REVIEW Physical Principles of Nanoparticle Cellular Endocytosis Sulin Zhang,†,‡,* Huajian Gao,§,* and Gang Bao ,) * †Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, Pennsylvania 16802, United States, ‡Department of Biomedical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States, §School of Engineering, Brown University, Providence, Rhode Island 02912, United States, and Department) of Bioengineering, Rice University, Houston, Texas 77005, United States ABSTRACT This review article focuses on the physiochemical mechanisms underlying nanoparticle uptake into cells. When nanoparticles are in close vicinity to a cell, the interactions between the nanoparticles and the cell membrane generate forces from different origins. This leads to the membrane wrapping of the nanoparticles followed by cellular uptake. This article discusses how the kinetics, energetics, and forces are related to these interactions and dependent on the size, shape, and stiffness of nanoparticles, the biomechanical properties of the cell membrane, as well as the local environment of the cells. The discussed fundamental principles of the physiochemical causes for nanoparticleÀcell interaction may guide new studies of nanoparticle endocytosis and lead to better strategies to design nanoparticle-based approaches for biomedical applications. KEYWORDS: nanoparticles . endocytosis . nanomedicine . cellular uptake . ligandÀreceptor binding . coarse-grained model . membrane bending . membrane tension he past decade has witnessed enormous for the biophysics of NPs entry into the cells research efforts undertaken in the de- via endocytosis. The theoretical focus Tvelopment of nanosized particulate renders this review distinctive from and platforms for biological studies, early stage complementary to several existing reviews cancer detection, simultaneous diagnosis on similar topics that are primarily focused and treatment of pathological conditions, on either the novel concepts and experi- and targeted therapy with minimal toxicity mental observations13À15 or computational to achieve “personalized” medicine.1À12 To simulations.16 It also significantly extends enable cell-type specific targeting, nano- previous reviews on the mechanics of cellÀ particles (NPs) are often surface-coated with NP interactions17 and of vesicle wrapping biopolymers or macromolecules, bioconju- NPs.18 In particular, we probe the mechan- gated with targeting ligands that bind spe- ical forces generated at the NPÀcell inter- cifically to the complementary receptors on face and the kinetics and energetics of the the cell membrane. Armed with drug mol- NPÀcell interactions. We then describe how ecules and/or diagnostic reporters, the NPs the size, shape, elastic modulus and surface may transport in the bloodstream, adhere to chemistry of NPs affect the interactions and the endothelium, diffuse through the inter- consequently dictate the cellular uptake pro- fi cellular space, speci cally adhere to the dis- perties. The integrated theories presented * Address correspondence to eased cells, enter the cells via different here may form a basis for the rational design [email protected], pathways (Figure 1), and release the drug of NP-based diagnostic and therapeutic [email protected], [email protected]. molecules effectively. Accordingly, the effi- agents with improved targeting efficiency. cacy of NP-based agents depends on the Depending on the particle size and surface Received for review May 26, 2015 efficiency of these subprocesses, many of treatment, engineered particles may enter and accepted August 8, 2015. which remain poorly understood. Instead of cells via different pathways, as schematically Published online giving an exhaustive review on the research shown in Figure 1. Micrometer-sized particles 10.1021/acsnano.5b03184 progress in all the subprocesess, this review can enter the cells through phagocytosis19 or 20,21 sets forth to provide a theoretical foundation macropinocytosis. Phagocytosis (Figure 1a) C XXXX American Chemical Society ZHANG ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX A www.acsnano.org REVIEW directs the formation of cup-shaped membrane pro- VOCABULARY: Membrane wrapping - the action that trusions that gradually surround and close the parti- cell membrane curves to wrap around an object; Endocy- cles. The shape and size of the phagosomes, i.e., the tosis - the process that cell membrane wraps around an closed membrane protrusions, are dictated by the object and takes it inside the cell; Adhesion Strength - particles being taken up (typically a few micrometers). measures how strong two surfaces cling together; Ligand Phagocytosis is primarily used to uptake dead cells, cell density - refers to the number of ligands per unit