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Internal benchmarking of a human blood-brain barrier cell model for screening of nanoparticle uptake and transcytosis
Abstract
Transport of drugs across the blood-brain barrier, which protects the brain from harmful agents, is considered the holy grail of targeted delivery, due to the extreme effectiveness of this barrier at preventing passage of non-essential molecules through to the brain. This has caused severe limitations for therapeutics for many brain-associated diseases, such as HIV and neurodegenerative diseases. Nanomaterials, as a result of their small size (in the order of many protein-lipid clusters routinely transported by cells) and their large surface area (which acts as a scaffold for proteins thereby rendering nanoparticles as biological entities) offer great promise for neuro-therapeutics. However, in parallel with developing neuro-therapeutic applications based on nanotechnology, it is essential to ensure their safety and long-term consequences upon reaching the brain. One approach to determining safe application of nanomaterials in biology is to obtain a deep mechanistic understanding of the interactions between nanomaterials and living systems (bionanointeractions). To this end, we report here on the establishment and internal round robin validation of a human cell model of the blood-brain barrier for use as a tool for screening nanoparticles interactions, and assessing the critical nanoscale parameters that determine transcytosis.
... The BBB is a physical barrier composed of a tightly packed layer of endothelial cells surrounding the brain, which separates the blood from the cerebrospinal fluid, allowing the transfer of oxygen and essential nutrients but preventing the access of most molecules. NMs function at the nanoscale; bind critical transport proteins such as apolipoproteins, including the brain transporter apolipoprotein E (13); and thus may have better ability to be transported across the BBB via endocytosis or diffusion (14,15). Moreover, their biotransformation may dynamically alter their mobility and transport pathways. ...
... For all NMs, increasing the exposure concentration to 2.5 mg/L did not cause significant increase in the percentage of transported elements, suggesting that the turnover rate of the endocytotic receptors is a rate-limiting step, as shown previously for a range of other human cells (28). It is also likely that the increase in the concentration of NMs facilitates the agglomeration of NMs, which in turn reduces the transport of the agglomerates across the BBB (15). Another potential explanation is that particle agglomeration may lead to low dissolution and thus the low transport of the released ions. ...
Significance
Although the brain is protected by a tight physiological guardian named the blood–brain barrier (BBB), deposition of engineered nanomaterials (ENMs) in the brain and consequent neurotoxicity has been reported. To date, it is still unclear whether and how ENMs enter the brain by crossing the BBB. In this study, we found that metallic ENMs transform in the BBB as affected by their shape, size, and intrinsic solubility, which in turn modulates their transport form, efficiency, and pathways through the BBB and, consequently, their neurotoxicity. The library of quantitative data on the chemical transformations presented here will support in silico modeling and prediction of the neurotoxicity of NMs and facilitate the tailored design of safe NMs.
... For the in vitro screening of NPs translocation the static simple model of bENd.3 (mouse brain vascular endothelial cells) was selected. bEnd.3 cells grow as a monolayer, establishing a low permeability barrier to macromolecules [59] and expressing high level of TJ proteins [30]. ...
... Studies using smaller particles are showing translocation rates more consistent with the present results. For instance, Ragnaill et al. reported that about 7% of the initial dose of 50 nm silica NPs was translocated across a hCMEC/D3 monolayer on a 0.4 μm pore filter [59]. Khan et al. demonstrated that about 15% of the 25-nm polymeric NPs dose was translocated after 12 h across a bEnd.3 ...
Treatments of neurodegenerative diseases (NDDs) are severely hampered by the presence of the blood-brain barrier (BBB) precluding efficient brain drug delivery. The development of drug nanocarriers aims at increasing the brain therapeutic index would represent a real progress in brain disease management. PEGylated polyester nanoparticles (NPs) are intensively tested in clinical trials for improved drug delivery. Our working hypothesis was that some surface parameters and size of NPs could favor their penetration across the BBB and their neuronal uptake. Polymeric material PEG-b-PLA diblocks were synthesized by ring opening polymerisation (ROP) with PEG2000 or PEG5000. A library of polymeric PEG-b-PLA diblocks NPs with different physicochemical properties was produced. The toxicity, endocytosis and transcytosis through the brain microvascular endothelial cells were monitored as well as the neuronal cells uptake. In vitro results lead to the identification of favourable surface parameters for the NPs endocytosis into vascular endothelial cells. NPs endocytosis took place mainly by macropinocytosis while transcytosis was partially controlled by their surface chemistry and size. In vivo assays on a zebrafish model showed that the kinetic of NPs in circulation is dependent on PEG coating properties. In vivo findings also showed a low but similar translocation of PEG-b-PLA diblocks NPs to the CNS, regardless of their properties. In conclusion, modulation of surface PEG chain length and NPs size impact the endocytosis rate of NPs but have little influence on cell barriers translocation; while in vivo biodistribution is influenced by surface PEG chain density.
... Some illustrative images for 50 nm and 150 nm SiO 2 -NPs are shown in Figure 3 and Figure 4, respectively. The particles are clearly visible in electron microscopy due to their high density, as also observed in our previous work [14,15,37,51]. Overall, consistent with the above results, very few nanoparticles were found inside the cells. ...
... The low degree of internalisation is particularly striking in comparison to our previous observations of a substantial uptake of silica nanoparticles into (single) lung cells [37]. Moreover, we have previously found sizable uptake of silica and polystyrene nanoparticles into another type of barrier, namely an in vitro model of the human endothelial blood brain barrier [14,15,51,52]. Thus, the low degree of uptake observed in the Caco-2 barrier may be a characteristic of this type of barrier and could be related to the more complex polarised nature of thicker epithelial layers. ...
Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.
... In contrast to other described procedures we do not use growth factor depleted medium but regular cell culture medium supplemented with FCS (Ragnaill et al 2011). However, the composition of FCS may vary and thus affect reproducibility, but the effect of FCS exclusion on cell layer integrity was verified. ...
... These methods allow an indirect detection of nanoparticles only and dependable results may be hindered by the disintegration of the fluorescent dye from the nanoparticles. Furthermore, dye leakage may be a serious event leading to critical consideration of uptake and transcytosis studies (Ragnaill et al 2011, Salvati et al 2011. ...
Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles' shape, material, size, and coating.
... The TEER values obtained were lower compared to the in vivo situation (1500-8000 V cm 2 ). However, the lower TEER values obtained are in agreement with recent reports, for which, under static culture conditions, these values were around 40 V cm 2 (Ragnaill et al., 2011). The monoculture model conditions (4.6 Â 10 4 cells/cm 2 for 7 days) were used in the following procedures. ...
... There is a negative linear relationship between the values of Papp and molecular weight (Papp = À1,64 Â 10 À10 Â MW + 1.20 Â 10 À5 ; R 2 = 0.999; p = 0.021). These values are similar to previously published data, namely for FD 4 kDa compound, for which Ragnaill et al. (2011) achieved values at around 5.5 Â 10 À6 cm/s, the Wekler group around 6 Â 10 À6 cm/s (Weksler et al., 2005) and finally Forster and colleagues achieved higher permeability values around 13 Â 10 À6 cm/s (Förster et al., 2008). In comparison with rat cells using the same compound, values as high as 16.3 Â 10 À6 cm/s were achieved. ...
... The combination of transmission electron microscopy (TEM) with traditional Transwell ™ systems has revealed some crucial limitations of these models (18,44,45), which bring into question the reliability and reproducibility of these systems. These limitations include barrier imperfections, such as formation of multiple layers of endothelial cells on the filter and holes (see Figure 3); nanoparticle adhesion to the microporous filter pore; nanoparticle agglomeration in the culture medium and inside the cell; and leakage of the fluorescent dye. ...
... Based on previous observations (18,44,45), imperfections exist in all in vitro barriers and monolayers models. Furthermore, the traditional Transwell ™ systems suffer from numerous limitations, preventing their application for accurate and reproducible nanoparticle-BBB studies. ...
In the past decade, the importance of quality and reproducibility within research has been re-emphasized, consequently becoming a crucial part of scientific experiments. Their implementation into in vitro and in vivo biological experiments is challenging due to various parameters that can influence the final scientific outcome. In parallel to these activities, there is a huge scientific effort to improve today's medicines to make them safer and more efficient, and to cure untreatable diseases, such as many neurodegenerative diseases. Nanosized materials have been recognized as potential drug delivery systems in this arena due to their small size and surface properties, which enable the design and synthesis of safe and efficient delivery vehicles that might be able to cross the blood-brain barrier. However, the fundamental understanding behind their uptake mechanism and intracellular trafficking remains unknown. Simple and cost-effective in vitro blood-brain barrier models are widely used to address these questions. This paper aims to critically evaluate the current in vitro models using Transwell™ systems and to discuss alternative approaches towards more reproducible in vivo features.
