Labeling nanoparticles: Dye leakage and altered cellular uptake

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence‐based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP–cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37°C. Cell–NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP‐dye retention is confirmed when no cellular fluorescence is detected at 4°C. Three different NP‐platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye–NP combination before pursuing NP‐based applications.

Related papers:

title: Nanoparticles in modern medicine: State of the art and future challenges
abstract: Nanoparticles are materials with overall dimensions in the nanoscale, ie, under 100 nm. In recent years, these materials have emerged as important players in modern medicine, with clinical applications ranging from contrast agents in imaging to carriers for drug and gene delivery into tumors. Indeed, there are some instances where nanoparticles enable analyses and therapies that simply cannot be performed otherwise. However, nanoparticles also bring with them unique environmental and societal challenges, particularly in regard to toxicity. This review aims to highlight the major contributions of nanoparticles to modern medicine and also discuss environmental and societal aspects of their use.

title: Biocompatibility of engineered nanoparticles for drug delivery.
abstract: The rapid advancement of nanotechnology has raised the possibility of using engineered nanoparticles that interact within biological environments for treatment of diseases. Nanoparticles interacting with cells and the extracellular environment can trigger a sequence of biological effects. These effects largely depend on the dynamic physicochemical characteristics of nanoparticles, which determine the biocompatibility and efficacy of the intended outcomes. Understanding the mechanisms behind these different outcomes will allow prediction of the relationship between nanostructures and their interactions with the biological milieu. At present, almost no standard biocompatibility evaluation criteria have been established, in particular for nanoparticles used in drug delivery systems. Therefore, an appropriate safety guideline of nanoparticles on human health with assessable endpoints is needed. In this review, we discuss the data existing in the literature regarding biocompatibility of nanoparticles for drug delivery applications. We also review the various types of nanoparticles used in drug delivery systems while addressing new challenges and research directions. Presenting the aforementioned information will aid in getting one step closer to formulating compatibility criteria for biological systems under exposure to different nanoparticles.

title: Applications of nanoparticles in biology and medicine
abstract: Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. Their unique size-dependent properties make these materials superior and indispensable in many areas of human activity. This brief review tries to summarise the most recent developments in the field of applied nanomaterials, in particular their application in biology and medicine, and discusses their commercialisation prospects.

title: The big picture on nanomedicine: the state of investigational and approved nanomedicine products.
abstract: Developments in nanomedicine are expected to provide solutions to many of modern medicine's unsolved problems, so it is no surprise that the literature contains many articles discussing the subject. However, existing reviews tend to focus on specific sectors of nanomedicine or to take a very forward-looking stance and fail to provide a complete perspective on the current landscape. This article provides a more comprehensive and contemporary inventory of nanomedicine products. A keyword search of literature, clinical trial registries, and the Web yielded 247 nanomedicine products that are approved or in various stages of clinical study. Specific information on each was gathered, so the overall field could be described based on various dimensions, including FDA classification, approval status, nanoscale size, treated condition, nanostructure, and others. In addition to documenting the many nanomedicine products already in use in humans, this study identifies several interesting trends forecasting the future of nanomedicine.


FROM THE CLINICAL EDITOR
In this one of a kind review, the state of nanomedicine commercialization is discussed, concentrating only on nanomedicine-based developments and products that are either in clinical trials or have already been approved for use.

title: Development of nanoparticles for clinical use.
abstract: During recent years, there has been much interest in using nanoparticles (NPs) for delivery of therapeutic drugs or contrast agents. Many interesting in vitro and animal studies have been reported [1–3], and some NP-based products are already in clinical use. Iron-oxide containing NPs have been used as iron supplement or contrast agents in MRI for more than 20 years . Albumin and liposomal-based products containing anticancer drugs have also been available for several years, and new NP-based products are in clinical trials [2,6]. The discrepancy between the large number of papers published about NPs and other drug candidates versus the few new drugs entering the market is heavily debated, and important discussions regarding what is needed to bring new products to the market have been published [6–9]. No doubt, there are many challenges to overcome before NPs may become common tools in clinical practice.

