Form Follows Function: Nanoparticle Shape and Its Implications for

Review pubs.acs.org/CR

Form Follows Function: Nanoparticle Shape and Its Implications for Nanomedicine Calum Kinnear,† Thomas L. Moore,‡ Laura Rodriguez-Lorenzo,‡ Barbara Rothen-Rutishauser,‡ and Alke Petri-Fink*,‡,§ †

Bio21 Institute & School of Chemistry, University of Melbourne, Parkville 3010, Australia Adolphe Merkle Institute and §Chemistry Department, University of Fribourg, Fribourg 1700, Switzerland

‡

ABSTRACT: This review is a comprehensive description of the past decade of research into understanding how the geometry and size of nanoparticles affect their interaction with biological systems: from single cells to whole organisms. Recently, there has been a great deal of effort to use both the shape and the size of nanoparticles to target specific cellular uptake mechanisms, biodistribution patterns, and pharmacokinetics. While the successes of spherical lipid-based nanoparticles have heralded marked changes in chemotherapy worldwide, the history of asbestos-induced lung disease casts a long shadow over fibrous materials to date. The impact of particle morphology is known to be intertwined with many physicochemical parameters, namely, size, elasticity, surface chemistry, and biopersistence. In this review, we first highlight some of the morphologies observed in nature as well as shapes available to us through synthetic strategies. Following this we discuss attempts to understand the cellular uptake of nanoparticles through various theoretical models before comparing this with observations from in vitro and in vivo experiments. In addition, we consider the impact of nanoparticle shape at different size regimes on targeting, cytotoxicity, and cellular mechanics.

CONTENTS 1. Introduction 2. Morphologies Observed in Nature 2.1. Viruses 2.2. Bacteria 2.2.1. Filaments 2.2.2. Helical Rods 2.2.3. Rods 3. Engineered Nanoparticle Morphologies 3.1. Organic Materials 3.2. Inorganic Materials 3.2.1. Metal Nanoparticles and Seeded Growth Methods 3.2.2. Alloys and Galvanic Replacement 3.2.3. Hollow Structures 3.2.4. Templated Routes in Nanoparticle Preparation 3.2.5. Synthesis of Two-Dimensional Materials: The Case of Graphene 3.2.6. Green Nanoparticle Synthesis Using Deep-Eutectic Solvents 4. Atomic- and Molecular-Scale Shape-Dependent Interactions 5. Theoretical Approaches 5.1. Analytical and Continuum Models 5.2. Numerical Simulations 5.3. Cooperative Endocytosis 5.4. Shapes under Flow 5.5. Summary of Theoretical Approaches

© 2017 American Chemical Society

6. Influence of Differently Shaped NPs on Their Interaction with in Vitro Systems 6.1. Large, Mostly Organic Particles 6.2. Small Inorganic and Organic Particles 6.3. Cellular Targeting 6.4. Cytotoxicity and Cell Mechanics 6.5. Particle Behavior and Uptake under Flow 6.6. Summary of Shape Effects in Vitro 7. Influence of Differently Shaped NPs on Their Interaction with in Vivo Systems 7.1. Pharmacokinetics and Biodistribution 7.2. Targeting in Vivo 7.3. Toxicity 7.4. Two-Dimensional Materials 7.5. Summary of Shape Effects in Vivo 8. Lessons and Future Challenges Author Information Corresponding Author ORCID Author Contributions Notes Biographies Acknowledgments Abbreviations References

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Received: April 7, 2017 Published: September 1, 2017 11476

DOI: 10.1021/acs.chemrev.7b00194 Chem. Rev. 2017, 117, 11476−11521

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1. INTRODUCTION The excitement surrounding nanomedicines stems from the possibility to precisely engineer their physicochemical properties such as size, shape, elasticity, surface charge, and surface functionalization to achieve desired behaviors in vivo.1,2 Moreover, the ability to use innovative materials or create composite particles, e.g., made from inorganic and polymeric materials, enables theranostic modalities which provide simultaneous diagnostic and therapeutic functionality. In drug delivery applications, particle-based platforms are exciting due to their ability to prolong circulation times, undergo controlled drug release, overcome biological barriers (e.g., passive tumor accumulation via the enhanced permeability and retention effect or active targeting of overexpressed cancerous markers), and reduce nonspecific drug toxicity.3 In fact, a nanoparticle (NP) formulation for drug delivery, Doxil, has been clinically approved for systemic use since 1995 as a liposomal formulation of doxorubicin which decreased doxorubicinrelated cardiotoxicity.4,5 To date, most nanomedicines in clinical trials or lab-scale studies are spherical. However, over the past two decades we have gained a greater understanding of the growth of inorganic NPs and the synthesis of nonspherical shapes (Figure 1).6 Advances in self-assembly strategies have also led to the development of anisotropic polymeric nano- and microparticles,7 which are now finding applications in drug delivery, imaging, therapy, and diagnostics. This incredible progress in the shape control of inorganic NPs has led to the generation of various kinds of rods, 2D prisms, and branched structures (Figure 1).8 The standard route to most of these materials is through wet chemical syntheses in the case of metals and their oxides. Polymeric particles, on the other hand, typically require templates or postsynthesis manipulation to alter their shapes. We direct the reader to a number of more comprehensive reviews on synthetic routes for shape control of both inorganic and organic NPs.6,7 The attractive and oft-superior physicochemical characteristics of NPs are due to their small size.17 Often different shapes impact these properties in a beneficial manner; however, it is not always clear how it will impact the performance of the material upon interacting with biological interfaces and entities.18 When we look to nature, an abundance of shapes persist: from the spherical HIV virus to rod-shaped tobacco mosaic viruses and star-shaped bacteria.19 Bacterial morphology is known to confer evolutionary advantages based on specific mechanisms such as their interaction with surfaces, passive diffusion, and active motility.20 In Darwinian evolution, form precedes function due to random mutations and natural selectionbacteria and viruses are now observed to have these shapes for good reasons. As scientists, we can ensure the future NPs in medicine have a form that follows their function. However, currently, the vast majority of systems are spherical due to both their ease of synthesis and a lack of understanding in their structure−activity relationships. The elucidation of specific structure−activity relationships between NP shape and certain common biological end points, such as uptake, toxicity, biodistribution, and inflammatory response, is nontrivial.21 The difficulty stems from two main factors: First, the variety of potential variables to alter is huge, such as material composition, mechanical properties of the material, cell types, and surface ligands,22 which mean that

Figure 1. Metallic NPs with various morphologies: (a) nanosphere, (b) nanorod, (c) nanobelt, (d) nanowires, (e) 2D triangle, (f) 2D hexagon, (g) disc, (h) nanocube, (i) octahedron, (j) tripod, (k) nanostar, (l) nanothron, (m) tetrapod. (c, d, e, f, g, j, and m) Adapted with permission from refs 9, 10, 11, 1213, and 14, respectively. Copyright 2008, 2003, 2007, 2005, 2005, and 2003 American Chemical Society, respectively. (k, l) Adapted with permission from ref 15. Copyright 2008 John Wiley and Sons. (i) Reprinted by permission from Macmillan Publishers Ltd.: ref 16, copyright 2007. Some figures have had the background removed for clarity; for original figures and scale bars, see the relevant references.

there are few comparable studies from which robust conclusions can be drawn. Second, crucial variables are often correlated and entangled, meaning isolating and studying only shape as a parameter is often near-impossible.23 As an example, tuning the shape of silver NPs requires an alteration in the ratio of different crystal facet surface areas. These facets are known to have different dissolution rates, and considering it has been shown that silver ions are often the source of observed cytotoxicity, these different rates mean any observed shape effect may not actually be due to a physical effect, rather a chemical one.24,25 In this review, we summarize the past decade of research around NP interactions with biological systems, e.g., single cells, tissues, or organs, with a specific focus on the impact of NP shape. There has been a huge volume of published data on this topic, and due to the interdisciplinary nature of the research, it is often distributed across journals with dissimilar tags and keywords; therefore, forming guidelines in the design 11477


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of NPs for biomedical applications is rather challenging.26 Herein, we are concerned with both inorganic (primarily metals/semiconductors and their oxides) and organic (polymeric or carbon allotropes) NPs and split their interaction into three broad areas: (1) simulations and theoretical models of NP interactions with cells, (2) experimental observations from in vitro experiments, and (3) from in vivo experiments. The greatest focus within these three areas is given to endocytosis rates, i.e., cellular uptake of NPs, generally within in vitro settings, biodistribution, and adverse biological reactions such as cytotoxicity or (pro-)inflammation. Several principles are outlined, depending on the desired biological outcome, to help rationally design future NPs for drug delivery and theranostics. In addition, some critical challenges and open questions are posed that need addressing such as how to cross from in vitro to in vivo and how to ensure only shape-dependent effects are probed.

found in spherical (coccus), rod-like (bacillus), crescent (vibrio), and twisted (spirilla) forms (Figure 2b). There are few controlled studies investigating the relationship between the form of viruses and bacteria and their function; however, examples have hinted at the importance of different shapes on their survival and proliferation. Before delving into the interactions between viruses, bacteria, and NPs with mammalian cells, it is pertinent to discuss some terminology used throughout this review when we talk about these “interactions”. Specifically, we will focus on the cellular uptake of NPs. Most cells can secrete and ingest macromolecules and particles by exocytosis and endocytosis, respectively, with the initiation of these processes occurring at the outer cell membrane.27 This plasma membrane is a dynamic structure that segregates the chemically distinct intracellular milieu (the cytoplasm) from the extracellular environment by coordinating the entry and exit of both small and large molecules. All mammalian cellular membranes have a common structure consisting of a thin film of lipid and protein molecules, primarily held together by noncovalent interactions.28 The lipid molecules are arranged as a continuous double layer about 4−5 nm thick, creating a relatively impermeable barrier for most water-soluble molecules. Transmembrane protein molecules, on the other hand, mediate specific functions such as pumps across the bilayer or catalyzing membrane-associated reactions. They can also serve as structural links to connect the cytoskeleton through the lipid bilayer to the extracellular matrix or an adjacent cell by integrins and cadherins. Others serve as receptors to detect and transduce chemical signals into the cell’s environment.29 It is important to note that the membrane is not a rigid structure but rather a dynamic system composed of fluid and gel-like regions, described as a twodimensional fluid in which lipid and protein molecules can diffuse (for a review see ref 30). The uptake of NPs by mammalian cells occurs mainly via endocytotic pathways as shown in Figure 3. Two types of endocytosis are distinguished: pinocytosis (“cellular drinking”) which involves the ingestion of fluid and molecules often via small vesicles (0.25 μm in diameter) (for more detailed reviews on this topic, see refs 31 and 32). The term pinocytosis includes macropinocytosis, clathrin- and caveolinmediated endocytosis, and clathrin- and caveolin-independent endocytosis.31 Additionally, studies have shown that some NPs with at least one very small dimension have the potential to passively diffuse across the plasma membrane (Figure 3).33 Phagocytosis is carried out by professional phagocytes (i.e., monocytes/macrophages, neutrophils, dendritic cells) and usually involves the ingestion of large particles such as microorganisms and cell debris through the formation of large intracellular vesicles called phagosomes (generally >0.25 μm in diameter). Macromolecule or particle internalization is initiated by the interaction of specific receptors on the surface of the phagocyte. This leads to the polymerization of actin at the site of ingestion, i.e., membrane ruffling, and after internalization the phagosome matures by a series of fusion and fission events with components of the endocytic pathway, culminating in the formation of the mature phagolysosome.34 Macropinocytosis triggers actin formation, and the macropinosomes form large intracellular vesicles. However, instead of

2. MORPHOLOGIES OBSERVED IN NATURE If one was to observe seawater with an optical microscope, a multitude of different shaped organisms would appear. If electrons are exchanged for photons then these organisms will appear to have strongly scale-dependent morphologies. While the impact of morphology at a molecular scale is evident, for example, in the form of an antibody, we are concerned with the scales from 10 to 1000 nm, which can be roughly sectioned into viral sizes of ∼10−500 nm and bacterial sizes above this. Within these two ranges there are various shapes observed with specific functional consequences. The majority of viruses appear to be spherical in shape; however, filamentous and bullet- and rodshaped forms, among others, have all been observed in nature (Figure 2a). At the larger end of the scale, bacteria are often

Figure 2. Various morphologies of viruses and bacteria in nature. (a) Schematic of viruses to scale, including brick-shaped or pleomorphic, spherical, bullet-shaped, icosahedral, and filamentous forms. Reprinted with permission from ViralZone, SIB Swiss Institute of Bioinformatics. (b) Common bacterial forms such as spherical (coccus), rod like (bacillus), crescent (vibrio), and twisted (spirillum). 11478


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Figure 3. Schematic representation of different endocytotic mechanisms. Large (micrometer-sized) particles may be actively incorporated via phagocytosis. Areas of high curvature on anisotropic particles, such as large ellipsoids, can contact cells and be more favorably phagocytosed. Smaller particles can be internalized through multiple distinct mechanisms, namely, macropinocytosis (>1 μm), clathrin-mediated endocytosis (∼120 nm), clathrin- and caveolae-independent endocytosis, or caveolae-mediated endocytosis (∼60 nm). Besides active transport, nanoparticles may also enter the cell passively via diffusion or passive uptake by van der Waals or steric interactions through the plasma membrane. This can include piercing of the cell membrane by areas of very high curvature (e.g., carbon nanotubes or graphene edges).

membrane, the clathrin coat is disassembled and the clathrin triskelia are recycled back to the plasma membrane where they assemble again around a new vesicle bud.41 Clathrin- and caveolin-independent endocytotic mechanisms are poorly understood. It is generally related to the cholesterolrich microdomains, called rafts, with a diameter of 40−50 nm in the cell membrane. Their unique lipid composition provides a physical basis for specific sorting of membrane proteins, glycoproteins, and/or glycolipids. These small rafts can presumably be captured by and internalized within any endocytic vesicle.36,37 All of the previously presented endocytic pathways result in particles located in membrane-bound compartments. However, there are studies which have reported that nanoparticles of different materials can be found free in the cytoplasm, indicating alternative pathways for particles to enter the cells or that they have been released from the vesicles.42−44 The uptake of nanoparticles via such alternative mechanisms, by passive and active (receptor mediated) diffusion through membrane pores and passive uptake by van der Waals or steric interactions (subsumed as adhesive interactions),45 need, however, to be explored further. Contrary to some viruses and bacteria, the endocytosis of (nano)materials rarely follows just one mechanism and depends on the physical interaction of the material with the cell wall, the material itself, and the cell type. This physical interaction has been experimented with over millennia by viruses and bacteria, leading to the plethora of surface chemistries, targeting strategies, and morphologies observed in nature.

invaginating a ligand-coated particle, they collapse onto and fuse with the plasma membrane to generate large endocytic vesicles called macropinosomes that sample large volumes of extracellular milieu. However, little is known about the nature of the entire uptake process, and many questions remain unanswered.35 Caveolin-mediated endocytosis is mostly used for the transport of serum proteins. Caveolae are static flask-shaped invaginations of the plasma membrane, and are observed in several cell types, including capillary endothelium, type I alveolar epithelial cells, smooth muscle cells, and fibroblasts, and are slow in uptake.31 This mechanism is generally referred to as dependent on cholesterol-rich microdomains, called lipid rafts with a diameter of 40−50 nm.36,37 The protein which gives shape and structure to the invaginations is called caveolin-1, which is a dimeric protein and binds cholesterol onto the cellular surface for intracellular trafficking (lipid homeostasis).38 Clathrin-mediated endocytosis is very well studied and it is, like most pinocytic pathways, a form of receptor-mediated endocytosis that is generally fast. It constitutively occurs in all mammalian cells and carries out the continuous uptake of essential nutrients, such as the cholesterol-laden low-density lipoprotein particles that bind to the low-density lipoprotein receptor and iron-laden transferrin that binds to transferrin receptors.39,40 This receptor-mediated process is very well studied and results in vesicles of about 100 nm in diameter which are then coated by a protein complex consisting of clathrin. In contrast to caveolin-mediated transport, the vesicle coat does not remain stable during clathrin-dependent endocytosis. After the vesicles have detached from the plasma 11479


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2.1. Viruses

The geometry-dependent uptake route of viruses was further shown by Rossman et al.49 for the case of influenza where spherical forms (100 nm) were internalized by clathrinmediated endocytosis, while the filamentous ones (100 nm × 20 μm) followed a macropinocytosis route. Interestingly, once the filamentous viruses had been trafficked to the acidic late endosome, they fragmented into spherical virions enabling more efficient viral-endosome membrane fusion. Sieczkarski and Whittaker50 found that filamentous influenza viruses, similar to those found in the lungs of infected individuals, presented a much delayed internalization than their spherical lab-grown analogs. Nanbo et al.51 as well as Saeed et al.52 have both shown how the filovirus Ebola enters cells through using the actin-dependent cellular machinery via macropinocytosis, in contrast to the smaller spherical viruses. Tobacco mosaic virus (TMV), a plant RNA virus, is considered a model self-assembled structure with dimensions of around 300 × 18 nm. When the virus was fragmented into shorter rods with ultrasonication to aspect ratios of 4 or 8, faster ingestion was observed by both HeLa and HUVEC cells compared with rods of aspect ratio 17.53 However, no uptake by macropinocytosis was found, contrary to the studies on filamentous influenza viruses by Rossman et al. as well as others.49,51,52 Smaller TMV formulations were also predicted and shown experimentally to diffuse further into simulated tumor spheroids compared with longer virus particles.54,55 These are a handful of examples on the endocytosis of viruses; however, there are far fewer studies on the impact of shape on the extracellular behavior of viruses in flow, in viscoelastic fluids, or interaction with various cellular barriers. Nevertheless, these findings are a great source of insights to guide the design of NPs for drug delivery, theranostics, and imaging. The membrane of these structural analogs often contains the same proteins, as identified for the glycoprotein spikes on pleomorphic influenza virions.56 This means that the effect of different ligand/receptor densities on the surface is less of a compounding problema factor not easily controlled for in the case of engineered nanomaterials.

