Toward Understanding in Vivo Sequestration of Nanoparticles at the

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Toward Understanding in Vivo Sequestration of Nanoparticles at the Molecular Level Bohan Yin,†,⊥ Kin Hei Kelvin Li,‡,⊥ Lok Wai Cola Ho,† Cecilia Ka Wing Chan,§ and Chung Hang Jonathan Choi*,† †

Department of Biomedical Engineering, ‡Department of Medicine and Therapeutics, and §Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

ABSTRACT: A longstanding and widely accepted bottleneck in the targeted delivery of intravenously injected nanoparticles lies in their clearance by macrophages in the liver and spleen. In this Perspective, we call for deeper understanding of the critical role of endothelial cells in the sequestration of nanoparticles in vivo. In this issue of ACS Nano, Campbell et al. used a combination of real-time imaging and genome-editing methods to demonstrate that stabilin-2 is an important receptor for removing anionic liposomes from blood circulation in a zebrafish model. Such mechanistic insights at the molecular level will provide a more holistic picture of the in vivo sequestration of administered nanoparticles beyond the cellular level and pose valuable design considerations for redistributing nanoparticles in vivo. n the field of nanomedicine, targeted delivery to specific biological sites in vivo is critical for nanoparticles to induce their intended therapeutic outcomes. Because a sizable portion of the injected dose naturally accumulates in the liver, intravenous injection is a particularly convenient method for delivering nanomedicines for tackling diseases that arise from the liver. For example, Sato et al. systemically delivered liposomes loaded with small interfering RNAs (siRNAs) against gp46, a protein that mediates fibrosis, into rats with lethal liver cirrhosis. Remarkably, they observed the accumulation of more than half of the injected liposomes in the cirrhotic liver, full resolution of liver fibrosis, and prolonged survival of the rats.1 Nevertheless, after decades of intense research, intravenous delivery of nanoparticles to internal organs other than the liver remains inefficient. In the context of cancer targeting, Choi et al. observed the accumulation of only ∼2% of systemically injected transferrin-targeting gold nanoparticles in the tumor, 10 times lower than the accumulation in liver (17−21% of the injected dose).2 For kidney targeting, Choi et al. demonstrated that no more than 5% of intravenously injected polyethylene glycol (PEG)-coated gold nanoparticles accumulate in the kidney, 12-fold lower than the levels found in the liver.3 In the context of targeting atherosclerotic plaques, Tang et al. showed that for 17 different types of nanoparticles, including high-density lipoprotein mimicking nanoparticles, micelles, and liposomes,

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less than 0.1% of the injected dose accumulates in the plaquecontaining aortic root, 10 times lower than liver accumulation in terms of injected dose per gram of tissue.4 Naturally, sequestration of nanoparticles by the liver represents an enormous delivery bottleneck, hampering the development of effective nanomedicines for treating diseases in other organs. What happens at the tissue and cellular levels once the circulating nanoparticles arrive at the liver? The nanoparticles reach the liver through the hepatic artery or hepatic portal vein and then traverse along the hepatic sinusoidal capillaries, which are composed of liver sinusoidal endothelial cells (LSECs). Interspersed with the sinusoidal endothelium are Kupffer cells (KCs), resident macrophages of the liver that can effectively scavenge circulating nanoparticles (Figure 1A,B).5 Unlike the capillary endothelium in other organs, the liver sinusoidal endothelium is full of fenestrae with pore sizes ranging from 100 to 150 nm. Therefore, sub-100 nm nanoparticles can easily penetrate through the layer of LSECs, diffuse through the space of Disse, and cross the hepatocytes via transcytosis. Such transcytosed nanoparticles reach the bile canaliculus (Figure 1C) and are later excreted through the bile ducts to the duodenum.5 Apart from the physiological processes and anatomical features of the liver, blood hemodynamics also play a significant role in the

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Figure 1. Schematic illustration of our current understanding of the in vivo sequestration of intravenously injected nanoparticles by the liver at the (A) organ, (B) tissue, and (C) cellular levels. The nanoparticles (1) enter the liver through the hepatic artery and (2) arrive at the liver sinusoids through branches of the hepatic artery. Inside the liver, the three possible fates of the nanoparticles include (3(i)) phagocytosis by Kupffer cells (KCs), (3(ii)) permeation through the fenestrae of the liver sinusoidal endothelial cells (LSECs), transcytosis via hepatocytes and excretion to the duodenum through the hepatic bile ducts, and (3(iii)) passing through the liver sinusoids and departure from the liver through the hepatic vein. Modified and reproduced with permission from ref 14. Copyright 2006 Springer Nature.