title: Optical imaging in drug discovery and diagnostic applications.
abstract: Optical imaging combines a variety of different diagnostic modalities which have shown great promise for biomedical imaging and as a tool in drug discovery. Several different principles to identify and characterize fundamental processes at the organ, tissue, cellular and molecular level have been exploited, supported by the design of novel imaging agents and biomolecular reporter systems. New optical imaging procedures will contribute considerably to the improvement of the knowledge of disease processes and the more efficient evaluation of drug effects in living laboratory animals. They may also find new diagnostic and therapeutic applications in human clinical practices. These techniques can be used in the field of molecular imaging to allow both visualization and quantification of molecular events associated with disease in a non-invasive and radiation-free manner using relatively simple equipment. The different aspects of imaging instrumentation and methods; the achievements in synthesis and evaluation of novel imaging agents and biochemical reporters; as well as the opportunities of optical imaging in drug delivery, drug discovery and imaging diagnostics will be discussed in this review article.

title: Synthetic pathways to make nanoparticles fluorescent.
abstract: In biosciences, it is often necessary to follow the pathway of nanoparticles within cells or tissues. The nanoparticles can be used as labeled sensors which may, e.g., address functionalities within a cell, carry other specific agents like drugs or be magnetic for tumor thermotherapy. In the context of nanotoxicology, the fate of a given nanoparticle is of interest. As many methods in cell biology are based on fluorescence detection, there is a strong demand to make nanoparticles fluorescent. Different ways to introduce fluorescence are reviewed and exemplified with typical kinds of nanoparticles, i.e. polymers, silica and calcium phosphate.

title: Design of polymeric nanoparticles for biomedical delivery applications.
abstract: Polymeric nanoparticles-based therapeutics show great promise in the treatment of a wide range of diseases, due to the flexibility in which their structures can be modified, with intricate definition over their compositions, structures and properties. Advances in polymerization chemistries and the application of reactive, efficient and orthogonal chemical modification reactions have enabled the engineering of multifunctional polymeric nanoparticles with precise control over the architectures of the individual polymer components, to direct their assembly and subsequent transformations into nanoparticles of selective overall shapes, sizes, internal morphologies, external surface charges and functionalizations. In addition, incorporation of certain functionalities can modulate the responsiveness of these nanostructures to specific stimuli through the use of remote activation. Furthermore, they can be equipped with smart components to allow their delivery beyond certain biological barriers, such as skin, mucus, blood, extracellular matrix, cellular and subcellular organelles. This tutorial review highlights the importance of well-defined chemistries, with detailed ties to specific biological hurdles and opportunities, in the design of nanostructures for various biomedical delivery applications.

title: Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery.
abstract: Drug delivery, magnetic resonance and fluorescence imaging, magnetic manipulation, and cell targeting are simultaneously possible using a multifunctional mesoporous silica nanoparticle. Superparamagnetic iron oxide nanocrystals were encapsulated inside mesostructured silica spheres that were labeled with fluorescent dye molecules and coated with hydrophilic groups to prevent aggregation. Water-insoluble anticancer drugs were delivered into human cancer cells; surface conjugation with cancer-specific targeting agents increased the uptake into cancer cells relative to that in non-cancerous fibroblasts. The highly versatile multifunctional nanoparticles could potentially be used for simultaneous imaging and therapeutic applications.

title: Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate.
abstract: Fluorescence microscopy may provide important information regarding interactions between nanoparticulate drugs carriers, such as liposomes and micelles, with target cells as well as their intracellular fate. Current paper describes various applications of fluorescence microscopy to investigate specific targeting of antibody-modified drug carriers to cancer cells. The enhanced antibody-mediated targeting of drug-loaded immunomicelles confirmed by fluorescence microscopy resulted in enhanced cancer cell killing compared to free drug or drug-loaded nontargeted micelles. Fluorescence microscopy was also used to prove the endosomal escape of properly assembled polymeric micelles (based on polyethylene glycol-phosphatidylethanolamine conjugate, PEG-PE) containing various additives destabilizing the endosomal membrane. When loaded with the anticancer drug (paclitaxel or vitamin K3), such micelles demonstrate increased cytotoxicity. Fluorescence microscopy was also applied to investigate the capture of cell-penetrating TAT peptide-modified liposomes by various cells and stability and intracellular trafficking of captured TAT-liposomes inside cells. It was also used to confirm the successful transfection of cells with TAT-liposomes bearing the plasmid encoding for the Green Fluorescent Protein (GFP).

