Influence of Size and Shape on the Anatomical Distribution of

May 30, 2017 - Influence of Size and Shape on the Anatomical Distribution of Endotoxin-Free Gold Nanoparticles ... To this goal, quantitative data ach...
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Influence of Size and Shape on the Anatomical Distribution of Endotoxin-Free Gold Nanoparticles Laura Talamini,†,° Martina B. Violatto,†,° Qi Cai,‡,° Marco P. Monopoli,‡,⊥ Karsten Kantner,§ Ž eljka Krpetić,‡,# André Perez-Potti,‡ Jennifer Cookman,‡ David Garry,‡ Camila P. Silveira,‡ Luca Boselli,‡ Beatriz Pelaz,§ Tommaso Serchi,∥ Sébastien Cambier,∥ Arno C. Gutleb,∥ Neus Feliu,§,∇,¶ Yan Yan,‡ Mario Salmona,† Wolfgang J. Parak,*,§ Kenneth A. Dawson,*,‡ and Paolo Bigini*,† †

IRCCS-Istituto di Ricerche Farmacologiche Mario, Negri, Milan, 20156, Italy Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Dublin 4, Ireland § Fachbereich Physik, Philipps University of Marburg, Marburg, 35037, Germany ∥ Environmental Health group, Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology (LIST), L-4362, Luxembourg ⊥ RCSI Pharmaceutical and Medical Chemistry, Royal College of Surgeons in Ireland, St. Stephen’s Green, Dublin, Dublin 2, Ireland # School of Environment and Life Sciences, University of Salford Manchester, Salford, M5 4WT, United Kingdom ∇ Department of Laboratory Medicine (LABMED), Karolinska Institutet, Stockholm, 171 77, Sweden ¶ Medcom Advance S.A., Barcelona, 08840, Spain ‡

S Supporting Information *

ABSTRACT: The transport and the delivery of drugs through nanocarriers is a great challenge of pharmacology. Since the production of liposomes to reduce the toxicity of doxorubicin in patients, a plethora of nanomaterials have been produced and characterized. Although it is widely known that elementary properties of nanomaterials influence their in vivo kinetics, such interaction is often poorly investigated in many preclinical studies. The present study aims to evaluate the actual effect of size and shape on the biodistribution of a set of gold nanoparticles (GNPs) after intravenous administration in mice. To this goal, quantitative data achieved by inductively coupled plasma mass spectrometry and observational results emerging from histochemistry (autometallography and enhanced dark-field hyperspectral microscopy) were combined. Since the immune system plays a role in bionano-interaction we used healthy immune-competent mice. To keep the immune surveillance on the physiological levels we synthesized endotoxin-free GNPs to be tested in specific pathogen-free animals. Our study mainly reveals that (a) the size and the shape greatly influence the kinetics of accumulation and excretion of GNPs in filter organs; (b) spherical and star-like GNPs showed the same percentage of accumulation, but a different localization in liver; (c) only star-like GNPs are able to accumulate in lung; (d) changes in the geometry did not improve the passage of the blood brain barrier. Overall, this study can be considered as a reliable starting point to drive the synthesis and the functionalization of potential candidates for theranostic purposes in many fields of research. KEYWORDS: gold nanoparticles, biological barriers, biodistribution, accumulation, particle size, particle shape when tested in patients.3,4 The main obstacles that these molecules encounter once administered in vivo are their low ability to reach the pathological target, and the dangerous

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n recent years much progress in understanding cellular and molecular processes that lead to disease progression has been clarified. These discoveries have allowed the development of many compounds with a strong effectiveness toward specific cellular targets. Unfortunately, very often, the encouraging results emerging from in vitro assays have not been confirmed through in vivo studies,1,2 or completely failed © 2017 American Chemical Society

Received: January 23, 2017 Accepted: May 30, 2017 Published: May 30, 2017 5519

DOI: 10.1021/acsnano.7b00497 ACS Nano 2017, 11, 5519−5529

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Figure 1. (A) Transmission electron microscopy (TEM) micrographs of the GNP library. (B) Differential centrifugal sedimentation (DCS) analysis (on the left) and absorption spectra (on the right) of the four different GNPs. In the case of anisotropy, DCS does not represent the real particle diameter. Therefore, a representative particle distribution is reported rather than the real size because of the incorrect approximation assumed by the DCS software during measurements.

In this context, in vitro studies have highlighted the potential role of the NP external surface functionalization with antibodies, receptors, and/or peptides to both promote the passage through biological barriers and improve the uptake of specific cell targets.14,15 Unfortunately, these results have not been confirmed in preclinical models of human disorders. For example, processes, such as the binding of plasmatic proteins to the NPs, the uptake from immune-competent cells, the capture by filter organs, and finally the excretion, may influence their tropism from the site of administration to the final destination. The recent comparison on the fate of three different nanomaterials in terms of biodistribution, neutralization, and clearance9 furthermore increases the urgency to create a platform to assess the nature of the interaction between nanocarriers and hosts before the loading with therapeutic and/ or diagnostic cargo and the consequent development of each kind of nanodrug/nanodiagnostic. Since the aim of pharmacologists is to generate a reliable body of evidence for a responsible translation from mice to patients, it is therefore important to design the most appropriate carriers (in terms of material, size, and shape) to favor the bioavailability and the interaction with the target, even before the loading with drugs and/or ligands. To this goal, the reproducibility of highly characterized protocols of NP synthesis will be basilar to be tested before any preclinical pharmacological approach, and a fine-tuning of each individual parameter of the synthesis should be carefully defined.

accumulation in vital organs. In the last decades, several strategies have been undertaken to improve the therapeutic index of many classes of drugs.5,6 Among them, the loading of compounds to biocompatible and biodegradable nanoparticles (NPs) is considered one of the most promising and effective approaches to improve the targeted delivery.7−10 The Food and Drug Administration’s (FDA) approval of the doxorubicin liposomal nanoformulation (Doxil)11 and the superparamagnetic iron-oxide NPs as contrast agent12 seemed to guarantee the massive entry of nanotechnology on the clinical practice. However, many other classes of extensively investigated NPs did not reach the bedside.13 This very low translational impact is apparently in contrast to the huge and even more increasing number of manuscripts and patents appearing in the past decade. One of the main reason for this “lost in translation” from mice to humans is the poor characterization of the carrier physicochemical properties (in terms of size, shape, surface, biosafety), and the extremely weak evaluation of “if and how” these parameters may influence NP kinetics after administration. An extensive knowledge of the carrier, before its conjugation to any kind of molecule, is essential to think about a responsible good manufacture practice (GMP) production in nanomedicine. Many preliminary studies have been performed almost exclusively considering the effect of these parameters on cell uptake, accumulation, and toxicity. However, the validation of these analyses in multicellular and multiorgan systems often lacks or has produced controversial results. 5520