The relatively high surface-to-volume ratios of viruses mean they pay a high energy penalty for deviating from isometric forms. Viruses are commonly found with icosahedral, spherical, or faceted morphologies; however, as described by the International Committee on Taxonomy of Viruses, several other shapes are found in nature such as rods, helices, filaments, bullet shapes, and pleomorphic forms (Figure 2a).46,47 One of the few studies undertaken in the area of form and function was by Kirchhausen and colleagues,48 who took the bullet-shaped vesicular stomatitis virus and engineered a spherical analog or defective interfering particle. They found that when the surface proteins were maintained constant, the elongated virus, 70 × 200 nm versus the 75 nm spherical analog, could not be sufficiently internalized by only clathrinmediated endocytosis and required actin assembly. In other words, because the virus is elongated it is too large to fit within a solely clathrin-coated vesicle and requires actin assembly to form a larger vesicle (Figure 4).

2.2. Bacteria

In contrast to viruses, bacteria can be found in an abundance of different morphologies and sizes. The increase in energy for a bacterium to transition from spherical to rod-like and to maintain their form against the internal osmotic pressure means that great evolutionary advantages must be gained from this transition. Due to the changes in the bacterial cell wall necessary to alter their morphology, it has been difficult in the past to deconvolute the shape effects with the consequences of varying surface proteins. Indeed, the exact mechanics and mechanisms that underpin the cell shape−function relationship are still elusive in most cases. We direct the reader to several excellent reviews addressing this topic, specifically around the area of host−pathogen interactions and the evolutionary advantage of morphology.20,57−59 Most bacteria have little desire to be taken up by mammalian cells, with a few notable exceptions, and have evolved to survive and proliferate extracellularly. This means that their forms are specifically designed to help them avoid predation, disperse and attach, move more efficiently, or uptake nutrients. Herein, we will only highlight the physical advantages gained by various shapes and how these could apply to designing future NPs for medicine.

Figure 4. Differential uptake mechanisms for bullet-shaped and spherical vesicular stomatitis virus (VSV) particles. (a) Electron micrograph of spherical defective interfering (DI-T) particles undergoing clathrin-dependent endocytosis at early (left) and late (right) stages. (b) Clathrin- and actin-dependent endocytosis of rod-like vesicular stomatitis virus sequentially from left to right and top to bottom. Adapted with permission from ref 48. Copyright 2010 PLOS. 11480


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2.2.1. Filaments. Given that nutrient uptake is so important for bacteria, it is not surprising that there are so many filamentous morphologies found in nature. If the surfaceto-volume ratio was the only driving force then a thin disc would maximize this value; however, there are very few flat disc-shaped bacteria in nature.60 Therefore, other driving factors should be considered. Predation, or conversely immune resistance, of filamentous bacteria is an aspect that has gathered a great deal of attention, particularly because the filament forms were considered, for a long time, weaker members of the population. However, there is now a consensus that filaments can avoid killing by professional phagocytotic cells due to the difficulty of completely engulfing a long filament, compared with bacillus or coccus forms.59,61 An example of this was observed by Prashar et al., who found Legionella pneumophila’s filamentous form, above 5 μm in length, could not be easily engulfed by phagocytes, allowing the bacteria to escape killing in a lengthdependent manner.62 Additionally, phagocytosis was only successfully completed when it was initiated at the bacterial pole, as opposed to the lateral sides of the filaments. In another case, Escherichia coli (E. coli) in urinary tract infections in mouse bladders was observed to form filaments that helped it avoid phagocytosis by polymorphonuclear leukocytes.63 In vitro studies have shown that macrophages can only successfully engulf filamentous E. coli when they can access the pole of the filament.64 If access to the pole is not possible then macrophages must reorient the bacteria in order to engulf them, something addressed in more detail later in this review. These observations have led to proposals that the pleomorphic ability of various bacteria is a survival strategy whereby the transition from small, single, entities to filaments is down to environmental triggers.65 The enhanced adherence of bacteria to surfaces conferred by their large surface areas is another important aspect. Intuitively, filaments can have a larger surface area in contact with a surface compared with spheres, and therefore, detachment at one point does not necessarily mean complete removal. Streptococcus pneumoniae has been shown to adhere stronger to A549 lung epithelial cells in vitro upon forming chains, as well as in vivo, indicating that morphological heterogeneity could promote colonization of the upper respiratory tract.66 The normally spherical Staphylococcus aureus has also been found to form filamentous biofilm streamers under flow.67 These observations point to two major evolutionary advantages for filamentous forms: avoiding killing by the innate immune system and increased surface adherence, which are both properties that can be leveraged in designing next-generation nanomedicines. 2.2.2. Helical Rods. The relationship between motion at the nano/micrometer scale and particle morphology has been studied for decades.68 There have been pioneering biophysical examinations on the energy of chemotaxis and bacterial propulsion, particularly how this depends on geometry.69 Helical bacteria have been found in environments of high viscosity, such as intestinal mucus,70 indicating the helical form confers some advantage in these fluids. Notably, the Nobel Prize in Medicine in 2005 was awarded for the discovery of the short helical bacterium, Helicobacter pylori, which causes inflammation and stomach ulcers upon colonizing the gastric mucosa.71 Early experiments indicated helical bacteria had far higher motility in a viscous solution of the polymer polyvinylpyrrolidone (PVP) than less coiled forms.72 More recent research has shown that these shapes impart significant

benefits in biofilm formation, motility, and movement through viscoelastic fluids.73,74 A key property seems to be the ability of the helical bacterium to move through mobile fluid channels within gel-like networks, something rod-like forms are unable to do with ease: “like a corkscrew through a cork”.59,75 2.2.3. Rods. E. coli and Bacillus subtilis are the two most studied rod-like bacteria to date and are thought to possess the same shape as the first bacteria on earth.76 The correlation between structure and motility for E. coli was elegantly shown, without any biochemical alterations, by Takeuchi et al.77 who forced the cells to grow into embossed templates with a specific shape. They found that short crescents moved in straight lines as did helical cells with a long pitch, whereas those with short pitches were only able to move in tight circles. The impact of these morphologies, particularly where the biochemical homeostasis is maintained across different shapes, on pathogenicity was not addressed. Another organism that takes advantage of anisotropy in shape, and trans-membrane proteins, is Listeria monocytogenes which, once in the cytosol, anisotropically polymerizes actin to propel itself toward the cell surface.78 Once at the surface, it maintains its orientation to protrude into a cellular extension, which is then engulfed by a neighboring cell. While rods have advantages in motility, evidence also points to another benefit: surface adhesion under flow. In a similar vein to that of filamentous bacteria, rods can attach to a surface either via their pole or along their long axis and typically orient themselves with the flow, thereby minimizing shear stresses which threaten to rip them from the surface.79 The same is true of the crescent-shaped Caulobacter crescentus, which attaches at one pole and orients in the flow placing the other pole proximal to the surface, thus enabling easier division and surface colonization.80 However, the situation is not as simple as thisrather counterintuitively, rod-like Bacillus subtilis and Pseudomonas aeruginosa were found to concentrate in highshear regions, with up to 70% depletion from low-shear regions, of microfluidic channels.81 This was confirmed, through analytical models, to originate from competition between the orientation of the rods with the flow and their stochastic swimming direction, resulting in the trapping of bacteria in high-shear regions near the walls of channels or vessels. It would be disingenuous to suggest that shape is the major, or only, factor when considering the interactions between bacteria and surfaces or mammalian cells as the biochemistry of these organisms plays a major role. As an example, consider the Gram-negative bacterium Caulobacter crescentus which, while crescent shaped, attaches to surfaces through a long stalk and holdfast that has amazingly large detachment forces in the μN range.82 The advantage of a crescent shape for this bacterium has little to do with surface adhesion and likely more to do with swimming motility and nutrient access. Nevertheless, from these studies on naturally occurring shapes, several guiding principles can be suggested. First, filamentous forms appear to confer an advantage in avoiding predation through frustrating their endocytosis. Ingestion of these higher aspect ratio particles commonly occurs via macropinocytosis, whereas smaller spherical particles are endocytosed by clathrin-dependent mechanisms, which may require actin polymerization if the particle is slightly too long. Micrometer-sized rod-like particles may also adsorb stronger to vessel endotheliums under flow. Interestingly, motile particles gain advantages from helical forms in viscoelastic fluids or from margination dynamics of rods under flow. 11481


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3. ENGINEERED NANOPARTICLE MORPHOLOGIES 3.1. Organic Materials

Given the wide variety of organic and flexible shapes of bacteria and viruses in nature, the question arises of how close can we mimic these through synthetic chemistry strategies? Regarding NPs synthesized via bottom-up routes, we still have a long way to go to have full control over self-assembled structures at the nanoscale; however, recently several impressive top-down approaches have enabled us to custom design bacteria-scale particles and study their interactions with various biological systems. For wet-chemically-synthesized inorganic NPs, shape control is driven by altering the kinetics of surface facet growth, often through passivation with various surfactants or polymers, to form rods, wires, stars, spheres, or various other structures.6 For in-depth reviews in the chemistry of anisotropic NP synthesis, we direct the reader to several comprehensive reviews.6,83−86 Large organic structures of almost any morphology can be made through UV or laser-based lithography of photoresists;87 however, designing scalable means of producing complex structures remains a challenge, especially at smaller scales. Recently, Huang and co-workers developed a flexible organic− inorganic hybrid microrobot through UV-lithography to cure three different layers of swelling or nonswelling polymers with magnetic particles embedded.88 Upon folding, a flagellated bacteria-like structure was generated that could “swim” under applied magnetic fields; however, it was still around 100 times larger than a typical bacterium. An alternative route to topdown custom-designed shapes was pioneered by DeSimone’s group, termed PRINT (polymer replication in nonwetting templates).89 Here, a precursor solution is forced into a fluorinated mold with nano/micro features which is then cured and subsequently released by placing the mold in contact with an adhesive release layer (Figure 5a). Via this route, many polymeric nano- and microparticles could be synthesized with great control over geometry.90 A widely used approach to form ellipsoidal, or barrel-shaped, polymeric NPs involves embedding spherical particles formed via emulsion polymerization, nanoprecipitation, or other routes91,92 in a film, such as poly(vinyl alcohol) (PVA). If the film is heated above the glass transition temperature of the particles, stretched, and then cooled back down under strain, the spheres will reform as ellipsoids (Figure 5b).93 Alternatively, self-assembly of polymers has been widely used to form micelles in the shape of cylinders, ellipsoids, filaments, or simply spheres through a wide variety of synthetic strategies.94−96 As an example, amphiphilic DNA-block-poly(propylene)oxide (DNA-b-PPO) micelles with single-stranded (ss) and double-stranded (ds) DNA shells of different shapes can be synthesized by an automated grafting onto strategy on a solid support. The shape modification is achieved by hybridizing spherical DNA-b-PPO micelles with long ss-DNA template molecules that encode the complementary sequence of the micelle shell multiple times. Upon this hybridization event, the shape of the micelles changes from spheres to uniform rods, where the length is defined by the template.95 Rod-like micelles can also be prepared using fructose-based block copolymers poly(1-O-methacryloyl-β-D-fructopyranose)b-ply(methyl methacrylate). The critical water content, temperature, and stirring rate play an important role on the morphological transition from sphere to rods of various aspect ratios, allowing the generation of different kinetically trapped

Figure 5. Methods to synthesize anisotropic polymeric particles. (a) PRINT process. (Top row) True solution (red) is cast and cured onto a PET substrate using a mayer rod. (Middle row) Perfluoropolyether elastomeric mold (green) is brought into contact with a delivery sheet (red), passed through a heated nip (gray), and split. Cavities of the mold are filled. (Lower row) Filled mold is brought into contact with a high-energy film or excipient layer (yellow) and passed through the heated nip without splitting. After cooling, the mold is removed to reveal an array of particles. Adapted with permission from ref 89. Copyright 2011 American Chemical Society. (b) (Top) This scheme involves liquefaction of particles by using heat or toluene, stretching the film in one or two dimensions, and solidifying the particles by extracting toluene or cooling. The example shown here produces elliptical discs. (Bottom) This scheme involves stretching the film in air to create voids around the particle, followed by liquefaction using heat or toluene and solidification. The example shown here produces barrels. Reprinted with permission from ref 93. Copyright 2007 National Academy of Sciences.

shapes.96 Similar filaments have also been formed through the use of the plant virus Potato virus X as a soft template.97 The self-assembly of lipids is a relatively well-studied system, with the formation of spheres, vesicles, discs, and tubes being regularly attainable.98−100 Tekobo et al. have shown how these self-assembled structures can be used as soft templates to form small, fairly rigid NPs through the polymerization of monomers such as styrene in the hydrophobic interior of bicelles (discoidal lipid aggregates).101 Similarly, wormlike micelles formed from either surfactants or amphiphilic polymers can be used as soft templates to synthesize other rod-shaped nanoparticles of various materials.102,103 3.2. Inorganic Materials

The study of shape control of inorganic NPs is a massive subject and has many dedicated reviews all to its own.6 One of the original materials used to study anisotropic nanocrystal 11482