in vivo sequestration of nanoparticles. According to a recent study by Tsoi et al., the local blood flow rate in the sinusoidal endothelium is 1000 times slower than that in the hepatic portal vein, giving rise to 7.5-fold more interactions between the nanoparticles and the liver cells, along with a concomitantly higher probability of sequestration.6 It should come as no surprise that only a minute portion of the “lucky” nanoparticles that escape sequestration by the constituent tissues and cells of the liver can re-enter the blood circulation through the hepatic vein. Considering the interactions between intravenously injected nanoparticles and the liver, what approaches did nanomaterials scientists adopt to avoid liver sequestration? In the early 1990s, pioneers in the nanomedicine field showed that attachment of PEG strands to nanoparticles can significantly prolong their blood circulation and reduce their sequestration by the liver.7,8 In the 2000s, researchers further managed to control the organlevel and cellular-level distribution of nanoparticles by tuning their physicochemical properties. For instance, nanoparticles with smaller hydrodynamic diameters experience less severe sequestration by the liver3 and KCs9 than their larger counterparts. In addition, positively charged nanoparticles accumulate in the liver (especially in KCs) more abundantly than neutral and negatively charged nanoparticles.10 Reflecting on these strategies, the “trial-and-error” philosophy appears to be the mainstream guiding principle for tackling the challenge of in vivo nanoparticle sequestration, emphasizing the optimization of nanomaterials design parameters while passively observing how these engineered nanoparticles eventually accumulate in different organs and their constituent tissues and cells. From a biological perspective, this “trial-and-error” philosophy treat the in vivo system as a “conceptual blackbox”, characterized by the disregard of how the physiological processes, anatomical features, and molecular mechanisms of an organ (most prominently the liver in the context of nanoparticle sequestration) may collectively influence the delivery of nanoparticles to that organ. A more holistic approach to dealing with the challenge of nanoparticle sequestration requires in-depth knowledge in not only how nanoparticle parameters govern in vivo distribution (already elucidated in detail in the past decade) but also how tissue structures, cellular processes, and molecular mechanisms dictate the distribution of engineered nanoparticles in vivo. A recent study by Tavares et al. explored the specific role of KCs

in modulating nanoparticle sequestration by the liver. By pharmacologically depleting the KCs before injecting nanoparticles into mice, the authors observed a 60% reduction in liver accumulation. Surprisingly, these authors did not detect a comparable increase in nanoparticle contents in blood and other internal organs.11 These results preliminarily indicate KC depletion as a viable strategy for ameliorating liver sequestration and, more generally, reveal the potential of perturbing the biological system (by pharmacological or even genetic means) to improve nanoparticle distribution. Thus, it is imperative that the in vivo interactions of nanoparticles with the liver are comprehensively elucidated at the organ, tissue, cellular, and molecular levels, yet our current understanding remains at the cellular level, often pinpointing KCs as the main “culprit”. Efforts in addressing “bio−nano” interactions at the molecular level are thus far limited to in vitro studies,12 but fall short of validating whether these in vitro observations are physiologically relevant. In this issue of ACS Nano, Campbell et al. report mechanistic insights into the clearance of nanoparticles in zebrafish at the molecular level, showing a critical role of the stabilin-2 scavenger receptor (stab-2) in mediating the uptake of anionic liposomes by venous endothelial cells (ECs).13 These data not only alert the nanomedicine community to pay closer attention to the role of ECs in nanoparticle sequestration, but also enhance our understanding of the in vivo molecular mechanism for sequestration. The authors utilized whole-body confocal imaging to monitor the real-time distribution of fluorescently labeled nanoparticles in a zebrafish model, taking advantage of its transparency and compatibility with real-time imaging at a spatiotemporal resolution down to the cellular level and at various time points.15 In addition to zebrafish’s transparency, which is not shared by higher order mammals such as mice and monkeys, zebrafish also exhibit decent reproducibility and genetic homology to humans, and its husbandry and experimental costs are low.16 Specifically, Campbell et al. chose liposomes as their model nanomaterial. To analyze the effect of surface charge on distribution, they systemically injected fluorescently labeled Myocet, endoTAG-1, and Ambisome (i.e., neutral, positively charged, and negatively charged liposomes, respectively) into the duct of Cuvier, which carries blood from the cardinal veins to the sinus venosus in zebrafish. Although the effect of surface charge on in vivo distribution at the organ and cellular levels was previously B

