Hicheme Hadji

Kawthar Bouchemal
email: kawthar.bouchemal@universite-paris-saclay.fr

Effect of Micro-and Nanoparticle Shape on Biological Processes

Keywords: Nanoparticle, Shape, Cytotoxicity, Cell internalization, Biodistribution, Pharmacokinetics

In the drug delivery field, there is beyond doubt that the shape of micro-and nanoparticles (M&NPs) critically affects their biological fate. Herein, following an introduction describing recent technological advances for designing nonspherical M&NPs, we highlight the role of particle shape in cell capture, subcellular distribution, intracellular drug delivery, and cytotoxicity. Then, we discuss theoretical approaches for understanding the shape effect on particle internalization by the cell membrane. Subsequently, recent advances on shape-dependent behaviors of M&NPs in the systemic circulation are detailed. In particular, the interaction of M&NPs with blood proteins, biodistribution, and circulation under flow conditions are analyzed. Finally, the hurdles and future directions for developing nonspherical M&NPs are underscored.

Introduction

Since the 60', micro-and nanoparticles (M&NPs) have had a powerful impact in biomedical applications such as imaging, diagnosis, and drug delivery. Since then, various classes of spherical M&NPs have been designed, mainly by tuning compositions, sizes, and surface properties. More recently, a new criterion to control biological processes has been added by conceiving M&NPs with specific shapes. Synthetic nonspherical M&NPs have been generated using different fabrication techniques divided into two categories: bottom-up and top-down approaches. Bottom-up fabrication relies on the association of specifically designed molecules into ordered structures. Chemical synthesis of particles with complex architectures (e.g., carbon nanotubes, inorganic particles) is based on bottom-up fabrication. Self-assembly of amphiphilic molecules constitutes the second group of bottom-up approaches. For example, disk liposomes 1 and filamentous micelles 2 were obtained by self-assembly processes. More recently, polymerization-induced self-assembly (PISA) [START_REF] Agosto | RAFT-Mediated Polymerization-Induced Self-Assembly[END_REF][START_REF] Rieger | Guidelines for the synthesis of block copolymer particles of various morphologies by RAFT dispersion polymerization[END_REF] offered the possibility to tune particle shape, size, and glass transition temperature [START_REF] Mellot | Combinaison de la chimie supramoléculaire et de la PISA contrôlée par RAFT pour synthétiser des nanofibres dans l'eau[END_REF] by varying the composition of starting materials during polymerization. A novel class of particles was recently designed by combining nonspherical particle shape to Janus properties. [START_REF] Han | Straightforward preparation of supramolecular Janus nanorods by hydrogen bonding of end-functionalized polymers[END_REF] Each particle face can be functionalized differently and independently. [START_REF] Han | Straightforward preparation of supramolecular Janus nanorods by hydrogen bonding of end-functionalized polymers[END_REF] In another embodiment, the self-assembly of hydrophobically modified polysaccharide and -cyclodextrin in water resulted from hexagonalshaped M&NPs, referred to as micro-and nanoplatelets. [START_REF] Galus | Hexagonal-shaped chondroitin sulfate self-assemblies have exalted anti-HSV-2 activity[END_REF][8][START_REF] Diaz-Salmeron | Pickering emulsions with α-cyclodextrin inclusions: Structure and thermal stability[END_REF][START_REF] Diaz-Salmeron | Real-Time Visualization of Morphology-Dependent Self-Motion of Hyaluronic Acid Nanomaterials in Water[END_REF][START_REF] Diaz-Salmeron | Role of the interactions with CD44 and supported bilayer membranes in the cellular uptake of soft multivalent hyaluronan nanoparticles[END_REF][START_REF] Diaz-Salmeron | Hierarchically built hyaluronan nano-platelets have symmetrical hexagonal shape, flattened surfaces and controlled size[END_REF][START_REF] Diaz-Salmeron | Hierarchical supramolecular platelets from hydrophobically-modified polysaccharides and α-cyclodextrin: Effect of hydrophobization and α-cyclodextrin concentration on platelet formation[END_REF][START_REF] Lembo | Auto-associative heparin nanoassemblies: a biomimetic platform against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV[END_REF] It was possible to modify surface composition by changing the nature of the polysaccharide, while the size was adjusted by acting on the stirring duration of the starting materials. [START_REF] Diaz-Salmeron | Hierarchically built hyaluronan nano-platelets have symmetrical hexagonal shape, flattened surfaces and controlled size[END_REF] Top-down approaches, on the other hand, are based on a controlled shaping of starting materials using different techniques such as lithography, [START_REF] Enlow | Potent engineered PLGA nanoparticles by virtue of exceptionally high chemotherapeutic loadings[END_REF][START_REF] Gratton | Microfabricated particles for engineered drug therapies: elucidation into the mechanisms of cellular internalization of PRINT particles[END_REF][START_REF] Gratton | Nanofabricated particles for engineered drug therapies: A preliminary biodistribution study of PRINT™ nanoparticles[END_REF][START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] and physical stretching of spherical particles. [START_REF] Florez | How shape influences uptake: interactions of anisotropic polymer nanoparticles and human mesenchymal stem cells[END_REF][START_REF] Champion | Making polymeric micro-and nanoparticles of complex shapes[END_REF][START_REF] Champion | Role of target geometry in phagocytosis[END_REF][START_REF] Palazzo | Obtaining nonspherical poly (alkylcyanoacrylate) nanoparticles by the stretching method applied with a marketed water-soluble film[END_REF][START_REF] Ahmed | Shape stability of ellipsoidal nanomaterials prepared by physical deformation[END_REF] All the available techniques have offered the possibility for a precise modification of particle shape, in addition to chemical, physicochemical, and physical properties. Consequently, the researchers considered particle shape as a novel parameter for controlling biological processes. Over the last decade, we have learned that particle shape mediates the motion in biological fluids in static conditions, [START_REF] Diaz-Salmeron | Real-Time Visualization of Morphology-Dependent Self-Motion of Hyaluronic Acid Nanomaterials in Water[END_REF][START_REF] Fakhri | Brownian motion of stiff filaments in a crowded environment[END_REF][START_REF] Han | Brownian motion of an ellipsoid[END_REF] and under a shear flow, [START_REF] Anselmo | Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries[END_REF][START_REF] Vahidkhah | Microparticle shape effects on margination, near-wall dynamics and adhesion in a three-dimensional simulation of red blood cell suspension[END_REF][START_REF] Gentile | The effect of shape on the margination dynamics of non-neutrally buoyant particles in two-dimensional shear flows[END_REF][START_REF] Gentile | The margination propensity of spherical particles for vascular targeting in the microcirculation[END_REF][START_REF] Anselmo | Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting[END_REF] interactions with the cell membrane, [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF][START_REF] Bahrami | The role of membrane curvature for the wrapping of nanoparticles[END_REF][START_REF] Van Der Wel | Lipid membranemediated attraction between curvature inducing objects[END_REF][START_REF] Huang | Role of nanoparticle geometry in endocytosis: laying down to stand up[END_REF] endocytosis pathway, [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF][START_REF] Florez | How shape influences uptake: interactions of anisotropic polymer nanoparticles and human mesenchymal stem cells[END_REF][START_REF] Champion | Making polymeric micro-and nanoparticles of complex shapes[END_REF][START_REF] Champion | Role of target geometry in phagocytosis[END_REF][START_REF] Doshi | Macrophages recognize size and shape of their targets[END_REF][START_REF] Sharma | Polymer particle shape independently influences binding and internalization by macrophages[END_REF][START_REF] Zhang | Graphene: a versatile nanoplatform for biomedical applications[END_REF][START_REF] Zhang | Permission to enter cell by shape: nanodisk vs nanosphere[END_REF][START_REF] Agarwal | Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms[END_REF][START_REF] He | Effects of the microparticle shape on cellular uptake[END_REF][START_REF] Hu | Polyprodrug amphiphiles: hierarchical assemblies for shape-regulated cellular internalization, trafficking, and drug delivery[END_REF] and intracellular distribution. [START_REF] Chu | Unambiguous observation of shape effects on cellular fate of nanoparticles[END_REF][START_REF] Chu | Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery[END_REF][START_REF] Chaturbedy | Shape-directed compartmentalized delivery of a nanoparticleconjugated small-molecule activator of an epigenetic enzyme in the brain[END_REF][START_REF] Xu | Subcellular compartment targeting of layered double hydroxide nanoparticles[END_REF] Herein, we will first review how M&NP shape impacts endocytosis. Both phagocytic and nonphagocytic pathways were underscored. Then, we will give a comprehensive analysis of whether particle shape impacts subcellular distribution and cytotoxicity. As an endocytosis process begins with the interaction of the particle with the cell membrane followed by the membrane deformation, a section will be dedicated to the multiscale modeling of the mechanics and energies occurring during a wrapping episode. The complexity of the interaction between a particle and the cell membrane due to the shape effect will be discussed.

In this review, special attention was paid to the events occurring immediately after administering a particle in the body, even before interacting with the cell membrane. Immediately after their administration, the biomolecules present in the biological media (e.g., proteins, lipids, and sugars) will competitively bind to the M&NP surface leading to a biomolecular corona. Adsorption of blood proteins is most relevant since blood contains thousands of different proteins, each of which may potentially interact with a particle through non-covalent interactions. [START_REF] Walkey | Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment[END_REF][START_REF] Nel | Understanding biophysicochemical interactions at the nano-bio interface[END_REF] The protein corona controls pharmacokinetics, biodistribution, interaction with cells, intracellular trafficking, targeting capabilities, [START_REF] Corbo | The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery[END_REF][START_REF] Salvati | Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface[END_REF] biological activity, immunological reactions, [START_REF] Barbero | Formation of the protein corona: the interface between nanoparticles and the immune system[END_REF] and toxicity. [START_REF] Corbo | The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery[END_REF] In this review, we will explore how nonspherical shape impacts particle behaviors in the systemic circulation, including interaction with blood protein, biodistribution, and circulation under a shear flow. We will analyze the possibility of manipulating the M&NP shape to escape the complement cascade's activation and prolong the circulation duration in the blood. Finally, we will discuss whether particle shape could dictate targeting a specific organ and increase therapeutic efficacy by encapsulating a drug.