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different shapes, such as cubes, nanostars, triangles, and prisms, can all be prepared in conditions analogous to those used for the synthesis of nanorods.115 They showed that a fine control of the shape is possible by simple modification of the concentration of the reagents: Au seed, CTAB, gold salt, ascorbic acid, and silver nitrate, which stimulates the anisotropic growth of the NPs. 3.2.2. Alloys and Galvanic Replacement. Nanoscale galvanic replacement, based on the difference in the redox potentials between two metal species, is a versatile and elegant approach that has been well developed by transforming solid metal NPs into multimetallic hollow NPs with complex morphologies, thereby tuning their optical and catalytic properties.116 In this process, the final cage-like nanostructures typically preserve the shape of the original sacrificial templates. For example, Ag cubes are converted into either hollow M−Ag nanocubes (M = Au, Pt, or Pd) or cubic nanoframes.112,117 This approach can also be applied to solid bimetallic NPs: gold− silver core−shell NPs lead to the formation of plasmonic nanorattles, showing enhancement in their surface-enhanced Raman scattering (SERS) activity.118 Recently, deviation from the parent template shape has been reported which may be very promising in tuning NP properties through shape modification. An example of this unconventional transformation via roomtemperature galvanic replacement is the conversion of cubic Ag NPs and Au−Ag core−shell nanorods/cubes into octahedral nanocages119 and octahedral nanorattles,120 respectively. To date, nonactive-metal nanostructures (such as Ag and Cu) and noble-metal ions (i.e., AuCl4−, Ag+) have been commonly used as sacrificial templates and reactants, respectively, as shown in the above examples. Several groups have investigated more active-metal templates (such as Co, Fe, Ni, and NiCo) with the goal of extending the galvanic replacement, thereby producing composite nanostructures.121,122 However, the number of such studies is very limited, and the capabilities of this methodology as a preparation technique are still far from being fully exploited. 3.2.3. Hollow Structures. Inorganic and organic hollow synthetic micro- and nanostructures show an incredible potential for storage and release application. In recent years layer-by-layer self-assembly has proven to be the method of choice to construct organic microcapsules123 as well as the templating procedure for inorganic hollow capsules.124−126 However, anisotropic nanocapsules are scarcely reported, even though they play an important role in nature, i.e., cell−NP interaction. For example, Shchepelina et al.127 fabricated anisotropic, ultrathin organic micro- and nanocapsules using a layer-by-layer approach. The anisotropic shape of these capsules was provided by CdCO3 and SnS micro- and nanocrystals as an inorganic template. Inorganic hollow-capsule shape anisotropy can be categorized by the overall structure and surface structure. The overall structure refers to the outline of hollow capsules, such as spherical, cubic, and spindle shapes, while surface structure involves the shape and orientation of building units like porous capsules with an urchin-like surface.128 The overall structure of capsules is generally determined by the shape of the template used during the synthetic procedure. Hyeon et al. reported the synthesis of biocompatible spindlelike iron oxide nanocapsules using a wrap−bake−peel process.129 This process consists in wrapping akageneite (βFeOOH) NPs with a silica coating, heat treatment, and etching of the silica layer to produce hollow iron oxide capsule.

growth was gold, whereby the capping of different facets with the surfactant cetyltrimethylammonium bromide (CTAB), and some silver, lead to growth in one dimension.104 This spawned a whole field of research where various surface active agents, both small molecules such as CTAB and polymers such as PVP, were used to generate wires, rods, stars, cubes, octahedrons, discs, plates, among others in materials as diverse as silver, gold, platinum, palladium, copper, iron, cobalt, tin, lead, iodine, rhodium, and silica, to name but a few (Figure 1). Such bottom-up strategies lend themselves to applications requiring scale with, often, low technology barriers to obtaining various morphologies. However, a compounding problem is the need for capping ligands and polymers resulting in variable and complicated surface chemistries.23,105 Furthermore, it is difficult to draw analogies between hard, inorganic, NPs with the softer self-assembled structures formed in nature. This is most obvious in the role that NP elasticity, or stiffness, has in their uptake mechanisms and kinetics.106 3.2.1. Metal Nanoparticles and Seeded Growth Methods. Herein, we have chosen to highlight published synthetic approaches that we consider more reliable and well understood for controlling NP shape. Among the large number of synthetic approaches that have been reported, only a few provide us with the ability to tune a NPs shape. The most relevant of these are the so-called seeded-growth procedures. These methods are based on the controlled growth of material onto preformed seed particles, which is normally carried out in different reaction to that of the seed particle synthesis. For example, the polyol process is based on the reduction of inorganic salts by ethylene glycol or higher polyols.107 On the other hand, N,N-dimethylformamide (DMF)-assisted reduction relies on the use of DMF as both solvent and reducing agent.108 Most of these methods require the presence of surfactants or polymers to avoid the aggregation and flocculation processes typical of colloidal dispersions. These capping agents provide colloidal stability while also playing an important role in directing the particle morphology. Among the common capping agents reported in the literature, PVP108 and CTAB109 are clearly the most popular for the synthesis of anisotropic NPs. It has additionally been reported that PVP can act as a mild reducing agent110 and strongly affects the final particle shape due to its different adsorption affinities toward the various crystal facets of a crystalline NP. For example, decahedral and octahedral Au NPs have been synthesized successfully using a DMF/PVP procedure with ultrasound as an energy source,111,112 a method subsequently extended to silver by Tsuji et al.113 The control of NP shape in these cases is assisted by Ostwald ripening through the selective growth of certain crystal facets guided by PVP. Interestingly, though Ag+ can be directly reduced to Ag0 by DMF,108 the formation of anisotropic Au NPs requires the presence of a catalyst (under an external energy source such as temperature, ultrasound, etc.). This catalyst can be comprised of small metal seed NPs or high concentrations of PVP that act as nucleation centers. Notwithstanding, it has been demonstrated that the external energy source is only required when the PVP concentration is low. However, when the PVP concentration is increased (>2 mM), the reduction reaction leads to the reproducible formation of star-shaped NPs even at room temperature.114 Several synthetic procedures have been established for various aspect ratios of Ag and Au nanorods, Ag nanowires, and cubic Cu2O particles in aqueous solution in the presence of CTAB. In fact, Sau and Murphy reported that AuNPs with 11483


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Nonspherical hematite (α-Fe2O3) colloidal particles provide a general template for the preparation of uniform silica130 and TiO2131 hollow capsules with a wide range of shapes. 3.2.4. Templated Routes in Nanoparticle Preparation. Template-driven routes have been widely used to synthesize different inorganic anisotropic NPs. These routes are commonly employed for the controlled production of nanomaterials with ordered structure and a specific shape. Templated routes are generally divided into three steps: (1) template preparation, (2) synthetic approaches such as hydrothermal method or precipitation to synthesize the NP material, and (3) template removal. The key to this method is an apt choice of templates, which are generally divided into hard and soft depending on their structures.132 Porous anodic aluminum oxide (AAO) is an example of a hard template which is widely used because of the adjustable and quantized pore size. AAO has been used in the preparation of 1-D nanomaterials (i.e., ZnS nanowires with a diameter of 40 nm46 and carbon nanotubes133) and mesoporous films (i.e., Al nanomesh thin films134). Surfactants and polymers are typically used as soft templates. For example, sheet-like iron−cobalt alloyed magnetic NPs were prepared using a CTAB/water/ hexanol system as soft template.135 Platinum nanowire networks have been also synthesized in the presence of soft template formed by CTAB.136 Recently, cellulose grafted with amphiphilic poly(acrylic acid)-b-polystyrene (PAA-b-PS) block copolymers was used to reduce different materials into the core or shell to form Au nanorods, upconverting rods, Fe3O4 rods, CdSe rods, and various core−shell morphologies among other materials (Figure 6).137 3.2.5. Synthesis of Two-Dimensional Materials: The Case of Graphene. One the most promising 2D nanomaterials is nanographene (nG) due to its remarkable electrical, chemical, and mechanical properties. nG does not contain intrinsic surface charges and also lacks suspension stability in aqueous media; therefore, it inevitably requires additional steps in order to make it colloidally stable for biological studies. There are a number of approaches to achieve this, such as direct surface modification, surfactant-assisted dispersion, π−π stacking interactions with aromatic compounds, and functionalization with polymers.138 One of the most common methods is the chemical oxidation of graphitic layers followed by sonication, which can then be simply exfoliated as individual sheets of graphene oxide (nGO).139,140 The chemically exfoliated nGO sheets are well dispersed in aqueous solution due to charged functional groups on the surface, such as carboxylic acids. nGO is, however, electrically insulating, but it can be converted back to conducting graphene by chemical reduction (e.g., using hydrazine). Hollow graphene capsules have also been prepared based on the electrostatic interactions by the repetitive depositions of amine-modified graphene and carboxylic-modified graphene on polystyrene (PS) templates. 141 After the layer-by-layer deposition with graphene sheets, hollow capsules were recovered by removing the PS colloidal templates. These graphene hollow capsules are an example of surface structure anisotropy. 3.2.6. Green Nanoparticle Synthesis Using DeepEutectic Solvents. Currently, a great deal of resources and time are being invested in developing a simple, seedless, repeatable, eco-friendly, and low-cost synthetic approach for anisotropic NPs. Recently, deep-eutectic solvents (DESs) were introduced by Abbott and co-workers142 as a new generation of

Figure 6. (a) Synthetic strategy for nanotubes using cellulose-g-(PS-bPAA-b-PS) as a template. (b−e) TEM images at different magnification of hollow Au nanotubes synthesized following the strategy shown in a. Adapted with permission from ref 137. Copyright 2016 AAAS.

solvent that can prepare anisotropic NPs via green chemistry.143,144 DESs are an extended class of ionic liquids made by complexing a (typically ammonium halide) salt with hydrogenbond-donor molecules and depressing the glass transition temperature at the eutectic molar ratio.143 DESs are green solvents with low vapor pressure and a tunable nature; the hydrophobicity and physicochemical properties of the solvent can be altered by changing the salt or hydrogen-bond donor or by addition of various additive compounds.145 DESs are prepared from many species, including metal ions and plant metabolites. Choline chloride (ChCl) systems have gathered the most interest, and 1:2 ChCl:urea DES (reline) is the most popular due to it being particularly tractable, low cost, biodegradable, and noncytotoxic.144 Therefore, DESs are promising solvents to be used in the shape-controlled green synthesis of NPs. However, there are as yet very few reports of the use of these green solvents in the NP synthesis ( cube > rod > disc due to a larger membrane deformation energy of the discs and rods (Figure 13c). However, in these studies the

Figure 12. CGMD simulation of a nanotube interacting with a lipid bilayer. (a) Models of DPPC lipid and receptor molecules formed by one hydrophilic head−bead and two hydrophobic tail−beads and a capped MWCNT with diameter d = 20 nm and length L = 46 nm consisting of three concentric walls. Membrane bilayer consisting of lipid and receptor molecules spans the simulation box. (b) Time sequence of CGMD simulation results showing a MWCNT penetrating the cell membrane at an initial entry angle of 45°. At a receptor density of 25% (left), the MWCNT rotates to 90° before being fully wrapped. At a receptor density of 33% (middle), the tube is fully wrapped before reaching the 90° entry angle. At a receptor density of 100% (right), the tube rotates toward a low entry angle. Reprinted with permission from ref 231. Copyright 2011 Macmillan Publishers Ltd.

of receptor diffusion between the tips and the sides of the rod. This means that shorter rods would first lie parallel to the membrane before rotating to perpendicular during wrapping, as indicated by other studies.230 Through a Monte Carlo-based energy minimization simulation, intended to mimic phagocytosis of large micrometer-sized objects, the orientation of particles on a membrane was found to be key.234 Prolate spheroids with their tip first in contact with the membrane were engulfed much faster than spheroids with the long axis parallel to the surface. Curiously, spiral particles were wrapped even slower than either; however, spiral bacteria in nature such as Helicobacter pylori or Campylobacter jejuni seem to evade killing by the innate immune response through various biochemical means rather than any shape-dependent uptake.235,236 The above membrane-tension-dependent findings should not be too surprising given the known importance of membrane tension on endocytosis and exocytosis rates.237 When the plasma membrane of cells is under tension due to osmotic swelling or mechanical stretching, clathrin-mediated endocytosis may need to engage actin assembly in order to form 11491


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be observed experimentally. In addition, the 50% ratio of lipids to receptors greatly overestimates the real receptor density; however, it is necessary to ensure wrapping occurs on time scales that are computationally accessible. Experimentally, the consequence of lower PEGylation densities is often larger degrees of opsonization with complement activation proteins and consequently a larger degree of uptakethe opposite of the DPD simulations which could not include serum protein interactions.249 An interesting experiment that could test these predictions would be similar to that of Walkey et al.249except with targeting groups at the distal end of the grafted polymers. While the particle shape and polymer ligand properties are important, the surface chemistry and interaction with serum proteins still seems to be dominant. Contrary to the above materials, 2-dimensional nanomaterials have one dimension on the atomic scale and have very recently garnered a massive amount of interest from graphene to boron nitrides and metal dichalcogenides. The interaction of graphene with lipid bilayers was studied through CGMD simulations, and an edge/corner-piercing mechanism was found where wrapping was hindered for long defect-free edges while any defects or edges caused membrane penetration and initiation of wrapping.250 The wrapping proceeded when the orientation of the nanosheet was orthogonal to the membrane. However, the orientation was later shown to depend on the surface chemistry of the nanosheet.251,252 Further all-atom MD simulations found that the piercing of the membrane by these sharp edges is near spontaneous, with an energy barrier comparable to kBT, which was qualitatively confirmed by in vitro experiments with macrophages and epithelial cells.250 5.3. Cooperative Endocytosis

Simulations of RME typically consider the cellular membrane as homogeneous in receptor density. The case, in reality, can be rather different: receptors can cluster at different locations with multidentate ligands inducing receptor clustering or pore formation.253−255 Therefore, it is feasible that NPs with multiple ligands are mobile on the membrane and are preferentially endocytosed upon forming larger clusters. Indeed, some rod-shaped viruses have been observed to form perfectly radially symmetric spoke wheels surrounded by a membrane upon uptake.256 An example of this was shown by the kinetic model of Jin et al. that explained the uptake of NPs below the critical uptake radius through clustering and uptake.201 Additionally, membrane curvature can be generated by specifically shaped proteins that act cooperatively to form the wide variety of shapes observed in bacteria and cells in nature.257 One of the earliest CGMD simulations considered the impact of multiple membrane curvature-inducing proteins or NPs on their wrapping.227 It was shown that beyond a certain curvature imprint, i.e., the amount of curvature induced by the binding of one protein or NP, there exists an attractive interaction between adsorbed objects. These result in clustering and subsequent invagination of the membrane and budding, as observed for the Mason−Pfizer monkey virus which lacks the ability to bud individually (Figure 14a−d). Similar cooperative budding effects were found through a theoretical approach considering the thermodynamics alone between two scenarios: wrapping each particle individually or coating a membrane invagination with the particles (Figure 14e).258 Compared with spherical NPs, oblate ellipsoids gain a significant energy advantage when being wrapped cooperatively

Figure 13. DPD simulations of NPs coated with proteins or PEG. (a) Simulation snapshots of interactions between a positively charged NP (with a radius of 5 nm) and a cell membrane in the absence (above) and presence (below) of serum proteins at pH 7.4. Reprinted with permission from ref 223. Copyright 2014 Elsevier. (b) Side view of the internalization pathway for PEGylated NPs of different shapes with a grafting density of 1.6 chains/nm2. The whole process can be classified into three stages: membrane bending stage (0 < t < 0.24 μs), membrane monolayer protruding stage (0.24 < t < 2.24−2.77 μs), and an equilibrium stage (t > 2.24−2.77 μs). (c) Wrapping time τw required for various shapes of PEGylated NPs to be fully wrapped by the membrane. Adapted with permission from ref 224. Copyright 2015 The Royal Society of Chemistry.

particle becomes essentially embedded symmetrically in the membrane due to the high binding energies, something yet to 11492


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Figure 14. Cooperative modes of uptake. (a) Attraction and cooperative budding driven by 16 capsids on a membrane containing 40 960 lipids. (a− c) Series of simulation snapshots, with the last corresponding to roughly 0.3 ms. Arrows in b point to formed capsid pairs. Slices in c indicate cooperative budding, a phenomenon also seen in the electron micrograph d. Reprinted with permission from ref 227. Copyright 2007 Macmillan Publishers Ltd. (e) Schematic of cellular uptake of multiple spherical NPs. (f) Variation of total energy with the vesicle size for different aspect ratios, λ, of cooperatively wrapped ellipsoids in 3-dimensions. Reprinted with permission from ref 258. Copyright 2014 Elsevier. (g and h) Endocytosis of two identical NPs. Two smaller NPs of 3.8 nm (g) or two larger NPs of 9.0 nm (h) were placed on a membrane, and the initial (final) structures are shown on the left (right). Initial inter-NP distances were 6.46 (g) and 15.8 nm (h), respectively. Reprinted with permission from ref 261. Copyright 2012 American Chemical Society.