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Anionic liposomes only associated with veins, such as the caudal vein (CV), posterior cardinal vein (PCV), and caudal hematopoietic tissue (CHT), throughout the experiments (Figure 2). Cationic liposomes associated with both arteries and veins at initial stages, but associated exclusively with veins at later time points. At the cellular level, neutral liposomes translocated through blood vessel walls and entered macrophages. Positively charged liposomes associated with ECs of both arteries and veins at early time points, whereas negatively charged liposomes associated with the ECs of veins only. On the effect of composition, the authors also detected intricate differences in the biodistribution of liposomes with different combinations of surface charges and levels of saturation. Specifically, liposomes made of DOPC (a neutral, unsaturated lipid) freely circulated in the bloodstream, whereas DSPC (a neutral saturated lipid)and DOPG (a negatively charged, unsaturated lipid)-based liposomes associated with venous ECs most strongly. Intriguingly, the authors also claimed that DSPC- and DOPC-based liposomes accumulated in macrophages within the CHT, although they did not specifically address the interactions of the macrophages with the positively charged endoTAG-1 liposomes or the negatively charged Ambisome liposomes. At any rate, these results suggest design considerations for liposomes for targeting or evading uptake by ECs in zebrafish. To address the translatability of the mechanistic insights derived from zebrafish to mammalian models, Campbell et al. next established the functional homology between the ECs and macrophages in zebrafish to the LSECs and KCs in mice in terms of their ability to sequester nanoparticles.13 By intravenously injecting fluorescent DOPG-based liposomes into mice (because such liposomes are found to associate with ECs in zebrafish), they observed the association of the liposomes in both LSECs and KCs inside the liver by ex vivo multiphoton imaging. To test their results, the authors subsequently injected lithium carmine (Li-Car), a type of colloidal stain for delineating the mammalian reticuloendothelial system (encompassing

In this issue of ACS Nano, Campbell et al. report mechanistic insights into the clearance of nanoparticles in zebrafish at the molecular level, showing a critical role of the stabilin-2 scavenger receptor (stab-2) in mediating the uptake of anionic liposomes by venous endothelial cells. determined in mice,10 how these differently charged nanoparticles engage specific receptors responsible for in vivo clearance remains unknown. Because liposomes are composed of a mixture of phospholipids and cholesterol, their chemical composition (e.g., level of saturation and length of alkyl chains in fatty acids) and cholesterol content together determine the physicochemical properties and thus their “nano−bio” interactions. For instance, at the in vitro level, Ho et al. showed that, with a constant number of alkyl chains per nanoparticle, nanoparticles bearing dodecyl chains or octadecyl chains can enter keratinocytes 30-fold more abundantly than those containing hexyl chains.17 In Campbell et al., the authors ascertained the effects of chemical composition on the biodistribution in zebrafish by incorporating several additional types of liposomes made of individual phospholipid derivatives in their comparative studies, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). At the organism level, the authors identified no significant differences in distribution between neutral, positively charged, and negatively charged liposomes. Liposomes of all surface charges were found associated with blood vasculature over time. At the tissue level, however, the authors noticed interesting observations as a function of surface charge. In particular, neutral liposomes were mostly found within the blood vessel lumen.

Figure 2. Schematic illustration of the mechanism for the in vivo sequestration of intravenously injected liposomes in a zebrafish model at the organism, tissue, cellular, and molecular levels. At the organism level, Campbell et al. used confocal imaging to track the distribution of fluorescent liposomes. At the tissue level, negatively charged liposomes (Ambisome) primarily accumulate in the caudal vein (CV) and caudal hematopoietic tissue (CHT). At the cellular level, the anionic liposomes experience in vivo clearance by not only macrophages but also venous endothelial cells (ECs). At the molecular level, the anionic liposomes do not readily enter ECs with expression levels of stabilin-2 (stab-2) silenced by CRISPR/Cas9-based genome editing. Modified and reproduced from ref 13. Copyright 2018 American Chemical Society. C