... The hCMEC/D3 cell line has been used to investigate nanoparticle trancytosis by various research groups, establishing it as a well-validated model (162,163). For example, transport of SiO 2 nanoparticles across the hCMEC/D3 BBB model has been reported (164). Through transcytosis studies and electron microscopy imaging, it was shown that, although the nanoparticles moved through the BBB model via the transcellular route, the filter used impeded the progress of the nanoparticles, despite the pore size being far larger than the nanoparticle diameter (0.4 μm pore PET filter with 50 nm nanoparticles) (164). ...
... For example, transport of SiO 2 nanoparticles across the hCMEC/D3 BBB model has been reported (164). Through transcytosis studies and electron microscopy imaging, it was shown that, although the nanoparticles moved through the BBB model via the transcellular route, the filter used impeded the progress of the nanoparticles, despite the pore size being far larger than the nanoparticle diameter (0.4 μm pore PET filter with 50 nm nanoparticles) (164). ...
The blood-brain barrier is a unique cell-based restrictive barrier that prevents the entry of many substances, including most therapeutics, into the central nervous system. A wide range of nanoparticulate delivery systems have been investigated with the aim of targeting therapeutics (drugs, nucleic acids, proteins) to the brain following administration by various routes. This review provides a comprehensive description of the design and formulation of these nanoparticles including the rationale behind individual approaches. In addition, the ability of currently available in-vitro BBB models to accurately predict the in-vivo performance of targeted nanoparticles is critically assessed.
... Ligands are often employed to traverse the nanoparticles across the BBB by the mechanism of receptor-mediated transcytosis [90]. Transferrin (Tf ), lactoferrin (Lf ), and LDL receptors LDL, among other ligands, have been employed for specific targeting of receptors which are expressed on the membrane of BBB for transport by receptormediated transcytosis [91]. ...
Nanotechnological advancements over the past few years have led to the development of newer treatment strategies in brain cancer therapy which leads to the establishment of nano oncology. Nanostructures with high specificity, are best suitable to penetrate the blood-brain barrier (BBB). Their desired physicochemical properties, such as small sizes, shape, higher surface area to volume ratio, distinctive structural features, and the possibility to attach various substances on their surface transform them into potential transport carriers able to cross various cellular and tissue barriers, including the BBB. The review emphasizes nanotechnology-based treatment strategies for the exploration of brain tumors and highlights the current progress of different nanomaterials for the effective delivery of drugs for brain tumor therapy.
... Caco-2 cells were seeded in the apical compartment of a diffusion system equipped with a permeable support of Transwell PET membrane [50] (12-well with 0.33 cm 2 , 0.4 µm pore size; Corning Inc., New York, USA). A density of 2 × 10 5 cells per chamber was reached. ...
Polymer nanoparticles are promising candidates for drug encapsulation and drug delivery systems. Particle surface functionalization allows for targeting specific sites, improving the drug distribution and hence potentializing the treatment of a wide range of diseases. In the present work, both the ability of polymeric nanoparticles to cross tissue membranes in Caco-2 cell cultures and the nanoparticle distribution among different tissues after intravenous infusion in mice are analyzed. Poly(methyl methacrylate-co-acrylic acid) and poly(methyl methacrylate-co-methacrylic acid) nanoparticles were prepared through miniemulsion polymerizations and functionalized with a fluorescence dye through covalent bonding. Cytotoxic effects were not observed in concentrations below of 1689 µg/mL for methacrylic particles and 2411 µg/mL for acrylic particles. Half-lives of nanoparticle circulation in animals were short, as particles were removed from the bloodstream after 30 min. Fluorescence analyses showed that polymer nanoparticles can reach different organs with very heterogeneous distributions, reaching maximum concentration in the kidneys.
Graphical Abstract
... There have been examples in the case of central nervous system cancers in which the drug is faced by an obstacle in passing through the blood-brain roadblock while attacking the tumor. 322,323 It has been proved in clinical trials that drugs that are loaded with nanoparticles have the capacity to cross this roadblock and hence enhance the drug efficacy. 324,325 To minimize the toxic levels of an anticancer drug, we should give a proper direction to the drug toward its target. ...
Cancer cannot be controlled by the usage of drugs alone, and thus, nanotechnology is an important technique that can provide the drug with an impetus to act more effectively. There is adequate availability of anticancer drugs that are classified as alkylating agents, hormones, or antimetabolites. Nanoparticle (NP) carriers increase the residence time of the drug, thereby enhancing the survival rate of the drug, which otherwise gets washed off owing to the small size of the drug particles by the excretory system. For example, for enhancing the circulation, a coating of nonfouling polymers like PEG and dextran is done. Famous drugs such as doxorubicin (DOX) are commonly encapsulated inside the nanocomposite. The various classes of nanoparticles are used to enhance drug delivery by aiding it to fight against the tumor. Targeted therapy aims to attack the cells with features common to the cancer cells while minimizing damage to the normal cell, and these therapies work in one in four ways. Some block the cancer cells from reproducing newer cells, others release toxic substances to kill the cancer cells, some stimulate the immune system to destroy the cancer cells, and some block the growth of more blood vessels around cancer cells, which starve the cells of the nutrients, which is needed for their growth. This review aims to testify the advancements nanotechnology has brought in cancer therapy, and its statements are supported with recent research findings and clinical trial results.
... Nanocarriers such as polymeric nanoparticles, liposomes, solid lipid nanoparticles (SLNs), and micelles can facilitate drug transmigration into the brain through endocytosis by inhibiting ABC transporters of the BBB [97]. Nanocarriers, with its small size and increased surface area, offer great potential in therapeutic delivery [118]. Besides, they can be manipulated in terms of size, shape, and surface engineering to favour the drug uptake, release, and ingress across the BBB [107,119]. ...
NeuroAIDS (Neuro Acquired Immunodeficiency Syndrome) or HIV (Human Immunodeficiency Virus) associated neuronal abnormality is continuing to be a significant health issue among AIDS patients even under the treatment of combined antiretroviral therapy (cART). Injury and damage to neurons of the brain are the prime causes of neuroAIDS, which happens due to the ingress of HIV by direct permeation across the blood-brain barrier (BBB) or else via peripherally infected macrophage into the central nervous system (CNS). The BBB performs as a stringent barricade for the delivery of therapeutics drugs. The intranasal route of drug administration exhibits as a non-invasive technique to bypass the BBB for the delivery of antiretroviral drugs and other active pharmaceutical ingredients inside the brain and CNS. This method is fruitful for the drugs that are unable to invade the BBB to show its action in the CNS and thus erase the demand of systemic delivery and thereby shrink systemic side effects. Drug delivery from the nose to the brain/CNS takes very less time through both olfactory and trigeminal nerves. Intranasal delivery does not require the involvement of any receptor as it occurs by an extracellular route. Nose to brain delivery also involves nasal associated lymphatic tissues (NALT) and deep cervical lymph nodes. However, very little research has been done to explore the utility of nose to brain delivery of antiretroviral drugs in the treatment of neuroAIDS. This review focuses on the potential of nasal route for the effective delivery of antiretroviral nanoformulations directly from nose to the brain.
... NMs could not penetrate into the brain due to the tight junctions between the endothelial cells. However, the blood-brain barrier could not prevent from penetrating all NMs [38,39]. It has been suggested that biodistribution and biological half-life are affected by NMs shape and surface characteristics. ...
... Surface-modified poly(lactide-co-glycolide) NPs decreased TEER and increased permeability in a HBMEC-human astrocyte co-culture model [39]. Compared to a BBB model using hCMEC/D3 cells, TEER and P app values in our model, albeit being slightly lower and higher, respectively, were comparable [40]. However, models using primary ECs resulted in higher TEER and lower overall permeability [19,23,24]. ...
Nanomedicine is a constantly expanding field, facilitating and improving diagnosis and treatment of diseases. As nanomaterials are
foreign objects, careful evaluation of their toxicological and functional aspects prior to medical application is imperative. In this
study, we aimed to determine the effects of gold and polymer-coated silica nanoparticles used in laser tissue soldering on brain endothelial
cells and the blood–brain barrier using rat brain capillary endothelial cells (rBCEC4). All types of nanoparticles were
taken up time-dependently by the rBCEC4 cells, albeit to a different extent, causing a time- and concentration-dependent decrease
in cell viability. Nanoparticle exposure did not change cell proliferation, differentiation, nor did it induce inflammation. rBCEC4
cells showed blood–brain barrier characteristics including tight junctions. None of the nanoparticles altered the expression of tight
junctions or impaired the blood–brain barrier permeability. The findings suggest that effects of these nanoparticles on the metabolic
state of cells have to be further characterized before use for medical purposes.
... However, the primary pathway of incorporation of CeO 2 -NPs by living organisms is by ingestion (Collin et al., 2014), as evidenced in zebrafish (Johnston et al., 2010), being unlikely that these NPs enter the organism through the gills of aquatic species, as opposed to many dissolved compounds (Baker et al., 2014). Furthermore, it has also been shown that NPs have the capacity to cross biological membranes, from the gastrointestinal tract into blood vessels and other parts of the body, such as the brain (Ragnaill et al., 2011). The surface charge of NPs may alter their capacity to be transported through the blood-brain barrier and change their permeability to cells, but systemic effects are likely to occur (Saraiva et al., 2016). ...