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With this library of GNPs it is possible to gain important insights about the role of size (using spherical GNPs with different size) and shape (using comparable sizes). GNPs were subsequently functionalized using ligand exchange reaction with a short carboxy-terminated mercaptopolyethylene glycol derivative (HS−C11−EG6−OCH2−COOH). This allowed the stabilization of the particles against aggregation and normalization of their surface chemistry, without losing their distinct shapes, since longer PEG chains might compromise the overall particle shape interfering with geometrical features.28 Noticeably, traditional batch-to-batch synthesis generates subtle differences in the nanomaterials size and shape, especially in the case of complex nanostructures, which represents an unsolved issue despite efforts to achieve good control of synthetic methods. GNP concentration was normalized by particle number, as this should represent the most meaningful concentration representative of this kind of experiments. The mass normalization indeed could lead to a large difference in particle number, for example in the case of GNP1 and GNP4.29 Therefore, nanoparticle concentration was quantified using the nanoparticles tracking analysis (NTA) technique, which represents the most reliable method to directly establish the NP concentration (here expressed as NPs/mL), with the exception of GNP1, for which the concentration was calculated by using the ultraviolet−visible (UV−vis) absorption spectra,30 because of the size limit of the NTA analysis. For a better comparison, GNP2 concentration was analyzed by both UV− vis spectrophotometry and NTA (see Table SI 1). All GNPs were synthesized following an endotoxin-free approach operating in a laminar flow hood, normally used for cell/tissue culture. Note that simple sterile filtration would not be capable of removing endotoxins for the GNP samples, and other more vigorous purification methods may impact the colloidal stability of the GNPs. Thus, in this work emphasis was given to endotoxin-free synthesis of the GNPs.31 The absence of endotoxins (e.g., lipopolysaccharide, LPS) is important for an accurate evaluation of NP biological behavior32 as the LPS can be recognized by receptors, such as scavenger receptors and toll-like receptor 4, abundant in the mononuclear phagocyte system.33 For example, the LPS adsorbed onto the NP surface can be recognized by the aforementioned receptors, modifying their “natural” binding cells,34,35 and potentially influence their biodistribution. Herein, both Limulus Amebocyte Lysate (LAL assay, the golden standard assay for detection of LPS) and Western Blot (WB) were employed to evaluate the presence of LPS in NP dispersion (see Figure SI 15, SI 16). The Influence of the Size on GNP Kinetics. It is widely known that subtle modifications of physicochemical parameters alter the overall kinetics of NPs.36 It is also ascertained that a different kinetics largely influences the permanence in the bloodstream, the uptake from immune-competent cells, the renal filtration, the escape from endothelial junctions and many other factors that play a key-role on the overall efficacy of drugs.37 In the first part of this in vivo study we focused our attention on the size effect comparing the fate of GNP1 and GNP2 that share almost all physicochemical parameters, but exclusively differ from their diameter. The gold content in tissues was carried out by ICP-MS analysis. It was expressed as percentage of injected dose (%ID), calculated as mass of gold found in each organ, divided by the

Among the large number of materials proposed for nanomedicine, gold nanoparticles (GNPs) are one of the most extensively investigated for their tunable features and for their potential application both in diagnosis and in therapy. Certainly, while not being a “revolutionary” system, GNPs, in particular those coated with polyethylene glycol (PEG), are one of the best studied particle systems. Extensive information about the correlation of the interaction of those particles with cells in vitro are available in the literature.16,17 In addition, several recent studies have revealed some correlation between NP physicochemical parameters and their in vivo behavior. Size hereby is the classical example.18 On the other hand, it has been shown that the surface charge of GNPs dictates the rate of penetration on ovarian tumor parenchyma in mice,19 whereas in a murine model of breast cancer, it has been found that spherical GNPs more efficiently accumulated in the tumor mass compared to other geometrical shapes.20 The present work moves from these first interesting premises to provide an overall view on how size and shape of GNPs influence their pharmacokinetics in healthy, immune-competent, and pathogen-free mice. The influence of size was evaluated by comparing the distribution of two spherical GNPs with a diameter of about 10 and 50 nm, respectively. To assess the role of shape, animals were treated with spherical, rod-like, and star-like GNPs of a similar size of about 50 nm with similar surface chemistry. Although the organ accumulation is a crucial parameter of interaction, it is extremely important to evaluate where NPs are localized in organs and, above all, if there is a relevant entrapment of them in immune-competent cells. This is needed to actually hypothesize their potential role in the host tissue. For this reason, inductively coupled plasma mass spectrometry (ICP-MS) was used to quantitatively measure the total gold concentration. Autometallography (AMG) staining and enhanced dark-field hyperspectral microscopy were selected to qualitatively assess gold localization in organs. Our combined approach demonstrates that both size and shape are key determinants in in vivo behavior and provides an interesting scenario to better exploit further functionalization of these GNPs.

RESULTS AND DISCUSSION Endotoxin-Free Preparation of the Gold Nanoparticles Library. Shape, size and surface functionalization are factors that can lead to important differences in the behavior of nanomaterials in biological environment.21 Therefore, it is fundamental to investigate the effects of these physicochemical parameters in vitro and in vivo in order to exploit the complexities of these NPs. GNPs represent the most known family of nanomaterials and different methods have been recently reported for their synthesis.22−25 For this study, we synthesized a set of GNPs (Figure 1) including 10 and 50 nm spheres (GNP1 and GNP2, respectively), rods 60 × 30 nm (GNP3), and stars (GNP4) with 55 nm as average of the longest tip-to-tip distance. GNP1, GNP2, and GNP3 have been prepared using selected procedures reported in the literature.24,26,27 GNP4 was prepared following an in-house preparation method, similar to a reported literature method.25 The suitability of all GNPs for biological applications was tested, ensuring solubility in water and stability in biologically relevant media (see paragraph 2 in the Supporting Information). 5521

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Figure 2. Percentage of injected dose measured by ICP-MS in collected organs (brain, lung, heart, liver, kidney, spleen) (A), liver (B), spleen (C), kidney (D), lung (E), urine (F), feces (G), and whole blood (H) in mice treated with GNP1 (pink) or GNP2 (green) at 1−24−120 h after treatment. A group of vehicle treated mice was recruited, and the levels of gold content measured in these organs were used as background. The data are reported as mean ± SE. One-way ANOVA followed by Bonferroni post hoc test was carried out. Significant difference (∗ p < 0.05, ∗∗ p ≤ 0.005) ∗ = GNP1 compared to GNP2 at the same time point.