bilayeronly possible due to the small dimensions of the rods studied. When flexible tubular polymersome particles were studied, a variety of interactions were observed depending on the strength of interaction with the membrane and the membrane tension.263 At weak interaction strengths, the tube could be partially wrapped via membrane monolayer protrusion that wraps the top from the top side and is heterogeneous along with the length of the tubethe reason the authors postulate as to why tubular polymersomes are internalized differently compared with their spherical counterparts. At higher interaction strengths, fusion with the membrane and pearling were observed, although, as far as we are aware, the fusion mechanism has not been definitively proven to occur in experiments with polymersomes. An alternative structure to that considered above is membrane tubules where NPs or viruses are completely wrapped inside an elongated tube composed of the cellular membrane. The bending energy to form these tubes can be offset by the electrostatic interaction of curvature inducing proteins; however, independent energy minimization approaches264 and Monte Carlo simulations265 have both predicted the wrapping of particles in tubes when interacting with vesicles without any specific protein interactions.258 When the vesicle volume is taken as a control parameter, determined by osmotic conditions, tubular invagination into the vesicle that included 2−3 particles was found.264 For three spheres, the lowest total energy for a bound state was of a nearly linear orientation of the spheres (Figure 15a and 15b). In this study, the size of the particle was not negligible compared with the size of the vesicle; therefore, the controlling parameter was the reduced volume of the vesicle which depends on the ratio of

as shown in Figure 14f. This is because the membrane does not need to wrap the end of the ellipse with high curvature. The approach the authors used was not extendable to prolate ellipsoids due to a lack of rotational symmetry about the short axis. As previously discussed, soft elastic particles can deform upon interaction with the membrane, making their individual wrapping more difficult.209,259 However, considering cooperative uptake routes, soft and anisotropic NPs could be easier endocytosed together rather than individually.258,260 Yue and Zhang261 used DPD simulations to examine the cooperative uptake of small NPs, diffusing on a lipid bilayer composed of a distinct number of mobile receptors. It was shown that small, ca. 2 nm, NPs pack into close arrangements and are internalized. Intermediate, ca. 4 nm, NPs arrange into pearl-like chains which are internalized, while larger particles were internalized individually (Figure 14g and 14h). This behavior stems from curvature-induced attractions and kinetics: the larger particles diffuse slower on the membrane and, therefore, are less likely to form aggregates. Therefore, as the authors show, this is dependent on the strength of the ligand− receptor interaction. The authors followed this study with an examination of other shapes such as rods or nanotubes. They found that when small rods were placed in close proximity, their wrapping and orientation depended on the strength of adhesion between the rod and the membrane.262 For low adhesion strengths, the asymmetric membrane curvature induced by the anisotropic rods caused the rotation of neighboring particles into either chain of rods or into parallel assemblies. At higher adhesion strengths, the nanorods are wrapped asymmetrically from the top and eventually become embedded in the core of the 11493


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Figure 15. continued conformation (B) that occurs at large D0. Bottom region of the phase diagram is the gaseous phase (G). Radius of the membrane is R = 30σ, and the particle surface fraction is kept constant at 0.15. (Right) Snapshots of the linear (L) and tubular (T) phases. (d) Hysteresis associated with the tubulation of a linear aggregate in terms of the surface coverage χ and D0 for the extrusion of a four-particle-long aggregate. χ is computed as the ratio between the number of membrane beads in contact with the particles and the same number when the surface completely envelops the particles. Red crosses show the results of simulations that start form a linear aggregate, while black circles show simulations that start from a tube. Here Rp = 4σ and R = 15σ. Reprinted with permission from ref 265. Copyright 2012 The American Physical Society.

surface area to volume. Similarly, in the Monte Carlo simulation adsorption of linear chains of spherical particles on the membrane led to tubule formation (Figure 15c and 15d). Given that the tubulation is strongly dependent on the binding energy between the particle and the membrane, it would be expected that this process had a strong dependence on the shape of the NP. Indeed, this was exactly what was predicted by Wang et al.258 in a theoretical approach of free energy minimization using oblate particles. They found that the orientation of the ellipse within the tube would have an impact on the energy cost for tubulation. In a similar study, Weikl and co-workers calculated the energy gain from wrapping particles individually versus in a long tube. Larger energy gains were expected for wrapping of both prolate and oblate ellipsoids in tubular structures, as their regions of high curvature do not necessary need to be wrapped in this state. 5.4. Shapes under Flow

While we have focused primarily on the endocytosis of various shaped NPs, their biodistribution and fate in vivo is an important topic. Typically, systemically introduced NPs need to undergo margination toward the walls of blood vessels, concentrate at the targeted site, be it a tumor, site of inflammation, or specific organ, and avoid uptake by the reticuloendothelial system (RES) organs and immune cells. Upon injection, NPs are in either a laminar or pulsatile flow in a complex fluid composed of up to 45% red blood cells by volume. This flow and complex fluid affects the motion of the particles and is, essentially, a problem of hydrodynamics whereby the consequence of size and shape can be predicted. Under a classical continuum approach, where an object interacts with a certain number of receptors on a surface under an external flow, Decuzzi and Ferrari266 evaluated different shapes and their likelihood of adhering to a surface. They showed that under the typical shear stress of a capillary wall, around 1−10 Pa, oblate spheroids had a scale-dependent larger adhesive strength and therefore probability of adhesion than spheres of equivalent volume. However, this calculation presumes the spheroid is flat on the membrane. Nevertheless, this predicted behavior has been experimentally verified by several groups using microfluidic approaches.267 When the Brownian, and convective, dynamics of NPs in a capillary were modeled by Shah et al.268 and Tan et al.269 it was found that rods typically adhered with a higher probability to vessel walls than spheres of equivalent volume (1000 × 200 nm,268 and 189−522 × 52−126 nm,269 respectively) due to their tumbling motion and larger contact area, with the process being orientation dependent. Initially, the rods contact the membrane

Figure 15. Cooperative wrapping of NPs in tubes. (a) Bound minimum-energy state of two particles for a vesicle with a reduced volume of ν = 0.96 (v ≡ 3 4π V /A3/2 ≤ 1) and the rescaled adhesion energy ≡ UR2p/κ = 2, where U is the adhesion energy per unit area, Rp is the particle radius, and κ is the bending rigidity of the vesicle membrane (left). (Middle) Bound minimum-energy state of two particles for ν = 0.92 and u = 2.33. (Right) Bound state of three particles for ν = 0.88 and u = 2. (b) Rescaled total energy of a vesicle with three adsorbed particles bound together by membrane tubes as a function of the angle between the particles for the reduced volume for ν = 0.88 and u = 2. Four snapshots depict minimum-energy conformations at angles φ = 0°, 45°, 90°, and 120°. Reprinted with permission from ref 264. Copyright 2012 the American Physical Society. (c) (Left) D0−Rp phase diagram of the membrane aggregates and protrusions induced by colloidal particles. D0 is the membrane− particle binding constant. Inset shows a typical single-particle bud 11494


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tip first before rotating to lie flat, whereby they can resist detachment by shear forces better than adsorbed spheres of equivalent volume. Before adhering to a surface, particles must first migrate from the blood flow to the edge of a vessel. The propensity to do this is called margination and is a well-known mechanism for leukocytes to accumulate at an endothelium. Larger red blood cells tend to concentrate at the center of blood vessels, leaving a so-called cell-free layer near the wall. Particles, white blood cells, and platelets can migrate heretermed margination. In an early study, Gavze and Shapiro270 showed how prolate spheroids have a drift velocity toward the vessel wall under inertial and hydrodynamic effects. The lateral drift velocity increases with the size, density, and rotational inertia of the particles, with nonspherical particles able to marginate better than spherical ones.271−273 Additionally, a number of further in vitro, in vivo, and in silico experiments have all highlighted the increasing margination with size of spherical particles.274−276 This effect is largely due to interactions of particles with the red blood cells, which occupy a large fraction of the volume of the vessel. Smaller particles can occupy the space between the red blood cells and remain relatively confined to the core of the flow, whereas larger particles are pushed to the edges. Additionally, due to collisions with red blood cells, oblate particles have a greater lateral drift than microspheres.277 Prolate particles conversely have fewer collisions with red blood cells and a reduced lateral drift. Once marginated, prolate ellipsoids have a higher probability of contacting the vessel wall than spheres or oblate particles, while disc-like particles have the highest likelihood of firm adhesion. Combining the high adhesion probabilities of nonspherical particles, such as discs, and their propensity to marginate along with a large size has been used by Decuzzi and colleagues, as well as others, to target tumors and endotheliums in vivo.

NPs on the membrane can overcome this barrier. Some studies have also postulated that clustering and wrapping of multiple ellipsoids is far more favorable than cooperative uptake of equivalent spheres. Experimental observations of nonlinear concentration-dependent endocytosis could potentially corroborate some of these cooperative-uptake-based hypotheses. Interestingly, the orientation of anisotropic NPs has been shown by many groups to dictate the extent and speed of wrapping. Typically, wrapping proceeds from the end of filaments or rods, and stiff cylinders rotate during uptake to a perpendicular, or rocket-like, orientation. This prediction is similar to the observation of bacterial phagocytosis which is commonly initiated at the pole of the bacterium. Another surprising result from MD simulations was the spontaneous piercing of membranes by atomically sharp edges of nanomaterials. Finally, hydrodynamic simulations of particles in flow have predicted higher lateral drift velocities, or more margination, for both larger particles and those with high rotational inertia such as ellipsoids. Collisions with red blood cells also increase the margination of anisotropic particles such as oblate ellipsoids. Currently, the case for filamentous NPs is less clear. Nevertheless, many of these predictions have been tested, to some extent, both in vitro and in vivo with a broad agreement between theory and experiment resulting, particularly for larger particle sizes.

6. INFLUENCE OF DIFFERENTLY SHAPED NPS ON THEIR INTERACTION WITH IN VITRO SYSTEMS 6.1. Large, Mostly Organic Particles

Just as uptake mechanisms and sizes of viruses and bacteria are split roughly into micrometer-sized and nanosized regimes, studies on (nano)materials can be similarly divided. Indeed, most polymeric particles described within this review have characteristic dimensions that range from 200 to 20 000 nm, while inorganic materials are primarily from 10 to 500 nm. One of the first investigations on the shape-dependent endocytosis of polymeric particles was by Champion and Mitragotri (Figure 16a).278 They utilized the film-stretching method of Ho et al.279 to morph PS spheres into oblate and prolate ellipsoids as well as various discs. The speed at which J774A.1 murine and NR8383 rat macrophages wrapped their membranes around the various particles was then measured. They found that the radius of curvature at the initial point of contact between the particle and the macrophage determined the speed and success of phagocytosis. In other words, regions of higher curvature were associated with a larger degree of wrapping or phagocytosis. This revolutionary idea that physical mechanisms could play such a dominant role in the phagocytosis of large objects led to numerous follow-up studies. In a similar approach, Sharma et al.195 stretched three different volumes of PS spheres, ranging from 0.5 to 3.6 μm in diameter to form prolate or oblate ellipsoids and studied their attachment and internalization in RAW 264.7 murine macrophages. Independent of volume, particles adhered to the cell surface in the order prolate > oblate > spheres. However, internalization proceeded in the order oblate ≫ spheres > prolate. These findings are roughly in agreement with the theoretical work of Dasgupta described earlier (Figure 8a), where oblate ellipsoids are easier, energetically, to wrap than prolate ellipsoids and spheroids partially wrap easier than spheres.202 However, few studies predicted spheroids would

5.5. Summary of Theoretical Approaches

Approaching the problem from a theoretical standpoint allows us to simplify complex systems, as described in section 4, and identify morphologically driven trends in the adhesion of NPs to membranes, their wrapping, and eventual endocytosis under various external shear stresses. In all studies, wrapping is driven by the enthalpy of binding between a NP surface, or ligands, and a fluid bilayer, or receptors. The entropic penalty of clustering receptors at the NP−membrane interface competes against the enthalpic gain of forming receptor−ligand bonds. It appears that short ellipsoids may translocate across a cellular membrane via endocytosis faster than spheres of an equivalent diameter; however, this trend is reversed for even longer rods or filaments. Additionally, due to the large contact area of anisotropic NPs with membranes, their partial wrapping is often favorable compared with spheres. It should be noted, however, that this depends on the comparison metric: radius vs surface area vs volume. Additionally, softer spheres and ellipsoids are normally wrapped less favorably than rigid NPs. In many of these studies, NPs with sharp edges, such as cubes or cylinders, have higher energy barriers to endocytosis due to the high bending energy required to wrap these edges. However, the atomic-scale sharpness is, in most experimental setups, likely hidden below a polymeric, or protein, corona that surrounds the particle. Models have also shown that below a certain size, roughly 30−40 nm, wrapping of individual NPs is not possible due to the associated high bending energy. In this case, clustering of 11495


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J774A.1 cells.283 This is a particularly attractive route to drug delivery whereby the shape transition could be triggered either externally or by the NPs local environment, consequently increasing the likelihood for endocytosis.284 There have been a wide array of synthetic strategies recently developed to trigger the transformation of, normally polymeric or hydrogel, particles from one shape to another with the aim to control their fate in vivo.285 Mitragotri’s group has also investigated the uptake and transport of smaller stretched PS particles in a triple-cell coculture model of the intestine.286 They found that rod- and disc-like particles, stretched from 200 nm spheres, were both taken up and transported across the epithelium more than spheres. This difference was more pronounced when the particles were conjugated with the targeting ligand biotin: the larger contact areas between particle/ligands and cells/ receptors promoted internalization. However, there was essentially no translocation without the addition of the Raji-B immune cells to the coculture, highlighting the importance of advanced in vitro systems to mimic conditions in vivo. Mesoporous silica rods (80 × 240 nm) as well as calcium phosphate rods have also been shown to penetrate deeper into the mucosal tissue of the gastrointestinal tract ex vivo compared with spheres (80 and 140 nm).287 This effect originated from their rotational diffusion, combined with shear flows and the mesh-like network of mucus enabling deep penetration of the viscoelastic layer. Combined, these two studies suggest anisotropic and rod-like particles are far superior to targeting the epithelium of the GI tract than their spherical equivalents. At a similarly small scale, Florez et al.288 stretched PS particles that were copolymerized with sulfonate groups after observing aggregation of the particles once removed from the PVA film-stretching medium without any sulfonate groups added. From an initial diameter of 100 nm, these fluorescent particles were stretched into prolate ellipsoids with an aspect ratio from 2 to 6 and incubated in excess with Mesenchymal stem cells or HeLa cells. They found that the higher the aspect ratio, the fewer NPs were internalized. Additionally, the ellipsoids were found to orient with their major axis parallel to the cell surface, indicating a strong binding along the area of lowest curvature, which is known to correlate with reduced uptake.278 However, residual PVA, which can act similarly to PEG in reducing opsonization and consequently uptake,289−291 and the influence of the stretching method on the surface charge distribution from sulfonate groups on the surface complicated the interpretation. A number of theoretical investigations have pointed to the difficulty in wrapping particles that present sharp edges or extremely high curvature as present on cubes. A discontinuous phase boundary between partial and complete wrapping was observed by Dasgupta and colleagues for cubesnot present for ellipsoids.203 MD simulations also found wrapping of cubes takes longer than other shapes.220,222 This was confirmed experimentally for the case of negatively charged, micrometersized CaCO3 particles where cubes of 2.5 × 3.2 μm were internalized by HeLa cells far less than spheres and ellipsoids that were both smaller and larger in total volume.292 The smallest ellipsoids were taken up the most. This appears to contrast with the findings of Doshi and Mitragorti,280 who showed a strong correlation between the longest particle dimension and the degree of association with macrophagesa contradiction likely due to the difference in uptake mechanisms of professional phagocytes with those of HeLa cells.