title: The effect of nanoparticle polyethylene glycol surface density on ligand-directed tumor targeting studied in vivo by dual modality imaging.
abstract: The development and application of nanoparticles as in vivo delivery vehicles for therapeutic and/or diagnostic agents has seen a drastic growth over the last decades. Novel imaging techniques allow real-time in vivo study of nanoparticle accumulation kinetics at the level of the cell and targeted tissue. Successful intravenous application of such nanocarriers requires a hydrophilic particle surface coating, of which polyethylene glycol (PEG) has become the most widely studied and applied. In the current study, the effect of nanoparticle PEG surface density on the targeting efficiency of ligand-functionalized nanoemulsions was investigated. We synthesized 100 nm nanoemulsions with a PEG surface density varying from 5 to 50 mol %. Fluorescent and paramagnetic lipids were included to allow their multimodal detection, while RGD peptides were conjugated to the PEG coating to obtain specificity for the α(v)β(3)-integrin. The development of a unique experimental imaging setup allowed us to study, in real time, nanoparticle accumulation kinetics at (sub)-cellular resolution in tumors that were grown in a window chamber model with confocal microscopy imaging, and at the macroscopic tumor level in subcutaneously grown xenografts with magnetic resonance imaging. Accumulation in the tumor occurred more rapidly for the targeted nanoemulsions than for the nontargeted versions, and the PEG surface density had a strong effect on nanoparticle targeting efficiency. Counterintuitively, yet consistent with the PEG density conformation models, the highest specificity and targeting efficiency was observed at a low PEG surface density.

title: Biodegradable nanoparticles for drug and gene delivery to cells and tissue.
abstract: Biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide) (PLGA) have been extensively investigated for sustained and targeted/localized delivery of different agents including plasmid DNA, proteins and peptides and low molecular weight compounds. Research about the mechanism of intracellular uptake of nanoparticles, their trafficking and sorting into different intracellular compartments, and the mechanism of enhanced therapeutic efficacy of nanoparticle-encapsulated agent at cellular level is more recent and is the primary focus of the review. Recent studies in our laboratory demonstrated rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytosol following their uptake. Based on the above mechanism, various potential applications of nanoparticles for delivery of therapeutic agents to the cells and tissue are discussed.

title: Residual polyvinyl alcohol associated with poly (D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake.
abstract: Polyvinyl alcohol (PVA) is the most commonly used emulsifier in the formulation of poly lactide and poly (D,L-lactide-co-glycolide) (PLGA) polymeric nanoparticles. A fraction of PVA remains associated with the nanoparticles despite repeated washing because PVA forms an interconnected network with the polymer at the interface. The objective of this study was to determine the parameters that influence the amount of residual PVA associated with PLGA nanoparticles and its effect on the physical properties and cellular uptake of nanoparticles. Nanoparticles were formulated by a multiple emulsion-solvent evaporation technique using bovine serum albumin (BSA) as a model protein. The parameters that affected the amount of residual PVA include the concentration of PVA and the type of organic solvent used in the emulsion. The residual PVA, in turn, influenced different pharmaceutical properties of nanoparticles such as particle size, zeta potential, polydispersity index, surface hydrophobicity, protein loading and also slightly influenced the in vitro release of the encapsulated protein. Importantly, nanoparticles with higher amount of residual PVA had relatively lower cellular uptake despite their smaller particle size. It is proposed that the lower intracellular uptake of nanoparticles with higher amount of residual PVA could be related to the higher hydrophilicity of the nanoparticle surface. In conclusion, the residual PVA associated with nanoparticles is an important formulation parameter that can be used to modulate the pharmaceutical properties of PLGA nanoparticles.