Cerium dioxide nanoparticles (CeO2-NPs) have a variety of uses, especially in the production of solar panels, oxygen pumps, gas sensors, computer chips and catalytic converters. Despite their worldwide use, the few published studies demonstrate that metallic nanoparticles, in general, are still not properly characterized in terms of their potencial ecotoxicological effects. CeO2-NPs, in particular, have demonstrated extreme antioxidant activity, but their in vivo toxicity is still unknown. This work intended to characterize the chronic toxicity (28 days) of three different ecologically relevant concentrations (0.1, 0.01, and 0.001 μg/L) of CeO2-NPs in the rainbow trout (Oncorhynchus mykiss), in terms of biomarkers of oxidative stress [activity of the enzymes glutathione S-transferases (GSTs) and catalase (CAT)] and neurotoxicity [activity of the enzyme acetylcholinesterase (AChE)], as well as histological alterations in liver and gills. In the hereby study, GSTs activity was increased in gills of fish exposed to the highest CeO2–NPs level. Moreover, a potential anti-oxidant response was also reported, with a significant increase of CAT activity observed in livers of the same fish. AChE, however, was not significantly altered in fish eyes. Individuals exposed to CeO2-NPs also presented marked changes in the gills (e.g. epithelial lifting, intercellular edema, lamellar hypertrophy and hyperplasia, secondary lamella fusion and aneurysms) and liver (e.g. hepatocyte vacuolization, pyknotic nucleus, enlargement of sinusoids and hyperemia). The semi-quantitative analysis (organs pathological index) also showed the establishment of a dose effect relationship. Further studies about the ecotoxicological effects of the CeO2-NPs have yet to be conducted, considering their properties, as the aggregation chemistry and the ratio of its redox state, which may affect their availability to the organism and their toxicity in the environment and biota.
... Regardless of the route of exposure, NPs could reach the blood vessels and translocate to the brain [8,9]. The distribution of NPs in the bloodstream also raises a particular concern of NP transfer from placenta to the fetal CNS [7] with serious damage as a consequence of direct exposure to NPs in utero [10]. ...
... Regardless of the route of exposure, NPs could reach the blood vessels and translocate to the brain [8,9]. The distribution of NPs in the bloodstream also raises a particular concern of NP transfer from placenta to the fetal CNS [7] with serious damage as a consequence of direct exposure to NPs in utero [10]. ...
Since nanoparticles (NPs) can translocate to the brain and impact the highly vulnerable central nervous system (CNS), novel in vitro tools for the assessment of NP-induced neurotoxicity are advocated. In this study, two types of CNS spheroids have been developed from human D384 astrocyte- and SH-SY5Y neuronal-like cells, and optimized in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by Fe₃O₄NPs, as NP-model, after short- (24⁻48 h; 1⁻100µg/ml) and long-term repeated exposure (30days; 0.1⁻25µg/ml). Short-term exposure of 3D-spheroids to Fe₃O₄NP induced cytotoxicity at 10 µg/mL in astrocytes and 25 µg/mL neurons. After long-term repeated dose regimen, spheroids showed concentration- and time-dependent cell mortality at 10 µg/mL for D384 and 0.5 µg/mL for SH-SY5Y, indicating a higher susceptibility of neurons than astrocytes. Both spheroid types displayed cell disaggregation after the first week of treatment at ≥0.1 µg/mL and becoming considerably evident at higher concentrations and over time. Recreating the 3D-spatial environment of the CNS allows cells to behave in vitro more closely to the in vivo situations, therefore providing a model that can be used as a stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.
... In vivo studies showed that NP could be found in the CNS upon various ways of administration (Semmler-Behnke et al., 2008;Zensi et al., 2009Zensi et al., , 2010. In parallel, in vitro models of human and murine BBB have been used and developed for the investigation of NP translocation (Andrieux and Couvreur, 2009;Ragnaill et al., 2011;Bramini et al., 2014;Herda et al., 2014;Raghnaill et al., 2014). ...
The scientific community has witnessed an exponential increase in the applications of graphene and graphene-based materials in a wide range of fields. For what concerns neuroscience, the interest raised by these materials is two-fold. On one side, nanosheets made of graphene or graphene derivatives (graphene oxide, or its reduced form) can be used as carriers for drug delivery. Here, an important aspect is to evaluate their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. On the other side, graphene can be exploited as a substrate for tissue engineering. In this case, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation. In this review, we try to give a comprehensive view of the accomplishments and new challenges of the field, as well as which in our view are the most exciting directions to take in the immediate future. These include the need to engineer multifunctional nanoparticles able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. We describe the state-of-the-art in the use of graphene materials to engineer three-dimensional scaffolds to drive neuronal growth and regeneration in vivo, and the possibility of using graphene as a component of hybrid composites/multi-layer organic electronics devices. Last but not least, we address the need of an accurate theoretical modeling of the interface between graphene and biological material, by modeling the interaction of graphene with proteins and cell membranes at the nanoscale, and describing the physical mechanism(s) of charge transfer by which the various graphene materials can influence the excitability and physiology of neural cells.
... Regardless of the route of exposure, it would seem that nanoparticles could quickly reach the blood vessels, (Ragnaill et al., 2011;Sharma and Sharma, 2007;Zensi et al., 2009) via transcytosis through endothelial cells rather than between endothelial cells. Translocation from blood compartment through endothelial cells to reach the brain has been demonstrated in vivo in the mouse after intravenous injection of nanoparticles functionalized with Apo-E molecules (Kreuter et al., 2002;Zensi et al., 2009). ...
The present critical review analyzes the question of how nanoparticles from continuously growing industrial production and use of nanomaterials may impact human brain health.
Available evidence suggests incomplete effectiveness of protective barriers of the brain against nanoparticles translocation to the brain. This raises concerns of potential effects of manufactured nanoparticles on brain functions, given that nanoparticle’s potential to induce oxidative stress, inflammation, death by apoptosis, or changes in the level of expression of certain neurotransmitters. Most concerns have not been studied sufficiently and many questions are still open: Are the findings in animals transposable to humans? What happens when exposure is chronic or protracted? What happens to the developing brain when exposure occurs in utero? Are some nanoparticles more deleterious, given their ability to alter protein conformations and aggregation? Aside from developments in nanomedicine, the evidence already available fully justifies the need to specifically evaluate the interactions between nanoparticles and the nervous system. The available data clearly indicates the need for original dedicated experimental models and tools for neurotoxicological research on the one hand, and the need for epidemiological studies of neurodegenerative diseases in manufactured nanoparticle-exposed populations, on the other. A combination of nanotoxicology with neurology in a novel discipline, with its specific tools and methods of investigation, should enable answering still unresolved questions.
... Fluorescence intensity of labelled particles 42 or light absorption through a non-fluorescent NP suspension can be detected by spectrophotometric methods 43 . Spectrophotometry is a simple and non-invasive method to determine particle concentration, but limited in terms of sensitivity. ...
Existing in vitro and in vivo tests used to assess the toxicity of chemicals can also be applied to engineered nanomaterials. However, shrinking a material to the nanoscale introduces new features that need to be taken into consideration when toxicity testing is conducted. Thus, validated toxicity tests are highly desirable. OECD recommended or scientifically validated tests may require adaptation for testing of nanomaterials. Nanoparticles as well as their aggregates/agglomerates can transfer across biological barriers and thus methods for detection and uptake must be an integral part of the testing strategy. In this chapter, an overview of in vitro and in vivo test methods for the evaluation of nanomaterial toxicity is provided and methods for assessment of cellular uptake and biodistribution are discussed. Care should be taken to the selection of in vitro endpoints to avoid interference with the assay and appropriate controls should be used.
... Collectively, the mechanism of particle movement in the brain endothelial cells and the information above suggest that such barrier crossing events occur for both blank-and gH625-NPs, but with different mechanisms related to the ability of gH625 to interact with membranes containing a high percentage of cholesterol. In particular, our results show that the majority of blank-NPs ended up in intracellular organelles, many of them lysosomes, similarly to several other NP-cell systems [41,42]. As far as we can tell, they accumulated within the lysosomes. ...
The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.
... One of the most exciting prospects that have emerged from the field of nanoscience is nanoparticle (NP) technology that are currently being incorporated and utilized to solve many intricate technical problems in modern science, chiefly in the field of medicine and biomedical science, which has given birth to the term nanomedicine. Application of NP in medical biology arises from their ability to encounter cellular machinery and potentially access to unreachable targets like the brain due to their small size [6,7]. Consequently, they have shown promising application in various branches of biomedical science such as drug delivery [8], gene delivery [9][10][11], tissue repair [12], cancer therapy, [13] disease diagnoses and therapy [14], hyperthermia [15], magnetic resonance spectroscopy [16], and as contrast agents for magnetic resonance imaging (MRI) [17]. ...