Figure 16. (a) Scanning electron microscopy (SEM) images show macrophages attempting to phagocytose (left, middle) ellipsoidal disks or (right) spherical particles. Particles can be seen as pseudocolored purple particles, and cell membrane are colored brown. Bottom panels show bright field and fluorescent microscopy of analogous situations where the actin rings were live stained and can be seen in red. Actin filaments form rings around the particle at the site of engulfment. Reproduced with permission from ref 278. Copyright 2006 National Academy of Sciences, USA. (b) SEM images of macrophages attempting to phagocytose high aspect ratio, flexible worm-like particles. Macrophages attach to the worm-like particles at multiple points. Reproduced with permission from ref 295. Copyright 2008 Springer. (c) SEM of highly monodisperse nanoparticles synthesized via a top-down, lithography approach. (d) NP uptake by HeLa and HEK 293 human epithelial cells showed strong shape-dependent uptake of different-shaped NP. Adapted with permission from ref 298. Copyright 2013 National Academy of Sciences, USA.

out-compete spheres for complete wrapping. Similar observations were made with J774A.1 macrophages and opsonized PS particles; however, the trend reversed for the largest volume particles (3 μm spheres).280 The authors of this study postulated that the distance between membrane ruffles, which are actin-filament structured extensions of the membrane, influenced the observed shape dependence. Additionally, when the longest dimension of the stretched particle was in the range of 2−3 μm, maximal adhesion was observed. The film-stretching method has also been applied to poly(lactide-co-glycolide) (PLGA) particles by Yoo and Mitragotri,281 who compared the internalization of 1.8 μm spheres with elliptical discs with a major axis of 7 μm in HUVEC. Unsurprisingly, given the results of other studies282 and the large major axis, the discs were internalized slower than the spheres. Once internalized, the particles localized to the perinuclear region of the cell with the discs oriented parallel to the nuclear envelope. The authors then showed how these high aspect ratio discs could undergo a temperature-induced shape transformation to a sphere, triggering their phagocytosis by 11496


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remain there, while spheres were internalized.300 This stronger binding with the cellular membrane may explain why the larger discs in Agarwal’s study were internalized faster: larger contact area and stronger binding with the membrane triggering macropinocytosis. However, in other cases, the binding of rods with low curvature can mean a lower probability of internalization.283,295,299 As opposed to the anionic polymeric particles described above, rod-like cationic oligolysine brush polymeric NPs have been synthesized in different lengths for transfection purposes: 25 × 74 nm or 18 × 102 nm.301 Despite both associating similarly with HeLa cells, the shorter nanorods transfected with a much higher efficiency, although the authors could not explain this observation. Other more complex self-assembled polymeric structures as well as siliceous materials have been investigated where uptake and efficacy has correlated well with the roughness of the surface similar to spikes on viruses or, in other words, the surface curvature.302,303 The case for mesoporous silica particles (MSP), a promising drug delivery agent, seems to be clearer. Meng et al.304 observed that MSP with an intermediate aspect ratio of ca. 2.3 were internalized to a larger degree and faster than either shorter or longer rods in both HeLa and A549 cells. This was linked to the shape-dependent activation of the small GTPase Rac1, inducing actin assembly and the formation of filopodia. The authors postulate that this shape-sensing mechanism occurs through recognition of patterns and distributions of silanol groups on the surface of the NP. When slightly larger diameter MSP, with aspect ratios of 1, 2, and 4, were apparently functionalized with PEG, a similar trend was observed.305 The higher aspect ratio particles were internalized, primarily by caveolin-mediated endocytosis, faster by HeLa cells than shorter and smaller ones. At first glance, this may indicate the silanol group spacing is not critical to the shape-dependent uptake. However, the authors supposedly PEGylated the amine-coated silica particles with a succinimidyl carbonate functional PEG in Tris buffera buffer that itself reacts with the PEG, quenching the reaction; therefore, the impact of PEG functionalization in this study is unknown. In another study, the authors found increased cellular association, measured via fluorescence-assisted cell sorting (FACS), of aspect ratio 4 and to a lesser extent 2 MSP with A375 cells.306 Again, the fluorescence functionalization was surprisingly made with FITC in a Tris buffer. More recently, magnetic MSP were shown to elicit shapedependent cellular association and biodistribution both in vivo and in HepG2, and HL-7702 cells in vitro.307 Utilizing similarly fluorescent particles, with diameters of roughly 100 nm, the quantity of MSP internalized increased with increasing aspect ratio as 1 < 2 < 4. While all MSP were endocytosed by a combination of clathrin-mediated and macropinocytosis routes, the longer particles were internalized preferentially by macropinocytosis, in line with other studies.304 In another study with MSP, spherical (200 nm) NPs were found to associate less with A549 and RAW 264.7 cells than rod- or worm-like ones (200 × 400, 200 × 1300 nm).308 Subsequently, the anisotropic particles, having one dimension much longer than the spheres, were shown to be internalized primarily by macropinocytosis or phagocytosis, while the spheres proceeded via clathrin-mediated endocytosis. When neutral or negatively charged silica rods and spheres were incubated with HeLa and Caco-2 cells over 4 h, the rods were again endocytosed to a greater extent.309 However,

A particle’s longest dimension is undoubtedly worth discussing when we consider extremely elongated forms, often termed worm-like, needle-like, or filamentous. As an example, when PS or PLGA particles were stretched to aspect ratios above 10 from micrometer-sized sphere, they could pierce cellular membranes releasing the intercellular enzyme LDH and delivering larger quantities of siRNA to the cells than spheres or discs of equivalent volume.293,294 When the spheres were stretched even longer to form semiflexible worm-like particles, NR8383 macrophages could not completely phagocytose them despite them interacting strongly with the cell surface (Figure 16b).295 This is due to the low lengthnormalized surface curvature of the particle, with the most common interactions along the side of the worm rather than at the tips, a strategy bacteria use to avoid killing by professional phagocytes as described earlier. Interestingly, when polyelectrolyte-assembled bowl-like particles were compared with spherical particles of equivalent diameter, the bowls with regions of higher curvature were internalized faster.296 Self-assembled amphiphilic block copolymers have also been used to study the translocation rates across cellular barriers and the intercellular diffusion of differently shaped NPs.297 When these particles were loaded with doxorubicin, rods and worms (200 × 7 nm and 550 × 7 nm, respectively) but not spheres (20 nm) or vesicles (100 nm) entered the nucleus by passive diffusion and consequently induced higher cytotoxicity through the delivery of doxorubicin. However, spheres diffused fastest within the cytosol, and all shapes escaped endosomes equally fast. More worms and rods diffused into the nucleus with a nuclear localization signal, while no change was observed for the spherical particles. It may be that the nuclear pore complexes are too small to allow diffusion of the spheres across the membrane, while the high aspect ratio NPs are thin enough to enter. A question therefore remains: it is a size or geometry effect? 6.2. Small Inorganic and Organic Particles

The findings outlined in the section above roughly agree with predictions from theory on shape-dependent uptake mechanisms. However, the case is less clear for small NPs. A multitude of studies with seemingly contradictory findings persist in the literature, originating from the difficulty of controlling surface chemistry and other physicochemical characteristics, the various uptake mechanisms that particles in this size range span, and variability in cell types. A top-down lithographic approach was used by Agarwal et al.298 to synthesize highly anionic, PEG-diacrylate based disc and rod-like NPs which were incubated with HeLa, BMDC, HUVEC, and HEK 293 cells (Figure 16c). The largest particle dimensions ranged from 100 to 800 nm. In all cases, the discs were endocytosed to a greater extent and more rapidly over 24−50 h, typically via macropinocytosis (Figure 16d). Rather uniquely, the authors also accounted for sedimentation through an inverted setup that showed the same trends as the upright cell culture. Similarly, anionic hydrogel NPs, composed of poly(methacrylic acid) and ranging from 400 to 1300 nm, showed decreased cellular association with increasing anisotropy. The longer rods, additionally, were more often found adhered to the membrane, while spherical NPs were internalized.299 At a smaller scale, when anionic PS spheres (20 nm) and discs (20 × 2 nm) were incubated with HeLa cells, the discs were also found to associate strongly with the membrane and 11497


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exposure. A follow-up study then investigated transferrin-coated spheres and nanorods with STO, HeLa, and SNB19 cells.199 Again, less cell-associated gold was measured, by ICP-OES, for nanorods with larger aspect ratios. The difference was extraordinary: 100-fold less 7 × 42 nm nanorods were detected per cell than 50 nm nanospheres. However, in Figure S3 of ref199 the extinction spectrum shows that this nanorod sample is composed mostly of spheres and byproducts, not nanorods: the transverse mode of a nanorod with a diameter of 7 nm should only weakly absorb and scatter around 518 nm, whereas in Figure S3 it dominates the spectrum. Therefore, given the sample is mostly composed of spheres, the 100× decreased uptake is unexpected and must originate from the difference in surface chemistry: the nanorods are originally coated with CTAB, which is not easily displaced, while the spheres are coated with citrate, an easily displaced ligand. An example of the complication of CTAB when assessing the uptake of gold nanorods was presented by Qiu et al., who studied polyelectrolyte-coated particles.314 With only CTAB present, increasing the aspect ratio from 1 to 4 monotonically decreased the number of particles per cell. However, once the nanorods were coated with either PSS or PDADMAC the shape-dependent uptake was either eliminated in the case of the former or minimized for the latter. Additionally, the authors observed loose aggregates of nanorods in suspension, although whether these are formed only in close proximity to the cell or isolated in media is unknown. Bartczak et al.315 also observed similar uptake efficiencies between negatively charged gold nanospheres (15 nm) and nanorods (17 × 47 nm) functionalized with short thiolated ethylene glycol ligands when incubated with primary HUVEC over 4 h. When comparing CTAB coated NPs of different shapes, care needs to be taken to ensure no aggregation occurs and alterations in the biochemical homeostasis of the cells due to excess ligands are minimized.316−318 Nevertheless, CTABcoated spheres have been shown, by two completely orthogonal methods, to be preferentially internalized over rods in MDCK II cells.319,320 When the CTAB was replaced with PEG-NH2, the trend reversed and slightly more rods were internalized.319 In another study, gold nanorods were coated with PVP and thermally reshaped to maintain the same particle volume, diffusion constants, and surface chemistry across samples. Upon incubation with A549 and HeLa cells, nanorods with aspect ratios between 2 and 5 were endocytosed equally, as in other studies.315 However, longer rods with an aspect ratio of 7.2 were taken up significantly more after both 4 and 24 h. This likely originates from stronger orientation-mediated nanorod− membrane binding energies. From TEM, long nanorods were observed to cluster on the cell surfaces and be endocytosed by a macropinocytosis-like mechanism, similar to observations by Qiu et al.314 J774A.1 macrophages, however, displayed no shape preference. Interestingly, immune cells, such as neutrophils, mast cells, monocytes, and macrophages, are known to form extracellular traps composed of DNA and protein that can trap pathogens at sites of infection.321 It was shown by Bartneck et al. that these networks trapped gold nanospheres and nanorods depending on their surface functionalization (CTAB vs PEG) but not shape.322 Additionally, primary macrophages and monocytes internalized roughly equal amounts of PEGylated gold nanorods (15 × 50 nm) as nanospheres (15 or 50 nm).323 However, when CTAB-coated particles were tested, the uptake of rods was 230 times more efficient than spheres with the same

grafting of positively charged primary, secondary, and tertiary amine groups on the surface appeared to partially suppress the shape-dependent uptake in HeLa cells and to a lesser extent in Caco-2 cells. Yu et al.310 also found that increasing the anisotropy of silica NPs led to a higher association with A549 and RAW 264.1 cells after 1 h incubation, although the effect of grafting primary amines, in this case, to the surface enhanced the observed shape dependence. A likely explanation for this contradiction is the difference in pore sizes and surface silanol densities in the two studies, as evidenced by the large difference in uptake of nonporous vs mesoporous silica NPs.310 PRINT has been used to generate a wide array of cationic PEG-hydrogel NPs and study their shape-dependent uptake with cells. High aspect ratio cylinders (450 × 150 nm) were found to internalize in HeLa cells quicker than smaller cylinders (300 × 100 nm) or isotropic cylinders (200 × 200 nm) and much faster than large micrometer-sized cubes.311 The particles were associated with clathrin-coated pits as well as phagocytotic and macropinocytotic uptake mechanisms, which was conserved across various cell lines.312 Upon chemical inhibition it was shown that the HeLa cells engage multiple uptake mechanisms for all particles, and interestingly, the longer cylinders were internalized strongly by all pathways, explaining their rapid uptake. When the cationic surface groups were passivated with acetic anhydride to form an anionic surface, isotropic particle internalization was significantly reduced. However, when PLGA particles were formed by the PRINT method and coated via a spray-based layer-by-layer technique with anionic targeting ligands, rod-like particles (80 × 320 nm) were internalized 10× more than cubes (200 × 200 nm) by BT20 cells.313 This seems to imply that stronger interactions, e.g., via targeting ligands, drastically changes the morphologydependent uptake patterns of NPs in cells: surface chemistry, geometry, and size are all strongly interdependent parameters. Aside from organic and silica NPs, over the last 10 years a great deal of effort has been dedicated to understanding the interaction of Au NPs with proteins and cells. This is due to their ease of synthesis, shape control, optical properties, and biocompatibility. While there is a mountain of data and results, a consensus is still lacking. We attribute this to many reasons: First, a lack of adequate material characterization data means that it is difficult to compare results. Second, gold is a dense material with a high surface energy and strong van der Waals interactions meaning in high salt environments, such as in vitro, gold NPs experience strong gravitational and interparticle attractive forces. This can easily result in sedimentation or aggregation, thus affecting the dose and effective identity of the presumed nanomaterial. Third, given the importance of adsorbed ligands in dictating NP−cell interactions, adequate control over the identity of ligands on Au NPs of different shapes is challenging. To date, the most highly cited and widely disseminated findings on the size and shape dependence on Au NP uptake was published by Chithrani and colleagues.200 They found that 14 and 74 nm citrate-coated spherical particles were associated with HeLa cells far more than 14 × 40 nm and 14 × 70 nm gold nanorods coated with CTAB. However, given the known cytotoxicity and pro-inflammatory effect of CTAB it is likely that the altered biochemical homeostasis of the cells partially explains these observations. Additionally, the UV−vis spectrum of gold particles in serum and media was broadened which typically indicates some degree of aggregation as well as the larger NPs would be expected to partially sediment over the 6 h 11498


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they were also internalized both faster and in greater quantities by A-375 and HepG2 cells.336 There have also been studies pointing to other less common shape-dependent biological outcomes. For example, PEGylated rod-like gold nanorods were found to penetrate and remain in skin at higher concentrations than spherical particles.337 High aspect ratio gold nanorods have also been found to alter the properties of the extracellular matrix, thereby changing the migration, adhesion, and protease expression of MDA-MB-231 cells.338 Some interesting results have been observed with sharp gold nanostars and nanodiamonds, whereupon on wrapping and localization to an endosome these sharp NPs can pierce the endosome envelope and escape directly into the cytosol.339 Such piercing of a plasma membrane has also been observed for nanoneedles293,340 and theorized to occur for graphene sheets, as described in section 5.2.250 On a molecular scale, the sharp tips of ZnO nanopyramids have been shown to penetrate and disrupt β-galactosidase through competitive and noncompetitive (or mixed) inhibition.341

diameter (15 nm) and 6 times more efficient than spheres with a diameter the same as the length of the rod (50 nm). Arnida et al.324 found that 50 nm gold spheres were associated with RAW 264.7 macrophages 4 times more than 10 × 45 nm rods; however, there is a 20-fold difference in volume/mass per NP, and particokinetics/sedimentation is likely to have a strong effect here.325,326 Conversely, weakly positive PEG−PEI MSP resulted in only slight shape-dependent cellular association in vitro and minimal transport across a simulated blood−brain barrier.327 These results begin to paint a picture whereby particles of different shapes that only weakly interact with proteins and cell membranes, such as PEG- or PVA-coated particles, are, generally, internalized equally well by professional phagocytes and some epithelial and endothelial cell lines, particularly at smaller scales. A counter example is when the Au NPs are conjugated with carbohydrates that target mannose or galactose receptors to strongly interact with cellular membranes. In HeLa, MDA-MB231, and HepG2 cells, nanorods were found to associate far more than nanospheres or nanostars when conjugated with mannose or galactose.328 TEM images showed internalized nanorods packed into ordered lattices within the cells. Yang et al.329 also found that gold nanorods coated in targeting DNA strands were internalized more in endothelial cells, C166, than spheres. Interestingly, from TEM images they observed longer nanorods undergo rotation to a perpendicular angle of entry as the predictions describe in section 5.2.230,231,233 Similar to the case of anisotropic Au NPs, folate-conjugated cylindrical micelles were preferentially taken up by KB cells compared with spherical micelles.330 However, when the folate groups were replaced with cell-penetrating peptides, which interact with the membrane in a receptor-independent fashion, thereby not benefiting from any polyvalent multireceptor effects, the spherical micelles had a higher rate of entry into CHO cells.331 Along these same lines, rod-like mannose-coated block copolymer micelles were internalized by RAW 264.7 cells more than spherical micelles.332 Spherical DNA block copolymer micelles (5 nm) were also internalized less than DNA rods (30 × 3.5 nm) in Caco-2 cells, although spherical micelles shielded their hydrophobic interior better.95 Additionally, rod-like mPEG−PCL block copolymer micelles, despite lacking any targeting moieties, were internalized by HeLa and HepG2 faster than spherical micelles.333 Contrasting with the above, mannose-functionalized poly(D,L-lactide)-b-poly(acrylic acid) spherical micelles (50 nm) were associated more with RAW 264.7 macrophages than rodlike micelles (100 × 50 nm or 230 × 50 nm).334 The two rodlike particles were endocytosed to a similar extent, indicating two separate uptake mechanisms for the spheres vs rods. When fructose was used on cylindrical micelles, shorter rods were also preferentially internalized and penetrated deeper into multicellular tumor spheroids compared with longer ones.96 As described earlier in this section, theoretical approaches and experimental findings at the micrometer scale roughly agree that it is more difficult for a cell to wrap a cube than a sphere.203,220,222,292 As an example of this at the nanoscale, when gold nanocubes and nanospheres, ranging from 15 to 55 nm, were conjugated with either PEG or PEG-anti-HER2, CKBR-3 cells internalized the spheres to a greater degree.335 A similar agreement between theory and experiment was obtained for short PEGylated ellipsoids, with a core of lanthanide-doped NaYF4, which displayed a stronger membrane association to liposomes than spheres or hexagonal prisms. Consequently,