area; debris, and pathogens. Macropinocytosis (Figure 1b) is Entropy - a thermodynamical quantity that measures the an actin-regulated process that involves engulfment of disorder of a system; Enthalpy -defined as a thermody- a large quantity of extracellular fluid and particles namic potential that consists of the internal energy of the through plasma membrane ruffling. The membrane system plus the product of pressure and volume of the ruffles exhibit different shapes, and when close, form system; Coarse-grained model - a simulation model that large organelles called macropinosomes.19 Because uses pseudoatoms (coarse grains) to represent groups of of the micrometer length scale of phagocytosis and atoms, instead of explicitly representing every atom in the macropinocytosis, actin assembly plays an imperative system; Molecular dynamics simulation - a computer role in the uptake process.21,22 In clathrin-mediated simulation of physical movements of atoms and mol- endocytosis (Figure 1d), receptorÀligand binding ecules; Bending energy - the energy stored in an object triggers the recruitment and formation of “coated when it is curved; Deformation - the action or process of pits” (clathrin) on the cytosolic side of the plasma changing in shape of an object through the application of membrane.23 The pits self-assemble into closed poly- force gonal cages that facilitate the endocytosis. Clathrin assembly is also responsible for the formation of vesicle necking and the pinch-off process in the late non-specific interactions as well (Figure 1f). For large stage of membrane wrapping of NPs. Clathrin- NPs, transmembrane penetration may occur provided mediated endocytosis is the most common endocytic the interaction force is sufficiently large.31À35 This pathway exploited by viruses.24,25 Caveolin-dependent process, however, may be harmful to cells since large endocytosis (Figure 1c) involves the assembly of the membrane pores must open for particle entry. Small hairpin-like caveolin coats on the cytosolic side of the NPs and molecules (<1 nm) may enter the cell by plasma membrane, forming a flask-shaped caveolae of simply diffusing across the lipid bilayer (Figure 1g). ∼50À80 nm in diameter.26,27 It is generally known that With the rapid advance of nanotechnology, NPs Clathrin- and Caveolin-dependent endocytosis in- of different types can now be synthesized with well- volves complex biochemical signaling cascades.28 controlled size uniformity and shape (Table 1), many of However, to what extent the entry of engineered NPs which have manifested a great potential for a broad is regulated by the biochemical signaling remains range of biomedical applications including in vitro/vivo poorly understood. While clathrin and caveolin provide diagnostics,36 cell tracking,37 molecular imaging,38À41 additional driving force for endocytosis, clathrin and and drug/gene delivery.42À44 These NPs have a common caveolin independent endocytosis29,30 (Figure 1e) can core/coating structure. The cores are either inorganic or also occur through receptorÀligand binding. NPs with- organic, while the coating layer is generally formed by out conjugated ligands may be endocytosed through natural macromolecules, synthetic biopolymers or their Figure 1. Possible internalization pathways of NPs. ZHANG ET AL. VOL. XXX ’ NO. XX ’ 000–000 ’ XXXX B www.acsnano.org REVIEW entry? Answering similar fundamental questions in TABLE 1. Various Types of NPs as Diagnostic and/or the cellular uptake of synthetic NPs will pave the way Therapeutic Agents toward the development of better principles for the NP type size range typical shapes biomimetic design of highly effective NPs. À Metallic 5 500 nm Sphere/cube/rod NPÀCELL MEMBRANE INTERACTIONS: FORCES Oxides 5À200 nm Sphere/cube AND ENERGETICS Quantum dot 2À30 nm Sphere/ellipsoid Silica 10À100 nm Sphere When a NP docks on cell membrane, it forms a highly Carbon nanotube 1À10 nm Cylinder heterogeneous NPÀcell interface and initiates dy- Graphene 10À1000 nm 2D sheet namic physiochemical interactions and a sequence of À Polymer 10 1000 nm Spherical/cylindrical/rod-like/elliptical/ kinetic processes. The interaction forces of different cubic/disk-like origins shape the interactions, modify the associated Nanogel 10À1000 nm Cylinder Liposome 100À1000 nm Spherical energy landscapes, and dictate the endocytosis of 55 Bacteria 500À5000 nm Rod/spirals/ellipsoid the NPs. Wrapping NPs necessitates curving the cell Dendrimers 1À100 nm Spherical membrane and pulling the membrane against mem- Micelles 10À100 nm Spherical/rod-like/worm-like/cylindrical/ brane tension to the wrapping site, presenting resis- elliptical tance to endocytosis. All the other forces, including À fi Virus 10 300 nm Icosahedral/spherical/