Protein corona has became a prevalent subject in the field of nanomedicine owing to its diverse role in determining the efficiency, efficacy, and the ultimate biological fate of the nanomaterials used as a tool to treat and diagnose various diseases. For instance, protein corona formation on the surface of nanoparticles can modify its physicochemical properties and interfere with its intended functionalities in the biological microenvironments. As such, much emphasis should be placed in understanding these complex phenomena that occur at the bio-nano interface. The main aim of this review is to present different factors that are influencing protein-nanoparticle interaction such as physicochemical properties of nanoparticle (i.e., size and size distribution, shape, composition, surface chemistry, and coatings) and the effect of biological microenvironments. Apart from that, the effect of ignored factors at the bio-nano interface such as temperature, plasma concentration, plasma gradient effect, administration route, and cell observer were also addressed.
... They are also limited due to lack of analytical methods and by the physical properties of the membranes used to develop two-chamber models as Transwell Õ inserts. Permeable membranes are available from a number of manufacturers and can differ widely in terms of composition, coating and pore size, all of which have the potential to introduce artefacts in cell seeding and NP interactions (Ragnaill et al., 2011;Saunders, 2009). ...
In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.
... The second aim in this report was to investigate the transport of the oxcarbazepine-loaded nanoformulations and fluorescently-labeled nanoparticles (loaded with coumarin-6) across in vitro models of human placenta (BeWo b30 cells) and the BBB (hCMEC/D3 cells) using well-established protocols. [27][28][29] Methods Materials Preparation of oxcarbazepineand coumarin-6-loaded nanoparticles ...
Encapsulation of antiepileptic drugs (AEDs) into nanoparticles may offer promise for treating pregnant women with epilepsy by improving brain delivery and limiting the transplacental permeability of AEDs to avoid fetal exposure and its consequent undesirable adverse effects. Oxcarbazepine-loaded nanoparticles were prepared by a modified solvent displacement method from biocompatible polymers (poly(lactic-co-glycolic acid) [PLGA] with or without surfactant and PEGylated PLGA [Resomer(®) RGPd5055]). The physical properties of the developed nanoparticles were determined with subsequent evaluation of their permeability across in vitro models of the blood-brain barrier (hCMEC/D3 cells) and human placental trophoblast cells (BeWo b30 cells). Oxcarbazepine-loaded nanoparticles with encapsulation efficiency above 69% were prepared with sizes ranging from 140-170 nm, polydispersity indices below 0.3, and zeta potential values below -34 mV. Differential scanning calorimetry and X-ray diffraction studies confirmed the amorphous state of the nanoencapsulated drug. The apparent permeability (Pe ) values of the free and nanoencapsulated oxcarbazepine were comparable across both cell types, likely due to rapid drug release kinetics. Transport studies using fluorescently-labeled nanoparticles (loaded with coumarin-6) demonstrated increased permeability of surfactant-coated nanoparticles. Future developments in enzyme-prodrug therapy and targeted delivery are expected to provide improved options for pregnant patients with epilepsy.
... It is plausible to assume that this dynamic protein exchange occurred during the translocation across membrane barriers by either transcellular endo-and exocytosis, or by paracellular transport mechanisms. While there are some in vitro studies suggesting protein exchange on NP surfaces during membrane crossing we are not aware of any in vivo studies having shown such exchange [17][18][19]. Hence, our in vivo studies together with our in vitro studies as well as other in vitro studies support the notion that protein binding on NP surfaces does undergo dynamic protein exchange when NP cross organ membranes. ...
When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP) this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumu-lation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon) for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm) of gold spheres (AuNP), surface-modified with the same ionic ligand; as well as 5 nm AuNP
Glioblastoma multiforme is a primary brain tumor whose diagnosis carries with it a dismal prognosis for survival. The development of nanomedicine would lay a path to cross the hurdles that current treatments fail to overcome: the blood-brain barrier (BBB) and the tumor’s immune microenvironment. Targeted drug delivery systems are responsible for releasing the chemotherapeutic drug into specific tumor cells, which in addition to allowing crossing the BBB, reduces the damage caused to healthy cells in conventional chemotherapy. However, this type of therapy is still in its infancy and its health effects are still being studied using murine models. The present project aims to determine whether the use of nanoparticles in targeted drug delivery for the treatment of glioblastoma has an inhibitory effect on tumor cell growth, so a systematic review was developed using a defined search strategy using the key terms focused on the research question. The steps and guidelines defined in the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) for systematic reviews were followed. The analysis of the data extracted from the articles included in the review indicates that there is an inhibitory effect on the proliferative activity of tumor cells and a reduction in tumor size when nanoparticles are used to encapsulate drugs in targeted delivery.KeywordsNanoparticlesDrug deliveryCancer
Nanoparticle systems are often used to facilitate drug delivery to the central nervous system (CNS). There are many clinical situations in which CNS tissue might be removed prior to administration of a therapeutic nanoparticle; however, the iatrogenic effects of surgical resection on nanoparticle deposition in the brain remain unknown. We hypothesized that resection would facilitate nanoparticle delivery to peri-resection tissue as a function of timing of nanoparticle administration after removal of tissue. To test this hypothesis polystyrene nanoparticles surface modified with poly(ethylene glycol) (PEG) were administered either immediately, 2 hours, 24 hours, 4 days, or 7 days after resection of murine cortex. Fluorescence microscopy revealed that minimal nanoparticle delivery to brain vasculature was observed in healthy mice, yet significant nanoparticle delivery was observed in mice that received resection. Spatially, nanoparticles were confined to the vascular compartment and did not enter the parenchyma. Nanoparticle delivery was high near the resection boundary and declined with distance into the peri-resection tissue. The highest level of delivery was observed when nanoparticles were administered immediately after resection, and FNPs could be detected in the CNS when nanoparticles were administered up to 24 hours after resection. The diameter of blood vessels that contained nanoparticles was significantly greater than the diameter of blood vessels that did not contain nanoparticles, and larger vessels contained brighter clusters of nanoparticles. These relationships depended on time after resection, suggesting that a dynamic vascular response. These studies highlight important considerations that can be used to develop nanotechnology for neurosurgical applications.
The administration of drugs to the CNS has become a difficult task because several barriers in the brain, i.e. the blood-brain barrier (BBB) and a blood-cerebrospinal fluid barrier must be overcome. Several methods to assist in overcoming the BBB have been implemented in recent years, such as osmotic obstruction with the BBB and chemical amplification of prodrugs. Brain targeting creates new doors for scientists to move forward for better investigations so that people with brain diseases can be treated. The current demand is to establish drug delivery techniques to allow the molecules of the drug to efficiently cross the BBB and this study discusses these strategies that efficiently improve the delivery of brain-targeted drugs. Therefore, drugs powered by nanoparticles are being developed via advanced non-invasive methods to control neurological disorders. This study will explore and examine the anatomy and physiology of the BBB, the concept of Magic Bullet, the pathways for drug transport, drug permeability assessment strategies for BBB, and endogenous cells possible interventions as nanoparticles supply cells.
The blood–brain barrier (BBB), a unique structure in the central nervous system (CNS), protects the brain from bloodborne pathogens by its excellent barrier properties. Nevertheless, this barrier limits therapeutic efficacy and becomes one of the biggest challenges in new drug development for neurodegenerative disease and brain cancer. Recent breakthroughs in nanotechnology have resulted in various nanoparticles (NPs) as drug carriers to cross the BBB by different methods. This review presents the current understanding of advanced NP-mediated non-invasive drug delivery for the treatment of neurological disorders. Herein, the complex compositions and special characteristics of BBB are elucidated exhaustively. Moreover, versatile drug nanocarriers with their recent applications and their pathways on different drug delivery strategies to overcome the formidable BBB obstacle are briefly discussed. In terms of significance, this paper provides a general understanding of how various properties of nanoparticles aid in drug delivery through BBB and usher the development of novel nanotechnology-based nanomaterials for cerebral disease therapies.
Predicting the therapeutic efficacy of a nanocarrier, in a rapid and cost-effective way, is pivotal for the drug delivery to the central nervous systems (CNS). In this context, in vitro testing platforms, like the transwell systems, offer numerous advantages to study the passage through the blood-brain barrier (BBB), such as overcoming ethical and methodological issues of in vivo models. However, the use of different transwell filters and nanocarriers with various physical-chemical features makes difficult to assess the nanocarrier efficacy and achieve data reproducibility. In this work, we performed a systematic study to elucidate the role of the most widely used transwell filters in affecting the passage of nanocarriers, as a function of filter pore size and density. In particular, the transport of carboxyl- and amine-modified 100 nm polystyrene nanoparticles (NPs), chosen as model nanocarriers, was quantified and compared to the behavior of Lucifer yellow (LY), a molecular marker of paracellular transport. Results indicate that the filter type affects the growth and formation of the confluent endothelial barrier, as well as the transport of NPs. Interestingly, the in situ dispersion of NPs was found to play a key role in governing their passage through the filters, both in absence and in presence of the cellular barrier. By framing the underlying nanobiointeractions, we found that particle-specific effects modulated cellular uptake and barrier intracellular distribution, eventually governing transcytosis through their interplay with “size exclusion effects” by the porous filters. This study highlights the importance of a careful evaluation of the physical-chemical profile of the tested nanocarrier along with filter parameters, for a correct methodological approach to test BBB permeability in nanomedicine.