Endocytosis

General overview

Endocytosis is a general term describing a fundamental cellular process by which the eukaryotic cells internalize substances from the extracellular environment. The substances to be engulfed are surrounded by pieces of the cell membrane and carried into the cell in vesicular structures that eventually pinch off the membrane inside the cell (Figure 1). Endocytosis plays a critical role in nutrient uptake, surface receptor regulation, cell motility, cell adhesion and orchestrates several cell signalling pathways. Viruses and bacteria exploit this process to enter the cells.

Endocytosis is usually subdivided into two internalization mechanisms: 'phagocytosis' or cell eating and 'pinocytosis' or cell drinking. Pinocytosis is also referred to as a nonphagocytic pathway.

Phagocytosis is mainly undertaken by professional phagocytes of the immune system, including neutrophils, macrophages, and mature dendritic cells. [START_REF] Flaherty | Immunology for Pharmacy-E-Book[END_REF] In addition to professional phagocytes, the engulfment of cells by nonprofessional phagocytic cells has been described for decades. In this process, living cells engulf other living or dying cells. High rates of nonprofessional phagocytic pathways were reported in cancer cells. This phenomenon was called cell cannibalism, [START_REF] Fais | Cannibalism: a way to feed on metastatic tumors[END_REF][START_REF] Brill-Karniely | Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability[END_REF] which is defined as 'a cell that is contained within another bigger cell with a crescent-shaped nucleus'.

Pinocytosis occurs in all cells according to four main engulfment mechanisms depending on the different proteins and lipids involved: macropinocytosis, clathrin-mediated endocytosis, caveolaemediated endocytosis, and clathrin-and caveolae-independent endocytosis. Clathrin-mediated endocytosis is the most elucidated mechanism for nonphagocytic pathways of M&NPs and macromolecules. Unlike phagocytosis, all mammalian cells can ensure this uptake pathway, [START_REF] Kaksonen | Mechanisms of clathrin-mediated endocytosis[END_REF] which mainly engages receptor-ligand complexes. Endocytic coat proteins in the cytosol start to cluster on the inner plasma membrane upon binding of a ligand to its receptor on the cell membrane. The process continues with further recruitment of coat proteins from the cytosol. The process involves a triskelion-shaped protein called clathrin. The flat membrane transforms on a 'clathrin-coated pit' following the self-organization of clathrin into cage-like structures (Figure 1). Subsequently, the pit becomes deeper until it pinches inside the cytoplasm as vesicles (120 nm size) that are typically delivered to the early endosomes (See Transmission electron microscopy (TEM) images in Figure 2).

(For review on clathrin-mediated endocytosis pathway see [START_REF] Kaksonen | Mechanisms of clathrin-mediated endocytosis[END_REF] Caveolae-mediated endocytosis is a clathrin-independent endocytosis pathway. It is present in different cells, mainly endothelial cells. Over this internalization process, a dimeric protein called caveolin triggers the invagination of the cell membrane in the cytosolic side and forms small flaskshaped caveolae ( ) (Figures 1 and2). Then, the cargo containing caveolar vesicle is detached from the cell membrane and released into the cytosol after a GTPase dynamin ensures the fission from the cell membrane. Although this endocytic pathway is described to be relatively slow (half-time >20 min), the internalized materials could bypass the endosomal and/or the lysosomal compartments. [START_REF] Bareford | Endocytic mechanisms for targeted drug delivery[END_REF] Macropinocytosis, also called big drink, is clathrin-and caveolin-independent endocytosis pathway. It occurs in most mammalian cells, including professional phagocytes [START_REF] Liu | Macropinocytosis in phagocytes: regulation of MHC class-II-restricted antigen presentation in dendritic cells[END_REF] and cancer cells under stimuli such as growth hormones. [START_REF] Haigler | Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor[END_REF] Macropinocytosis is mostly initiated by actin-driven membrane ruffles (Figures 1 and2). These laters often fuse with the cell membrane to form large vesicles (macropinosomes), whose size varies from 0.2 µm to more than 5 µm (Figure 1). For review, see reference [START_REF] Swanson | Shaping cups into phagosomes and macropinosomes[END_REF] . Once inside the cytoplasmic environment, unmatured macropinosomes shrink through a series of tubulations and fissions. [START_REF] Buckley | Drinking problems: mechanisms of macropinosome formation and maturation[END_REF] Then, they sequentially fuse with endosomes and lysosomes.

Shape-dependent endocytosis

While it is beyond doubt that particle size and surface properties significantly impact their cellular internalization pathway, shape-dependent cell uptake has been studied more recently. Investigating the impact of particle shape on phagocytosis determines whether undesirable capture by the mononuclear phagocytic system (MPS) can be limited. In contrast, the cell capture of M&NPs can dictate therapeutic applications for intracellular drug delivery. The mechanism by which the M&NPs are captured dictates intracellular transport and the possibility to target specific intracellular compartments by acting on particle shape, as detailed in section 2.3. Although there is evidence that M&NP shape affects the rate and the mechanism of particle endocytosis, [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF][START_REF] Muro | Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers[END_REF][START_REF] Yoo | Endocytosis and intracellular distribution of PLGA particles in endothelial cells: effect of particle geometry[END_REF][START_REF] Decuzzi | The receptor-mediated endocytosis of nonspherical particles[END_REF][START_REF] Zhao | Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity[END_REF][START_REF] Huang | The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function[END_REF][START_REF] Meng | Aspect ratio determines the quantity of mesoporous silica nanoparticle uptake by a small GTPasedependent macropinocytosis mechanism[END_REF] contradictory data were reported on the shape-dependency of M&NPs cellular uptake. Previous works showed that the shape of M&NPs affects phagocytosis, [START_REF] Champion | Role of target geometry in phagocytosis[END_REF] internalization by nonphagocytic cells, [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] and intracellular transport. [START_REF] Yoo | Endocytosis and intracellular distribution of PLGA particles in endothelial cells: effect of particle geometry[END_REF] In contrast, other works reported that M&NP geometry does not play a dominant role in their cellular uptake. [START_REF] Herd | Silica nanoconstruct cellular toleration threshold in vitro[END_REF] Hereafter, the influence of M&NPs shape on cellular uptake was analyzed and discussed.

Phagocytosis of nonspherical particles

The phagocytosis process is critical in eliminating invading pathogens such as viruses, bacteria, yeasts, and dead cells. [START_REF] Yoon | Dead cell phagocytosis and innate immune checkpoint[END_REF] It is also involved in immune regulation, inflammation, and cancer. In the drug delivery context, the phagocytic mechanism was extensively studied for exogenous M&NPs as it regulates their biological fate and toxicological behaviors. [START_REF] Gustafson | Nanoparticle uptake: the phagocyte problem[END_REF] One crucial step before particle phagocytosis is their opsonization. This process consists of attaching proteins, called opsonins, on the surface of the particles to be engulfed. Opsonized particles are thus tagged and will be recognized by the phagocyte membrane receptors. To date, several opsonins have been reported, including complement components, [START_REF] Vonarbourg | Parameters influencing the stealthiness of colloidal drug delivery systems[END_REF] immunoglobulins (e.g., IgM, IgG), type one collagen, and C-reactive protein. [START_REF] Bodman-Smith | C-reactive protein-mediated phagocytosis and phospholipase D signalling through the high-affinity receptor for immunoglobulin G (FcγRI)[END_REF] Conventional receptors for opsonized particles include the Fc region of immunoglobulins, complement receptors, and scavenger receptors. [START_REF] Gordon | Macrophage Activation in: Encyclopaedia of Immunology 2[END_REF] Others investigated the effect of other receptors such as CD44 in the opsonization process. [START_REF] Vachon | CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages[END_REF] Importantly, unopsonized particles can also be recognized by phagocytes, either as a consequence of the presence of specific patterns in their surface such as carbohydrates residues (Mannose, I-fructose, glucose) or via the CD11/CD18

integrins receptors that can bind to both opsonized and unopsonized particles. 75 Once introduced to the phagocyte's membrane receptors, a signalling cascade will trigger the assembly of actin. [START_REF] May | Phagocytosis and the actin cytoskeleton[END_REF] This event provides a force necessary for forming cell membrane extensions that engulf the foreign particle and create a phagosome, 77 (Figure 3.A) which will carry the uptaken particles in the cytoplasm. After actin depolymerization, the phagosome transforms into an intracellular vacuole membrane. This later fuses with the endosomes and eventually lysosomes forming phagolysosomes, [START_REF] Kaufmann | Immune responses to intracellular bacteria[END_REF] which acidic environment is responsible for the engulfed particle's degradation. [START_REF] Wong | Lysosomal degradation is required for sustained phagocytosis of bacteria by macrophages[END_REF] Although M&NP uptake by macrophages is usually undesirable, it was also exploited to target reticuloendothelial system for the delivery of drugs (e.g., cytotoxic, antiparasitic, antifungal, antibacterial, antiviral, and anti-inflammatory drugs). In the drug delivery context, the impact of M&NPs size and surface chemistry on phagocytosis was investigated by several research groups.

Studies have suggested that macrophage phagocytes can engulf a wide range of particle sizes from 250 nm to several micrometers. [START_REF] Brill-Karniely | Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability[END_REF][START_REF] Paul | Phagocytosis dynamics depends on target shape[END_REF] It is also known that macrophage phagocytes can 'eat bigger than their head'. [START_REF] Aderem | How to eat something bigger than your head[END_REF] The engulfment of large particles is essential for host defense against infection and removing dead cells.

Along with the size, the shape of M&NPs has emerged as a critical attribute of their internalization by macrophages. The Mitragotri group revealed that particle shape plays a crucial role in macrophage phagocytosis. [START_REF] Champion | Making polymeric micro-and nanoparticles of complex shapes[END_REF][START_REF] Champion | Role of target geometry in phagocytosis[END_REF][START_REF] Doshi | Macrophages recognize size and shape of their targets[END_REF][START_REF] Sharma | Polymer particle shape independently influences binding and internalization by macrophages[END_REF] Polystyrene particles were prepared by the film stretching method. They had variables sizes and shapes: spheres (radius varied from 1 to 12.5 m), oblate ellipsoids (1 4 m, AR 4), prolate ellipsoids (major axis  , AR ), elliptical disks (major axis  , AR ) and UFO-shaped particles (sphere radius 1.5 m, ring radius 4 m). [START_REF] Champion | Role of target geometry in phagocytosis[END_REF] Particle internalization revealed that the phagocytosis kinetic was controlled by the local geometry of the particle-cell interface. In this regard, ellipsoidal disks (3 14 m) were internalized within a few minutes ( ) when the tangent angle ( ) of the particle surface with macrophages was smaller than 45° (Figure 3.A). In contrast, for (Figure 3.B), the macrophages, attached to the flat side of the particle, were able to spread on the particle surface but did not internalize it even after 12 h. [START_REF] Champion | Role of target geometry in phagocytosis[END_REF] Importantly, those results were related to the differences in the actin structure formed when the particle interacted with the macrophage surface. Indeed, when the particle was attached to the cell with its flat side, actin polymerized at points of contact but failed to create the adequate actin structure necessary to engulf the particle (Figure 3.B).