with the capacity of smaller NPs to escape from organs passing through endothelial cells.38,39 In detail, the NP accumulation in filter organs is shown in Figure 2B,C. In liver and spleen GNP2 particles rapidly accumulate, and their levels did not significantly increase along time. On the other hand, a progressive increase of gold levels occurred in animals treated with GNP1. It has been already demonstrated that, many types of NPs, including GNPs, are efficiently transported into cell cytoplasm through a vesicle-dependent process and then segregated in endosomes or in other vacuoles.40,41 Even if other parameters, such as the stiffness of the material, the coating, or the ζ-potential, are the main players for the efficiency of vesicle-dependent endocytosis, it has been clearly demonstrated that a progressive improvement of this process

mass of gold injected in each mouse: %ID = 100%·mAu (organ)/mAu (injected). The amount of GNP1 and GNP2 penetrated in selected organs is shown in Figure 2. The measurements revealed that (1) the size influences the kinetics of NPs; (2) GNP1 has a slower penetration, but the particles are able to progressively accumulate passing from the 20 to the 70% from the 24th to the 120th h after their administration; (3) GNP2 penetrates faster in organs, but the particle amount remains stable along time. The different behavior between the two sizes may be somehow explained by the hypothesis that larger NPs (GNP2) are most avidly retained by filter organs. This process may be associated both with a higher ability of larger NPs to be internalized in cells and 5522

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Figure 3. Percentage of injected dose measured by ICP-MS in collected organs (brain, lung, heart, liver, kidney, spleen) (A), liver (B), spleen (C), kidney (D), lung (E), urine (F), feces (G), and whole blood (H) in mice treated with GNP2 (green), GNP3 (purple), or GNP4 (red) at different time points. A group of vehicle treated mice was recruited, and the levels of gold content measured in these organs were used as background. The data were reported as mean ± SE. One-way ANOVA followed by Bonferroni post hoc test was carried out. Significant difference (∗ p < 0.05, ∗∗ p ≤ 0.005, ∗∗∗ p ≤ 0.0005), ^ = GNP2 compared to GNP3, # = GNP3 compared to GNP4, § = GNP2 compared to GNP4 at the same time point.

occurs for GNPs in a range from 10 to 60 nm of diameter.42 This could be, in part, the reason for the different kinetics of the two sizes.43 The accumulation of both GNPs in kidneys was extremely low, and the kidney filtration was very fast and efficient (Figure 2D). A little increase of the % ID was found in animals receiving GNP1, sacrificed 120 h after administration. However, this value was not statistically different compared to the level of GNP2. No particular difference was observed comparing the levels of both GNPs in lung parenchyma (Figure 2E). The overall values

never overcame the 1% of ID and they cannot account for any size-dependent influence in terms of biological relevance. The percentage of ID in brain was lower than the threshold of detectability for both GNP1 and GNP2 at any time-points (data not shown). This suggests that the size does not play any role in influencing the passage through the blood brain barrier. The levels of GNPs in urines, feces, and bloodstream are shown in Figure 2F−H. Since our goal was not the determination of the gold mass balance in treated animals, a low percentage of urine and blood volume and feces mass was collected for this study. The expected underestimation of these three latter values does not represent a bias on the overall 5523

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Figure 4. Histological evaluation of gold tissue localization (black spots) by AMG. Representative images of liver and spleen from mice treated with GNP2 and GNP4, respectively, and sacrificed 1, 24, and 120 h after NP administration. R.P. = red pulp; L.N. = lymphatic nodules. Asterisk refers to the signal in loops of lobular arterioles and veins, arrow refers to the signal inside vessels and arrowhead shows the signal in endothelium of large vessels. Scale bars: (A, B, E, G, I, J, M, N, O, P, Q, R) = 200 μm, (C, D, F, H, K, L) = 20 μm.

gold NPs with a similar size range was evaluated in healthy mice (Figure 3). However, it is important to keep in mind that each sample can contain a relatively small number of “exceptionally” shaped, or otherwise organized nanoparticles. In particular, heterogeneity in between nanoparticles of the same batch is especially common for anisotropic complex nanostructures, such as star-like NPs.46 Similarly to the size, the shape greatly influenced the overall accumulation of NPs. Whereas GNP4 had a similar behavior to the one shown by GNP2, GNP3 showed a lower penetration and a very fast body clearance (Figure 3A). The kinetics of the three different GNPs in filter organs is reported in Figure 3B,C. Similarly to the overall data, the trend of accumulation of GNP4 does not differ from that observed for GNP2 in both liver and spleen. In the same organs, the percentage of ID of GNP3 was lower since the first hour and, opposite to the two other shapes, it drastically decreased along the time. The lower ability of GNP3 to penetrate in organs was further confirmed by the results recorded in kidneys (Figure 3D), where they rapidly disappeared. It is possible to argue that the surface/ volume ratio may play a role on the interaction with vessels and the penetration in tissue.47 No significant differences among the three groups were found, demonstrating that kidneys have a very poor accumulation of GNPs independent of their geometry. Differently from other organs, GNP4 exclusively showed a high tropism for the lungs, for which their levels were many-folds higher than those of animals treated with both GNP2 and GNP3, respectively (Figure 3E). GNP4 rapidly penetrated into lung parenchyma, reaching the 4% of ID 1 h

meaning of the study. The rate of GNPs in urines and feces after 8, 24, and 120 h of treatment (Figure 2F-G) demonstrates that GNP2 is efficiently filtered through the renal structures and then excreted by urine. On the contrary, GNP1 seems to follow a different way of clearance, mostly associated with intestinal excretion. The content of NPs in the whole blood is reported in Figure 2H. Interestingly, GNP1 showed a progressive rate of increase, whereas GNP2 had a drop from the first to the 24th h, rising again at the 120th h of measurement. Because circulating immune-competent cells may actively interact with NPs through a cellular uptake,44 and their efficiency can be influenced by NP-geometry, we decided to collect the whole blood tissue (serum and cellular components) instead of the exclusive evaluation of plasma levels. Similarly to urines and feces, which were not entirely collected along the experiment, the blood withdrawal was around 500 μL for each animal at the sacrifice. Data from the literature45 indicate that circulating blood volume in mice is about 72 mL/kg; considering a mouse of 30 g, the expected volume should be more than 2 mL/ mouse. It is therefore possible that the physiological ID % were at least 4 times higher than those we measured in this study. The Influence of the Shape on GNP Kinetics. It has been recently reported how the shape may influence the distribution of GNPs in tumor-bearing mice. In particular the attention was focused on the penetration and accumulation inside the tumor parenchyma via enhanced permeation and retention (EPR) effect.19,20 Here, the biodistribution of spherical (GNP2), rod-like (GNP3), and star-like (GNP4) 5524