6.3. Cellular Targeting

As shape has a direct effect on particle uptake by cells, it would be expected that particle geometry similarly influences active cellular targeting. Moreover, shape determines the particle specific surface area, thereby yielding more “real estate” to attach targeting ligands. In addition, as described above, the formation of multiple ligand−receptor complexes, or polyvalency, can increase the adhesion energy between particle and cell, thereby facilitating endocytosis. Investigation into ICAM-1, a glycoprotein typically on the surface of epithelial and immune cells, targeted microspheres (5 μm diameter) versus ellipsoidal discs (3 μm long axis, 1 μm short axis, 0.1 μm thickness) showed that ellipsoidal discs are taken up slower compared to the spherical particles by HUVEC in vitro.342 Comparatively, negative control IgG-labeled particles were not significantly taken up. Shape has also been shown to play a role in the targeting of human epidermal growth factor receptor 2 (HER2).343 In some breast cancers, HER2 is overexpressed and has been identified as a promising target for NP-based therapies. Trastuzumab (TZB), an antiHER2 monoclonal antibody (mAb), can therefore be utilized to target HER2 positive (HER2+) cancer cells.344,345 The uptake of PS nano/microspheres, rods, and discs, synthesized via the film stretching method, was tested by Barua et al.343 in BT-474 (HER2+), SK-BR-3 (HER2+), and MDA-MB-231 (HER2-) human breast cancer lines. Without TZB, spherical NPs (200 nm diameter) were taken up more favorably by BT-474 when compared to rods (367 nm length, 126 nm width) and discs (236 nm diameter, 88 nm thickness). With BT-474 and SK-BR3 cells, both HER2+ cell lines, the uptake of TZB-coated nanorods was greater compared to spheres and discs. Importantly, normalizing HER2-targeted particle uptake by nontargeted particle uptake showed that nanorods’ specificity in BT-474 was highest (6-fold) compared to discs (5-fold) and spheres (2-fold). Rods and discs also showed higher targeting affinity for SK-BR-3, but there was no increase in targeting for the negative control MDA-MB-231. Microrods (2.5 μm length, 0.68 μm width) showed a higher uptake in SK-BR-3 instead of BT-474, which differs from the trend observed on the nanoscale. However, in both BT-474 and SK-BR-3, microrods were taken up more compared to spheres (1 μm diameter) and discs (1.39 μm diameter, 0.3 μm thickness). 11499


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shape of a particle become extreme enough to cause adverse biological reactions? Using silica NPs as a model, Huang et al.306 studied the impact of silica particles on cell viability and mechanics. Particles were spheres (100 nm diameter), short rods (240 nm length, diameter ≈ 100 nm, aspect ratio 2), and long rods (450 nm length, diameter ≈ 100 nm, aspect ratio 4). In vitro studies were conducted with A375 human melanoma cells, and no NP showed signs of cytotoxicity when evaluated with a standard MTT assay. Flow cytometry was used to evaluate cellular apoptosis, and it was shown that higher aspect ratio particles resulted in more apoptosis. This could be related to shape directly or indirectly by the increased uptake of the high aspect ratio NPs. Similarly, Meng et al.304 showed that while silica NPs aspect ratio changed cellular uptake, it did not exhibit any toxicity in HeLa cells at 200 μg/mL. Investigations into mesoporous silica particle toxicity claimed concentration, cell type, and shape-dependent cytotoxicity in RAW 264.7 murine macrophages and A549 human lung epithelial cells.310 At a concentration of 500 μg/mL, both bare silica NPs and aminemodified particles were not toxic to A549 cells. However, in RAW 264.7 cells, the amine-modified NPs were significantly less toxic than their bare counterparts. It is worth considering that this toxicity could be due to residual CTAB, a surfactant used in synthesis that has been shown to be highly toxic, although the authors did apply an acid extraction in this particular study.355−357 Nevertheless, it is worth mentioning that rigorous procedures should be taken to remove this and other surfactants from the suspension prior to use.304 Indeed, when cytotoxicity is concerned, careful controls should ideally be run such as exposing the cells to the suspension solvent after removing only the particles to correct for any residual reactants or endotoxins. Adnan et al.358 showed that there was no significant toxicity for spherical, rod, or star-shaped gold NPs up to concentrations of 100 μg/mL in MCF-7 human breast adenocarcinoma. However, another study investigating the cytotoxicity of different aspect ratio TiO2 NPs using primary mouse (C57BL/6) lung macrophages found shape-dependent cytotoxicity for larger nanofibers.359 The TiO2 NPs tested were spherical (60−200 nm diameter), short nanofibers (60−300 nm diameter, 0.8−4 μm length), and long nanofibers (60−300 nm diameter, 15−30 μm length). The long nanofibers incubated with alveolar macrophages resulted in the release of cathepsin B, a lysosomal protease, indicating the disruption of the lysosomal membrane. These nanofibers also resulted in drastically elevated levels of interleukin-1 (IL-1) and IL-18, cytokine indicators of an inflammatory response. Similarly, Stoehr et al.360 showed the dose-dependent cytotoxicity of high aspect ratio silver particles in A549 cells. Silver nanospheres (30 nm diameter) did not show any toxicity, whereas silver nanowires (1.5−25 μm length, 100−150 nm diameter) showed shape-dependent toxicity. Media incubated with particles and then exposed to cells (without NPs) did not show any toxicity, indicating that toxicity was mediated by the actual particle uptake by cells rather than the release of Ag+ ions. High aspect ratio nanomaterials have long been investigated for cytotoxicity and other end points such as oxidative stress, proinflammatory responses, and genotoxicity due to their resemblance to longfiber asbestos, a known carcinogen. The high aspect ratio leads to an impaired macrophage phagocytosis (frustrated phagocytosis) which can result in acute or chronic inflammation and systemic effects.361

The effect of shape on cellular targeting has been further confirmed by comparing anti-epidermal growth factor receptor (EGFR) affibody-targeted PEG hydrogel nanorods (80 × 320 nm) and cuboidal NPs (55 × 60 nm).346 A431 human epidermoid carcinoma cells which overexpress EGFR, a transmembrane receptor overexpressed in a number of cancers,347 were exposed to NPs with differing amounts of targeting ligands. While there was a direct relationship between the particle−cell association and targeting ligand density, they observed an optimal concentration of targeting ligand at which nanorods were maximally internalized. Above and below this optimal level particle internalization decreased. For the cuboidal NPs, this ligand density-dependent internalization was not observed; however, targeted cuboidal NP were taken up by A431 significantly more compared to PEGylated only (nontargeted) or wild-type affibody controls. Wang et al.348 further showed that targeting ligand density was directly related to the cellular association for cylindrical NPs (200 nm diameter, 200 nm height) targeted to Ramos human Burkitt’s lymphoma cells via human transferrin. However, as these cylinders are low aspect ratio, it is unclear the role geometry plays in this situation as one must consider that size also has an influence in particle targeting/internalization. Nevertheless, in a study using elongated NPs as artificial antigen presenting cells, Sunshine et al.349 showed that higher aspect ratio particles increased T-cell proliferation and activation. Ellipsoidal PLGA microparticles coated with a major histocompatibility complex-IgG dimer and anti-CD28 antibody, irrespective of antigen concentration or density, were shown to increase T-cell activation compared to analogous spherical particles. The theory is that the high aspect ratio increases cell membrane-contacting surface area and thereby increases targeting ligand valency. Sailor and colleagues350 investigated the difference between single iron oxide NPs coated with dextran (30 nm hydrodynamic diameter) and nanoworms comprised of 5−10 aligned iron oxide NPs (50−80 nm hydrodynamic diameter). Not only did nanoworms have more desirable magnetic properties for T2-weighted magnetic resonance imaging, but they also showed increased targeting to MDA-MB-435 human metastatic melanoma via a conjugated peptide.351 This increase in uptake was attributed to the special nanoworm geometry presenting multiple ligands for cellular attachment. Cellular targeting and uptake, therefore, does appear to be mediated by particle geometry if simply for the fact that higher aspect ratio particles have a higher surface area that can interact with the cell membrane. Discerning the exact role of particle geometry on cellular targeting is confounded by a number of factors: particle size (which plays its own role in cellular uptake), targeting ligand density and valency, molecular weight of the linking molecule connecting the targeting ligand and particle surface,352 stability of the anchoring molecule (targeting ligands can be detached in biological media),353 and protein adsorption that can mask targeting ligands.354 Thus, while geometry appears to play a significant role in cellular targeting, the role of numerous other physicochemical and biological factors cannot be ignored. 6.4. Cytotoxicity and Cell Mechanics

The cytotoxicity of nanomaterials is known to be affected by the delivered particle dose to the cell surface, particle uptake, material, colloidal stability, and dissolution among other physicochemical factors.245 Given that shape is also known to influence uptake, the question arises at what point does the 11500


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It is important to add that most studies regarding cytotoxicity and cell mechanics are done with 2D cell cultures. However, during the last 5 years the design of sophisticated 3D culture models have emerged that mimic in vivo biological systems much closer than flat 2D cultures.371−373 Such organotypic cultures have been established to study the liver, nervous system, skin, respiratory system, intestine, and heart, among others, and can also be designed to model tumor tissues.374 The improvement of such systems facilitates their application in toxicology testing and drug discovery, especially since recently published results demonstrate correlations between advanced 3D cell culture systems and in vivo models.375,376 Nevertheless, there are few studies on the impact of nanomaterial shape and size on uptake and toxicity in 3D cell culture systems, particularly how they relate to the findings from equivalent flat 2D cultures.

It is important to note the significance of in vitro methodology when evaluating cytotoxic response. For example, Clift et al.362 evaluated the toxicity of MWCNT and SWCNT with a triple-cell coculture system that mimics the human lung tissue barrier. Monoculture tests for cytotoxicity and proinflammatory response with 16HBE14o- human epithelial cells, human monocyte-derived macrophages, and human monocytederived dendritic cells showed significantly different responses when compared to the coculture system. Thus, it is important to consider more nuanced in vitro models that incorporate rationally determined cell types where intercellular interactions can provide a more robust understanding of particle-induced cytotoxicity. Comparatively, a study with cellulose nanocrystals (CNC) of two different high aspect ratio dimensions showed no cytotoxicity when exposed to the coculture system,363 while a previous study comparing CNC, MWCNT, and crocidolite asbestos fibers showed that CNC elicited a lower cytotoxic response compared to MWCNT and asbestos.364 Importantly, CNC and MWCNT had similar diameters and stiffness and differed mostly in length with aspect ratios of 15 and 200−250 for CNC and MWCNT, respectively. Others have postulated that the shape of endocytosed NPs can play a role in mediating the cellular migration. Mesoporous silica spherical particles were shown to promote migration of epithelial A375 cells in comparison to short or longer rod-like NPs of equivalent diameter, i.e., the longer rods had a larger volume.306 However, these data contradict conventional wisdom regarding particle effects on cell migration where it has been understood that particle internalization can disrupt microtubule or cytoskeletal function and therefore retard cell migration.365−367 The authors contribute this cell migration phenomenon to the effect of NPs on cell cytoskeletal organization and protein expression.306 Long rods appear to affect the cytoskeletal organization, as shown by confocal laser scanning microscopy. Western blot analysis revealed that while ICAM-1 levels stayed consistent, high aspect ratio NPs led to a decrease in Melanoma Cell Adhesion Molecule expression. This was in turn linked to weaker surface adhesion of A375 cells when exposed to higher aspect ratio NPs. Conversely, as previously discussed, it has also been shown using different aspect ratio silica NPs that particle shape affects actin organization and filopodia development in HeLa cells.304 Particles with an aspect ratio of 2 significantly increased the number of filopodia per cell and were shown to increase the activation of Rac1, a GTPase involved in actin organization and filopodia development.368,369 NP-mediated cytotoxicity appears to be governed by several factors: quantity of NP uptake, particle physicochemical properties, particle−cell interactions, and cell type. NP geometry can influence, depending on the cell type, the quantity of particles ingested. Too high levels of NP uptake can disrupt cellular functions and lead to adverse cytotoxic effects. Notably, the concentration of NPs used for in vitro exposures is often more than what would be predicted in vivo, and this high NP to cell ratio can induce excessive endocytosis rates which result in oxidative stress-mediated mitochondrial damage.370 Particle geometry can also affect cytoskeletal organization, which in turn can either disrupt or increase cell mechanics (again depending on cell and particle type). Finally, particle anisotropy in the micrometer scale can lead to disruption of lipid bilayers and induce frustrated uptake leading to cytotoxic and pro-inflammatory responses.