Those findings could be interesting for different applications, including the possibility of acting on particle shape to inhibit particle phagocytosis by macrophages. [START_REF] Champion | Shape induced inhibition of phagocytosis of polymer particles[END_REF] It was demonstrated that wormlike particles with high AR ( ) exhibited negligible phagocytosis compared to spherical particles with equivalent volume. [START_REF] Champion | Shape induced inhibition of phagocytosis of polymer particles[END_REF] Videomicroscopy and fluorescence microscopy techniques revealed that worm-like microparticles interacted with macrophages at the two-particle endpoints (Figure 3.C).

Nonphagocytic pathways of nonspherical particles

Improved cell uptake of nonspherical particles

Several studies suggested that nonspherical M&NPs were taken up by cells at faster rates and in more significant amounts than spheres. Comparing the cellular uptake of mesoporous silica M&NPs with different aspect ratios (Ars) showed that particles having larger ARs were taken up by cells (e.g., HeLa cells, human melanoma cells, and adenocarcinomic human alveolar basal epithelial cells) in large amounts and had faster internalization rates. [START_REF] Huang | The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function[END_REF][START_REF] Meng | Aspect ratio determines the quantity of mesoporous silica nanoparticle uptake by a small GTPasedependent macropinocytosis mechanism[END_REF] The particles had similar equivalent diameter, surface composition, and surface charge but different AR: nanospheres (AR of 1, diameter 100 nm), short rods (AR of 2 and 240 nm in length), and long rods (AR of 4 and 450 nm in length). [START_REF] Huang | The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function[END_REF] The number of long rods internalized by cells was almost twice that of short rods.

Similar results were reported earlier by Gratton and coworkers. [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] In this investigation, hydrogel particles with variable size, shape, and surface charge were designed by a top-down lithographic fabrication method called PRINT TM (Particle Replication In Nonwetting Templates). [START_REF] Rolland | Direct fabrication and harvesting of monodisperse, shape-specific nanobiomaterials[END_REF][START_REF] Kelly | Shape-specific, monodisperse nano-molding of protein particles[END_REF] While all the particles were internalized by HeLa cells, high AR cylindrical particles (150 450 nm) were internalized faster than cubic particles and short cylinders. Those cylindrical particles had an AR of 3 and were taken up by cells 4 times faster than spheres. The particles were positively charged and had a similar volume.

In agreement with those findings, other investigations showed that rod-like polymeric micelles were taken up by Caco-2 cells 12 times more efficiently than spherical particles with similar compositions. [START_REF] Alemdaroglu | DNA block copolymer micelles-a combinatorial tool for cancer nanotechnology[END_REF] The exact mechanism for faster and higher internalization rates of nonspherical M&NPs is not well elucidated. However, the increased surface area of a long rod and disk particles with the cell membrane is believed to play a relevant role in cell internalization. [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF][START_REF] Florez | How shape influences uptake: interactions of anisotropic polymer nanoparticles and human mesenchymal stem cells[END_REF][START_REF] Zhang | Graphene: a versatile nanoplatform for biomedical applications[END_REF][START_REF] Zhang | Permission to enter cell by shape: nanodisk vs nanosphere[END_REF][START_REF] Agarwal | Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms[END_REF] In addition to AR, the sharpness of the edges was found to be a relevant parameter for cell internalization. For instance, it was found that microparticles that had sharp edges were faster internalized by cells than round-shaped particles. [START_REF] He | Effects of the microparticle shape on cellular uptake[END_REF] While the exact mechanism was not yet studied; the authors explained those differences by the possible difficulty to recruit enough actin filaments for round-shaped particles compared to particles with sharp edges. [START_REF] He | Effects of the microparticle shape on cellular uptake[END_REF] Similarly, Hu et al., [START_REF] Hu | Polyprodrug amphiphiles: hierarchical assemblies for shape-regulated cellular internalization, trafficking, and drug delivery[END_REF] revealed that particles with irregular spiked surfaces exhibited faster cellular uptake than spheres and smooth disks. The shape-controlled nanostructures were formed by the hierarchical self-assembly approach of amphiphilic block copolymers.

Decreased cell uptake of nonspherical particles

Several studies suggested that cellular internalization of nonspherical M&NPs through nonphagocytic pathways was reduced compared to spherical particles. In particular, Au nanospheres were taken up by cells to a larger extent than chemically similar nanorods. [START_REF] Zhao | Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity[END_REF] In this experiment, uptake efficiency was inversely proportional to AR. The cellular internalization of Au nonspherical particles tends to be reduced compared to spherical particles. [START_REF] Zhao | Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity[END_REF][START_REF] Chithrani | Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells[END_REF] However, it is worth noting that the surface properties of spherical and rod-like M&NPs are different. Indeed, the surface of the Au spherical particles is coated with citric acid, while the nanorod surface was stabilized with cetyl trimethylammonium bromide (CTAB). The differences in surface composition of rods and spheres may partially explain the contradictory results obtained with other particles.

Nevertheless, similar trends were also reported for polystyrene disk particles. [START_REF] Florez | How shape influences uptake: interactions of anisotropic polymer nanoparticles and human mesenchymal stem cells[END_REF][START_REF] Zhang | Permission to enter cell by shape: nanodisk vs nanosphere[END_REF] Indeed, while spherical particles enter the cells and perturb the cellular function, the disks bind only to the cell membrane with a significantly reduced cellular function such as reactive oxygen species (ROS) generation, apoptosis, and cell cycle progression. [START_REF] Zhang | Permission to enter cell by shape: nanodisk vs nanosphere[END_REF] The nanospheres and the disks had similar diameters ( 20 nm) and surface potentials ( in serum). As the disks had a 2 nm thickness, the volume was not constant. Furthermore, the nanoparticles used in this investigation are about 10 to 50 times smaller than those used in further experiments revealing preferential cell uptake for nonspherical particles.

Lower cell uptake for nonspherical M&NPs agrees with theoretical models based on free energy minimization. In a model developed by Decuzzi and Ferrari, [START_REF] Decuzzi | The receptor-mediated endocytosis of nonspherical particles[END_REF] it was predicted that nanoparticles with extremely low or high AR were not endocytosed. The AR should be in an intermediate range for complete wrapping by the cell membrane.

Subcellular distribution

We have detailed how the M&NPs interact with cells and how they are internalized depending on their shape. While the correlation between the M&NP shape, their interactions with cells, and the internalization mechanism has been thoroughly investigated, only a few studies addressed their distribution inside the cells. Beyond the internalization mechanism, understanding the intracellular distribution of M&NPs is essential for the subcellular drug delivery to the target compartment, cell imaging, and understanding cytotoxicity behaviors. The intracellular distribution of a drug or its carrier is mediated by the internalization route (Figure 1). After their cell uptake, most M&NPs are confined in early endosomes, which would mature into late endosomes and then lysosomes before exocytosis. [START_REF] Yanes | Involvement of lysosomal exocytosis in the excretion of mesoporous silica nanoparticles and enhancement of the drug delivery effect by exocytosis inhibition[END_REF] Several strategies have been envisioned for endosome targeting through receptormediated internalization. Ligands such as folate, transferrin, and low-density lipoproteins have been exploited for their ability to bind receptors that are overexpressed in malignant tumors for therapy or imaging. [START_REF] Kim | In vivo evaluation of doxorubicin-loaded polymeric micelles targeting folate receptors and early endosomal pH in drug-resistant ovarian cancer[END_REF][START_REF] Fernández | Advances in targeting the folate receptor in the treatment/imaging of cancers[END_REF] The pH of early endosome ( ) was exploited to design pH-sensitive delivery systems to treat severe diseases such as cancer or Alzheimer's disease. [START_REF] Marshall | Misfolded amyloid-β-42 impairs the endosomal-lysosomal pathway[END_REF][START_REF] Moku | Delivering anti-cancer drugs with endosomal pH-sensitive anti-cancer liposomes[END_REF] However, the internalization of the early endosome could represent a hurdle in developing M&NPs for targeting other subcellular compartments such as the cytosol or the nucleus. Most of the drug-loaded particles that are endocytosed accumulate in late endosomes and lysosomes, where they are degraded due to harsh acidic pH or removed from the cell before reaching the target subcellular compartment (Figure 1). [START_REF] Xu | Escaping the endosome: Assessing cellular trafficking mechanisms of non-viral vehicles[END_REF] For example, mRNA or siRNA carriers must escape the endosome to be released into the cytosol, where they interact with the RNA machinery. To release the drugs in the cytosol and avoid endosomal degradation, ionizable lipids were used in designing lipid-based nanoparticles. These lipids are neutral at physiological pH but protonated in endosomes. In this way, lipid ionization facilitates the fusion of their lipids with the endosomal membrane and enables cytosolic RNA delivery (For reviews on mRNA delivery, see references [START_REF] Chaudhary | mRNA vaccines for infectious diseases: principles, delivery and clinical translation[END_REF][START_REF] Hajj | Tools for translation: non-viral materials for therapeutic mRNA delivery[END_REF] ). In another embodiment, it was demonstrated that particles with sharp corners and edges offered a rapid endosomal escape to the cytosol by breaking the endosome's membrane. [START_REF] Chu | Unambiguous observation of shape effects on cellular fate of nanoparticles[END_REF][START_REF] Chu | Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery[END_REF] Soft-shaped particles failed to escape the endosome, regardless of their surface charge, size, and composition. The AR of the particles used in this study was , the average size was , and most of the particles had at least 1 or 2 sharp corners. [START_REF] Chu | Unambiguous observation of shape effects on cellular fate of nanoparticles[END_REF] Shape-directed subcellular compartmentalization was already demonstrated with iron oxide nanoparticles. [START_REF] Chaturbedy | Shape-directed compartmentalized delivery of a nanoparticleconjugated small-molecule activator of an epigenetic enzyme in the brain[END_REF] The particles had similar composition and surface charge but differed by their shape (spheres, spindles, biconcave particles, and nanotubes). The intracellular distribution revealed that the biconcave particles were preferably localized in the nucleus, whereas spheres were distributed in the cytoplasm and nucleus. The spindle and nanotube geometries were observed mainly in the cytoplasm. In another investigation, Xu et al., [START_REF] Xu | Subcellular compartment targeting of layered double hydroxide nanoparticles[END_REF] revealed that layered double hydroxide nanorods ( in width and in length) and nanosheets ( in thickness and in lateral wide) were internalized through clathrin-mediated endocytosis, and they rapidly escape the endosome. The authors revealed that the intracellular distribution is shapedependent since the nanorods quickly translocated into the nucleus while nanosheets were retained in the cytoplasm. The authors hypothesized that the width of layered double hydroxide nanorods reduced from to after deacidification within the endosome. Indeed, layered double hydroxides are a family of anionic materials that can buffer endosome acidification.