title: Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.
abstract: To elucidate the effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles (NPs), rhodamine B (RhB) labeled carboxymethyl chitosan grafted NPs (RhB-CMCNP) and chitosan hydrochloride grafted NPs (RhB-CHNP) were developed as the model negatively and positively charged polymeric NPs, respectively. These NPs owned well defined particle sizes (150-500 nm) and Zeta potentials (-40 mV - +35 mV). FITC labeled protamine sulfate (FITC-PS) loaded RhB-CMCNP and camptothecin (CPT) loaded RhB-CHNP with high encapsulation efficiency were prepared. The fluorescence stability in plasma and towards I(-) was investigated, and the result indicated it was sufficient for qualitative and quantitative analysis. NPs with high surface charge and large particle size were phagocytized more efficiently by murine macrophage. Slight particle size and surface charge differences and different cell lines had significant implications in the cellular uptake of NPs, and various mechanisms were involved in the uptake process. In vivo biodistribution suggested that NPs with slight negative charges and particle size of 150 nm were tended to accumulate in tumor more efficiently. These results could serve as a guideline in the rational design of drug nanocarriers with maximized therapeutic efficacy and predictable in vivo properties, in which the control of particle size and surface charge was of significance.

title: Particle shape: a new design parameter for micro- and nanoscale drug delivery carriers.
abstract: Encapsulation of therapeutic agents in polymer particles has been successfully used in the development of new drug carriers. A number of design parameters that govern the functional behavior of carriers, including the choice of polymer, particle size and surface chemistry, have been tuned to optimize their performance in vivo. However, particle shape, which may also have a strong impact on carrier performance, has not been thoroughly investigated. This is perhaps due to the limited availability of techniques to produce non-spherical polymer particles. In recent years, a number of reports have emerged to directly address this bottleneck and initial studies have indeed confirmed that particle shape can significantly impact the performance of polymer drug carriers. This article provides a review of this field with respect to methods of particle preparation and the role of particle shape in drug delivery.

title: Influence of nanoparticle shape, size, and surface functionalization on cellular uptake.
abstract: With the rapid development of biotechnology and nanomedicine, extensive research has focused on the investigations of delivering large-cargo molecules using nanoparticles through the cell membrane for disease diagnosis and treatment. Various inorganic and polymeric nanoparticles with optimized surface properties have been developed to carry these active cargo molecules such as organic molecules, oligonucleotides and proteins. Phagocytosis and pinocytosis have been suggested as the two major uptake mechanisms for nanoparticles to enter into cellular interior, but such mechanisms are still under debate. In order to enhance the efficiency of cellular uptake of nanoparticles and further understand the physiological process, it is important to investigate detailed interaction mechanisms between nanoparticles and cell membranes. Here, we will review the recent advances of the effect of nanoparticle properties (e.g., nanoparticle shape, size, charge, surface modification, etc.) on cellular uptake mechanisms. These will aid in the future design and development of nanoparticles with improved surface properties for drug and biomolecule delivery. Up to now, novel analytical techniques have been used to examine nanoparticle-cell membrane interactions, but their detailed uptake mechanisms and pathways still need more in-depth research. It is suggested that developing appropriate analytical techniques to study cellular uptake mechanisms of nanoparticles in real time is urgently desired.

title: Highly lipophilic fluorescent dyes in nano-emulsions: towards bright non-leaking nano-droplets.
abstract: Dye-loaded lipid nano-droplets present an attractive alternative to inorganic nanoparticles, as they are composed of non-toxic biodegradable materials and easy to prepare. However, to achieve high fluorescence brightness, the nano-droplets have to be heavily loaded with the dyes avoiding fluorescence self-quenching and release (leakage) of the encapsulated dyes from the nano-droplets in biological media. In the present work, we have designed highly lipophilic fluorescent derivatives of 3-alkoxyflavone (F888) and Nile Red (NR668) that can be encapsulated in the lipophilic core of stable nano-emulsion droplets at exceptionally high concentrations in the oil core, i.e. up to 170 mM and 17 mM, respectively, corresponding to ~ 830 and 80 dyes per 40-nm droplet. Despite this high loading, these dyes keep high fluorescence quantum yield and thus, provide high nano-droplet brightness, probably due to their bulky structure preventing self-quenching. Moreover, simultaneous encapsulation of both dyes at high concentrations in single nano-droplets allows observation of FRET. FRET and fluorescence correlation spectroscopy (FCS) studies showed that NR668 release in the serum-containing medium is very slow, while the reference hydrophobic dye Nile Red leaks immediately. This drastic difference in the leakage profile between NR668 and Nile Red was confirmed by in vitro cellular studies as well as by in vivo angiography imaging on zebrafish model, where the NR668-loaded nano-droplets remained in the blood circulation, while the parent Nile Red leaked rapidly from the droplets distributing all over the animal body. This study suggests new molecular design strategies for obtaining bright nano-droplets without dye leakage and their use as efficient and stable optical contrast agents in vitro and in vivo.