In recent years, the scientific community has witnessed an exponential increase in the use of nanomaterials for biomedical applications. In particular, the interest of graphene and graphene-based materials has rapidly risen in the neuroscience field due to the properties of this material, such as high conductivity, transparency and flexibility. As for any new material that aims to play a role in the biomedical area, a fundamental aspect is the evaluation of its toxicity, which strongly depends on material composition, chemical functionalization and dimensions. Furthermore, a wide variety of three-dimensional scaffolds have also started to be exploited as a substrate for tissue engineering. In this application, the topography is probably the most relevant amongst the various properties of the different materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation.
Cerium dioxide nanoparticles (CeO2-NPs) have a variety of uses, especially in the
production of solar panels, oxygen pumps, gas sensors, computer chips and catalytic
converters. Despite their worldwide use, the few published studies demonstrate that
metallic nanoparticles, in general, are still not properly characterized in terms of their
potencial ecotoxicological effects. CeO2-NPs, in particular, have demonstrated extreme
antioxidant activity, but their in vivo toxicity is still unknown. This work intended to
characterize the chronic toxicity (28 days) of three different ecologically relevant
concentrations (0.1, 0.01, and 0.001 µg/L) of CeO2-NPs in the rainbow trout
(Oncorhynchus mykiss), in terms of biomarkers of oxidative stress [activity of the
enzymes glutathione S-transferases (GSTs) and catalase (CAT)] and neurotoxicity
[activity of the enzyme acetylcholinesterase (AChE)], as well as histological alterations
in liver and gills. In the hereby study, GSTs activity was increased in gills of fish exposed
to the highest CeO2–NPs level. Moreover, a potential anti-oxidant response was also
reported, with a significant increase of CAT activity observed in livers of the same fish.
AChE, however, was not significantly altered in fish eyes. Individuals exposed to CeO2-
NPs also presented marked changes in the gills (e.g. epithelial lifting, intercellular edema,
lamellar hypertrophy and hyperplasia, secondary lamella fusion and aneurysms) and liver
(e.g. hepatocyte vacuolization, pyknotic nucleus, enlargement of sinusoids and
hyperemia). The semi-quantitative analysis (organs pathological index) also showed the
establishment of a dose-effect relationship. Further studies about the ecotoxicological
effects of the CeO2-NPs have yet to be conducted, considering their properties, as the
aggregation chemistry and the ratio of its redox state, which may affect their availability
to the organism and their toxicity in the environment and biota.
The blood brain barrier (BBB) offers protection to the central nervous system (CNS) by maintaining homeostasis. Besides restricting the entry of harmful xenobiotics and endogenous molecules to CNS, BBB also limits the entry of therapeutic agents. Nanotechnology offers the possibility to deliver small molecules/drugs against CNS disorders across BBB. Recently, due to excellent biocompatibility, functional versatility and unique surface electrostatics properties nanodiamonds are also being explored for their drug delivery potential against BBB. Although, studies has been done to understand the mechanisms involved in drug delivery across BBB, but still, progress in the development of any functional drug delivery systems across BBB is in juvenile stages. For this purpose, the strategic developments of more potent drug delivery systems to CNS via endocytic pathways appears to be the most promising approach. Endocytic pathways are essential for communication and transport of nutrients across BBB in endothelial cells. Moreover, modifications of the nanotherapeutic agents with specific brain capillary endothelial cells targeting molecules or ligands have recently shown impressive results of successful delivery of drugs to the brain via receptor-mediated transcytosis. The current review provides a comprehensive insight in the advancements made in nanocarriers-based successful drug delivery to the CNS and their availability in clinics.
Mucus is an endogenous viscoelastic biopolymer barrier that limits the entry of foreign pathogens and therapeutic carriers to the underlying mucosal cells. This could be overcome with a hydrophilic and nonpositively charged carrier surface that minimizes interactions with the mucin glycoprotein fibers. Although PEGylation remains an attractive surface strategy to enhance mucopenetration, cell uptake of PEGylated nanoparticles (NPs) often remains poor. Here, we demonstrated polydopamine (PDA) coating to enhance both mucopenetration and cell uptake of NPs. PDA was polymerized on carboxylated polystyrene (PS) NPs to form a PDA coating, and the resulting PS-PDA achieved a similar level of mucopenetration as our PEGylated PS (PS-PEG) positive control in three separate studies: NP-mucin interaction test, transwell assay, and multiple particle tracking. Compared to water, the diffusions of PS-PDA and PS-PEG in reconstituted mucus solution were only 3.5 and 2.4 times slower, respectively, whereas the diffusion of bare PS was slowed by up to 250 times. However, the uptake of PS-PDA (61.2 ± 6.1%) was almost three times higher than PS-PEG (24.6 ± 5.4%) in T24 cells, which were used as a model for underlying mucosal cells. Our results showed a novel unreported functionality of PDA coating in enhancing both mucopenetration and cell uptake of NPs for mucosal drug delivery applications, not possible with conventional PEGylation strategies.
In order to improve the current success of nanomedicine, a better understanding of how nano-sized materials interact with and are processed by cells is required. Typical in vitro nanoparticle-cell interaction studies often make use of cells cultured at different cell densities. However, in vivo, for their successful delivery to the target tissue, nanomedicines need to overcome several barriers, such as endothelial and epithelial cell barriers. Unlike sub-confluent or confluent cell cultures, cell barriers are tight cell monolayers, expressing a series of specialized tight junction proteins between adjacent cells to limit paracellular transport and ensure close cell-to-cell interactions. A clear understanding on how development of cells into a cell barrier may affect uptake of nano-sized drug carriers, is still missing. To this aim, here, human primary umbilical vein endothelial cells (HUVEC) are used as a model cell line to form endothelial cell barriers. Then, nanoparticle uptake is assessed in the developed endothelial barriers and compared to to the uptake in sub-confluent or confluent HUVEC cultures. The results clearly show that the organization of cells into a cell barrier leads to a differential gene expression of endocytic markers, and – interestingly - this is accompanied by reduced nanoparticle uptake levels. Transport inhibitors are used to characterise the mechanisms involved in the uptake. However we show that some of them can strongly compromise barrier integrity, thus impairing the interpretation of the outcomes, and overall, only a partial inhibition of nanoparticle uptake could be obtained.
Nanoparticles have distinct properties and these properties are rapidly revolutionizing the biomedical applications. Despite of development in nanoscience, there is quite little understanding about the interaction of nanomaterials with living systems for the safe and proficient application. It is believed that in a biological medium, proteins interact with nanoparticles and compete for the surface of nanoparticles leading to formation of protein capping which modify the physicochemical properties of nanoparticles. The biological fluid observes nanoparticles with modified surface hence composition of capping proteins become responsible for the further cellular response. In this chapter, we present different factors which are responsible for variations in corona composition such as physicochemical properties of NPs (e.g., size, shape, surface charge, composition, surface functional groups, coatings and hydrophilicity/hydrophobicity) and influence of biological environment. Aside from that impact of ignored issues at bionano interface like administration route, temperature, cell observer, plasma concentration and plasma gradient effect will also be discussed.
The interface between the Central Nervous System (CNS) and the blood, known as the Blood Brain Barrier (BBB), must possess highly effective mechanisms for facilitating the transport of specific nutrients and limiting the transport of xenobiotics. The endothelial cells are major constituents of the BBB; they are characterized by tight junctions and the presence of the ATP Binding Cassette (ABC) transporter family. These transporters regulate the exit of compounds from the endothelial cells. An understanding of how these compounds could interact with the ABC transporters expressed at the BBB, could play a key role in drug research and development. Currently, there is neither a standard BBB pharmacological model nor a method for studying drug permeability and ABC transporter interactions. A pharmacological model must be available, quick to implement and inexpensive.
The main parameters useful for evaluating the interaction of the BBB with ABC transporters are bilateral studies and the efflux ratio determination. These parameters could be influenced by different parameters, such as cell lines, apparent permeability, choice of inserts, and other parameters, which would be detailed in this review.
Two major hurdles in nanomedicine are the limited strategies for synthesizing stealth nanoparticles and poor efficacy of the nanoparticles in translocating across the blood brain barrier (BBB). Here we examined the uptake and transcytosis of iron oxide nanoparticles (IONPs) grafted with biomimetic phosphorylcholine (PC) brushes in an in vitro BBB model system, and compared it with bare, PEG or PC-PEG mixture grafted IONPs. Hyperspectral imaging indicated IONP co-localization with cells. Quantitative analysis with total reflection X-ray fluorescence spectrometry showed that after 24 h, 78% of PC grafted, 68-69% of PEG or PC-PEG grafted, and 30% of bare IONPs were taken up by the BBB. Transcytosis of IONPs was time-dependent and after 24 h, 16-17% of PC or PC-PEG mixture grafted IONPs had passed the model BBB, significantly more than PEG grafted or bare IONPs. These findings point to PC as a viable grafting strategy for the uptake and transcytosis of nanoparticles.