6.5. Particle Behavior and Uptake under Flow

In order to optimize the delivery of nanomedicines and biomedical NPs to their pathological targets, particles must overcome certain biological barriers.377 When considering systemically administered microparticles, shape will affect the behavior of microparticles within circulation, as discussed earlier in section 5.4. Under shear stress, shape factors will in part determine microparticle circulation time, biodistribution/ organ accumulation, and clearance. At the microscale, hydrodynamic shear forces will more substantially affect particle behavior compared with smaller NPs. Gentile et al.274 studied the margination, the accumulation and adhesion of bodies to the endothelium, of microparticles with different shapes. They compared nonporous silica spheres (1 μm diameter), discoidal polysilicon (1.5 μm diameter, 0.3 μm height), and porous quasi-hemispherical silicon particles (1.6 μm diameter) and investigated particle accumulation onto the bottom of a rectangular flow chamber coated with type I collagen. It is intuitive that denser particles would exhibit a greater propensity for margination; however, it was also shown that discoidal particles exhibited a 5× greater margination at all shear rates (γ) compared to spherical particles of similar density and volume. At the nanoscale, Toy et al.378 similarly showed margination was more common with rod-shaped gold NPs (26 nm diameter, 56 nm length) compared to gold nanospheres (60 nm diameter). These data were further explained by mathematical simulations, which confirmed that particle geometry dominates the lateral drift and results in greater particle margination for discoidal particles compared to spherical particles.271,379,380 In a similar study, the effect of geometry on microparticle targeting was investigated using PS microparticles functionalized with an antibovine serum albumin mAb.267 Particles were spheres, ellipsoidal and circular discs, or rod-like particles. Within a bifurcating microfluidic system coated with BSA, intended as a simulated microvascular network, particles were administered at γ ranging from 15 to 250 s−1. They analyzed the accumulation of particles at the inlet section (area of margination) and at the site of bifurcation where the channel diverged. At lower γ, elliptical discs (aspect ratio 6.5) accumulated 2.5-fold more compared to spheres of a similar volume (the elliptical discs were fabricated from the spheres via a stretching procedure). At the bifurcation site, elliptical disc accumulation was 6.5-fold higher compared to the spheres. This trend continued for particles of different sizes as well. The different shaped microparticles were made by stretching “base” spherical 11501


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particles of different sizes (1, 3, or 6 μm) in uni- or biaxial directions (Figure 5b). The resulting margination within the inlet section showed that the base 1 μm spheres had a higher margination propensity compared to discs or rods. However, with the larger base particles the trend was opposite, and rodlike particles were more likely to marginate compared to the spherical ones. At the bifurcation site, the rod-like particles displayed significantly higher accumulation compared to the spherical particles for all base particles. In essence, particle targeting under flow was influenced by particle surface chemistry, shear rate, and particle geometry. Here, rod-shaped particles consistently had the highest margination and wall adhesion. This is largely due to the lower drag forces and higher contact surface area of elongated particles. In addition, the inertial and hydrodynamic forces on anisotropic particles induces a drift velocity toward the wall.270 Thompson et al.381 similarly showed that higher aspect ratio particles target vasculature better compared to spherical particles. They synthesized PS particles via a similar stretching approach from initial 500 nm, 1 μm, or 2 μm particles with aspect ratios of 1 (spherical), 2, 4, or 9. Particles were further functionalized with sialyl lewisA, a ligand that targets E-selectin overexpressed on inflamed endothelial cells. HUVEC activated to express E-selectin with IL-8 were exposed to particles in reconstituted blood (30% v/v hematocrit) at γ of 200, 500, and 1000 s−1. At first glance, all samples showed that particle binding (measured in number of particles per mm2) increased with increasing γ. However, when they evaluated binding efficiency by normalizing the number of particles bound by the number of particles introduced into the flow chamber (higher γ results in more total particles for the duration of the experiment), it was shown that increasing γ decreased binding efficiency for spherical, aspect ratio 2, and aspect ratio 4 particles. Only the particles with aspect ratio 9 maintained the same binding efficiency at all γ. Thus, the shape effect was the major factor for improving particle targeting toward the endothelium under flow.

factor into studying NP−cell interactions. For example, under hydrodynamic shear stress, i.e., flow, geometry plays a crucial role in the particle−cell interaction. In vitro studies with microfluidic devices as well as modeling studies have shown that anisotropic particles can align in flow. Furthermore, anisotropic particles and more complex geometries, such as platelet-like or discoidal particles, can marginate under flow leading to increased interaction between particles and cells on vessel walls. Particle cytotoxicity is also in part influenced by geometry. While in vitro toxicity is heavily influenced by particle material, colloidal stability, degree of particle uptake, and the in vitro model used, very high aspect ratio particles can mediate some pro-inflammatory or cytotoxic effects. This, of course, is heavily dependent on the cell type and material; however, high aspect ratio particles can induce frustrated phagocytosis in professional phagocytotic cells. Given these in vitro observations, the question then becomes do these behaviors follow in vivo and how do shape factors influence biodistribution and pharmacokinetics?

7. INFLUENCE OF DIFFERENTLY SHAPED NPS ON THEIR INTERACTION WITH IN VIVO SYSTEMS 7.1. Pharmacokinetics and Biodistribution

While microfluidic systems can provide insights into the fundamental behaviors of micro- or NPs under flow, it is exceedingly more difficult to predict sites of organ accumulation and pharmacokinetic (PK) behavior. One appeal of anisotropic nanomaterials is that the shape can prolong NP circulation time in vivo. In a seminal study, Geng et al.382 reported a decade ago that filamentous micelles, i.e., filomicelles made of block copolymer amphiphiles, persisted in circulation significantly longer than spherical particles. Filomicelles were approximately 20−60 nm in cross-sectional diameter with a length that could be tuned between 2 and 18 μm. Analogous spherical “stealth” vesicles presumably had diameters around 120 nm.383 NPs were administered via tail vein injection into male Sprague−Dawley rats or male/female C57 mice, and approximately 50% of the initial dose of 3.5 μm (initial length, L0) filomicelles persisted for 7 days. Comparatively, 50% of stealth vesicles were cleared in less than 48 h. Thus, higher aspect ratio NPs were shown to persist in circulation much longer than small, spherical NPs. This phenomenon was shown to be closely related to length, and below L0 = 8 μm filomicelle clearance from circulation was inversely related to NP length. In an earlier study, it was shown that the use of biotin turned these filomicelles from NPs that rarely attached to smooth muscle cells into strongly adsorbing ones that subsequently delivered hydrophobic drug molecule payloads.384 Other studies have further supported the finding that higher aspect ratio NPs, even of different materials, prolong systemic circulation time.324,385 PEGylated gold NPs, either spheres (50 nm diameter) or rods (10 nm diameter, 45 nm length), were injected into the tail vein of female nu/nu mice bearing orthotopic A2780 human ovarian cancer xenografts.324 Six hours after injection, gold nanorods persisted significantly more in circulation (11% initial injected dose) compared to spherical NPs (1%). Rods also accumulated more in the tumors compared to spheres. However, given the known complications of gold nanorod ligand exchange and the PEG grafting density-dependent opsonization with serum proteins, it

6.6. Summary of Shape Effects in Vitro

In vitro there appear to be several significant take-home messages where particle geometry influences particle−cell interactions. Initial studies showed that the radius of curvature where particles contact the cell surface were crucial in determining the amount and rate of particle phagocytosis. Modeling studies and subsequent in vitro experimental results have confirmed the importance of geometry in mediating cell uptake. In general, rod-shaped particles appear to be more favorably taken up compared to spherical particles. This is in part due to higher aspect ratio particles having more contact with the cell surface and presenting more “energetically favorable” contact for uptake. However, this is, of course, true to a point. Very high aspect ratio particles (e.g., nanowires and nanoworms with very high aspect ratio) can evade phagocytosis due to their length. Similarly, sharp edges or high-curvature particles are only moderately taken up. Cellular targeting is also greatly affected by particle geometry. Anisotropic particles increase the potential of targeting ligands to interact with cell surface receptors, and this increased valency leads to greater particle uptake. However, geometry is not the only factor when considering particle uptake, and other physicochemical properties such as particle coating and size will influence particle−cell interactions. In vitro models, i.e., monoculture experiments vs more physiologically relevant multicellular systems, will also 11502


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Figure 17. Particles can be synthesized in a wide range of shapes, sizes, and materials that can affect their pharmacokinetic (PK) properties and biodistribution. (a) Silicon-based microparticles can be hemispherical, discoidal, or cylindrical in shape. Reprinted with permission from ref 388. Copyright 2010 Elsevier. (b) Silica particles can also be rods of short or long aspect ratios. Adapted with permission from ref 385. Copyright 2011 American Chemical Society. (c) Gold NPs can be synthesized as spheres, discs, rods, or cubes. Reprinted with permission from ref 392. Copyright 2014 American Chemical Society. (d) PS particles can be stretched from spheres into a number of shapes including ellipsoids, discs, and rods. Reprinted with permission from ref 267. Copyright 2010 Elsevier. (e) PKs and biodistribution of systemically administered MP/NPs is mediated by particle size, shape, and colloidal stability.

been shown to prolong circulation time, other factors such as material choice and NP stabilization are important factors to consider. Regardless, altering PK properties through shape factors is expected to influence organ accumulation. There have been numerous studies into the biodistribution and PK of anisotropic particles in vivo. In the previously mentioned study by Perry et al.,386 PEG conformation altered not only the circulation time of particles but also the organ distribution. Bare particles accumulated primarily in the liver, while long-circulating brush-conformation PEGylated particles accumulated mainly in the spleen. In a different study, the biodistribution of MSP, both PEGylated and bare, was studied in mice following intravenous administration.387 Silica NPs had cross-sectional diameters of approximately 160 nm, and organ distribution was determined via inductively coupled plasma optical emission spectroscopy (ICP-OES). Two hours after injection, short nanorods (aspect ratio 1.5) accumulated more in the liver whereas long nanorods (aspect ratio 5) accumulated more in the spleen. PEGylation also influenced the biodistribution of MSNP, as long PEGylated nanorods accumulated more in the lung while short PEGylated nanorods accumulated more in the liver. Confocal microscopy showed the organ distribution of these NPs. In the liver, the NP distribution was diffuse throughout the tissue, while in the

is difficult to attribute the observed biodistribution solely to shape at this scale rather than surface chemistry.249,289,357 Circulation time is also influenced by other factors such as stabilizing molecule size, hydrophilicity, and flexibility. PK studies of intravenously administered SWCNT (length ≈ 100 nm) showed that blood circulation time was heavily dependent on the molecular weight (2−12 kDa) and structure (linear or branched) of the stabilizing PEG molecule. SWCNT circulation time was directly related to PEG length, but even more significant was the effect of PEG structure, where branched PEG significantly prolonged blood circulation. This is likely an effect of PEG conformation on the particle surface as PEG conformation has been directly related to grafting density, molecular weight, and structure.386 The PEG coating has ramifications for protein adsorption on the NP, opsonization, particle stability, and subsequently particle clearance and biological fate. Perry et al.386 showed that rectangular particles (80 nm width, 80 nm height, 320 nm length) with a brush conformation PEG coating significantly prolonged blood circulation (t1/2 ≈ 19.5 h) when compared to mushroom conformation (t1/2 ≈ 15.5 h) and bare (non-PEGylated) particles (t1/2 ≈ 0.89 h). This is linked to the process of opsonization at lower PEG grafting densities, as shown by Walkey et al.249 Thus, while higher aspect ratio particles have 11503


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When considering various routes of administration, the biological barriers which particles must overcome can differ. For example, oral administration necessitates that particles or pharmaceutical compounds persist through the gastrointestinal tract and cross mucosal barriers. Yu et al.287 investigated the mucosal penetration of two different mesoporous silica spheres (dH = 100 and 200 nm) and mesoporous silica rods (80 nm diameter, 240 nm length, aspect ratio 3, dH = 200 nm). Ex vivo studies with rat intestinal loops showed that rods exhibited approximately 5-fold more intestinal surface coverage compared to both spherical particles. Following oral administration into rats, the rod-shaped silica NPs were shown to penetrate and accumulate much more in the small intestines compared to spherical particles. The authors further investigated this phenomenon using CGMD. Simulations showed that the rotational movement of the nanorods around the “polymer” chains in the mucosal network fundamentally contributed to their translational movement through the fluid, with this conclusion supported by stimulated emission of depletion microscopy of the nanorods in mucus. The shape advantage for nanorods over spheres was, therefore, determined to be dictated by the porosity of the mucus and the shape-mediated diffusion/movement of anisotropic nanorods through the gellike network.

spleen, the NPs were mainly in the marginal zone: the interface between red and white pulp where particulate matter/antigens are filtered from circulation. After 24 h, bare short nanorods were more prevalent in the spleen compared to long nanorods, and short PEGylated nanorods were found more in the kidneys compared to the other formulations. Circulation times were investigated by measuring silicon content in the blood at 2 h, 24 h, and 7 days. At 24 h, short nanorods were significantly cleared from blood while longer nanorods persisted in circulation. Others have studied shapes that are more complex. Decuzzi et al.388 investigated the organ distribution of spherical, hemispherical, discoidal, and cylindrical silica or silicon microparticles after intravenous tail vein injection into female nu/nu mice bearing subcutaneous MDA-MB-231 breast cancer xenograft tumors. Particles were all approximately 0.6 μm3 in volume, and it was shown that discoidal particles accumulated significantly more (4−8×) in the lungs and heart compared to the cylindrical, hemispherical, or spherical particles. Alternatively, cylindrical particles were shown to accumulate significantly more (2−5×) in the liver. There was no significant difference in accumulation in the tumor, brain, or kidneys. It could well be that particle margination, as discussed previously, is responsible for the increased accumulation of discoidal particles in these highly vascularized tissues compared to the other shapes. Evidence of this was provided by Godin et al. with discoidal MSP that accumulated up to five times more than spherical particles of a similar diameter in breast tumors.389 It is, therefore, evident that shape plays an important role in particle PK and biodistribution. In all cases, it appears that higher aspect ratio particles persist longer in circulation. This is ostensibly due to the alignment of high aspect ratio particles under flow for larger particles as well as trapping of smaller particles in the center of the flow of red blood cells in vessels.275,382 For more complex shapes the biodistribution appears to be similarly governed by shape in circulation. Larger particles (dH > 200 nm) tend to be filtered out by the spleen, as do higher aspect ratio particles.350,390,391 Particles between dH = 15 and 200 nm tend to accumulate primarily in the liver. Discoidal/plate-like particles have been shown to accumulate in organs such as the heart and lungs.388,392 This phenomenon may be due to the margination propensity of discoidal particles under flow leading to the accumulation of particles on vascular walls. This would make sense considering the narrow diameter vessels in pulmonary capillaries. In a study with C57BL/6J mice bearing melanoma tumors, van de Ven et al. studied the biodistribution of discoidal and cylindrical NPs following systemic administration.393 Discoidal particles were three different sizes (600 nm diameter × 200 nm height, 1000 × 400 nm, and 1800 × 600 nm) compared to the two cylinders types (1500 nm length × 200 nm diameter and 1800 × 400 nm). The rigid cylindrical particles showed some morbidity in the mice following administration, likely due to the formation of lung emboli. However, for the discoidal particles the 1000 × 400 nm form showed the highest tumor accumulation at 5.1% injected dose. The size of these particles is higher than known fenestration sizes and therefore indicates another mechanism for particle accumulation in tumors. Aside from shape effects, there are certain other physicochemical properties such as stabilizing molecule conformation, adsorption of opsonizing proteins, and particle stability that will control where NPs will accumulate and the route/speed by which they will be cleared from systemic circulation (Figure 17).