title: Intracellular drug delivery by poly(lactic-co-glycolic acid) nanoparticles, revisited.
abstract: We reexamined the cellular drug delivery mechanism by poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to determine their utility and limitations as an intracellular drug delivery system. First, we prepared PLGA NPs which physically encapsulated Nile red (a hydrophobic fluorescent dye), in accordance with the usual procedure for labeling PLGA NPs, incubated them with mesothelial cells, and observed an increase in the intracellular fluorescence. We then prepared NPs from PLGA chemically conjugated to a fluorescent dye and observed their uptake by the mesothelial cells using confocal microscopy. We also used coherent anti-Stokes Raman scattering (CARS) microscopy to image cellular uptake of unlabeled PLGA NPs. Results of this study coherently suggest that PLGA NPs (i) are not readily taken up by cells, but (ii) deliver the payload to cells by extracellular drug release and/or direct drug transfer to contacting cells, which are contrasted with the prevalent view. From this alternative standpoint, we analyzed cytotoxicities of doxorubicin and paclitaxel delivered by PLGA NPs and compared with those of free drugs. Finally, we revisit previous findings in the literature and discuss potential strategies to achieve efficient drug delivery to the target tissues using PLGA NPs.

title: Erythrocyte incubation as a method for free-dye presence determination in fluorescently labeled nanoparticles.
abstract: The field of nanotheranostics encompasses the integration of nanosized carriers in cancer imaging, diagnosis, and therapy. The use of nanomedicines for theranostic application typically depends on direct visualization of the nanocarriers. Normally fluorescent probes are attached to nanocarriers for biodistribution measurement through fluorescence imaging. However continued, noninvasive assurance that the fluorescent probe remains bound to the carrier has proven elusive. Mature erythrocytes, also known as red blood cells, are incapable of endocytosis. As a consequence, when incubated with fluorescently labeled particles, they do not show any signal coming from the membrane or the cytoplasm. Yet, these cells readily take up free BODIPY fluorescent dyes into their membranes. Here we show that incubation of nanoparticles with erythrocytes is a rapid and reliable method for the detection of unbound dye present within a nanoparticle sample, as the detection of a fluorescent signal coming from the cells can only be due to unbound dye present in the sample. We test the method on both sulfonate and PEG terminated gold nanoparticles, and we determine the minimum concentration of detectable dye for a specific gold nanoparticle sample.

title: Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules.
abstract: Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided.

title: Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use
abstract: 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.

title: Flow cytometry as a new approach to investigate drug transfer between lipid particles.
abstract: Lipid nanoparticles and liposomal carrier systems are of growing interest for intravenous drug delivery due to their biocompatibility and targetability. It is, however, difficult to investigate their release behavior for lipophilic drugs under physiological conditions. This study describes a novel flow cytometric method studying drug transfer from such carrier systems to particles simulating physiological receptor sites. For this purpose, liquid and solid trimyristin nanoparticles or soybean phospholipid liposomes were loaded with the lipophilic fluorescent substances Nile red, temoporfin, and DiI. The transfer of these model drugs to large emulsion droplets was examined by flow cytometry. Transfer of DiI to differently sized acceptor emulsions was also monitored by separating donor and acceptor particles using ultracentrifugation. Flow cytometry revealed a completion of transfer within a few minutes for Nile red and temoporfin at considerable amounts of transferred dye. In contrast, the highly lipophilic DiI transferred over a period of weeks only for a small percentage of the dye. Ultracentrifugation results confirmed this for DiI and indicated a dependence of transfer characteristics on the acceptor surface area. Nile red transfer into a bulk oil phase as alternative acceptor system was also very slow. Flow cytometry seems to be well suited to study the intrinsic transfer of fluorescent lipophilic substances, as no kinetic hindrances like dialysis bags nor separation steps are required. Additional detailed experiments will, however, be necessary to elucidate the prevalent transfer mechanisms completely.