Consequently, protons were pumped from the cytosol, leading to particles releasing into the cytosol.

Considering that the cylindrical nuclear pores are in width, it is likely that the nanorods enter the nucleus after the endosomal escape. The size-fit of short single-walled carbon nanotubes (SWNTs) to the nuclear pores partly explained their rapid translocation into the nucleus. SWNTs, characterized by a diameter of and a length of , could cross the lipidic cell membrane through an energy-dependent nonendocytic pathway (Figure 1.4) and mainly distributes inside the nucleus. [START_REF] Pantarotto | Translocation of bioactive peptides across cell membranes by carbon nanotubes[END_REF] While others suggested that energy-dependent endocytosis pathways may occur for SWNTs. For example, SWNTs ( in length) were internalized by cells primarily by a clathrin-coated pit pathway. [START_REF] Kam | Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway[END_REF] While there is no doubt on the cell internalization and accumulation of SWNTs in the nucleus, a central question is whether those particles are excreted from the cells by exocytosis. Jin et al., [START_REF] Jin | Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells[END_REF] studied SWNT exocytosis by using single-particle tracking. The exocytosis rate closely matched the endocytosis rate.

From another perspective, several studies revealed that the shape and AR of M&NPs dictated the cell internalization mechanisms and significantly impacted intracellular trafficking (Table 1).

Macropinocytosis and clathrin-mediated endocytosis were the main mechanisms for cell entry of ellipsoidal polymeric particles. [START_REF] Desai | Self-assembled, ellipsoidal polymeric nanoparticles for intracellular delivery of therapeutics[END_REF] The ellipsoids were formed by the nanoprecipitation of poly(glycerol sebacate)-co-polyethylene glycol (PGS-co-PEG) copolymers. The dimensions of the ellipsoids were changed by acting on the molar ratio of PEG within the copolymer: AR 4.9 ( 23548 nm) or AR 2.4 (195 80 nm).

In other investigations, the uptake of rod-shaped mesoporous particles with an AR of ( ) was internalized through a macropinocytosis process. [START_REF] Meng | Aspect ratio determines the quantity of mesoporous silica nanoparticle uptake by a small GTPasedependent macropinocytosis mechanism[END_REF] The particles with an intermediate AR were endocytosed faster and in a higher amount than particles with low AR ( ) and high AR ( ). Interestingly the rod particles with an intermediate AR accumulated preferentially in the perinuclear region, while shorter and longer rods were more randomly distributed in the cell. Gratton et al., [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] revealed that hydrogel PRINT TM nanoparticles with an AR of 3 ( ) were rapidly internalized into cells and translocated close to the nuclear membrane. Those particles were internalized by cells through multiple modes of non-specific endocytosis, most notably by a clathrin-mediated mechanism, caveolae-mediated endocytosis, and to a lesser extent, macropinocytosis.

However, other findings revealed that nanoparticle shape and AR did not significantly affect intracellular trafficking. The impact of Au nanoparticle shape on intracellular trafficking was investigated by varying the ARs from 1 to 7. [START_REF] Yang | Mechanism for the cellular uptake of targeted gold nanorods of defined aspect ratios[END_REF] Both spherical and rod particles were functionalized with DNA oligonucleotides to target class A scavenger receptors. The particles entered the cells through a caveolae-mediated pathway regardless of the AR. Targeted nanorods reached the late endosome within 4 to 8 h post-incubation with cells, but they did not progress from the late endosome to the lysosome. In agreement with these findings, the lysosomal compartmentalization was independent of the shape of soft hydrogel capsules. 100 In this investigation, the AR varied from 1 to 3.8. Shi et al., 101 reported a higher accumulation of rod-like particles with high AR (

) in the endosome and the lysosome than low AR rods ( ). Intracellular trafficking experiments were performed on polyplexes of N-(2-hydroxypropyl) methacrylamideoligolysine brush polymers. When both polyplexes had the same molecular weight and chemical composition, they differed in the oligolysine length, which confers to them a different shape.

Furthermore, the preferential accumulation of high AR rod-like particles in the endosomal/lysosomal compartments delayed nuclear delivery. Those findings were attributed to the cationic polyplexes that could interact through multivalent interactions with the endosomal and lysosomal membranes.

1

Higher interactions are expected with a higher surface area offered by rod-shaped polyplexes.

Intracellular drug delivery

A key question is how to exploit shape-dependent cell uptake for efficient drug delivery? In this regard, it was demonstrated that rod-shaped mesoporous silica particles with an AR of were more efficient in delivering hydrophobic chemotherapeutic agents (e.g., camptothecin or paclitaxel) than spherical ones. [START_REF] Meng | Aspect ratio determines the quantity of mesoporous silica nanoparticle uptake by a small GTPasedependent macropinocytosis mechanism[END_REF] The cytotoxic potential of the encapsulated drugs was studied in HeLa cells. In a study conducted by Enlow et al., [START_REF] Enlow | Potent engineered PLGA nanoparticles by virtue of exceptionally high chemotherapeutic loadings[END_REF] cylindrical poly(lactic acid-co-glycolic acid)

(PLGA) particles ( ) prepared by PRINT TM process showed high and efficient loading of docetaxel. Particles containing up to 40 wt% of the drug showed higher cytotoxicity than

Taxotere. Similarly, the cell uptake and the cytotoxicity of tubular polymersome-loaded doxorubicin were higher than for spherical particles. 107 Nonspherical M&NPs showed promising results for the intracellular delivery of nucleic acids, which is challenging because of their negative charge and degradation in physiological conditions.

For instance, SWNT/DNA complexes resulted in an efficient gene delivery capacity in cells. 108 SWNTs had a diameter of 20 nm and a length of 200 nm. In another investigation, small interfering RNA (si-RNA)-encapsulated needle-shaped PLGA particles ( ) were generated by PRINT TM process. 109 Particle surface was coated with lipids DOTAP:DOPE ( ) to increase the transfection efficiency (DOTAP: 1,2-dioleoyl-3-trimethylammoniumpropane, DOPE: dioleoylphosphatidylethanolamine). The particles were efficiently internalized by different cell lines, and the knockdown of therapeutically relevant genes was demonstrated. 109 In a more recent investigation, 110 the si-RNA loading, release and internalization efficiencies, and effectiveness of post-transcriptional gene silencing of hollow Au particles ( size) with different shapes and dimensions were compared (nanoshells, nanocages, and nanorods). The three particles were internalized by cells, but nanoshells and nanocages displayed the highest knockdown efficiency.

Cytotoxicity

Among the different available approaches to evaluate the safety of M&NPs, in vitro experiments represent the first step before selecting the most promising preparation to be tested ex vivo and in vivo. A panel of cell viability tests is available to evaluate cytotoxicity, as summarized in Table 2.

Compared with M&NPs size and surface properties, the effect of particle shape on cytotoxicity was more recently studied. Efforts devoted to understand the relationship between particle shape and cell toxicity highlighted contradictory results (Table 3). For example, no significant cytotoxicity was observed with cylindrical PEGylated PRINT TM microparticles after 4 h or 72 h of incubation with four different cell lines, including HeLa and macrophages. [START_REF] Gratton | Microfabricated particles for engineered drug therapies: elucidation into the mechanisms of cellular internalization of PRINT particles[END_REF] The microparticles had a mean height of 0.68 µm, a mean width of 1 µm, and positive or negative zeta potentials. In another investigation, PEGylated nanoimprinted disks ( ), and rod-shaped particles (

) did not induce cell death on three different cell lines (HeLa, HEK 293, and HUVEC). A ratio of particles per cell was used. [START_REF] Agarwal | Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms[END_REF] As expected from the biocompatible and biodegradable PLGA, M&NPs were not toxic on cells regardless of their shape. [START_REF] Enlow | Potent engineered PLGA nanoparticles by virtue of exceptionally high chemotherapeutic loadings[END_REF] In this investigation, cylinders ( , , or ), 2 µm cubes, and 3 µm particles with center fenestrations were tested on ovarian carcinoma cells. In agreement with those findings, ellipsoidal particles composed of PGS-co-PEG copolymers with two different AR (AR , and AR , ) did not show any toxicity toward two cell lines preosteoblasts. [START_REF] Desai | Self-assembled, ellipsoidal polymeric nanoparticles for intracellular delivery of therapeutics[END_REF] Similarly, changing the shape from spherical to rods, cylinders, disks, or worms of silica, Au, or polystyrene nanoparticles did not affect cell viability. [START_REF] Zhang | Permission to enter cell by shape: nanodisk vs nanosphere[END_REF][START_REF] Herd | Silica nanoconstruct cellular toleration threshold in vitro[END_REF][START_REF] Chithrani | Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells[END_REF]104,111,112 Collectively, it was found that the cytotoxicity was rather related to particle concentration, surface charge, 109 particle size, and the cell type.

In opposition to these results, other studies showed that AR had a significant impact on cell death.