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for nanoparticle clearance in vivo, let alone the detailed mechanisms for clearance at the molecular level. In this issue of ACS Nano, Campbell et al. have demonstrated an important role of venous ECs in conjunction with macrophages in the sequestration on systemically injected liposomes in zebrafish.13 Importantly, the authors have shown that the stab-2 receptors of ECs are critical to the clearance of anionic liposomes by ECs. This work significantly extends our understanding in in vivo nanoparticle sequestration from the cellular level to the molecular level.

both LSECs and KCs), into zebrafish. Indeed, they detected localization of Li-Car in venous ECs and macrophages, the same locations as the DOPG- and DSPC-based liposomes. Furthermore, the authors repeated the intravenous injection experiment in zebrafish by replacing Li-Car with fluorescently labeled hyaluronic acid (HA), because HA is a ligand of scavenger receptors that are selectively expressed by mammalian LSECs but not macrophages. Again, HA enters the same set of venous ECs that internalize DOPG-based liposomes in zebrafish. These studies provide the necessary functional and conceptual connection between the LSECs and KCs in mice and the set of venous ECs and macrophages identified by the authors in zebrafish. Importantly, they establish a precedent for future studies on the homology between mice and higher order animals, such as monkeys and humans, when dissecting in vivo “bio−nano” interactions. Campbell et al. not only revealed the distribution of liposomes in zebrafish down to the cellular level but also elucidated the molecular-level relationships between liposomes of various physicochemical properties and specific endocytosis receptors. The authors searched for a receptor expressed by the venous ECs that primarily mediates the sequestration of liposomes from blood in zebrafish. They zeroed in on the stab-2 receptor due to its prominent role in internalizing HA and elevated expression level by LSECs as well as ECs found in the spleen and lymph nodes.18 By pretreating zebrafish with dextran sulfate, a competitive inhibitor of stab-2, and consequently injecting DOPG-based liposomes, the authors observed markedly attenuated uptake of liposomes by venous ECs and concomitantly increased uptake by macrophages. To provide direct evidence of the role of stab-2, the authors further employed CRISPR/Cas9-based genome editing to create zebrafish mutants with specifically silenced expression of the stab-2 gene and then injected DOPG-based liposomes into these mutants. Confocal imaging showed a significant reduction in the uptake of the DOPG-based liposomes by the same set of venous ECs previously determined by these authors, together with enhanced uptake by macrophages and higher amounts in circulation (Figure 2). Returning to their previous inquiry into the effect of surface charge on nanoparticle sequestration, the authors injected positively charged endoTAG-1 liposomes, neutral Myocet liposomes, and negatively charged Ambisome liposomes into the stab-2 mutants, concluding that genetic perturbation of stab-2 only affects the sequestration of Ambisome but not the other two counterparts. The results provide molecular evidence of at least one receptor (i.e., stab-2) that mediates the sequestration of negatively charged liposomes in zebrafish. Researchers should be cautious in using negatively charged nanoparticles if they desire to achieve prolonged blood circulation for targeted delivery. Also, these data suggest that a specific receptor is no less important than a specific cell type (EC versus macrophage) in biasing the fate of intravenously injected nanoparticles. Negatively charged liposomes may be attractive delivery shuttles to cell types that overexpress stab-2, such as ECs in the liver, spleen, and lymph nodes.