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little, but detectable signal, was also observed for both GNPs close to the endothelium of large vessels (blue arrowheads). Figure 4 panels E−H show the distribution of GNP2 and GNP4 in the liver of mice sacrificed 24 h after administration. At this time-point, the histology well matches the quantification data. No relevant difference can be observed among the two GNPs in terms of signal intensity, parenchyma distribution, and subcellular localization. Compared to the first time-point, in Figure 4F−H a marked accumulation of gold was found in celllike structures (green asterisks). This peculiar feature suggests an efficient uptake from Kupffer cells localized into the sinusoids of small vessels. Moreover, GNP2 and GNP4 were also distributed inside the vessels of liver lobules (green arrows) and, at a lesser extent, confined to the endothelium of larger veins (green arrowheads). At 120 h after GNP administration, the parenchyma from animals treated with GNP4 was still characterized by spots with a stronger intensity of signal compared to that of GNP2-treated mice (Figure 4I,J), in spite of a similar amount of gold detected by ICP-MS. High magnification pictures (Figure 4K,L) reveal that GNP4 is more efficiently retained into Kupffer cells (red asterisk), whereas GNP2 shows a reduction of signal intensity maybe due to a shift from Kupffer cells to the lobular vessels (red arrows). These results, suggesting a different ability of GNP2 and GNP4 to interact with immune-competent cells, could be potential information to select future nanocarriers depending on their shape. Similarly to liver, spleen may play an important role in NP filtering. It is known that debris from dying, or dead, leukocytes, erythrocytes, and platelets, are avidly taken up from immunecompetent cells in the red pulp and, after their complete catabolism, are drained by lymphatic nodules. Notably, our results highlighted a very low accumulation of GNPs, as % of ID. The images showed a confined staining just around the external border of lymphatic nodules, without any evidence of black spot in the red pulp, for both GNPs, at each time-point (Figure 4M−R). This peculiar localization strongly suggests that (a) GNPs are not actively taken-up from spleen resident macrophages (this explains the very low amount in terms of % ID); (b) GNPs are instead directed toward lymph nodes to be drained through the lymphatic system; (c) neither the shape nor the interval of time from the administration to the sacrifice influenced this process. The immunological purity of our conditions may explain this result. Many factors may influence the activation of resident spleen macrophages.52,53 The systemic injection of LPS strongly activates spleen functionality both enlarging the red pulp area and increasing the production of b-cells.54 The low penetration of endotoxin-free GNPs may have a relevant impact thinking about chronic or repeated treatments in many clinical areas. The selection of carriers which do not alter the morpho-functionality of spleen will be a great advantage, independently of their size and/or material. Figure SI 18 shows representative AMG images from liver and spleen of animals treated with GNP3 and sacrificed 1, 24, and 120 h after administration. In contrast to that shown in Figure 4 the presence of a very weak black staining associable with gold was found 1 h after the treatment and progressively faded away at the following time-points. This result furthermore confirmed the lack of tissue penetration of rodlike shapes in tissue parenchyma. The comparison among the levels of GNP2,4 and GNP3 is quite surprising. However, both quantification and histology confirmed this huge difference in terms of bioaccumulation. It is not easy to account for so great

after treatment and showing an accumulation higher than the 6% of ID 120 h after administration. While we are so far not able to correlate this behavior to the particular physicochemical properties of GNP4 we want to point out that other scenarios with preferential accumulation of particles inside lungs have been reported, for example, in the case of GNPs loaded into mesenchymal stem cells, using the stem cells as delivery vehicles.48 In dynamic light scattering (DLS) studies it was demonstrated that GNP4 particles as GNP1 do not agglomerate even at physiological NaCl concentrations (Figure SI 10). Thus, agglomeration cannot be the critical parameter which describes accumulation of GNP4 in the lung. For GNP2 and GNP3, onset of agglomeration was found after the 24th h at elevated NaCl concentrations. No evidence of gold accumulation was observed in brain from animals treated with GNP2, GNP3, and GNP4 (data not shown). This result, together with the previous data related to the size, suggests that further modifications of the external surface, such as functionalization with specific antibodies and/ or peptides, are requested to achieve relevant target delivery to the brain.49−51 The levels of GNP4 were significantly higher in urine compared to those of the two other shapes (Figure 3F). Opposite, very interestingly the excretion from feces of GNP4 was lower and less prolonger than that of the two other GNPs (Figure 3G). Finally, the levels of the gold content in the whole blood was measured (Figure 4H). Very interestingly both GNP3 and GNP4 did not show a progressive increase over time. This could be likely related to a lower ability to be internalized in blood cells. Overall, these results strongly suggest that the geometry of LPS-free GNPs not only influences the organ accumulation but also their clearance and their interaction with circulating cells. The combination between quantification and anatomical distribution is crucial for the estimation of both targeted delivery in diseased tissues and potential toxicity in filter organs. It is possible that two different NPs containing the same cytotoxic drug and showing the same penetration, may play a different effect depending on their anatomical localization. In this context, an efficient uptake from liver or spleen macrophages would hugely reduce the side-effects compared to a spread of NPs in hepatocytes or other functional cells. The results emerged from the accumulation of both GNP2 and GNP4 in liver and spleen (in terms of % ID) are overlapping. For this reason, we focused our attention on histological localization of these two types of GNPs in these two organs at the three time-points examined in our study (Figure 4). Low magnification pictures reveal that both types of GNPs are homogeneously diffused in the parenchyma 1 h after NP administration (Figure 4A,B). Differently from quantitative results, GNP4 staining was more intense than that of GNP2. Histological data suggest that GNPs rapidly reach the liver from the circulatory tree as well described in a recent study.9 Once penetrated inside the parenchyma from the portal triad, they are entrapped by Kupffer cells localized from the portal to the ductal vein. A faster accumulation of GNP4 inside these cells could somehow explain the more intense staining. In high magnification pictures (Figure 4C,D) blue asterisks clearly indicate the presence of signal accumulation in loops of lobular arterioles and veins, whereas blue arrows are related to the presence of thinner black spots localized inside the vessels. A 5525

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Figure 5. Histological evaluation of lung tissues from mice treated with GNP4 by Haematoxylin and Eosin staining (A,B) and by enhanced dark-field hyperspectral microscopy (CytoViva system) (C−E). In detail, (C) anatomical view; (D) spatial scanning of the gold content (light green), and (E) merge between the two previous fields of view. (F) GNP4 accumulation close to the external border of lung capillary lumen (light-green arrows). Scale bars: (A) = 200 μm, (B) = 20 μm, (C, D, E) = 5 μm, (F) = 1.5 μm.

CONCLUSION Biodistribution is one of the most important queries associated with nanoparticle-enabled drug delivery, determining efficacy and toxicity. Advances in nanoparticle synthesis give rise to a more complex nanostructure with different size and shape. It is now necessary to investigate their biodistribution and evaluate which are the key attributes that govern the biological outcomes. To do so, it is of utmost importance to carefully check the possible LPS contamination of the nanomaterials. The present study provides a careful analysis on the impact of endotoxin-free gold nanoparticles, with different size and shape on organ penetration, accumulation, and excretion in a healthy mouse model. While biodistribution of GNPs of different geometry have been reported before, in our study we present a direct comparison of a set of different GNPs, which rules out variations due to experimental procedures, exposure conditions, etc. Direct comparison of different GNPs with normalized surface chemistry has shown that particle size is determinant for mechanisms of nanoparticle clearance. In addition, the combination between quantification and histological localization makes this study even more innovative and consistent. In this context, demonstrating a shape-dependent effect on the interaction between GNPs and tissue residential macrophages (e.g., Kupffer cells), independently on the % of ID, may hold a key to unravelling the underlying mechanisms, providing the possibility to tailor distribution pattern. Investigations into these interactions are the core of our ongoing research endeavor. Last, it is extremely important to remember that the gold measurement achievable from this kind of analysis can reach about 70% of the total injected. Although in this study we exploit an ultrasensible method of measurement based on ICPMS, we were not aiming to determine the mass balance of injected material but at making a comparative evaluation of the behavior of GNPs in dependence on their geometry (size and shape). This approach could be applied, in future times, either for studies focused on the design of carriers for targeted delivery of pharmacological compounds or for toxicological analyses of bioaccumulation after unintentional exposure.