The cytotoxicity of high AR silica nanorods (AR 4, 450 nm in length) was higher than low AR nanorods (AR 2, 240 nm in length). [START_REF] Huang | The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function[END_REF] It was found that DNA-coated Au nanorods that had high AR ( 4 or 7) induced higher cytotoxicity than low AR nanorods. [START_REF] Yang | Mechanism for the cellular uptake of targeted gold nanorods of defined aspect ratios[END_REF] Similarly, nanorods and star-shaped particles had higher toxicity compared to spherical particles. 106 In another investigation, both diameter and AR of cerium oxide nanorods were varied to determine the critical AR that induced cell death. 113 The AR varied from 1 to (from 33 to in length and 7 to 9.5 nm in diameter). While no significant cell death was reported with low AR nanorods (up to 31), the highest AR induced significantly higher cell death and activated the NALP3 inflammasome. Subsequent activation of the multiprotein complex NALP3 inflammasome activates caspase 1, leading to proinflammatory cytokines including interleukin-1 (IL-1) and IL-18. For review, see reference 114 .

Similar conclusions were drawn for high AR multi-walled carbon nanotubes, which activated NALP3 inflammasome and led to cytokine release in a human monomyelocytic leukemia cell line. 115 Some researchers showed that the cytotoxicity of multi-walled carbon nanotubes could be decreased by acting on their length. 116,117 Indeed, short carbon nanotubes (600 nm in length) induced less ROS and inflammatory cytokines than long nanotubes (20 µm in length). Several studies showed that long ( in length) and more rigid multi-walled carbon tubes were not wrapped by macrophages.

Frustrated phagocytosis in macrophages induced chronic granulomatous inflammation due to the release of reactive oxygen species and hydrolytic enzymes. 118,119 Interestingly, spherically shaped carbon nanotubes induced lower cytotoxicity than high AR multi-walled carbon nanotubes. 120 In a recent study, Nahle et al.,121 studied the effect of the morphology of carbon nanotubes on their cytotoxicity on rat alveolar macrophage cell line. Their results showed higher toxicity of multiwalled carbon nanotubes than single-walled carbon nanotubes. 108 The nanotubes were ammonium functionalized and had a diameter of about 20 nm and an apparent length of about 200 nm. In addition to the shape and the length, other characteristics could contribute to adverse biological outcomes, including surface functionalization. Indeed, significant differences in cytotoxic behaviors were observed with multi-walled carbon nanotubes ( in length and in diameter), which induced less profibrogenic effects when functionalized with carboxylic groups. 115 In MTS assay, the formazan dye is soluble in cell culture media, and the optical density is measured at 490-500 nm.

Similar to MTT assay, the purple's color intensity is correlated to the cell viability. Lower optical density corresponds to higher cell death.

124,

125

LDH assay (or LDH release assay)

In the lactate dehydrogenase (LDH) assay, the level of plasma membrane damage in a cell population is assessed. LDH is an enzyme found in the cytoplasm of living cells. When the cell membrane is damaged, the LDH is released into the cell culture medium.

The assay is based on the reduction of tetrazolium salt into a red-colored formazan dye. The optical density is measured at 492 nm.

The cell death is proportional to the intensity of the red color, which is proportional to the reduction of tetrazolium salt by the LDH leaked into the cell culture. WST-8 ® (CCK-8 ® ) assay is based on the same principle as WST-1 ® or MTS assay but exhibits superior detection sensitivity by the tetrazolium salts are highly soluble. The optical density of formazan dye is measured at 450 nm.

Similar to MTT, MTS, and WST-1 ® assays, the formazan dye concentration is proportional to the intensity of the optical density and the cell viability.

130

Neutral red uptake assay

Neutral red uptake assay is based on the detection of neutral red (NR) dye inside the living cells. Viable cells can internalize the NR dye, which is taken up by the lysosome. This later is stained red. On the other hand, non-viable cells are not able to internalize this dye. After the lysosome staining, Neutral red uptake assay provides a quantitative estimation of the number of viable cells. The cell viability is positively correlated to the amount of NR dye released and thus the optical density intensity.

cells are washed, and the NR dye extracted. The optical density of NR dye is quantified at 540 nm.

Alamar blue ® assay

Alamar blue ® (AB) is a water-soluble dye, which is not toxic to the cells. AB enters the cytosol, where it is reduced by mitochondrial enzymes in living cells. This reduction is accompanied by a change in the culture medium color from indigo blue to fluorescent pink. The change in color is easily measured by colorimetric or fluorometric reading.

The magnitude of AB reduction is correlated to the optical density intensity (or the fluorescence), which is proportional to the number of viable cells.

127

PicoGreen ® assay

In the PicoGreen ® fluorescence enhancement assay, the fluorochrome (PicoGreen) selectively binds double-stranded DNA (dsDNA) in living cells, leading to a fluorescence enhancement. The samples are excited at 480 nm, and their emission is set at 520-530 nm.

Fluorescence intensity amplitude is a consequence of a high amount of DNA in the living cell.

Annexin V-affinity assay

Annexin V staining assay allows sensitive and nondestructive analysis of apoptosis. Annexin V has a high affinity for phosphatidylserine. In healthy cells, phosphatidylserine is found in the inner leaflet of the lipid bilayer of the plasma membrane. During an apoptotic episode, the phosphatidylserine becomes exposed to the cell surface and is subsequently detected by the Annexin V labeled kit. Reading can be done either by flow cytometry or microscopy techniques.

In flow cytometry, living cells emit in the window of the fluorescent conjugate of Annexin V (e.g., dark green for Annexin V-FITC). In contrast, the apoptotic cells emit in a different window (e.g., blue).

Caspase 3 assay

The activation of caspase 3, a mammalian cysteine protease, plays a significant role in the apoptotic process. Caspase 3 is detected in the cell lysate by means of a fluorogenic substrate (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin). This substrate is cleaved by caspase 3, generating a fluorescent component (7-amino-4-methylcoumarin) that can be detected in a fluorescent reader ( ) and ).

Higher fluorescence emitted after caspase 3 assay corresponds to a high apoptotic process.

Cell membrane surface molecules assessment

In addition to the release of LDH and cytokines, it was reported that cells may over-express some molecules on their surface after contact with M&NPs.

-CD11c: corresponds to an enhancement of macrophage maturation. -MHCII, CD1d: corresponds to an enhancement of antigen presentation. -CD40 and CD86: which translate a co-stimulation activity of the antigen-presenting cells. A way to access these molecules is by targeting them with specific staining after realizing an Fcblocking.

The high fluorescence toward a targeted surface molecule compared to a negative control is interpreted by an over-expression of this surface molecule after the cell's contact with M&NPs.

RT-PCR

Reverse transcript polymerase chain reaction (RT-PCR) is based on the detection of bcl-2 and bax, which are specific gene primers associated with apoptosis. The calculation of the bax/bcl-2 ratio determines the susceptibility of a cell to undergo apoptosis. Accordingly, high values of this ratio are When M&NPs are toxic to a cell, the apoptosis is translated by high values of bax/bcl-2 ratio.

highly correlated with an apoptotic episode.

CLSM

Confocal laser scanning microscopy is often used for analyzing the cell damage induced by the M&NPs. Appropriate staining of M&NPs and cells offers the possibility to visualize the internalization events and/or subcellular particle distribution. This technique was also used for visualizing cell shrinking and asymmetry during apoptosis.

CLSM allows the analysis of a cell apoptosis episode, the presence of white vesicles, and particle penetration inside the cell.

137

Electron microscopy techniques

Electron microscopy techniques are mainly used for the non-quantitative determination of cell apoptosis. They lead to the morphological characterization of dead cells and the determination of hallows regions in the dell membrane. Although very useful, these techniques are not often used as a first intention because, compared to other techniques, they are expensive, and they mainly allow a qualitative analysis.

Owing to their high resolution, electron microscopy techniques, when correctly set up, allow visualizing apoptotic events after cells treatment with M&NPs.

Theoretical approaches for particle endocytosis

Mechanical forces and energies over particle endocytosis

Forces and energies occurring during the interaction of a particle with a cell membrane are essential for cellular function and various biological processes. The creation of a vesicle wrapping the particle requires a mechanical force to deform the cell membrane and deviate from its spontaneous curvature denoted . Spontaneous curvature arises from combining several mechanisms, including membrane lipid composition and asymmetry, partitioning of transmembrane domains and protein crowding, insertion of hydrophobic protein motifs, the assembly of hydrophilic protein domains (e.g., clathrin, dynamin, caveolin), and macroscopic scaffolding by the cytoskeleton. For a review see, reference 143 .

Contacting a particle with the cell membrane is an essential step for the beginning of the endocytic cascade, which results in particle wrapping, either through specific or non-specific mechanisms. When a particle docks on the cell membrane, various new tensions are created which, are drained by electrostatic forces, van der Waals forces, hydrophobic forces, and receptor-ligand interactions. 144 All these forces cumulate to form an adhesion strength ( ) that creates the necessary imbalance to the resistance forces and thus starts the wrapping process.

Let us consider a simple example representing the interaction of a spherical particle with the cell membrane in Figure 4. Upon the interaction of a particle with the cell membrane surface, adhesion energy ( ) is generated at the particle-membrane interface. While unspecific is rapidly set up after particle interaction with the cell membrane, specific requires a longer time as it introduces a translational entropy. Once the particles are robustly attached to the cell membrane, the wrapping process begins, and the cell membrane deflects from its original curvature , relying primarily on a bending energy and a stretching energy (Figure 4). The wrapping extent (or the degree of wrapping) (with )

is calculated from the ratio of the wrapped particle area to the total particle area. Additional deformation energy stored in the curved membrane as represented in Figure 4. [145][146][147] Typically, for a fully wrapped particle, , the cell membrane comes back to its original curvature and . These two energies and the additional deformation energy are necessary for an effective wrapping of the particle (Figure 4). The total membrane deformation energy which size ranged from were endocytosed and exocytosed cells via wrapping events without implicating clathrin or caveolin coats. 154 According to the same authors, the optimal radius of spherical particles at which the endocytic time is the shortest was Those predictions are in good agreement with several experimental data, revealing that the optimal radius for a maximum cell uptake was . [START_REF] Chithrani | Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells[END_REF]155,156 Thermodynamic models developed for specific interactions of ligand-coated nanoparticles and receptors in the cell membrane also predicted an optimal size for nanoparticle wrapping of . 150,157 In the developed models, the ligands were assumed to be uniformly distributed on the particle surface with a density and were immobile. 154,157 The influence of ligand distribution on cellular uptake using coarse-grained molecular dynamins 158 or statistical dynamics model of endocytosis, 159 revealed that nanoparticles with homogenous ligand distribution are the most effectively wrapped.