We call for the nanomedicine field (1) to recognize that Kupffer cells may not be the only “culprits” for removing nanoparticles from blood and (2) to investigate the roles of other relevant cell types at the molecular level. However, the translational potential of the mechanistic insights derived from this work in mice, nonhuman primates, and even humans requires further validation. Future investigations are necessary to clarify the importance of the stab-2 gene in relation to other genes in the liver (and other major clearance organs like the spleen). Specifically, what is the relative expression level of stab-2 in hepatic KCs versus LSECs in mice or other animals? Does the expression level of stab-2 depend upon the specific disease, like cancer? What about the potential role of other members of the scavenger receptor superfamily in the in vivo sequestration of nanoparticles by ECs? For example, Choi et al. proved that Class A scavenger receptors (SR-A) are primarily responsible for the uptake of DNA-coated nanospheres by mouse ECs19 and that DNA-coated nanospheres and nanorods can both bind to SR-A.20 Vindigni et al. showed that oxidized low-density lipoprotein receptor-1 (LOX-1), another type of scavenger receptor, mediates the selective uptake of DNA nanocages by fibroblasts.21 Note that DNA nanostructures are polyanionic, analogous to the anionic liposomes used in this work. Because nanoparticles of different surface charges and compositions generally experience clearance by the liver, future studies should consider other receptors in addition to scavenger receptors. Careful characterization of the structural and functional homology of zebrafish to mice or humans at the cellular and molecular levels will encourage the use of zebrafish as a first-pass animal model in future studies to elucidate the roles of different receptors involved in the uptake of nanoparticles in vivo. This work highlights the merit of advanced imaging methods for tracking the distributions of nanoparticles in transparent animal models like zebrafish. What about opaque mammalian models? Past studies typically reported the distribution of nanoparticles as concentrations in different organs or the spatial distribution in two-dimensional stained histological sections. At the expense of collecting real-time imaging data that are affordable by zebrafish or other transparent animals, researchers are now expanding their imaging capabilities of capturing threedimensional (3D) nanoparticle distribution data in organ specimens ex vivo. In 2013, Chung et al. invented the CLARITY technique to generate 3D optical images of the mouse brain after immunostaining.22 Later, Sindhwani et al. applied this technique to perform 3D optical imaging of harvested internal organs, including the liver.23 Specifically, Sindhwani et al. showed

CONCLUSIONS AND OUTLOOK Over the past three decades, the field of nanomedicine has come to a consensus regarding a major delivery bottleneck for systemically administered nanoparticles: in vivo sequestration of nanoparticles by the liver. Whereas past researchers mostly focused on designing bionanomaterials that can evade uptake by hepatic KCs, rarely did they investigate the roles of other constituent cell types in the liver that may also be responsible D

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sequestration in higher order mammals with patience, commitment, and rigorous basic research.

that the 3D microvasculature, lymphatics, and spatial distribution of cells are critical to the local clearance of nanoparticles in tissues. We believe that real-time 3D imaging capabilities with cellular-level resolution in opaque animal models, albeit technologically challenging currently, will become an attractive opportunity for aspiring imaging experts to contribute to our understanding of in vivo “bio−nano” interactions. Genome editing, a powerful technique for modifying specific locations of the genome, enables us to examine the molecular basis of nanoparticle sequestration in vivo. In their work, Campbell et al. adopted the CRISPR/Cas9 technique to demonstrate the role of stab-2 receptor in clearing anionic liposomes from zebrafish.13 Recently, Yin et al. demonstrated the use of lipid nanoparticles to deliver single-guide RNAs and mRNA encoding the Cas9 protein to the liver in mice. Remarkably, they achieved more than 80% editing of the target gene Pcsk9 and reduced cholesterol levels by 35−40%.24 As the liver is a predominant organ of nanoparticle sequestration, we hypothesize that a similar CRISPR/Cas9-based lipid nanoparticle delivery system proposed by Yin et al. may facilitate editing of stab-2 (or other genes) in LSECs or KCs of mice with high efficiency, laying the technical groundwork for studying the roles of different receptors in nanoparticle sequestration in higher order animals than zebrafish. Mechanistic insights into the roles of genes responsible for in vivo nanoparticle clearance will create opportunities for designing advanced nanoparticle-based delivery systems that can not only carry therapeutic agents but also improve their targeted delivery. Campbell et al. presented an application of their insights to drug delivery.13 By systemically injecting liposomes that bear clodronic acid, a cytotoxic drug, into wild-type zebrafish embryos, the authors observed selective uptake of the drug-containing liposomes by ECs of the caudal vein, loss in venous ECs, disappearance of CVs, and rerouting of blood flow. In contrast, injection of the same drug-bearing liposomes to stab-2 zebrafish mutants did not cause damage to CVs or changes in blood flow patterns, evidence of the importance of stab-2 in the redistribution of nanoparticles. From the zebrafish model results, we postulate that blocking scavenger receptors of LSECs in higher order mammals will curb the transcytosis of nanoparticles through LSECs and limit their sequestration by the liver. Our proposed nanoparticle-based delivery system contains two parts, “therapeutic cargoes” for eliciting the intended therapeutic outcomes (e.g., small molecules or microRNAs) and “regulatory elements” for blocking their sequestration, mediated by stab-2 or other scavenging receptors (e.g., siRNA or sgRNA/mRNA against the stab-2 gene expressed by LSECs). One may refer to a conceptually similar example by Meng et al., who designed a multifunctional nanoparticle to codeliver chemotherapeutic drug molecules and siRNAs for silencing the P-glycoprotein efflux pump and enhance in vivo anticancer efficacy.25 With the breadth of nanomaterials fabrication tools available, we anticipate the birth of smart nanomedicines with extended blood circulation, reduced sequestration by the liver, improved targeting of key cell types, attenuated drug resistance, and improved therapeutic efficacy. To date, few studies have reported the mechanism for sequestration at the molecular level and strategies for redistribution of nanoparticles. The encouraging data in zebrafish reported by Campbell et al. represent an important step forward, inspiring the nanomedicine community to continue to tackle the longstanding delivery bottleneck of in vivo nanoparticle