a difference. It is possible to argue that a very fast clearance occurred in mice treated with this kind of NP. We repeated the experiments two times and no difference in the trend of rodlike NPs was observed. The measurement of GNP4 in lung by ICP-MS revealed a sustained and prolonged presence of gold in treated animals. The histological examination through AMG staining did not show any signal associated with a strong and focused accumulation of GNPs in specific cells. To overcome this limitation, an innovative approach was undertaken by evaluating the same samples through the application of enhanced dark field hyperspectral microscopy performed on a CytoViva system (CytoViva Inc., Auburn, AL, USA). In Figure 5A,B, the whole parenchyma and a higher magnification, highlighting epithelia, alveoli, and small capillaries, are respectively depicted. Figure 5C shows a representative image of lung from mice treated with GNP4 and acquired by CytoViva system. The analysis of the gold in this specific field of view pinpointed the presence of a very high number of events, revealed by light-green spots (Figure 5D). The overlap among Figures 5C,D is shown in Figure 5E, where a homogeneous distribution of GNP4 in the epithelial cells is easily detectable. Lastly, Figure 5F shows a higher magnification of a small vessel. Interestingly, a strong accumulation of green signal can be observed in correspondence with the edge of the vessel, very likely, matching the thin layer of endothelial cells. This result confirms the actual penetration of GNP4 into the parenchyma and gives a further indication on the relevant role of the geometry on the fate of nanocarriers, independently of their surface functionalization. To predict the behavior of nanocarriers and define how the fine-tuning of a single parameter may influence their kinetics would be a cleaver strategy to overcome some limitations toward the targeted delivery of cargoes. In our experimental condition, the use of endotoxin-free NPs and immunecompetent mice represents an added value for translational aims. The lack of confounding factors, such as the great inner variability of immune-response occurring in “conventional studies”, can lead to the achievement of a more reliable and robust body of evidence. 5526

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Histological Analysis. Briefly, animals belonging to GNP2, GNP3, and GNP4 group were used. Collected organs were fixed in 10% neutral buffered formalin (Bio-Optica, Italy) for 24 h at room temperature, routinely processed for paraffin embedding, and sectioned at 4 μm thickness. To visualize the presence of gold agglomerates in organ parenchyma, autometallography (AMG) staining was carried out.55 At the end of the staining, sections were visualized through light microscope (Olympus BX61VS), gold aggregates were visible as black granular pigment. Haematoxylin− Eosin staining (H&E) was carried out in the lungs of GNP4-treated mice and vehicle-treated mice, respectively, as previously described.55 GNP4 particles were imaged using enhanced dark field hyperspectral microscopy on a CytoViva system. Ultrafine cuts of exposed and nonexposed lung tissues, prepared as previously described, were imaged with a 60× oil immersion objective on an Olympus microscope and subjected to hyperspectral analysis (from 420 to 900 nm with a 2.5 nm spectral resolution). Spectral libraries of the exposed samples were generated by manually acquiring about 200 spectra per samples. Acquired spectral libraries were filtered against the nonexposed controls to filter out all spectra nonrelated to gold NPs, by using a spectral angle mapper (SAM) algorithm with a 0.08 radians tolerance. Filtered libraries were mapped onto images of exposed samples using a SAM with a 0.08 radians tolerance, which allows highlighting similarities between the spectra in the image and in the spectral library. All acquisitions and analyses were performed using the ENVI software (version 4.8 from Harris Corporation, Melbourne, FL, USA) and modified by CytoViva, Inc. ICP-MS. The measurement of gold by ICP-MS was performed from liver, spleen, kidneys, lungs, brain, heart, urine, feces, and blood. The amount of gold of different GNPs was quantified using ICP-MS (Agilent 7700 series). Additional information about the method and the calculation is provided in the Supporting Information.

METHODS Materials. The following chemicals were purchased from SigmaAldrich and were of highest available purity and used as received: Hydrogen tetrachoroaurate trihydrate (HAuCl4·3H2O, ≥ 99.9%), hexadecyltrimethylammonium bromide (CTAB, C19H42BrN, ≥ 99%), hexadecyltrimethylammonium chloride (CTAC, C19H42ClN, ≥ 98%), trisodium citrate dehydrate (C 6H9Na3O9, meets USP testing specifications), bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSSP, C18H17K2O8PS2, 97%), L-ascorbic acid (C6H8O6, ≥ 99%), sodium borohydride (NaBH4, ≥ 99%), potassium carbonate (K2CO3, ≥ 99%), tannic acid, (C76H52O46), silver nitrate (AgNO3, ≥ 99.9%), hydrochloric acid (HCl, 37%, AR grade), glycerol (C3H8O3, ≥ 99%), clean water (CHROMASOLV Plus, for HPLC) and (LC−MS Ultra CHROMASOLV, tested for UHPLC−MS). Sodium oleate (NaOL, C18H33O2Na, ≥ 97%) was purchased from Tokyo Chemical Industry CO., Ltd. Carboxy-terminated-PEG thiol (HS−C11−EG6−OCH2−COOH) was purchased from Prochimia Surfaces. General Information. The water used as solvent for NP preparation was purchased from Sigma-Aldrich, and was namely CHROMASOLV Plus, for HPLC for GNP1, GNP2, GNP3, and ultrapure water LC−MS Ultra CHROMASOLV, tested for UHPLC− MS for GNP4. To obtain endotoxin-free products, the NP syntheses were performed in a Class 2 laminar flow hood by following all the strict precautions normally adopted during cell culture. All the reagents stock solutions, except for PEG ligand (HS−C11−EG6−OCH2− COOH), were filtered through a 0.2 μm Millipore syringe filters prior to use. All of the plasticware used were endotoxin-free certified and all the glassware were previously cleaned with aqua regia and thoroughly rinsed with endotoxin-free water. All the solvents and the other reagents used for NP preparation in this work were strictly opened inside the laminar flow fumehood. Synthesis of GNPs. Four different samples of GNPs were prepared, as described in detail in the Supporting Information, following literature methods.24−27 Preparation was carried out in aqueous solution by reduction of a gold precursor. In the case of rodshaped GNPs, surfactant was used for shape control. After the preparation a ligand exchange reaction was used in order to normalize the surface of all particles with carboxy-PEG thiol ligand, providing the particles with the same surface chemistry. All samples were characterized by transmission electron microscopy (TEM), UV−vis absorption spectroscopy, differential centrifugal sedimentation (DCS), and other selected techniques for determining their concentration. Animals. Experiments involving mice and their care were conducted in conformity with the institutional guidelines at the IRCCSInstitute for Pharmacological Research “Mario Negri” in compliance with national (Legislative Decree n. 26, March 4, 2014; Authorization n.19/2008-A issued March 6, 2008, by the Italian Ministry of Health) and international laws and policies (EEC Council Directive 2010/63, August 6, 2013; Standards for the Care and Use of Laboratory Animals, U.S. National Research Council, Statement of Compliance A5023-01, October 28, 2008). This work was reviewed by IRCCS-IRFMN Animal Care and Use Committee (IACUC) and then approved by the Italian “Istituto Superiore di Sanità” (code: 42/2016PR). Adult male CD-1 mice, were bred at Charles River Italia (Calco, Lecco, Italy). Animals were housed in a specific pathogen free animal room at a constant temperature of 21 ± 1 °C, humidity of 55 ± 10% with a 12 h light/dark cycle and ad libitum access to food and water. Before the treatment, animals were randomly divided in four experimental groups (n = 12 mice each one). After characterization, all NPs were diluted in sterile water and a volume of 140 μL was injected in the tail vein with a dose of 1.1 × 1011 NPs/mL. This corresponds to the following masses of gold injected per animal, as determined by ICP−MS: GNP1, mAu = 0.2 μg; GNP2, mAu = 8.0 μg; GNP3, mAu = 14.3 μg; GNP4, mAu = 17.4 μg. To assess the biodistribution depending on GNP size and shape, three time points were selected (1, 24, and 120 h) and for each time 4 animals/ experimental group were sacrificed.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00497. GNP synthesis and characterization, LPS detection, analytical methods, histology, and nanoscopy (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. W.J.P., K.A.D., and P.B. equally contributed to the coordination of this study. ORCID