7.2. Targeting in Vivo

When introduced systemically, particles will distribute throughout the body. Optimizing factors such as particle shape can, therefore, be used to influence where particles tend to accumulate and help mediate biological response. For example, NPs have been investigated as vaccine adjuvants, and shape was shown to play a role in NP adjuvant efficacy. Gold particles were synthesized as small spheres (20 nm diameter), larger spheres (40 nm diameter), cubes (40 nm “diameter”), and rods (10 nm cross-sectional diameter, 36 nm length).394 Au NPs were coated with poly(4-styrenesulfonic acid-co-maleic acid) (PSS-MA), and then West Nile virus envelope (WNVE) was adsorbed as an antigen. C3H/HeNJc1 mice were immunized intraperitoneally twice over a 6-week span at equal doses of WNVE per particle, and anti-WNVE antibody production was measured. Particles enhanced antibody production over free WNVE injection (almost no Ab production), and shape played a role in the level of Ab production enhancement. Spheres of 40 nm in diameter enhanced the adjuvant effect the most compared to 20 nm spheres and rods. Analyzing specific particle parameters showed that the adjuvant effect was inversely related to particle specific surface area and directly related to the number of WNVE proteins adsorbed onto a single particle. Thus, from a practical standpoint shape can influence the loading density of antigens, targeting molecules, therapeutic payloads, etc., onto or within a particle. Physically, the shape also influences the biodistribution and PK of NPs in vivo, which in turn can influence their ability to target certain pathologies. NPs have been hyped as ideal vehicles for cancer therapy due to their ability to target tumors via passive or active targeting mechanisms (i.e., small molecule, aptamer, antibody targeting for cell−surface ligands).395 The Holy Grail of this endeavor, as coined by Paul Ehrlich, is to develop a “magic bullet” that is highly specific to the desired pathology while minimizing off-target or nondesirable biodistribution. Nonspecific targeting mechanisms rely on defective physiology leading to the “enhanced permeability 11504


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(e.g., targeting scavenger macrophages in atherosclerotic plaque).404 Active targeting is affected by ligand density,405,406 targeting ligand valency,348,407 length of the linker between the targeting molecule and the particle surface,352,408 and particle physicochemical properties (e.g., size, shape, particle rigidity).409,410 Kolhar et al.409 investigated the biodistribution and vascular targeting (specifically ICAM-1 in the lung endothelium) of nonspecific IgG-labeled (negative control) or anti-ICAM-1 mAb-targeted PS spherical NPs (diameter 200 nm) or nanorods (124 nm × 501 nm) following tail vein injection in female BALB/c mice. The accumulation of all particles was similar in the liver; however, nanorod accumulation was higher in the spleen. This difference was attributed to both the increased hydrodynamic size of rods and the elevated ICAM-1 expression in the spleen. There was no appreciable accumulation of IgG-labeled spheres or rods in the lungs; however approximately 2× more ICAM-1-targeted rods accumulated in the lungs compared to ICAM-1-targeted spheres. Compared to their IgG-coated nontargeting counterparts, ICAM-1-targeted rods and spheres accumulated more in the lungs: 8× and 3× respectively. Others have looked at the influence of particle shape and targeting ligand density on tumor targeting. Using particles similar to those used by Roode et al.,398 cuboidal or rod-like particles were targeted toward EGFR via an anti-EGFR affibody.346 Female Foxn1nu athymic nude mice bearing subcutaneous, EGFR-overexpressing human A431 epidermoid carcinoma xenografts were given cuboidal or rectangular NPs with varying targeting ligand densities. Without the targeting ligand, they showed the cuboidal NPs accumulated more in tumor xenografts compared to the rod-like NPs. Furthermore, cuboidal particles accumulated more in the liver compared to rod-like particles, and conversely rod-like particles accumulated more in the spleen, thereby further confirming the previously observed results.385,409 Interestingly, for the rod-shaped NPs increasing the targeting ligand density decreased NP circulation time. NP clearance was between 5- and 30-fold higher for low to high ligand densities compared to the PEG control. Poor NP PK were attributed to the nonspecific adsorption of proteins to the targeting ligand-bearing NP. Furthermore, liver accumulation of the rod-like particles was directly related to targeting ligand density, ostensibly due to the native expression of EGFR in the liver. Likewise, iron oxide nanoworms targeted toward tumors via a PEG-linked short peptide sequence showed an optimal peptide concentration where circulation time was not compromised and tumor-targeting efficiency was optimized.411 Taken together these studies paint a complex picture of targeting and NP shape. For one, targeting efficiency is closely tied to the tumor physiology. Passive targeting is dictated by tumor heterogeneitytumors differ in their vasculature, fenestration, porosity, extracellular matrix density, etc., based on cancer type and stage. Thus, whether a particle passively accumulates preferentially at a pathological target based on shape or size is highly variable. For active targeting, the myriad of factors dictating targeting efficacy becomes even more complex. Targeting becomes a balancing act between the choice of biological target, targeting ligand density, physiological expression of biological targets (i.e., receptors) in diseased and healthy/nontarget tissues, particle stability, and opsonization via nonspecific protein adsorption. In more straightforward cases (i.e., targeting vasculature with ICAM-1) higher aspect ratio particles can promote targeting because increased chance of particle margination to vascular walls and

and retention” (EPR) of macromolecules/NPs in tumors through abnormal vascular development, reduced lymphatic drainage, and porous vasculature.396,397 While there are limitations to relying on the EPR effect, it is intuitive that particle shape will in part dictate passive particle localization at tumor sites. Using FACS, Roode et al.398 investigated the subtumoral cellular distribution of PEG-based polymer particles synthesized via the PRINT method, where NPs were roughly cuboidal (55 × 70 nm) or rod-like in shape (80 × 320 nm). Tumor spheroids made from LKB498 murine melanoma cells were implanted intradermally in the ears of male Foxn1nu athymic nude mice, and particles were administered intravenously via the tail vein. Particles primarily accumulated in organs of the RES. Investigation of the cellular distribution of particles in the tumor showed that NPs were primarily associated with cancer cells compared to other tumorassociated cells such as macrophages, neutrophils endothelial cells, and fibroblasts. Furthermore, cuboidal NPs associated slightly more with cancer cells compared to rod-like NPs. From a drug delivery standpoint, NPs have been shown to improve tumor localization and PK of nanomedicine active pharmaceutical ingredients (i.e., drugs). Cylindrical PLGA NPs (80 nm diameter × 320 nm length or 200 nm diameter × 200 nm length) loaded with docetaxel were shown to increase drug plasma exposure by 20× compared to the free drug.399 The cylinders with the smallest diameter, or longest aspect ratio, resulted in the highest concentration of doxorubicin within the tumor and lower amounts in the spleen, liver, and lungs. The authors suggested that the smaller diameter enabled the NPs to easier escape from the vascularized RES, similar to findings from PEGylated quantum dots and nanorods.400 However, successful extravasation, the movement from tumor vasculature to the interstitium, is not universally guaranteed based on NP shape alone. Smith et al.174 investigated the tumoral extravasation of PEG-coated QD or dye-labeled, PEGylated SWCNT via intravital microscopy. While these are quite different materials, the surface areas were approximately the same and both NPs were PEGylated. SWCNT, however, had an aspect ratio of approximately 100:1 in comparison with the spherical QDs. NPs were administered via a tail vein injection in female nu/nu mice bearing SKOV3 human ovarian carcinoma, LS174T human colon adenocarcinoma, or U-87MG human glioblastoma xenografts in their ears. Interestingly, the rate of extravasation was closely related to particle shape. For SKOV3, QDs did not extravasate out of the tumor vasculature and into the tumor interstitium, whereas SWCNT did. Conversely, in LS174T the QD rapidly left tumor vasculature and accumulated within the tumors whereas SWCNT did not. In a third test, U-87MG showed significant SWCNT extravasation but little QD. Differences in extravasational capability were attributed to physiological differences such as endothelial porosity and vascular fenestrations. Studies using polymer-coated carbon nanotubes (CNT) and nG have shown similar results where fibrillar CNT preferentially accumulated in U-87MG intracranial xenografts compared to nG following intravenous administration.401 In fact, the EPR effect has been shown to be highly heterogeneous based on tumor type and stage,397 and therefore, more advanced approaches have attempted to augment or improve passive targeting via active mechanisms. Active targeting approaches are aimed at pathological cellular aberrations (e.g., upregulated cell surface proteins)402,403 or physiological differences between healthy and diseased tissue 11505


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Figure 18. Particle size and shape are influential in dictating the hydrodynamic behavior of particles in circulation. Higher aspect ratio particles can align in the direction of flow and prolong their circulation half-lives. Particle geometry, particularly oblate and discoidal shapes, can drive the margination of particles (represented by yellow lines) to vessel walls. Size and anisotropy of particles can also cause them to either concentrate in the central flow or marginate to the “cell-free” layer near the vessel wall through collisions with red blood cells. Upon contact with a cellular membrane, particle shape, elasticity, and contact angle can drive endocytotic mechanisms and uptake efficiency. Particle shape also mediates targeting, with high aspect ratio particles providing more “real estate” for targeting ligands to interact with receptors on cell surfaces.

7.3. Toxicity

higher aspect ratio particles improves receptor-targeting ligand interaction (Figure 18).193,412 However, when targeting specific pathologies such as tumors, the factors affecting targeting efficiency become far more complex. A recent meta-analysis by Warren Chan and colleagues evaluated the current state of nanomedicine delivery to tumors, and while the significance of intratumoral accumulation NPs compared to other metrics for nanomedicine efficiency is under debate, we can ascertain some details regarding shape and tumor targeting of nanomedicines.413−417 By their analysis, rodshaped particles accumulate more in tumors compared to sphere, platelet, or other shapes (median efficiency of 1.1%, 0.7%, 0.6%, and 0.9% of initial dose). However, their multivariate analysis also indicated that shape had a less significant effect on the tumoral distribution of particles compared to other factors such as cancer type, material, hydrodynamic diameter, and whether a particle was actively or passively targeted. While shape influences particle biodistribution and targeting in vivo, it is yet unclear whether there are significant benefits for therapeutic purposes (e.g., the treatment of cancer). Altering particle shape can influence the interaction with cellular or tissue structures and will change either the specific surface area or volume available for drug loading; however, few studies have either incorporated active pharmaceutical components into their NP formulations or accounted for the efficacy of such NPborne drugs. In one example following the initial work on paclitaxel-carrying filomicelles,382 Christian et al. showed significant inhibition of tumor growth with mice bearing A549 xenograft tumors following treatment with filomicelle− paclitaxel compared to paclitaxel carried in spherical NPs or free drug.418 This was associated with both increased relative delivery of filomicelles into leaky tumor vasculature rather than healthy organs and prolonged circulation.

Conventional wisdom contends that NP toxicity is influenced by particle physicochemical properties. When considering shape effects on adverse reactions in vivo, the most apparent example of shape-mediated toxicity comes from asbestos, a long-fibered silicate material that has been associated with pulmonary fibrosis and malignant mesothelioma development.419 Comparisons between the high aspect ratio shape of carbon nanotubes which are nanomaterials and micrometersized asbestos fibers has led to an intense investigation into high aspect ratio NP toxicity. In one study, CNTs of varying lengths were instilled into the peritoneal cavity of female C57Bl/6 mice, and histological analysis of their diaphragms revealed that long CNT displayed granulomatous inflammation comparable to long fiber amosite.420 Long fibers also resulted in significantly more recruitment of polymorphonuclear leukocytes, foreign body giant cells, and other indicators for asbestoslike pathology compared to short-fibered materials. In contrast to intraperitoneal instillation of CNT, intravenous injection of PEGylated CNT showed no significant signs of toxicity in nude mice.421 These studies point to the relevance of not only material shape but also the route of administration. Inhalation of high aspect ratio fibers has long been known to result, on a cellular level, in frustrated phagocytosis by pulmonary macrophages and can result in pathologies such as asbestosis, bronchogenic carcinoma, mesothelioma, pleural fibrosis, and pleural plaque formation.361,422 However, systemic toxicity due to high aspect ratio nanofibers following intravenous administration is less clear. It seems that with sufficiently stabilized CNT (e.g., with PEG/phospholipid) coatings, acute systemic toxicity can be mitigated.423−425 Considering other materials, Huang et al.385 studied the systemic toxicity and hematological effects of intravenously administered silica nanorods (short and long) at doses of 20 mg NP/kg body mass at 24 h and 18 days following injection. 11506


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significant accumulation in the lungs which is likely due to particle aggregation and entrapment within alveolar capillaries.429 Therefore, a stabilizing polymer coating such as PEG430−434 or dextran429 is generally attached. Yang et al.432 reported the biodistribution of nGO functionalized with PEG. nGO sheets were single or double layered, as determined by atomic force microscopy, and had dimensions between 10 and 50 nm. These nGO platelets were coated with branched PEG (MW = 10 kDa) and further functionalized at the PEG chain ends with the fluorophore Cy7. nGO-PEG were administered orally or systemically via intraperitoneal injection. Following oral administration, nGO-PEG remained within the gastrointestinal tract and were rapidly excreted via the feces. Systemically administered nGO-PEG accumulated within the spleen, which corresponds with other biodistribution studies showing that larger particulate matter is cleared via the spleen. Similarly, dextran-coated nGO was shown to accumulate in the liver and spleen and clear through the kidneys following intravenous administration.435 In vivo tumor xenograft models of BALB/c mice bearing 4T1 tumors (subcutaneous injection) showed that PEGylated nGO (10−50 nm) labeled with a fluorophore significantly accumulated in tumors.430 These studies have shown nG or nGO (bare or polymer coated) primarily accumulate in the organs of the RES following systemic administration. Others have further investigated potential systemic or hematological toxicity. Bare nGO (10−800 nm width, singlelayer) administered systemically at 1 or 10 mg/kg in Kunming mice showed severe pathological inflammation in lung tissue. Ex vivo hemolysis assays were further conducted on blood obtained from Sprague−Dawley rats and showed 18.5% hemolysis by nGO at 80 μg/mL (compared to 5% for a phosphate-buffered saline control). However, with a PEG coating there have been few reports of toxicity due to systemic nGO or nG administration. PEGylated nGO showed no hepatic toxicity, effect on kidney function, or hemotoxicity.431,432 Thus, for 2D nG it seems that NP biodistribution and toxicity is more heavily influenced by particle stability.241,245 Furthermore, graphene folding/conformational change may in part drive the changes in biodistribution (i.e., stabilized nGO vs nonstabilized nGO).436

Alanine aminotransferase and aspartate aminotransferase activity are typically used to evaluate liver function and were found to be within normal values at 24 h after injection. However, biliary function, measured by total bilirubin, was found to be different when compared to the control. Furthermore, creatinine and blood urea nitrogen activity, measures of glomerular filtration (i.e., kidney function), were different. They summarized that NPs could inhibit glomerular filtration and biliary excretion. Histopathological analysis of key tissues (liver, spleen, lung, kidney) was stated to reveal no adverse tissue changes. However, examination of the bare, nonPEGylated, nanorod images shows that there might be some granuloma formation as evidenced by the thickening of the alveolar walls and higher cell density. This could be expected from the aggregation of NPs in circulation resulting in the blockage of narrow alveolar capillaries. Particle in vivo toxicity, therefore, appears not only to be mediated by their physicochemical properties but also on the route of administration. It can be assumed that intravenously administered particles of a certain size, regardless of shape, will primarily accumulate in organs of the RES. Thus, the long-term toxicity is tied to particle biodistribution, fate (i.e., excretion), and degradation. Below 10 nm particles can be rapidly cleared via glomerular filtration by the kidneys and excreted in the urine.426 Evidence also suggests that sometimes high aspect ratio nanomaterials, where the cross-sectional diameter is low enough to pass through the glomerular pore, can also be filtered through the kidneys.427 This is, of course, dependent on particle stability. Particles that aggregate once in complex biological media will then take on a completely new “biological identity.” Particle aggregation can lead to rapid clearance by the RES due to the increased size but more seriously the blockage of circulatory vessels (e.g., pulmonary capillaries) by aggregated particles that can act as a synthetic embolism. Pulmonary instillation, a model relevant for occupational exposure scenarios to particles of different shapes, presents a more troubling view. High aspect ratio nanomaterials display biological effects similar to those of high aspect ratio asbestos. Thus, exposure to nanopowders or aerosolized particles with a high aspect ratio can potentially lead to serious long-term consequences (i.e., mesothelioma). 7.4. Two-Dimensional Materials

7.5. Summary of Shape Effects in Vivo

Until this point we have primarily focused the discussion on three-dimensional anisotropic materials. However, there is another class of materials, two-dimensional (2D) nanomaterials, that has a wholly different behavior and is similarly investigated for biomedical applications. Most prominent among these is graphene, a 2D honeycomb lattice monolayer of carbon which won Andre Geim and Konstantin Novoselov the Nobel Prize in Physics in 2010.139,140 In a biomedical context, graphene has primarily been investigated for its unique thermal/electrical properties but also as a drug delivery vehicle. Thus, there are many studies conducted on the cellular uptake, cytotoxicity, and in vivo biodistribution of these nanomaterials.428 Studies have primarily focused on the in vivo behavior of graphene, as its 2D nature is expected to significantly alter biological interactions. Uncoated, nanographene (nG), or nanographene oxide (nGO) has been administered systemically in vivo; however, it would be expected that uncoated nG/nGO would aggregate in biological media. Indeed, investigations into intravenously administered, bare nG, or nGO have shown

Understanding systemic particle interactions in a living system can be a complex, multifactorial undertaking. Biodistribution and PK are mediated by factors such as particle colloidal stability in complex biological media, stabilizing ligand and ligand density, material, size, geometry, surface charge, and route of administration. However, it is possible to understand general trends for particle geometry and biodistribution/PK. Elongated particles (e.g., filomicelles, rods) have prolonged circulation compared to their spherical counterparts, ostensibly due to flow alignment in circulation. Discoidal particles have been shown theoretically, in vitro, and in vivo to marginate under flow, and this is likely responsible for their localization in organs such as the lungs/heart. While most particulate matter will be trapped and cleared by the RES, larger particles and anisotropic particles are more likely to be localized in the spleen, whereas smaller particulate matter will be trapped and cleared by the liver. Ultrasmall particles ( 400 nm) particles, while at smaller scales the impact of different surface chemistries has a disproportionately stronger effect. The consequence of shape on the uptake of smaller (

Form Follows Function: Nanoparticle Shape and Its Implications for

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