Before the interaction, it was assumed that the receptors were uniformly distributed on the flat cell membrane with density , and that their total number remains constant over wrapping (Figure 5). Upon the contact of the nanoparticle with the cell membrane, the mobile receptors diffused toward the contact area. The receptor density interacting with the ligands on the nanoparticle surface ( ) became higher in the contact area. In contrast, a depletion zone with reduced receptor density ( ) is formed in the noninteracting region of the cell membrane (Figure 5). reaches a maximum value when each ligand interacts with one receptor ( ). In the model developed by Gao et al.,154 the nanoparticle was considered either cylindrical or spherical. Notably, if we assume that the cell size was much larger than nanoparticle size, the initial state of the cell membrane was presented as flat. Simulation results revealed that an optimal receptor density ratio ( ) is required for effective wrapping. As the increased, the optimal wrapping radius and optimal wrapping time decreased. In contrast, for small , the optimal particle radius and wrapping time increased. 154 is the receptor density in the remote region. is the receptor density in the depletion region. is the density of the receptor in the binding region. is the ligand density on the nanoparticle surface.

Shape effect

While the fundamental biophysical behaviors of cell membrane deformation over a wrapping episode remain applicable for nonspherical particles, they exhibited some differences in the wrapping mechanism compared with spherical ones. Understanding the energetics of a nonspherical particle is more complicated due to the various orientations possible upon the interaction with the cell membrane. Thanks to computational modeling at different length scales, the energetics for nonspherical particle wrapping were better understood. Typically, coarse-grained models and numerical calculations of membrane mechanics based on energy minimization revealed that the endocytosis mechanism of a nonspherical particle is defined as a sequence of four different steps: [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF][START_REF] Bahrami | The role of membrane curvature for the wrapping of nanoparticles[END_REF][START_REF] Van Der Wel | Lipid membranemediated attraction between curvature inducing objects[END_REF][START_REF] Huang | Role of nanoparticle geometry in endocytosis: laying down to stand up[END_REF] a nonwrapped state, a binding state, a partially wrapped state, and a complete wrapped state, where the particle is completely engulfed by the cell membrane (Figure 6). A partially wrapped state could be further subdivided into two other steps: the shallow wrapped state, which comes right after an effective particle binding to the surface area, and a deep-wrapped state mainly characterized by a deeper nanoparticle penetration and a higher surface tension of the cell (Figure 6). Interestingly, several groups agree that nonspherical particles can enter the cell through different modes (Figure 6): [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF][START_REF] Huang | Role of nanoparticle geometry in endocytosis: laying down to stand up[END_REF][160][161][162] a submarine mode (or parallel adhering mode), when the particle is presented to the cell membrane via its long axis and a rocket mode (or perpendicular entry mode) when the long axis is perpendicular to the cell membrane. Another case is also possible. It consists of the competition between both submarine and rocket modes, characterized by the rotation of the particle over a wrapping episode (Figure 6.a and 6.b). [START_REF] Huang | Role of nanoparticle geometry in endocytosis: laying down to stand up[END_REF]163 Within the same framework, modeling of particle membrane interaction revealed that the wrapping of cube-like and rod-like particles compared to the wrapping of spheres and ellipsoids is different. [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF] Complete wrapping of cube-like particles required increased adhesion energy in the lateral edges to back up the deformation energy of the lower edge and increased deformation energy of the upper edge. [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF] For cube-like particles, the adhesion strength to reach a deep wrapping state is times higher than for a sphere with an equal surface area. This was accompanied by increased membrane tension and adhesion strength. For a complete wrapping of Hauser's cube, the adhesion strength was found to be times higher than for a sphere. Moreover, very high adhesion strength is necessary for the complete wrapping of cube-like particles. On the other hand, particle shape dictated its internalization mode. For example, a nanorod with AR switched from a rocket position to a submarine position, but for sharper edges, rocket mode predominates. [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF] In another embodiment, molecular dynamic simulations on a coarse-grained model revealed that the local curvature radius and the sharpness of the edges of the nanoparticle in contact with the membrane played a significant role in the wrapping process. 161 Indeed, the passive endocytosis process of ligand-coated rod particles was thermodynamically favorable, while cylinders did not undergo complete wrapping, but they remained attached to the membrane. Notably, in this article, rod particles were referred to as spherocylinders (cylinders with hemispherical caps at both ends). The cylinders had a diameter , while the whole rod's length was . The AR was or , as calculated from the ratio . The results showed that, upon the interaction with the membrane, the rods changed their orientation from perpendicular to parallel (Figure 6.a). The same investigation revealed that the endocytosis of rods was more thermodynamically favorable than spheres. 161 However, opposite results were found with PEG-coated nanoparticles with variable shapes and using large-scale dissipative particle dynamics simulations. 164 The simulations revealed that, at low PEG density, spherical nanoparticles had the fastest internalization rate, followed by cubes, then rods and disks. 164 The molecular simulations showed that the entry pathway of nanoparticles with high AR (rods and disks) was highly dependent on the contact angle with the membrane. When the nanoparticles with high AR interacted with a cell membrane through their major axis, the large membrane deformation and time-consuming particle orientation delayed the internalization. The same study showed that star-shaped nanoparticles exhibited fast wrapping by the cell membrane, which is similar to spherical nanoparticles. It was noticed that when the nanoparticles were fully covered with PEG, the effective shapes of all the particles were close to spherical. In these conditions, the shape of the nanoparticle core is invisible to the cell membrane (see Figure 9 in reference 164 ).

Shape-dependent behaviors in the systemic circulation

Protein corona represents the first barrier a particle encounters when administrated in the blood because thousands of different blood proteins will competitively bind to the particle surface. 165,166 This phenomenon complicates any prediction of cell interactions, biodistribution, and toxicity because it is the protein corona/M&NP complex, rather than the bare M&NPs interacting with biological machinery. 167 Furthermore, targeting properties of M&NPs are hindered by protein adsorption on functional ligands, preventing their interaction with the corresponding cell receptors. [START_REF] Salvati | Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface[END_REF] After adsorption of opsonins on the M&NP surface and phagocytosis, the particles will be directed to MPS organs, such as the liver and spleen.

Indeed, of administered M&NPs accumulate and sequester in the liver. 168 To reduce non-specific adsorption of proteins on the M&NPs surface, a plethora of research works were focused on grafting hydrophilic polymers such as PEG on the particle surface. 169 This strategy reduced opsonization leading to the escape of immediate sequestration by the immune system after administration in the blood. Unfortunately, now, there is a piece of evidence that repeated administration of PEG-coated nanoparticles in the blood induces hypersensitivity reactions described as complement activation-related pseudoallergy (CARPA). 170 Several strategies have been envisioned so far to reduce the adsorption of opsonins on the particle surface. Unlike size and surface properties (e.g., hydrophilic/hydrophobic ratio, steric hindrance), the effect of M&NP shape on opsonization was understudied. Some data were reported on the impact of the radius of curvature of M&NPs in the composition and conformation of adsorbed proteins by using spherical particles with variable sizes. For example, previous works on spherical gold nanoparticles showed that big particle size, and consequently high radius of curvature, affects protein corona structure as proteins may stretch to fit the large particle surface. Smaller dimensions of isotropic nanoparticles resulted in decreased interaction with proteins, causing fewer structural changes. [171][172][173][174] For anisotropic nanoparticles with high AR, one expects large proteins aligning along the long axis to fit on the nanoparticle surface. 175,176 Large proteins, on the other hand, may not fit in small spaces and hence be excluded from the small dimension of the nanoparticles. Additionally, anisotropic particle geometry does affect complement activation. The IgM and the classical and lectin pathway convertase (C4b2a)

have cross-sectional diameters of and , respectively, making its assembly and deposition onto the surface of nanoparticles with high curvature rather tricky. [177][178][179] Therefore, particle dimensions lower than 30 nm, and depending on their surface chemistry, may not efficiently trigger calcium-sensitive pathways of complement activation, while a surface-bound C3b is expected to occupy an area of 40 nm. 180 Specific interactions of nonspherical M&NPs with blood proteins, with macrophages and polymorphonuclear neutrophils, as well as the dynamic behaviors of the particles under shear flow, significantly impact their biodistribution (Table 4). The most reported tendency is that nonspherical M&NPs had a prolonged circulation time in the blood, [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF][START_REF] Hu | Polyprodrug amphiphiles: hierarchical assemblies for shape-regulated cellular internalization, trafficking, and drug delivery[END_REF][START_REF] Muro | Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers[END_REF]181,182 and had a preferential accumulation in specific organs, [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF][START_REF] Gratton | Nanofabricated particles for engineered drug therapies: A preliminary biodistribution study of PRINT™ nanoparticles[END_REF][START_REF] Agarwal | Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms[END_REF]181,[183][184][185][186][187] compared to spherical particles.

For example, rod-shaped PEGylated Au particles (10 45 nm) had a longer circulation time in the blood than spherical particles (50 nm in diameter), which preferentially accumulated in the liver. 182 Exceptional long circulation times (up to 1 week) were reported with highly flexible filamentous micelles ( in cross-sectional diameter and a tuneable length

) after intravenous injection in rats. [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF] Comparatively, PEGylated spherical vesicles (120 nm) with similar surface properties to filomicelles were cleared within 48 h.

Several mechanisms are involved in the prolonged circulation time of filamentous micelles.

One possible explanation could be their low phagocytosis profiles. Indeed, high AR and high flexibility of the filamentous micelles reduced their interaction with macrophages, required for their internalization. Previous research on spherical nanoparticles with variable rigidity showed that rigid particles displayed higher uptake by macrophages than soft ones. [START_REF] Anselmo | Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting[END_REF]188 Rapid elimination of stiff nanoparticles was correlated to their higher macrophage capture. [START_REF] Anselmo | Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting[END_REF] When filomicelle rigidity was increased by crosslinking, they were cleared within a few hours.

On the other hand, the same study revealed that filomicelle size is a critical attribute for their interaction with macrophages and subsequent internalization. [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF] Long worms were less captured by macrophages than short filomicelles or spheres, thus reducing their clearance.