title: A new tool to ensure the fluorescent dye labeling stability of nanocarriers: a real challenge for fluorescence imaging.
abstract: Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.

title: Development of an in vitro drug release assay that accurately predicts in vivo drug retention for liposome-based delivery systems.
abstract: The therapeutic activity of numerous drugs can be dramatically improved by liposomal encapsulation. However, this requires that liposomes retain their encapsulated drugs following systemic administration. Often, in vitro drug release assays do not accurately predict the liposomal drug retention properties observed in vivo. We postulate that this discrepancy is due to the large membrane pool present in blood cells and tissues, into which drugs can distribute after in vivo administration. Herein we describe an in vitro drug release assay that more accurately predicts in vivo drug release from liposomes following systemic administration. Drug-encapsulated large unilamellar vesicles (LUVs) approximately 100 nm in diameter were incubated with a 100-fold excess of multilamellar vesicles (MLVs) containing 300 mM sucrose, which served as 'acceptors' for drug release and transfer from 'donor' LUVs. Following incubation at 37 degrees C, the donor and acceptor populations were separated with greater than 90% efficiency by centrifugation at 1600xg for 10 min. The amount of drug in the MLV pellet reflects the degree of drug leakage from the donor liposomes. Drug release profiles using this in vitro assay were compared to those obtained using dialysis-based assays and in vivo results following systemic administration to mice. Our results indicate that this release assay is a better predictor of in vivo drug transfer than dialysis-based systems. We also demonstrate its utility in measuring exchange of lipophilic components.

title: Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging
abstract: It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using dual-labeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Förster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC18(3) and DiOC18(3), was loaded into monomethoxy poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed.

title: Nile red: a selective fluorescent stain for intracellular lipid droplets
abstract: We report that the dye nile red, 9-diethylamino-5H- benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red- stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

title: Nile red staining of lysosomal phospholipid inclusions
abstract: SummaryWe have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.

title: Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles.
abstract: Nanoscale objects are typically internalized by cells into membrane-bounded endosomes and fail to access the cytosolic cell machinery. Whereas some biomacromolecules may penetrate or fuse with cell membranes without overt membrane disruption, no synthetic material of comparable size has shown this property yet. Cationic nano-objects pass through cell membranes by generating transient holes, a process associated with cytotoxicity. Studies aimed at generating cell-penetrating nanomaterials have focused on the effect of size, shape and composition. Here, we compare membrane penetration by two nanoparticle 'isomers' with similar composition (same hydrophobic content), one coated with subnanometre striations of alternating anionic and hydrophobic groups, and the other coated with the same moieties but in a random distribution. We show that the former particles penetrate the plasma membrane without bilayer disruption, whereas the latter are mostly trapped in endosomes. Our results offer a paradigm for analysing the fundamental problem of cell-membrane-penetrating bio- and macro-molecules.

title: Temperature: the "ignored" factor at the NanoBio interface.
abstract: Upon incorporation of nanoparticles (NPs) into the body, they are exposed to biological fluids, and their interaction with the dissolved biomolecules leads to the formation of the so-called protein corona on the surface of the NPs. The composition of the corona plays a crucial role in the biological fate of the NPs. While the effects of various physicochemical parameters on the composition of the corona have been explored in depth, the role of temperature upon its formation has received much less attention. In this work, we have probed the effect of temperature on the protein composition on the surface of a set of NPs with various surface chemistries and electric charges. Our results indicate that the degree of protein coverage and the composition of the adsorbed proteins on the NPs' surface depend on the temperature at which the protein corona is formed. Also, the uptake of NPs is affected by the temperature. Temperature is, thus, an important parameter that needs to be carefully controlled in quantitative studies of bionano interactions.

