro
International Journal of Molecular Sciences
The rather limited success of translation from basic research to clinical application has been highlighted as a major issue in the nanomedicine field. To identify the factors influencing the applicability of nanosystems as drug carriers and potential nanomedicine, we focused on following their fate through fluorescence-based assays, namely flow cytometry and imaging. These methods are often used to follow the nanocarrier internalization and targeting; however, the validity of the obtained results strictly depends on how much the nanosystem’s fate can be inferred from the fate of fluorescent dyes. To evaluate the parameters that affect the physicochemical and biological stability of the labeled nanosystems, we studied the versatility of two lipid dyes, TopFluor®-PC and Cy5-DSPE, in conventional liposomes utilizing well-defined in vitro assays. Our results suggest that the dye can affect the major characteristics of the system, such as vesicle size and zeta-potential. However, a nanoc...
Cytotoxicity of Poly(Alkyl Cyanoacrylate) Nanoparticles
International journal of molecular sciences, 2017
Although nanotoxicology has become a large research field, assessment of cytotoxicity is often reduced to analysis of one cell line only. Cytotoxicity of nanoparticles is complex and should, preferentially, be evaluated in several cell lines with different methods and on multiple nanoparticle batches. Here we report the toxicity of poly(alkyl cyanoacrylate) nanoparticles in 12 different cell lines after synthesizing and analyzing 19 different nanoparticle batches and report that large variations were obtained when using different cell lines or various toxicity assays. Surprisingly, we found that nanoparticles with intermediate degradation rates were less toxic than particles that were degraded faster or more slowly in a cell-free system. The toxicity did not vary significantly with either the three different combinations of polyethylene glycol surfactants or with particle size (range 100-200 nm). No acute pro- or anti-inflammatory activity on cells in whole blood was observed.
Epigenetics in Breast Cancer Therapy—New Strategies and Future Nanomedicine Perspectives
Cancers
Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identif...
Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)
2021
The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QP...
2021
We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (an...
Artificial Cells, Nanomedicine, and Biotechnology, 2017
In this study, it was aimed to investigate characteristics and intracellular delivery of two different-sized PLGA nanoparticles in ouzo region by considering number of nanoparticles. To determine the effect of formulation parameters on average particle size, Dil labeled nanoparticles were prepared using a threefactor, two-level full factorial statistical experimental design. PLGA 230 (230.8 ± 4.32 nm) and PLGA 160 (157.9 ± 6.16 nm) nanoparticles were obtained by altering polymer amount based on experimental design results and characterized. Same number of PLGA 230 and PLGA 160 nanoparticles per cell were applied onto HEK293 cells; then, cytotoxicity, uptake kinetics and mechanism were evaluated by flow cytometry and fluorescent microscopy. Also same weight of PLGA 230 and PLGA 160 nanoparticles were applied and cellular uptake of these nanoparticles was evaluated. It was found that PLGA 230 nanoparticles had higher encapsulation efficiency and slower dye release compared to PLGA 160 nanoparticles. When they were applied at same counts per cell, PLGA 230 nanoparticles displayed faster and higher intracellular dye transfer than PLGA 160 nanoparticles. On the other hand, PLGA 160 appeared to be a more effective vehicle than PLGA 230 when applied at the same weight concentration. It was also shown that for both nanoparticles, HEK293 cells employed macropinocytic, caveolae-and clathrin-mediated endocytic pathways.
Drug Delivery and Translational Research
The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF–SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF–SIMS, respective...
A comparative biodistribution study of polymeric and lipid-based nanoparticles
Drug Delivery and Translational Research
Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection. The studied nanoformulations were nanostructured lipid carriers incorporating the fluorescent dye IR780-oleyl, and polymeric nanoparticles containing the anticancer agent cabazitaxel. The biodistribution was approximated by quantification of the cargo in blood and relevant organs. Several clear and systematic differences in biodistribution were observed, with the most pronounced being a much higher (more than 50-fold)...
Journal of Colloid and Interface Science, 2021
Designing zeolites for medical applications is a challenging task requiring the introduction of new functionalities without altering their intrinsic properties such as morphology, crystallinity, colloidal stability, surface charge, and porosity. Herein, we present the encapsulation of luminescent ruthenium-tris(2,2′-bipyridyl) complex in FAU zeolite nanocrystals (Ru(bpy)3-FAU) and their use as an intracellular localization tracer. Upon exciting of the Ru(bpy)3-FAU zeolite at 450 nm, the sample gives rise to an orange-red emission at 628 nm thus permitting its use for cellular imaging and localization of the zeolite nanoparticles. The nanosized Ru(bpy)3-FAU zeolite is characterized in terms of size, charge, crystallinity, morphology, porosity, thermal stability, and sorption capacity. The potential toxicity of Ru(bpy)3-FAU on U251-MG 2 glioblastoma cells was evaluated. A safe concentration (50-100 µg/ml) for the Ru(bpy)3-FAU zeolite is identified. The luminescent properties of the ruthenium complex confined in the zeolite nanocrystals allow to detect their localization in the U251-MG cells with a main accumulation in the cytoplasm. The Ru(bpy)3-FAU nanosized zeolite is a potential candidate for biological applications for being stable, safe, capable of loading respiratory gases, and easily probed in the cells owing to its luminescent properties.
Dual-Labelled Nanoparticles Inform on the Stability of Fluorescent Labels In Vivo
Pharmaceutics
Fluorescent labelling is commonly used to monitor the biodistribution of nanomedicines. However, meaningful interpretation of the results requires that the fluorescent label remains attached to the nanomedicine. In this work, we explore the stability of three fluorophores (BODIPY650, Cyanine 5 and AZ647) attached to polymeric hydrophobic biodegradable anchors. Using dual-labelled poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) nanoparticles that are both radioactive and fluorescent, we investigated how the properties of the fluorophores impact the stability of the labelling in vitro and in vivo. Results suggest that the more hydrophilic dye (AZ647) is released faster from nanoparticles, and that this instability results in misinterpretation of in vivo data. While hydrophobic dyes are likely more suitable to track nanoparticles in biological environments, quenching of the fluorescence inside the nanoparticles can also introduce artefacts. Altogether, this work raises awareness ab...
Applied Microbiology and Biotechnology
Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phago...
International Journal of Pharmaceutics, 2021
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Scientific Reports, 2019
Galbanic acid (Gba), asesquiterpene coumarin, with strong antiangiogenic activity could serve as an excellent anti-cancer agent. However, Gba is a poor water-solube which hampered its clinical application. In this study, a pegylated liposomal Gba (PLGba) with HSPC/Cholesterol/mPEG2000-DSPE (56.2, 38.3, 5.3% molar ratio) was developed by the thin film hydration plus extrusion and calcium acetate gradient remote loading method, to address the issue of poor Gba solubility. Moreover, an integrin-targeting ligand (RGD peptide, cyclo[Arg-Gly-Asp-D-Tyr-Cys]) was post-inserted into liposomes in order to increase Gba cell delivery. Using fluorescently-labeled model liposomes, it was found that the targeting could improve the integrin-mediated cellular uptake of the liposomesin vitroin human umbilical vein endothelial cells (HUVECs), andin vivoas evidenced by chicken chorioallantoic membrane angiogenesis (CAM) model. It also could enrich the liposome accumulation in C26 tumor. Interestingly, ...
Cells
Background: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. Method: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by p...