Ž eljka Krpetić: 0000-0003-1129-6831 Beatriz Pelaz: 0000-0002-4626-4576 Yan Yan: 0000-0003-2938-4063 Wolfgang J. Parak: 0000-0003-1672-6650 Paolo Bigini: 0000-0002-0239-9532 Author Contributions °

L.T., M.B.V., and Q.C. are first coauthors.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the EU FP7 FutureNanoNeeds project (Grant Agreement No. 604602). K.D. and D.G. acknowledge the Science Foundation Ireland (SFI) Principal Investigator Award (Agreement No. 12/IA/1422). Y.Y. acknowledges the Science Foundation Ireland Starting Investigator Research Grant (Agreement No. 15/SIRG/3423) 5527

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Protein Coating of Gold Nanoparticles as Potential Tool for Organ Targeting. Biomaterials 2014, 35, 3455−3466. (16) Del Pino, P.; Yang, F.; Pelaz, B.; Zhang, Q.; Kantner, K.; Hartmann, R.; Martinez de Baroja, N.; Gallego, M.; Möller, M.; Manshian, B. B.; Soenen, S. J.; Riedel, R.; Hampp, N.; Parak, W. J. Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells. Angew. Chem., Int. Ed. 2016, 55, 5483−5487. (17) Polo, E.; Collado, M.; Pelaz, B.; Del Pino, P. Advances toward More Efficient Targeted Delivery of Nanoparticles in Vivo: Understanding Interactions between Nanoparticles and Cells. ACS Nano 2017, 11, 2397−2402. (18) Kreyling, W. G.; Hirn, S.; Möller, W.; Schleh, C.; Wenk, A.; Celik, G.; Lipka, J.; Schaffler, M.; Haberl, N.; Johnston, B. D.; Sperling, R.; Schmid, G.; Simon, U.; Parak, W. J.; Semmler-Behnke, M. AirBlood Barrier Translocation of Tracheally Instilled Gold Nanoparticles Inversely Depends on Particle Size. ACS Nano 2014, 8, 222−233. (19) Arvizo, R. R.; Miranda, O. R.; Moyano, D. F.; Walden, C. A.; Giri, K.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.; Reid, J. M.; Mukherjee, P. Modulating Pharmacokinetics, Tumor Uptake and Biodistribution by Engineered Nanoparticles. PLoS One 2011, 6, 24374. (20) Black, K. C.; Wang, Y.; Luehmann, H. P.; Cai, X.; Xing, W.; Pang, B.; Zhao, Y.; Cutler, C. S.; Wang, L. V.; Liu, Y.; et al. Radioactive 198Au-Doped Nanostructures with Different Shapes for in Vivo Analyses of Their Biodistribution, Tumor Uptake, and Intratumoral Distribution. ACS Nano 2014, 8, 4385−4394. (21) Rivera-Gil, P.; Jimenez de Aberasturi, D.; Wulf, V.; Pelaz, B.; del Pino, P.; Zhao, Y.; de la Fuente, J. M.; Ruiz de Larramendi, I.; Rojo, T.; Liang, X. J.; et al. The Challenge to Relate the Physicochemical Properties of Colloidal Nanoparticles to Their Cytotoxicity. Acc. Chem. Res. 2013, 46, 743−749. (22) Chen, S.; Wang, Z. L.; Ballato, J.; Foulger, S. H.; Carroll, D. L. Monopod, Bipod, Tripod, and Tetrapod Gold Nanocrystals. J. Am. Chem. Soc. 2003, 125, 16186−16187. (23) Sun, Y.; Xia, Y. Shape-Controlled Synthesis of Gold and Silver Nanoparticles. Science 2002, 298, 2176−2179. (24) Ye, X.; Gao, Y.; Chen, J.; Reifsnyder, D. C.; Zheng, C.; Murray, C. B. Seeded Growth of Monodisperse Gold Nanorods Using Bromide-Free Surfactant Mixtures. Nano Lett. 2013, 13, 2163−2171. (25) Yuan, H.; Khoury, C. G.; Hwang, H.; Wilson, C. M.; Grant, G. A.; Vo-Dinh, T. Gold Nanostars: Surfactant-Free Synthesis, 3D Modelling, and Two-Photon Photoluminescence Imaging. Nanotechnology 2012, 23, 075102. (26) Krpetic, Z.; Davidson, A. M.; Volk, M.; Levy, R.; Brust, M.; Cooper, D. L. High-Resolution Sizing of Monolayer-Protected Gold Clusters by Differential Centrifugal Sedimentation. ACS Nano 2013, 7, 8881−8890. (27) Krpetic, Z.; Guerrini, L.; Larmour, I. A.; Reglinski, J.; Faulds, K.; Graham, D. Importance of Nanoparticle Size in Colorimetric and SERS-Based Multimodal Trace Detection of Ni(II) Ions with Functional Gold Nanoparticles. Small 2012, 8, 707−714. (28) Tay, C. Y.; Setyawati, M. I.; Xie, J.; Parak, W. J.; Leong, D. T. Back to Basics: Exploiting the Innate Physico-Chemical Characteristics of Nanomaterials for Biomedical Applications. Adv. Funct. Mater. 2014, 24, 5936−5955. (29) Feliu, N.; Huhn, J.; Zyuzin, M. V.; Ashraf, S.; Valdeperez, D.; Masood, A.; Said, A. H.; Escudero, A.; Pelaz, B.; Gonzalez, E.; et al. Quantitative Uptake of Colloidal Particles by Cell Cultures. Sci. Total Environ. 2016, 568, 819−828. (30) Haiss, W.; Thanh, N. T.; Aveyard, J.; Fernig, D. G. Determination of Size and Concentration of Gold Nanoparticles from UV-Vis Spectra. Anal. Chem. 2007, 79, 4215−4221. (31) França, A.; Pelaz, B.; Moros, M.; Sánchez-Espinel, C.; Hernández, A.; Fernández-López, C.; Grazú, V.; de la Fuente, J. M.; Pastoriza-Santos, I.; Liz-Marzán, L. M.; González-Fernandez, A. Sterilization Matters: Consequences of Different SterilizationTechniques on Gold Nanoparticles. Small 2010, 6, 89−95.