title: Uptake mechanism of oppositely charged fluorescent nanoparticles in HeLa cells.
abstract: The endocytotic mechanisms involved in the uptake of charged polystyrene nanoparticles into HeLa cells were investigated. Uptake experiments were done in the presence or absence of drugs known to inhibit various factors in endocytosis. Independent of the particle charge, endocytosis is highly dependent on dynamin, F-actin, and tyrosine-specific protein kinases, which suggests a dynamin-dependent and lipid raft-dependent mechanism. However, cholesterol depletion did not hinder particle uptake. Regarding positively charged particles, macropinocytosis, the microtubule network, and cyclooxygenases are also involved. The clathrin-dependent pathway plays a minor role.

title: Design of fluorescently tagged poly(alkyl cyanoacrylate) nanoparticles for human brain endothelial cell imaging.
abstract: Rhodamine B-tagged poly(alkyl cyanoacrylate) amphiphilic copolymers have been synthesised, characterised and successfully used to prepare fluorescent nanoparticles for human brain endothelial cell imaging, allowing their uptake and intracellular trafficking to be finely observed.

title: Nanoparticle adhesion to the cell membrane and its effect on nanoparticle uptake efficiency.
abstract: The interactions between nanosized particles and living systems are commonly mediated by what adsorbs to the nanoparticle in the biological environment, its biomolecular corona, rather than the pristine surface. Here, we characterize the adhesion toward the cell membrane of nanoparticles of different material and size and study how this is modulated by the presence or absence of a corona on the nanoparticle surface. The results are corroborated with adsorption to simple model supported lipid bilayers using a quartz crystal microbalance. We conclude that the adsorption of proteins on the nanoparticle surface strongly reduces nanoparticle adhesion in comparison to what is observed for the bare material. Nanoparticle uptake is described as a two-step process, where the nanoparticles initially adhere to the cell membrane and subsequently are internalized by the cells via energy-dependent pathways. The lowered adhesion in the presence of proteins thereby causes a concomitant decrease in nanoparticle uptake efficiency. The presence of a biomolecular corona may confer specific interactions between the nanoparticle-corona complex and the cell surface including triggering of regulated cell uptake. An important effect of the corona is, however, a reduction in the purely unspecific interactions between the bare material and the cell membrane, which in itself disregarding specific interactions, causes a decrease in cellular uptake. We suggest that future nanoparticle-cell studies include, together with characterization of size, charge, and dispersion stability, an evaluation of the adhesion properties of the material to relevant membranes.

title: The First Step into the Brain: Uptake of NIO‐PBCA Nanoparticles by Endothelial Cells in vitro and in vivo, and Direct Evidence for their Blood–Brain Barrier Permeation
abstract: By using fluorescent polysorbate 80 coated poly(n‐butylcyanoacrylate) (PBCA) nanoparticles in an in vivo study, direct evidence was found for the presence of nanoparticles entering the brain and retina of rats. The nanoparticles, prepared with a miniemulsion process, were labeled in situ with a fluorescent dye and coated with polysorbate 80. After preparation the particle size, ζ potential, and the molecular weight distribution were determined. BMEC cells were used as an in vitro model for the BBB. The cells showed significant uptake of the particles, but no transcytosis could be observed in vitro. After applying the particles to the animals at two concentrations, cryosections of the brains and retinas were prepared. Regarding the sections of the rats that received the lower dose,  of the applied fluorescent particles and the stained endothelial cells could be detected in the brain and retina, indicating particle internalization in the endothelial cells. Applied at higher doses, the particles could be detected within the brain and retina with few co‐localized signals, suggesting passage through the blood–brain and blood–retina barriers.

title: Iron oxide core oil-in-water emulsions as a multifunctional nanoparticle platform for tumor targeting and imaging.
abstract: Nanoemulsions are increasingly investigated for the delivery of hydrophobic drugs to improve their bioavailability or make their administration possible. In the current study, oil-in-water emulsions with three different mean diameters (30, 60, and 95 nm) were developed as a new multimodality nanoparticle platform for tumor targeting and imaging. To that aim, hydrophobically coated iron oxide particles were included in the soybean oil core of the nanoemulsions to enable their detection with magnetic resonance imaging (MRI), while the conjugation of a near infrared fluorophore allowed optical imaging. The accumulation of this novel nanocomposite in subcutaneous human tumors in nude mice was demonstrated with MRI and fluorescence imaging in vivo, and with Perl's staining of histological tumor sections ex vivo.