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Chung Hang Jonathan Choi: 0000-0003-2935-7217 Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported in part by an Early Career Scheme grant (project no. 24300014) and a General Research Fund (project no. 14302916) from the Research Grants Council (RGC) of Hong Kong. It was also supported in part by a National Natural Science Foundation of China/RGC Joint Research Scheme grant (project no. N_CUHK434/16). C.H.J.C. acknowledges the Chow Yuk Ho Technology Centre for Innovative Medicine. He is also grateful to the Croucher Foundation for a Croucher Startup Allowance and a Croucher Innovation Award. REFERENCES (1) Sato, Y.; Murase, K.; Kato, J.; Kobune, M.; Sato, T.; Kawano, Y.; Takimoto, R.; Takada, K.; Miyanishi, K.; Matsunaga, T.; Takayama, T.; Niitsu, Y. Resolution of Liver Cirrhosis Using Vitamin A-Coupled Liposomes To Deliver siRNA against a Collagen-Specific Chaperone. Nat. Biotechnol. 2008, 26, 431−442. (2) Choi, C. H. J.; Alabi, C. A.; Webster, P.; Davis, M. E. Mechanism of Active Targeting in Solid Tumors with Transferrin-Containing Gold Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1235−1240. (3) Choi, C. H. J.; Zuckerman, J. E.; Webster, P.; Davis, M. E. Targeting Kidney Mesangium by Nanoparticles of Defined Size. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6656−6661. (4) Tang, J.; Baxter, S.; Menon, A.; Alaarg, A.; Sanchez-Gaytan, B. L.; Fay, F.; Zhao, Y.; Ouimet, M.; Braza, M. S.; Longo, V. A.; Abdel-Atti, D.; Duivenvoorden, R.; Calcagno, C.; Storm, G.; Tsimikas, S.; Moore, K. J.; Swirski, F. K.; Nahrendorf, M.; Fisher, E. A.; Pérez-Medina, C.; et al. Immune Cell Screening of a Nanoparticle Library Improves Atherosclerosis Therapy. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E6731−E6740. (5) Bertrand, N.; Leroux, J.-C. The Journey of a Drug-Carrier in the Body: An Anatomo-Physiological Perspective. J. Controlled Release 2012, 161, 152−163. (6) 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.; Conneely, J. B.; Alman, B. A.; Selzner, M.; Ostrowski, M. A.; Adeyi, O. A.; Zilman, A.; McGilvray, I. D.; Chan, W. C. W. Mechanism of Hard-Nanomaterial Clearance by the Liver. Nat. Mater. 2016, 15, 1212−1221. (7) Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Liposomes Containing Synthetic Lipid Derivatives of Poly(ethylene glycol) Show Prolonged Circulation Half-Lives in Vivo. Biochim. Biophys. Acta, Biomembr. 1991, 1066, 29−36. (8) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Biodegradable Long-Circulating Polymeric Nanospheres. Science 1994, 263, 1600−1603. (9) Ogawara, K.-I.; Yoshida, M.; Higaki, K.; Toshikiro, K.; Shiraishi, K.; Nishikawa, M.; Takakura, Y.; Hashida, M. Hepatic Uptake of Polystyrene Microspheres in Rats: Effect of Particle Size on Intrahepatic Distribution. J. Controlled Release 1999, 59, 15−22. (10) Xiao, K.; Li, Y.; Luo, J.; Lee, J. S.; Xiao, W.; Gonik, A. M.; Lam, K. S. The Effect of Surface Charge on in Vivo Biodistribution of PEGE

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