and the EU FP7-PEOPLE-2012- IAPP Marie Curie NanoClassifier (Agreement No. 324519). L.B. acknowledges the financial support of the EU H2020 Nanofacturing projects (Grant Agreement No. 646364). M.P.M. acknowledges the financial support of the Science Foundation Ireland Industry fellowship (Agreement No. 15/IFA/3057). Q.C. acknowledges Chinese Scholarship Council (Agreement No. 201408300003), and Irish Research Council under the Government of Ireland Postgraduate Scholarship scheme (GOIPG/2014/874). C.P.S. acknowledges the financial support of CNPqConselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico, of the Ministry of Science, Technology and Innovation of Brazil (Agreement No. 205146/2014-7)

REFERENCES (1) Horvath, P.; Aulner, N.; Bickle, M.; Davies, A. M.; Nery, E. D.; Ebner, D.; Montoya, M. C.; Ostling, P.; Pietiainen, V.; Price, L. S.; et al. Screening Out Irrelevant Cell-Based Models of Disease. Nat. Rev. Drug Discovery 2016, 15, 751−769. (2) Xu, R.; Zhang, G.; Mai, J.; Deng, X.; Segura-Ibarra, V.; Wu, S.; Shen, J.; Liu, H.; Hu, Z.; Chen, L.; et al. An Injectable Nanoparticle Generator Enhances Delivery of Cancer Therapeutics. Nat. Biotechnol. 2016, 34, 414−418. (3) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016, 1, 16014. (4) Winquist, R. J.; Boucher, D. M.; Wood, M.; Furey, B. F. Targeting Cancer Stem Cells for More Effective Therapies: Taking Out Cancer’s Locomotive Engine. Biochem. Pharmacol. 2009, 78, 326− 334. (5) Chang, E. H.; Harford, J. B.; Eaton, M. A.; Boisseau, P. M.; Dube, A.; Hayeshi, R.; Swai, H.; Lee, D. S. Nanomedicine: Past, Present and Future - A Global Perspective. Biochem. Biophys. Res. Commun. 2015, 468, 511−517. (6) Petros, R. A.; DeSimone, J. M. Strategies in the Design of Nanoparticles for Therapeutic Applications. Nat. Rev. Drug Discovery 2010, 9, 615−627. (7) Heidel, J. D.; Davis, M. E. Clinical Developments in Nanotechnology for Cancer Therapy. Pharm. Res. 2011, 28, 187−199. (8) Mukherjee, B.; Das, S.; Chakraborty, S.; Satapathy, B. S.; Das, P. J.; Mondal, L.; Hossain, C. M.; Dey, N. S.; Chaudhury, A. Potentials of Polymeric Nanoparticle as Drug Carrier for Cancer Therapy: with a Special Reference to Pharmacokinetic Parameters. Curr. Drug Metab. 2014, 15, 565−580. (9) Tsoi, K. M.; MacParland, S. A.; Ma, X. Z.; Spetzler, V. N.; Echeverri, J.; Ouyang, B.; Fadel, S. M.; Sykes, E. A.; Goldaracena, N.; Kaths, J. M.; et al. Mechanism of Hard-Nanomaterial Clearance by the Liver. Nat. Mater. 2016, 15, 1212−1221. (10) Weinstein, J. N.; Leserman, L. D. Liposomes as Drug Carriers in Cancer Chemotherapy. Pharmacol. Ther. 1984, 24, 207−233. (11) Gabizon, A.; Martin, F. Polyethylene Glycol-Coated (pegylated) Liposomal Doxorubicin. Rationale for Use in Solid Tumours. Drugs 1997, 54 (Suppl 4), 15−21. (12) Corot, C.; Warlin, D. Superparamagnetic Iron Oxide Nanoparticles for MRI: Contrast Media Pharmaceutical Company R&D Perspective. WIREs Nanomed. Nanobiotechnol. 2013, 5, 411−422. (13) Dawidczyk, C. M.; Kim, C.; Park, J. H.; Russell, L. M.; Lee, K. H.; Pomper, M. G.; Searson, P. C. State-of-the-Art in Design Rules for Drug Delivery Platforms: Lessons Learned from FDA-Approved Nanomedicines. J. Controlled Release 2014, 187, 133−144. (14) Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A. Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities when a Biomolecule Corona Adsorbs on the Surface. Nat. Nanotechnol. 2013, 8, 137−143. (15) Schaffler, M.; Sousa, F.; Wenk, A.; Sitia, L.; Hirn, S.; Schleh, C.; Haberl, N.; Violatto, M.; Canovi, M.; Andreozzi, P.; et al. Blood 5528