Brain microvessel endothelial cells (BMEC) are the main structural and dynamic component of the blood-brain barrier (BBB), preventing the majority of drugs from reaching the brain. Since BMEC are involved in a wide range of central nervous system diseases, the development of nanocarriers that trigger receptor-mediated uptake in these cells has been suggested as a promising approach to increase drug delivery to the brain. Here, we report the size and the bioconjugation effects of antibody-conjugated mesoporous silica nanoparticles (MSN) on in vitro and in vivo targeting ability to BMEC. For this, Ri7 antibody was conjugated to MSN of two different sizes (50 nm and 160 nm diameter) through a polyethylene glycol (PEG) linker. The particles were also functionalized with a MRI contrast agent (gadolinium chelate) and with a fluorescent label. The functionalized MSN suspensions showed good colloidal stability. The Ri7 antibody immobilized on the MSN surface maintained its high specific activity and high binding affinity, as demonstrated in vitro. Cells incubated with gadolinium-chelated Ri7-MSN showed a significant MRI positive contrast enhancement, highlighting the potential of such nanoparticles for theranostic applications. To measure the uptake and affinity of Ri7-MSN nanoparticles to brain endothelial and neuronal cells, cell uptake studies were performed and a quantitative cellular assay was developed. The results revealed that endocytosis of nanoparticles is mediated by transferrin receptors and that Ri7-MSN cellular uptake is size- and time-dependent. A highest specific uptake was found with the 50 nm Ri7-MSN. Upon intravenous injection, 50 nm Ri7-MSN were specifically accumulated in BMEC, suggesting the strong potential of antibody-coated nanoparticles for targeting BMEC in vivo. These findings open the door to therapeutic targeting of BMEC, enabling potential therapeutic drug delivery to the brain.
Aim:
Explore the use of transferrin-receptor peptide-functionalized nanoparticles (NPs) targeting blood-brain barrier (BBB) as siRNA carriers to silence P-glycoprotein (P-gp).
Materials & methods:
Permeability experiments were assessed through a developed BBB cell-based model; P-gp mRNA expression was evaluated in vitro; rhodamine 123 permeability was assessed after cell monolayer treatment with siRNA NPs.
Results:
Beyond their ability to improve siRNA permeability through the BBB by twofold, 96-h post-transfection, functionalized polymeric NPs successfully reduced P-gp mRNA expression up to 52%, compared with nonfunctionalized systems. Subsequently, the permeability of rhodamine 123 through the human BBB model increased up to 27%.
Conclusion:
Developed BBB-targeted NPs induced P-gp downregulation and consequent increase on P-gp substrate permeability, revealing their ability to modulate drug efflux at the BBB.
Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.
The blood-brain barrier accounts for the high attrition rate of the treatments of most brain disorders, which therefore remain one of the greatest health-care challenges of the twenty first century. Against this background of hindrance to brain delivery, nanomedicine takes advantage of the assembly at the nanoscale of available biomaterials to provide a delivery platform with potential to raising brain levels of either imaging or therapeutic agents. Nevertheless, to prevent later failure due to ineffective drug levels at the target site, researchers have been endeavoring to develop a battery of in vitro screening procedures that can predict earlier in the drug discovery process the ability of these cutting-edge drug delivery platforms to cross the blood-brain barrier for biomedical purposes.
Since its approval by the FDA, Abraxane™ has been established as a clinical standard of paclitaxel (PTX)-based therapy against a variety of cancers. Despite success, Abraxane™ is still limited by suboptimal biodistribution, unfavorable pharmacokinetics and chronic toxicities from chloroform used during preparation. Accordingly, a PTX-loaded nanosuspension based on human serum albumin (HSA) with PEG modifiers (PTX-PEG-HSA) has been developed to optimize the in-vivo biodistribution, pharmacokinetics and safety of PTX over traditional PTX-HSA nanosuspensions prepared using the accepted method for Abraxane™. Results of in-vivo pharmacokinetic (PK) studies indicated PTX-PEG-HSA achieved prolonged blood circulation, illustrated by a 8.8-fold and 4.8-fold increase in area-under-the-curve (AUC) of PTX over Taxol® and PTX-HSA, while the mean residence time (MRT) of PTX in PTX-PEG-HSA was increased by 3.2-fold and 1.5-fold, respectively. HSA mediated active targeting further suppressed non-specific distribution of PTX to normal tissues, which permitted enhanced antitumor efficacy in S180 mice over Taxol® and PTX-HSA. Safety of intravenously administered PTX-PEG-HSA was confirmed through lower hemolytic activity, a 2.2-fold and 1.2-fold increase in LD50 (113.4mg/kg) over Taxol® and PTX-HSA alongside the absence of local venous irritation. Studies herein suggest the therapeutic and clinical applicability of PTX-PEG-HSA for tumor specific therapy.
Size does matter! Shrinking a bulk material to the nanoscale introduces new features that need to be taken into consideration for toxicity testing of engineered nanomaterials (ENM). Validated toxicity tests are highly desirable in the field of nanotoxicology. Tests recommended or scientifically validated by the Organisation for Economic Cooperation and Development (OECD) need to be adapted to fit to the unique features related to the nano-size of ENM. The small size of nanoparticles enables transfer across biological barriers and thus methods for detection and uptake must be an integral part of the testing strategy. Toxicity can be influenced by interactions with macromolecules due to increased surface area and reactivity occurring concomitantly with a decrease in size. Careful thought should be given to selection of in vitro end-points to avoid interference between the nanoparticles and the assay, and appropriate controls and reference material should be considered. In vitro tests should always be closely linked with in vivo studies.
Drug delivery to the brain is one of the major challenges in the treatment of cerebral diseases and implies extensive understanding of nanomedicine transcytosis pathways across the Blood-Brain Barrier (BBB). In this study, we investigated the interaction of squalenoyl-adenosine nanoassemblies (SQAd NAs) with human brain endothelial cells, concerning their endocytotic pathway using chemical inhibitors, and nanostructure integrity using Förster Resonance Energy Transfer (FRET). Practically, SQAd NAs were labeled with two different organic dyes as a donor-acceptor FRET pair to form FRET SQAd NAs with diameters of ca. 120 nm. Using the human cerebral endothelial cell line, hCMEC/D3, as a well-recognized BBB model, we demonstrated that the NAs were internalized mainly by LDL receptors-mediated endocytosis, then progressively disassembled inside the cells, and finally exocytosed as single molecules. These observations allow explaining the previously described pharmacological efficiency of the SQAd NAs in both a cerebral ischemia model and a spinal cord injury model, confirming that the endothelial cells of the neurovascular unit may represent a very promising therapeutic target for the treatment of certain neurological diseases.
Poly(lactic acid), which has an inherent tendency to form colloidal systems of low polydispersity, and alkylglyceryl-modified dextran - a material designed to combine the non-immunogenic and stabilising properties of dextran with the demonstrated permeation enhancing ability of alkylglycerols - have been combined for the development of nanoparticulate, blood brain barrier-permeating, non-viral vectors. To this end, dextran, that had been functionalised via treatment with epoxide precursors of alkylglycerol, was covalently linked to poly(lactic acid) using a carbodiimide cross-linker to form alkylglyceryl-modified dextran-graft-poly(lactic acid). Solvent displacement and electrospray methods allowed the formulation of these materials into nanoparticles having a unimodal size distribution profile of about 100 - 200 nm and good stability at physiologically relevant pH (7.4). The nanoparticles were characterised in terms of hydrodynamic size (by Dynamic Light Scattering and Nanoparticle Tracking Analysis), morphology (by Scanning Electron Microscopy and Atomic Force Microscopy) and zeta potential, and their toxicity was evaluated using MTT and Presto Blue assays. Cellular uptake was evidenced by confocal microscopy employing nanoparticles that had been loaded with the easy-to-detect Rhodamine B fluorescent marker. Transwell-model experiments employing mouse (bEnd3) and human (hCMEC/D3) brain endothelial cells revealed enhanced permeation (statistically significant for hCMEC/D3) of the fluorescent markers in the presence of the nanoparticles. Results of studies using Electric Cell Substrate Impedance Sensing suggested a transient decrease of the barrier function in an in vitro blood-brain barrier model following incubation with these nanoformulations. An in ovo study using 3-day chicken embryos indicated the absence of whole-organism acute toxicity effects. The collective in vitro data suggest that these alkylglyceryl-modified dextran-graft-poly(lactic acid) nanoparticles are promising candidates for in vivo evaluations that would test their capability to transport therapeutic actives to the brain.
Copyright © 2015. Published by Elsevier Ltd.
Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, have generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organllel identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.
Nanomaterials are the main products of nanotechnology. In this paper we describe some of these nanomaterials, particularly Carbon based systems, their properties, manipulation strategies and applications. History of nanoparticles, as well as the comple-xity in defining nanomaterials, necessary for regulation and assessment of impacts on society, are also addressed.