Similarly, by tuning the shape of polystyrene or Au particles from spheres to rods or elliptical disks, macrophage uptake was reduced, circulation in mice blood was increased, while liver uptake was decreased. [START_REF] Muro | Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers[END_REF]182 Contrarily to spheres, the disks, targeted to intracellular adhesion molecule (ICAM-1), preferentially accumulated in the lungs. The lung to liver accumulation was 1.7-fold higher for anti-ICAM-coated polystyrene rods (125 500 nm) than for spherical particles (200 nm diameter). 189 Shape-specific tissue accumulation was also observed for anti-transferrinconjugated rod particles for brain targeting. Transferrin-coated rods exhibited fold higher accumulation in the brain compared with transferrin-coated spherical particles. 189 In vitro experiments performed on a synthetic microvascular network model showed that rod particles exhibited higher binding to the walls under shear flow than spherical particles. This phenomenon, known as margination, was tackled a decade ago for nonspherical M&NPs. 190- 192 In a theoretical study, Toy et al.,190 concluded that Au nanorods had an 8-fold higher margination rate than nanospheres. Another investigation revealed that both size and morphology had a consequent impact on the margination and adhesion of M&NPs. 193 Their experimental analysis, conducted on spherical, oblate, prolate, and rod M&NPs, revealed a higher margination and adhesion of micro-scale nonspherical particles than nano-scale spherical counterparts. Additionally, Da 194 found that rod particles have a more targeting efficiency to the endothelium receptors than spherical particles. Spherical particles are more likely to follow the shear forces' direction and continue their way in the streamline. Once they marginate, nonspherical particles display another characteristic which is their ability to set up a multi-point contact with the receptors in the vessel wall due to their bigger surface area and lower number of curvatures. Several experimental and theoretical studies confirmed the ability of elongated M&NPs to marginate to the vascular wall of the vessel and subsequent extravasation under shear and pressure forces. 195 For review articles, see references 196,197 .

The tendency of rod-like or disk-like particles to align the blood flow and to extravasate partly explains higher accumulation in tumoral tissues (Table 4). 182,184,187,[198][199][200] The biodistribution of porous silicon discoidal particles (400 600 nm and 400 1000 nm, height diameter) in a murine bearing cancer model was found to be up to 5 times higher than with spherical particles (600 nm diameter). 200 Spherical particles accumulated more prominently in the liver. Rod-like particles (80 320 nm) loaded with docetaxel showed reduced phagocytosis by macrophages and prolonged circulation time in the blood compared to spherical particles, resulting from higher tumor exposure to the drug. 181 Additionally, nonspherical M&NPs showed improved diffusion through tumoral tissues compared with spheroidal particles. 201 Chauhan et al.,199 revealed that nanorods (15 54 nm) diffused in a tumor-mimetic collagen gel 5 times faster than the nanospheres (35 nm). The same nanorods penetrated tumors 4 times faster than nanospheres in mice bearing orthotopic tumors. 199 The two particles had similar plasma and clearance in mice.

To sum up this section, although it is not possible to make a clear conclusion on the in vivo fate of nonspherical M&NPs particles compared to spherical ones, there is a piece of evidence that particles with a high AR and high flexibility had prolonged circulation in the blood. The increased accumulation of elongated particles in tumoral tissues is promising. It could lead to an increase in the efficacy of chemotherapeutics and a reduction of the side effects by limiting the accumulation in healthy tissues. Those findings should trigger further studies to design more effective nanomedicines by acting on M&NP shape. -The blood circulation of filomicelles was up to 6 days, while spherical vesicles of similar chemistry were cleared within two days after injection to rats. -Four days after injection, the filomicelles accumulated in the liver > spleen > kidney > lung. (bladder + liver + spleen). PEGylated short rods showed a peak tissue accumulation at 2 h, while PEGylated long rods showed a peak tissue accumulation at 6 h.

Clearance

-PEGylated long rods were cleared by the RE system as the nanoparticles were cleared via the liver and spleen. -PEGylated short rods were mainly cleared via the bladder suggesting a renal filtration elimination.

Accumulation in the tumors

-The accumulation of both stealth and targeted nanorods in the tumors was AR-dependent. PEGylated short rods showed the highest accumulation in tumors, followed by median and long nanorods. RGD-targeted median nanorods showed the best accumulation in tumors.

Biodistribution

-Worms displayed the highest accumulation in the spleen > liver > lungs > kidneys.

206 Clearance -Regardless of their morphology, the particles were cleared from the blood circulation 2 days post-delivery.

Accumulation in the tumors

-The highest tumor accumulation was obtained with small and spherical micelles, whereas the lowest tumor accumulation was observed for the worms.

Summary and future directions

While a consensus on the M&NPs shape dependence on biological processes was reached, some contradictory results should be pointed out at several levels. The major issue is related to a discrepancy in particle composition, size, shape, surface properties. In several investigations, the surface area and volume of M&NPs with different shapes are not always kept constant and the biological process may be rather driven by other particle parameters than geometry. The isolation of the parameters one by one should lead to a better understanding of the exact role of the shape on biological processes. Such understanding requires robust technological tools for designing particles with controlled properties in a reproducible manner. Not only by handling the shape but also 3D dimensions, surface properties, and particle mechanical behaviors, because those parameters are interconnected.

The development of novel technological tools for designing nonspherical M&NPs with a simultaneous control of their properties is still needed. Indeed, each technique used for preparing nonspherical particles has its advantages and drawbacks. For example, mainly micrometer sized particles with different shapes and AR can be obtained by photolithography 207 and microfluidics. 208 By self-assembly of small amphiphilic molecules, a variety of morphologies are harvested, including rods, nanoribbons, filomicelles, and worms. 209 Generally speaking, shape modification is mediated by the composition of the amphiphilic molecules. Ring-opened benzyl-L-glutamate led to particles with different AR (up to 3.5). 210 However, particles with different AR do not have comparable surface properties since the shape is controlled by polymer molecular weight. In some cases, nonspherical M&NPs did not always show a shape stability. For example, elliptical particles switch their shape to a sphere. 211 The particles were obtained by physical deformation of poly(lactide-co-glycolide) spheres contained in a film composed of poly(vinyl alcohol). In a recent investigation, Ahmed et al., [START_REF] Ahmed | Shape stability of ellipsoidal nanomaterials prepared by physical deformation[END_REF] succeeded in preparing shape-stable elliptical particles by film stretching technique. However, the same study highlighted some difficulties in removing poly(vinyl alcohol) from the particle preparation. [START_REF] Ahmed | Shape stability of ellipsoidal nanomaterials prepared by physical deformation[END_REF] In addition to surface modification following the adsorption of poly(vinyl alcohol), moving from spherical to elongated shape could also modify the particle mechanical properties, leading to mixed parameters. The exact implication of each parameter in the biological process could be complicated.

Modeling the interaction of M&NPs with the cell membrane offers the opportunity to isolate the parameters conferring a better understanding of fundamental events over particle wrapping. Such knowledge will pave the way toward the correct interpretation of the experimental data of the endocytosis process in different applications, including pathogen infections and drug delivery. Furthermore, the models developed for particle wrapping could be applicable to a wide range of biological processes such as viral entry to cells, intracellular trafficking, and exocytosis. The basic structures representing only the double bilayer membrane and receptors could be further improved by adding complex structures of the cell membrane. The effect of clathrin and other envelope proteins should also be considered.

Finally, it is necessary to consider physiologically relevant conditions such as osmotic pressure.

On the other hand, in several reports, it was found that the biological effect was correlated to the purification level of the preparations. For carbon nanotubes, cytotoxicity was rather due to metal traces contaminants than the carbon nanotube itself. 212 For Au particles, the cytotoxicity was mainly due to extrachemicals present in the preparation. The free detergent hexadecyltrimethylammonium bromide, due to incomplete purification of the Au nanorods, was shown to be toxic to the cell membrane. 112,213,214 Standardization of the properties of the preparations, their purification, and the conditions for cytotoxicity experiments is necessary to investigate the effect of particle shape accurately.

Furthermore, the experimental conditions for the biological evaluations, such as the cell lines, the ratio between the cells and the M&NPs, and the animal model used, are of crucial importance. For example, it was shown that the same preparation was more rapidly eliminated from the mice blood than rats. Comparisons between M&NPs can be reasonably drawn between experiments conducted on similar cells and animals.

Another challenge in the future is related to the lack of standardization of the physicochemical techniques for the characterization of M&NPs dimensions. In several studies, the particle size was given for spherical and nonspherical particles, while nonspherical particles are typically characterized by at least two dimensions (e.g., length, width, thickness). Furthermore, most of the investigations used dynamic light scattering techniques to characterize the size of nonspherical particles. However, this technique, based on the Stocks-Einstein equation, was not adapted to compare the size of particles with different diffusion coefficients. Indeed, in a recent investigation, Diaz-Salmeron et al., [START_REF] Diaz-Salmeron | Real-Time Visualization of Morphology-Dependent Self-Motion of Hyaluronic Acid Nanomaterials in Water[END_REF] revealed that, in static conditions, hyaluronan particles with a high AR, designated nanoplatelets, exhibited more linear trajectories and faster diffusion in water than nanospheres. Electron microscopy techniques could provide information on the particle shape and dimensions. However, imaging techniques that require the drying of the sample (e.g., TEM, SEM, and AFM imaging in the air), did not lead to a correct determination of the exact particle dimensions in liquid conditions, and in turn, it could lead to a misinterpretation of the results obtained in culture media or in vivo. A better analysis of M&NPs dimensions was provided by cryo-TEM, cryo-SEM, and AFM in liquid conditions. It was observed that the height cross-section of flat particles obtained by hierarchical self-assembly of α-cyclodextrin and hydrophobically modified hyaluronan in liquid were -fold lower than that measured in dry conditions. [START_REF] Diaz-Salmeron | Role of the interactions with CD44 and supported bilayer membranes in the cellular uptake of soft multivalent hyaluronan nanoparticles[END_REF] An accurate characterization thus is essential for understanding and the correct interpretation of the biological behaviors.

In short, despite the interest in particle shape in biological processes, the issues mentioned above should be addressed to speed-up the translation of nonspherical particles into the clinic.

Indeed, despite the efforts in developing shape-controlled M&NPs, only a few examples are commercially available. Besides inorganic particles, PRINT TM particles that are uniform in shape and bioresorbable (LIQ001) were tested in the clinic to improve immune response and efficacy of seasonal influenza vaccine. The therapeutic and commercial success of disk-like liposomes (Amphotec ® ) and Ribbon-like particles (Abelcet ® ) should pave the way to additional investigations for other applications.