DOI: 10.1021/acsnano.7b00497 ACS Nano 2017, 11, 5519−5529

Article

ACS Nano (32) Li, Y.; Boraschi, D. Endotoxin Contamination: a Key Element in the Interpretation of Nanosafety Studies. Nanomedicine (London, U. K.) 2016, 11, 269−287. (33) Vallhov, H.; Qin, J.; Johansson, S. M.; Ahlborg, N.; Muhammed, M. A.; Scheynius, A.; Gabrielsson, S. The Importance of an EndotoxinFree Environment During the Production of Nanoparticles Used in Medical Applications. Nano Lett. 2006, 6, 1682−1686. (34) Mano, S. S.; Kanehira, K.; Taniguchi, A. Comparison of Cellular Uptake and Inflammatory Response via Toll-Like Receptor 4 to Lipopolysaccharide and Titanium Dioxide Nanoparticles. Int. J. Mol. Sci. 2013, 14, 13154−13170. (35) Xu, W.; Zhou, Q.; Yao, Y.; Li, X.; Zhang, J. L.; Su, G. H.; Deng, A. P. Inhibitory Effect of Gardenblue Blueberry (Vaccinium Ashei Reade) Anthocyanin Extracts on Lipopolysaccharide-Stimulated Inflammatory Response in RAW 264.7 Cells. J. Zhejiang Univ., Sci., B 2016, 17, 425−436. (36) Ferrari, R.; Lupi, M.; Falcetta, F.; Bigini, P.; Paolella, K.; Fiordaliso, F.; Bisighini, C.; Salmona, M.; D’Incalci, M.; Morbidelli, M.; et al. Integrated Multiplatform Method for in Vitro Quantitative Assessment of Cellular Uptake for Fluorescent Polymer Nanoparticles. Nanotechnology 2014, 25, 045102. (37) Duncan, R.; Gaspar, R. Nanomedicine(s) under the Microscope. Mol. Pharmaceutics 2011, 8, 2101−2141. (38) Pegaz, B.; Debefve, E.; Ballini, J. P.; Konan-Kouakou, Y. N.; van den Bergh, H. Effect of Nanoparticle Size on the Extravasation and the Photothrombic Activity of Meso(p-tetracarboxyphenyl)porphyrin. J. Photochem. Photobiol., B 2006, 85, 216−222. (39) Vesterdal, L. K.; Mikkelsen, L.; Folkmann, J. K.; Sheykhzade, M.; Cao, Y.; Roursgaard, M.; Loft, S.; Möller, P. Carbon Black Nanoparticles and Vascular Dysfunction in Cultured Endothelial Cells and Artery Segments. Toxicol. Lett. 2012, 214, 19−26. (40) Brandenberger, C.; Muhlfeld, C.; Ali, Z.; Lenz, A. G.; Schmid, O.; Parak, W. J.; Gehr, P.; Rothen-Rutishauser, B. Quantitative Evaluation of Cellular Uptake and Trafficking of Plain and Polyethylene Glycol-Coated Gold Nanoparticles. Small 2010, 6, 1669−1678. (41) Schleh, C.; Semmler-Behnke, M.; Lipka, J.; Wenk, A.; Hirn, S.; Schaffler, M.; Schmid, G.; Simon, U.; Kreyling, W. G. Size and Surface Charge of Gold Nanoparticles Determine Absorption Across Intestinal Barriers and Accumulation in Secondary Target Organs After Oral Administration. Nanotoxicology 2012, 6, 36−46. (42) Chithrani, D. B. Intracellular Uptake, Transport, and Processing of Gold Nanostructures. Mol. Membr. Biol. 2010, 27, 299−311. (43) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. Determining the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Lett. 2006, 6, 662−668. (44) Hussain, S.; Vanoirbeek, J. A.; Hoet, P. H. Interactions of Nanomaterials with the Immune System. Wiley Interdiscip Rev. Nanomed Nanobiotechnol 2012, 4, 169−183. (45) Diehl, K. H.; Hull, R.; Morton, D.; Pfister, R.; Rabemampianina, Y.; Smith, D.; Vidal, J. M.; van de Vorstenbosch, C. European Federation of Pharmaceutical Industries Association; European Centre for the Validation of Alternative Methods. A Good Practice Guide to the Administration of Substances and Removal of Blood, Including Routes and Volumes. J. Appl. Toxicol. 2001, 21, 15−23. (46) Castagnola, V.; Cookman, J.; De Araujo, J. M.; Polo, E.; Cai, Q.; Silveira, C. P.; Krpetić, Ž .; Yan, Y.; Boselli, L.; Dawson, K. A. Towards a classification strategy for complex nanostructures Nanoscale Horiz., 2017, in press, DOI: 10.1039/C6NH00219F. (47) Decuzzi, P.; Godin, B.; Tanaka, T.; Lee, S. Y.; Chiappini, C.; Liu, X.; Ferrari, M. Size and Shape Effects in the Biodistribution of Intravascularly Injected Particles. J. Controlled Release 2010, 141, 320− 327. (48) Nold, P.; Hartmann, R.; Feliu, N.; Kantner, K.; Gamal, M.; Pelaz, B.; Huhn, J.; Sun, X.; Jungebluth, P.; Del Pino, P.; Hackstein, H.; Macchiarini, P.; Parak, W. J.; Brendel, C. Optimizing Conditions for Labeling of Mesenchymal Stromal Cells (MSCs) with Gold Nanoparticles: a Prerequisite for in Vivo Tracking of MSCs. J. Nanobiotechnol. 2017, 15, 24.

(49) Balducci, C.; Mancini, S.; Minniti, S.; La Vitola, P.; Zotti, M.; Sancini, G.; Mauri, M.; Cagnotto, A.; Colombo, L.; Fiordaliso, F.; et al. Multifunctional Liposomes Reduce Brain Beta-Amyloid Burden and Ameliorate Memory Impairment in Alzheimer’s Disease Mouse Models. J. Neurosci. 2014, 34, 14022−14031. (50) Bramini, M.; Ye, D.; Hallerbach, A.; Nic Raghnaill, M.; Salvati, A.; Aberg, C.; Dawson, K. A. Imaging Approach to Mechanistic Study of Nanoparticle Interactions with the Blood-Brain Barrier. ACS Nano 2014, 8, 4304−4312. (51) Tosi, G.; Fano, R. A.; Bondioli, L.; Badiali, L.; Benassi, R.; Rivasi, F.; Ruozi, B.; Forni, F.; Vandelli, M. A. Investigation on Mechanisms of Glycopeptide Nanoparticles for Drug Delivery across the BloodBrain Barrier. Nanomedicine (London, U. K.) 2011, 6, 423−436. (52) Lane, T. E.; Wu-Hsieh, B. A.; Howard, D. H. Gamma Interferon Cooperates with Lipopolysaccharide to Activate Mouse Splenic Macrophages to an Antihistoplasma State. Infect. Immun. 1993, 61, 1468−1473. (53) Wu-Hsieh, B. A.; Lee, G. S.; Franco, M.; Hofman, F. M. Early Activation of Splenic Macrophages by Tumor Necrosis Factor Alpha Is Important in Determining the Outcome of Experimental Histoplasmosis in Mice. Infect. Immun. 1992, 60, 4230−4238. (54) Xu, H.; Liew, L. N.; Kuo, I. C.; Huang, C. H.; Goh, D. L.; Chua, K. Y. The Modulatory Effects of Lipopolysaccharide-Stimulated B Cells on Differential T-Cell Polarization. Immunology 2008, 125, 218− 228. (55) Recordati, C.; De Maglie, M.; Bianchessi, S.; Argentiere, S.; Cella, C.; Mattiello, S.; Cubadda, F.; Aureli, F.; D’Amato, M.; Raggi, A.; Lenardi, C.; Milani, P.; Scanziani, E. Tissue Distribution and Acute Toxicity of Silver after Single Intravenous Administration in Mice: Nano-Specific and Size-Dependent Effects. Part. Fibre Toxicol. 2015, 13, 12.

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