title: Precise quantification of nanoparticle internalization.
abstract: Nanoparticles have opened new exciting avenues for both diagnostic and therapeutic applications in human disease, and targeted nanoparticles are increasingly used as specific drug delivery vehicles. The precise quantification of nanoparticle internalization is of importance to measure the impact of physical and chemical properties on the uptake of nanoparticles into target cells or into cells responsible for rapid clearance. Internalization of nanoparticles has been measured by various techniques, but comparability of data between different laboratories is impeded by lack of a generally accepted standardized assay. Furthermore, the distinction between associated and internalized particles has been a challenge for many years, although this distinction is critical for most research questions. Previously used methods to verify intracellular location are typically not quantitative and do not lend themselves to high-throughput analysis. Here, we developed a mathematical model which integrates the data from high-throughput flow cytometry measurements with data from quantitative confocal microscopy. The generic method described here will be a useful tool in biomedical nanotechnology studies. The method was then applied to measure the impact of surface coatings of vesosomes on their internalization by cells of the reticuloendothelial system (RES). RES cells are responsible for rapid clearance of nanoparticles, and the resulting fast blood clearance is one of the major challenges in biomedical applications of nanoparticles. Coating of vesosomes with long chain polyethylene glycol showed a trend for lower internalization by RES cells.

title: Fluorescent labeling of dendritic spines in cell cultures with the carbocyanine dye “DiI”
abstract: Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system.

title: Cellular uptake and elimination of lipophilic drug delivered by nanocarriers.
abstract: To investigate the cellular uptake and elimination process of a lipophilic drug loaded in different nanocarriers, emulsions, liposomes and poly (DL-lactide-co-glycolide) (PLGA) nanoparticles loaded with a model drug, coumarin 6, were prepared and their transportation in HeLa cells compared. After 4 h incubation, liposomes and nanoparticles mediated significantly higher intracellular drug levels, which were 3 or 2.5 times that of the emulsion group, into cells. A novel kinetic model was established to analyze the cellular elimination process. Emulsions had the longest intracellular mean residence time (MRT), which was about 1-2 times longer than other nanocarriers. The endocytosis inhibition experiment suggested that the coumarin 6 in liposomes and nanoparticles entered cells directly via diffusion, while part of the intracellular coumarin 6 was taken up through clathrin-mediated endocytosis in emulsions. Combined with the results on uptake pathway and kinetic parameters, it can be concluded that different nanocarriers bring about diverse mechanisms of cellular uptake and elimination.

title: Drug delivery without nanoparticle uptake: delivery by a kiss-and-run mechanism on the cell membrane.
abstract: Nearly all concepts of nanocarriers as drug delivery devices rely on intracellular uptake. Instead, we demonstrate an alternative concept for rapid and specific delivery of cargo by nanoparticles to TIP47+/ADRP+ lipid droplets. The model can serve as a novel strategy for the non-invasive delivery of drugs by releasing hydrophobic cargo, in our case a model dye, through a kiss-and-run mechanism between nanoparticles and the cell membrane.

title: Iron oxide nanoparticle-based magnetic resonance method to monitor release kinetics from polymeric particles with high resolution.
abstract: A new method to precisely monitor rapid release kinetics from polymeric particles using super paramagnetic iron oxide nanoparticles, specifically by measuring spin-spin relaxation time (T(2)), is reported. Previously, we have published the formulation of logic gate particles from an acid-sensitive poly-β-aminoester ketal-2 polymer. Here, a series of poly-β-aminoester ketal-2 polymers with varying hydrophobicities were synthesized and used to formulate particles. We attempted to measure fluorescence of released Nile red to determine whether the structural adjustments could finely tune the release kinetics in the range of minutes to hours; however, this standard technique did not differentiate each release rate of our series. Thus, a new method based on encapsulation of iron oxide nanoparticles was developed, which enabled us to resolve the release kinetics of our particles. Moreover, the kinetics matched the relative hydrophobicity order determined by octanol-water partition coefficients. To the best of our knowledge, this method provides the highest resolution of release kinetics to date.

title: Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery.
abstract: Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.