RNA interference (RNAi) holds one of the promising tools for Alzheimer's disease (AD) treatment by directly arresting the causative genes. For successful RNAi therapeutics for AD, limited access of therapeutic genes to the brain needs to be overcome by developing siRNA delivery system that could cross the blood–brain barrier (BBB). Here, we report a non-viral vector, rabies virus glycoprotein (RVG)-modified poly(mannitol-co-PEI) gene transporter (PMT), R-PEG-PMT. The RVG ligand directed the PMT/siRNA complexes toward the brain through binding to nicotinic acetylcholine receptors expressed on BBB. In mechanistic study using in vitro BBB model, we observed that osmotically-active PMT enhanced the receptor-mediated transcytosis by stimulating the caveolar endocytosis. The potential of RNAi therapeutics for AD using R-PEG-PMT/siBACE1 complexes was demonstrated in vitro and in vivo. Our results suggest that R-PEG-PMT is a powerful gene carrier system for brain targeted RNAi therapeutics with synergistic effect of RVG ligand and PMT on well-modulated receptor-mediated transcytosis through BBB.
Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimer's, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered.
The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood-brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para- or transcellular permeabilities (P(e)) of inulin (0.74 x 10(-3) cm/min), sucrose (1.60 x 10(-3) cm/min), lucifer yellow (1.33 x 10(-3) cm/min), morphine (5.36 x 10(-3) cm/min), propranolol (4.49 x 10(-3) cm/min) and midazolam (5.13 x 10(-3) cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1-5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1-mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p-glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood-brain barrier.
Nanoparticles in a biological fluid (plasma, or otherwise) associate with a range of biopolymers, especially proteins, organized into the “protein corona” that is associated with the nanoparticle and continuously exchanging with the proteins in the environment. Methodologies to determine the corona and to understand its dependence on nanomaterial properties are likely to become important in bionanoscience. Here, we study the long-lived (“hard”) protein corona formed from human plasma for a range of nanoparticles that differ in surface properties and size. Six different polystyrene nanoparticles were studied: three different surface chemistries (plain PS, carboxyl-modified, and amine-modified) and two sizes of each (50 and 100 nm), enabling us to perform systematic studies of the effect of surface properties and size on the detailed protein coronas. Proteins in the corona that are conserved and unique across the nanoparticle types were identified and classified according to the protein functional properties. Remarkably, both size and surface properties were found to play a very significant role in determining the nanoparticle coronas on the different particles of identical materials. We comment on the future need for scientific understanding, characterization, and possibly some additional emphasis on standards for the surfaces of nanoparticles.
• bionanoscience
• mass spectrometry
• interactions
• proteomics
• human plasma
The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.
Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.
Drug delivery to the brain is becoming more and more important but is severely restricted by the blood-brain barrier. Nanoparticles coated with polysorbates have previously been shown to enable the transport of several drugs across the blood-brain barrier, which under normal circumstances is impermeable to these compounds. Apolipoprotein E was suggested to mediate this drug transport across the blood-brain barrier. In the present study, apolipoprotein E was coupled by chemical methods to nanoparticles made of human serum albumin (HSA-NP). Loperamide, which does not cross the blood-brain barrier but exerts antinociceptive effects after direct injection into the brain, was used as model drug. Apolipoprotein E was chemically bound via linkers to loperamide-loaded HSA-NP. This preparation induced antinociceptive effects in the tail-flick test in ICR mice after i.v. injection. In contrast, nanoparticles linked to apolipoprotein E variants that do not recognize lipoprotein receptors failed to induce these effects. These results indicate that apolipoprotein E attached to the surface of nanoparticles facilitates transport of drugs across the blood-brain barrier, probably after interaction with lipoprotein receptors on the brain capillary endothelial cell membranes.
Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization.
Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles.
Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha.
Most research on the toxicology of nanomaterials has focused on the effects of nanoparticles that enter the body accidentally. There has been much less research on the toxicology of nanoparticles that are used for biomedical applications, such as drug delivery or imaging, in which the nanoparticles are deliberately placed in the body. Moreover, there are no harmonized standards for assessing the toxicity of nanoparticles to the immune system (immunotoxicity). Here we review recent research on immunotoxicity, along with data on a range of nanotechnology-based drugs that are at different stages in the approval process. Research shows that nanoparticles can stimulate and/or suppress the immune responses, and that their compatibility with the immune system is largely determined by their surface chemistry. Modifying these factors can significantly reduce the immunotoxicity of nanoparticles and make them useful platforms for drug delivery.
Although human polyomavirus JC (JCV) is known to cause progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, the mechanism by which JCV crosses the blood-brain barrier (BBB) remains unclear. To test our hypothesis that cell-free JCV gains entry into the brain by infecting endothelial cells, we inoculated human brain microvascular endothelial (HBMVE) cells with 50 HAU (1.33+/-0.27 x 10(7) genome copies) of JCV(Mad1) and analyzed the expression of early and late viral genes and proteins by immunocytochemistry, quantitative real-time PCR (qPCR), quantitative real-time reverse transcriptase PCR (qRT-PCR) and immunoprecipitation followed by Western blotting. JCV infected and replicated efficiently in HBMVE cells and produced infectious virions several hundred fold higher than the infecting inoculum. HBMVE cells in vitro did not express serotonin receptor 2A (5HT(2A)R), and 5HT(2A)R blockers did not prevent JCV infection of HBMVE cells. Collectively, our data indicate that the productive in vitro infection of HBMVE cells by JCV is independent of 5HT(2A)R.
Nanoparticles made of human serum albumin (HSA) and modified with apolipoproteins have previously been shown to transport drugs, which normally do not enter the brain, across the blood-brain barrier (BBB). However the precise mechanism by which nanoparticles with different apolipoproteins on their surface can target to the brain, as yet, has not been totally elucidated. In the present study, HSA nanoparticles with covalently bound apolipoprotein A-I (Apo A-I) as a targetor for brain capillary endothelial cells were injected intravenously into SV 129 mice and Wistar rats. The rodents were sacrificed after 15 or 30 min, and their brains were examined by transmission electron microscopy. Apo A-I nanoparticles could be found inside the endothelial cells of brain capillaries as well as within parenchymal brain tissue of both, mice and rats, whereas control particles without Apo A-I on their surface did not cross the BBB during our experiments. The maintenance of tight junction integrity and barrier function during treatment with nanoparticles was demonstrated by perfusion with a fixative containing lanthanum nitrate as an electron dense marker for the permeability of tight junctions.
Engineered nanoparticles (NPs) are in the same size category as atmospheric ultrafine particles, < 100 nm. Per given volume, both have high numbers and surface areas compared to larger particles. The high proportion of surface atoms/molecules can give rise to a greater chemical as well as biological activity, for example the induction of reactive oxygen species in cell-free medium as well as in cells. When inhaled as singlet particles, NPs of different sizes deposit efficiently in all regions of the respiratory tract by diffusion. A major difference to larger size particles is the propensity of NPs to translocate across cell barriers from the portal of entry (e.g., the respiratory tract) to secondary organs and to enter cells by various mechanisms and associate with subcellular structures. This makes NPs uniquely suitable for therapeutic and diagnostic uses, but it also leaves target organs such as the central nervous system (CNS) vulnerable to potential adverse effects (e.g., oxidative stress). Neuronal transport of NPs has been described, involving retrograde and anterograde movement in axons and dendrites as well as perineural translocation. This is of importance for access of inhaled NPs to the CNS via sensory nerves existing in the nasopharyngeal and tracheobronchial regions of the respiratory tract. The neuronal pathway circumvents the very tight blood brain barrier. In general, translocation rates of NP from the portal of entry into the blood compartment or the CNS are very low. Important modifiers of translocation are the physicochemical characteristics of NPs, most notably their size and surface properties, particularly surface chemistry. Primary surface coating (when NPs are manufactured) and secondary surface coating (adsorption of lipids/proteins occurring at the portal of entry and during subsequent translocation) can significantly alter NP biokinetics and their effects. Implications of species differences in respiratory tract anatomy, breathing pattern and brain anatomy for extrapolation to humans of NP effects observed in rodents need to be considered. Although there are anecdotal data indicating a causal relationship between long-term ultrafine particle exposures in ambient air (e.g., traffic related) or at the workplace (e.g., metal fumes) and resultant neurotoxic effects in humans, more studies are needed to test the hypothesis that inhaled nanoparticles cause neurodegenerative effects. Some but probably not the majority of NPs will have a significant toxicity (hazard) potential, and this will pose a significant risk if there is a sufficient exposure. The challenge is to identify such hazardous NPs and take appropriate measures to prevent exposure.
Fluorescence correlation spectroscopy is used as a quantitative method to understand the binding and exchange behaviour of proteins on the surfaces of nanoparticles.
In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull-down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high-density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A-I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions.
One of the most challenging problems, if not the most challenging, in drug development is not to develop drugs to treat diseases of the central nervous system (CNS), but to manage to distribute them to the CNS across the blood-brain barrier (BBB) using transvascular routes following intravenous administration. The development of BBB targeting technologies is a very active field of research and development. One goal is to develop chemically modified derivatives of drugs or chemically modified nanoparticulate vectors of drugs, capable of crossing biological barriers, in particular the BBB. This manuscript will review the approaches that have been explored to achieve these goals, using chemical functionalization of drugs or of drug vector systems and endogenous transporters at the BBB.
Elevated concentrations of particulate matter in the environmental atmosphere constitute a potential risk to human health. In vitro cell-based assays are therefore necessary to evaluate the toxicological potential of inhaled particulate emissions. In this study, the exposure of a co-culture cell model at the air-liquid interface was used to evaluate the dose-dependent biological effects of a test aerosol. The CULTEX system was used to expose human cells to an environmentally-relevant aerosol, generated from fly ash collected in a commercial municipal waste incinerator and resuspended in filtered air. Human bronchial epithelial cells, BEAS-2B, co-cultured with differentiated THP-1 macrophages growing on Transwell inserts, were employed in the bioassay. Analyses of cell viability, interleukin-8 (IL-8) release, intracellular glutathione, and haeme oxygenase-1 enzyme expression were performed. Transportation of the cells and exposure to humidified filtered air or the test aerosol, at 100 ml/min for 1 to 6 hours, were well tolerated by the cells and had no effect on their viability. Levels of IL-8 release and haeme oxygenase-1 expression were elevated by exposure to fly ash aerosol as a function of time, but not by exposure to clean air. For IL-8 release, a dose-dependent effect was demonstrated with the assumption that the deposited mass of the particles correlated with exposure time. Exposure to the test aerosol did not affect the intracellular glutathione concentration. This in vitro approach simulates particle deposition in the human lung more realistically than does submerged exposure, and it preserves the inherent properties of the particles. It shows promise for use to detect particulate emissions which are potentially detrimental to human health.
An in vitro model of the feline blood-brain barrier was developed using primary cultures of brain capillary endothelial cells derived from adult cats. They were grown in the presence of astrocytes obtained from newborn kittens. Feline endothelial cell cultures were characterised by uptake of DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) and expression of von Willebrand factor. Astrocytes were characterised based on their expression of glial fibrillary acidic protein (GFAP). Electron microscopy revealed junctional specialisation between endothelial cells. Occludin and ZO-1 expression by the endothelial cell cultures was detected by Western blot analysis. Barrier function of co-cultured endothelial cells and astrocytes was confirmed by a transendothelial electrical resistance (TEER) value of 30-35 Omegacm2 and apparent permeability coefficients (Papp) for FD-40 (FITC-dextran, 40 kDa) of 4x10(-6) cm/s and for FD-4 (4kDa) of 1.92x10(-5) cm/s. In endothelial cell monolayers grown with astrocyte-conditioned medium, the TEER value was lower (20-25 Omegacm2), and Papp of FD-40 and FD-4 was higher at 6.27x10(-6) and 3.96x10(-5) cm/s, respectively. This model should have useful applications in the examination of events occurring at the BBB early in FIV infection, and may provide knowledge applicable to HIV infection.
The blood-brain barrier, which is formed by the endothelial cells that line cerebral microvessels, has an important role in maintaining a precisely regulated microenvironment for reliable neuronal signalling. At present, there is great interest in the association of brain microvessels, astrocytes and neurons to form functional 'neurovascular units', and recent studies have highlighted the importance of brain endothelial cells in this modular organization. Here, we explore specific interactions between the brain endothelium, astrocytes and neurons that may regulate blood-brain barrier function. An understanding of how these interactions are disturbed in pathological conditions could lead to the development of new protective and restorative therapies.
(Figure Presented) Blood relations: Nanoparticles that enter the bloodstream become coated with proteins. Four apolipoproteins are consistently recovered on model copolymer nanoparticles by using a centrifugation procedure (see electropherogram); their interaction with the nanoparticles is stronger than that of other plasma proteins with higher abundance.
- M Lundqvist
- J Stigler
- T Cedervall
- G Elia
- I Lynch
- K A Dawson
M. Lundqvist, J. Stigler, T. Cedervall, G. Elia, I. Lynch, K.A. Dawson, PNAS 105
(2008) 14265-14270.
- B Poller
- H Gutmann
- S Krahenbuhl
- B Weksler
- I Romero
- P O Couraud
- G Tuffin
- J Drewe
- J Huwyler
B. Poller, H. Gutmann, S. Krahenbuhl, B. Weksler, I. Romero, P.O. Couraud, G.
Tuffin, J. Drewe, J. Huwyler, J. Neurochem. 107 (5) (2008) 1358-1368.
- N F Fletcher
- D J Brayden
- B Brankin
- S Worrall
- J J Callanan
N.F. Fletcher, D.J. Brayden, B. Brankin, S. Worrall, J.J. Callanan, Vet. Immunol.
Immunopathol. 109 (3-4) (2006) 233-244.
- S Diabaté
- S Mülhopt
- H R Paur
- H F Krug
S. Diabaté, S. Mülhopt, H.R. Paur, H.F. Krug, Altern. Lab. Anim. 36 (2008) 285-298.
- I Lynch
- A Salvati
- K A Dawson
I. Lynch, A. Salvati, K.A. Dawson, Nat. Nanotechnol. 4 (2009) 546-547.
- L M Dallasta
- L A Pisarov
- J E Esplen
- J V Werely
- A V Moses
- J A Nelson
L.M. Dallasta, L.A. Pisarov, J.E. Esplen, J.V. Werely, A.V. Moses, J.A. Nelson, Am. J.
Pathol. 155 (1999) 1915-1927.
- B Rothen-Rutishauser
- C Mühlfeld
- F Blank
- C Musso
- P Gehr
B. Rothen-Rutishauser, C. Mühlfeld, F. Blank, C. Musso, P. Gehr, Part. Fibre
Toxicol. 4 (2007) 9.
- D Walczyk
- F Baldelli-Bombelli
- A Campbell
- I Lynch
- K A Dawson
D. Walczyk, F. Baldelli-Bombelli, A. Campbell, I. Lynch, K.A. Dawson, JACS 132
(2010) 5761-5768.
- A Zensi
- D Begley
- C Pontikis
- C Legros
- L Mihoreanu
- C Büchel
- J Kreuter
A. Zensi, D. Begley, C. Pontikis, C. Legros, L. Mihoreanu, C. Büchel, J. Kreuter, J.
Drug Target. 18 (10) (2010) 842-848.
- E Hellstrand
- I Lynch
- A Andersson
- T Drakenberg
- B Dahlbäck
- K A Dawson
- S Linse
- T Cedervall
E. Hellstrand, I. Lynch, A. Andersson, T. Drakenberg, B. Dahlbäck, K.A. Dawson,
S. Linse, T. Cedervall, FEBS J. 276 (2009) 3372-3381.
Experimental and theoretical approach to comparative nanoparticle and small molecule intracellular import, trafficking, and export, nanomedicine
- A Salvati
- T Santos
- J Varela
- C Åberg
- P Pinto
- I Lynch
- K A Dawson
A. Salvati, T. Dos Santos, J. Varela, C. Åberg, P. Pinto, I. Lynch, K.A. Dawson,
Experimental and theoretical approach to comparative nanoparticle and small
molecule intracellular import, trafficking, and export, nanomedicine, in press.
- S Bhaskar
- F Tian
- T Stoeger
- W Kreyling
- J M De La Fuente
- V Grazú
- P Borm
- G Estrada
- V Ntziachristos
- D Razansky
S. Bhaskar, F. Tian, T. Stoeger, W. Kreyling, J.M. de la Fuente, V. Grazú, P. Borm,
G. Estrada, V. Ntziachristos, D. Razansky, Part. Fibre Toxicol. 7 (2010) 3.
- M A Dobrovolskaia
- S E Mcneil
M.A. Dobrovolskaia, S.E. McNeil, Nat. Nanotechnol. 2 (8) (2007) 469-478.
- B B Weksler
- E A Subileau
- N Perriere
- P Charneau
- K Holloway
- M Leveque
- H Tricoire-Leignel
- A Nicotra
- S Bourdoulous
- P Turowski
- D K Male
- F Roux
- J Greenwood
- I A Romero
- P O Couraud
B.B. Weksler, E.A. Subileau, N. Perriere, P. Charneau, K. Holloway, M. Leveque,
H. Tricoire-Leignel, A. Nicotra, S. Bourdoulous, P. Turowski, D.K. Male, F. Roux, J.
Greenwood, I.A. Romero, P.O. Couraud, FASEB J. 19 (13) (2005) 1872-1874.
- K Michaelis
- M M Hoffmann
- S Dreis
- E Herbert
- R N Alyautdin
- M Michaelis
- J Kreuter
- K Langer
K. Michaelis, M.M. Hoffmann, S. Dreis, E. Herbert, R.N. Alyautdin, M. Michaelis,
J. Kreuter, K. Langer, J. Pharm. 317 (2006) 1246-1253.