Funding

Author K.B. received funding from the Institut Universitaire de France and the ANR-17-CE09-0038-1.

1

 1

Figure 1 : 2 Figure 2 :

 122 Figure 1: (A) Illustration of the main nonphagocytic pathways in eukaryotic cells. 2

Figure 3 :

 3 Figure 3: Schematic representation of the different orientations of a particle during phagocytosis. The tangent angle between and wasin (A) and in (B). Dash lines in (C) represent the interaction of high AR particles at the two-particle endpoints. Adapted with permission. from references.[START_REF] Champion | Role of target geometry in phagocytosis[END_REF][START_REF] Champion | Shape induced inhibition of phagocytosis of polymer particles[END_REF]

67 a

 67 / aortic smooth muscle cell line, b/ macrophages, c/ human lung tumor epithelial cells, d/ human hepatocarcinoma cell line, e/ human gastric epithelial cell line, f/ mouse breast cancer cell line. # two different polymers were used for the nanoparticle design: poly(DL-lactic acid)-b-poly-(acrylic acid) for spherical micelles and poly(L-lactic acid)-bpoly(acrylic acid) for cylindrical micelles. CLSM: Confocal laser scanning microscopy. TEM: Transmission electron microscopy. AFM: Atomic force microscopy. IPC-AES: Inductively coupled plasma-atomic emission spectrometer.

  are produced inside the cells as a response to environmental stress. There are several assays for the detection of ROS:-Fluorescence dependent methods -Chemiluminescence dependent methods -Spectrometry methods -Chromatography methods -Electrochemical biosensors -Electron spin resonance -Fluorescent protein-based methods.Elevated ROS (e.g., elevated intensity in the fluorescence or spectrometry methods) is highly correlated with cell death by necrosis or apoptosis. iodophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium) is a water-soluble reagent. It does not penetrate the cell but is instead cleaved by the succinate-tetrazolium reductase at the cell surface to form a water-soluble formazan dye. The optical density of formazan dye is measured at 450 nm.Similar to MTT and MTS assays, the cell viability is correlated to the optical density

Figure 5 :

 5 Figure 5: Schematic representation of the different steps for receptor-mediated wrapping of a spherical nanoparticle. Step 1: Nonwrapped state. Step 2: binding state. Step 3: partially wrapped state.is the receptor density in the remote region. is the receptor density in the depletion region. is the density of the receptor in the binding region. is the ligand density on the nanoparticle surface.

Figure 6 :

 6 Figure 6: Illustration of the different entry modes of nonspherical nanoparticles according to numerical simulations.

2 Table 1 :

 21 Summary of the effect of the M&NP shape on the cell internalization pathway.

	Particle type	Shape	Endocytic pathway	Methods for	Dimensions		Cell	Ref
				characterizing	(nm)	(mV)	line
				particle cell uptake
	Hydroxyapatite	Plates	Macropinocytosis	CLSM	221 115	-8.45 A7R5	102
	crystals	Spheres	Clathrin-mediated		63 63	-2.57	a
			endocytosis
		Needles	Multiple pathways		227 44	-4.58
		Rods	Mainly		129 41	-7.02
			macropinocytosis
			followed by
			caveola-mediated
			endocytosis
	Mannose-	Spheres	Clathrin-and	CLSM, flow	46 46	-10.9 RAW	103
	functionalized micelles #		endocytosis caveola-mediated	cytometry			264.7 b
		Short	Clathrin-mediated		99 50	-13.9
		cylinders	endocytosis
		Long	Clathrin-mediated		215 47	-13.3
		cylinders	endocytosis
	Silica M&NPs	spheres	Clathrin-mediated	CLSM, TEM	178 178	87	A549 c	68,
			endocytosis				&	104
		worms	Macropinocytosis		232 1348	58	RAW 264.7 b
	Au	Stars	Clathrin-mediated		~68.55	-2.47 RAW	105
	nanoparticles		endocytosis	ICP-AES, TEM			264.7 b
		Rods	Clathrin-and		~70.49	40.53
			caveolae-mediated
			endocytosis
		Triangles	Clathrin		~61.33	36.07
			Dynamin dependent
			pathway
	Au	Stars	Clathrin and		15 15	-	SMM	106
	nanoparticles		Caveolae	ICP-AES, TEM, dark		13.87	C
		Rods	Clathrin	field microscopy	33 10	54.27	7721 d &
		(small) Sphere	Clathrin		15 15	-25.8	GES-1 e
		(medium) Sphere	Clathrin		45 45	-25.4	& 4T1 f
		Sphere	Macropinocytosis		80 80	-8.83
		(large)
	Silica particles	rod	Macropinocytosis	Flow cytometry,	60-90 160-	13.0	HeLa
				CLSM, TEM	190

Table 2 :

 2 Commonly used methods and assays to determine M&NPs cytotoxicity. 1

	Toxicity	Principle	Data interpretation	Ref
	test
	MTT assay MTT is a colorimetric assay, which determines the	The purple color's intensity (or optical
		mitochondrial activity in living cells. It is based on	density) is directly proportional to the
		the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-	mitochondrial oxidoreductase activity.
		2,5-diphenyl tetrazolium bromide) (yellow colored)	The healthy cells exhibit high rates of
		by the action of mitochondrial oxidoreductase	MTT reduction to formazan and high
		enzymes. This reduction results in insoluble purple	optical density, corresponding to lower
		formazan crystals that can be quantified after	cytotoxicity of the M&NPs at the
		solubilization in dimethyl sulfoxide and measuring	tested concentration.
		the optical density at 570 nm.

Table 3 :

 3 Cytotoxicity of nonspherical M&NPs prepared from different materials. at 72 h for WST-1 assay. 2/ incubation at 24 h for LDH assay. a/ macrophage; Abelson murine leukaemia virus-transformed cell line. b/Primary and immortalized human bronchial epithelial cells. c/ Human colon tumor epithelial cells. d/ star-shaped glial cells in the brain and spinal cord. e/ human lung tumor epithelial cells. f/ human hepatocarcinoma cell line. g/ human gastric epithelial cell line. h/ mouse breast cancer cell line.

	1
	M&NPs	Particle	Particle size	 (mV) Incubation	C	Cell line Cytotoxicity	Observation	Ref
		shape		(nm)			time	(µg/mL)		assay
		Rod Plates		96		-5.7 -9.7	24 h	0	RAW 264.7 a and	PicoGreen assay	PicoGreen assay revealed that the necrosis of BEAS-2B cells was tributary to the particle	141
									BEAS-	shape, which is in the order needles > plates >
									2B b	spheres > rods.
										Based on this assay, RAW264.7 was reported to
										be less sensitive to this test than BEAS-2B. Note
										that plate-shaped particles showed up to 15%
										reduction in dsDNA at 10 and 30 μg/mL.
										Overall, needles and plates were found to be
										more toxic according to this test.
	Hydroxyapatite	Spheres		92.3		-14.1				ROS assay (DCFDA)	DCFDA assay (2',7'-dichlorodihydrofluorescein) is a fluorescent-ROS. The assay showed that, in BEAS-2B cells, regardless of the nanoparticle shape, ROS levels were at the same level as controls without dependent assay that allows the detection of
										nanoparticles. However, in RAW264.7 cells,
										needle-shaped particles generated increased
										ROS levels than the other shapes.
		Needles		74.9		-4.8				Cytokine	Cytokine test revealed high expression of IL-6 in
										assay	needle-shaped and plates particles. The IL-6
										expression with the other nanoparticle shapes
										was comparable to the negative control. The
										same study did not reveal any difference in the
										TNF-α expression regardless of the particle
										shape.
		Long rods		278.1		-30	24 h		Caco2 c	MTT assay No significant changes in cell viability were
	Silica	Short rods	( (	289.0	) )					reported with the cells after incubation with the nanoparticles, compared to control cells that were not exposed to nanoparticles. The authors
		Spheres		250.9						explained the results by degrading the
										nanoparticles into nontoxic silicate ions,

Table 4 :

 4 Comparison of the effect of M&NP morphology on their behavior in vivo regarding their biodistribution, clearance, and therapeutic efficiency.

	1
	M&NPs	Route of	Shape and size		Studied property	Observations	Ref
		administration
		(Animal used)
	Mesoporous silica	Intravenous	Long rods (	in	Biodistribution (2	-Two hours after injection, both short and long	202
	nanoparticles	(mice)	length, AR )		h, 24 h, and 7 days	PEGylated nanorods accumulated mainly in the liver,
			Short rods (	in	after injection)	spleen, and lung (over 80% of the injected dose). Short
			length, AR	)		PEGylated nanorods were more easily trapped in the
						liver, while long PEGylated nanorods accumulated
						preferentially in the lung. However, 24 h after injection,
						both short and long PEGylated nanorods were trapped
						in the spleen > lung > liver.
					Excretion	-Short PEGylated nanorods had a faster clearance than
						long PEGylated nanorods in urine and feces.
					Biocompatibility	-There is no association between the shape and the
						toxicity of the nanoparticles.
	Micelles	Intravenous	Filomicelles (		Biodistribution (ap
		(mice and rats)	) (	)	to 6 days)

( ) required for an effective particle wrapping is the sum of , , and , as illustrated in Figure 4 and Eq.1.

Eq.1 Adapted with permission from references [145][146][147] . is the effective radius of the sphere.

Many theoretical studies were dedicated to understand how the cell membrane responds to external forces induced by the interaction of a particle. [START_REF] Bahrami | The role of membrane curvature for the wrapping of nanoparticles[END_REF][START_REF] Van Der Wel | Lipid membranemediated attraction between curvature inducing objects[END_REF][145][146][147][148][149][150][151][152] Although various parameters are recurrently linked with a wrapping process; particle size undeniably plays a significant role. Based on energetic and kinetic considerations, it was possible to predict the minimum radius ( ) upon which the endocytosis of a particle cannot be effective. If we consider a simple case in which a spherical particle interacts with a cell membrane through non-specific adhesion, the is expressed by Eq.2. 147

Eq.2

where is the membrane bending modulus, represents the adhesion strength, and the membrane tension. For biological membranes, membrane bending modulus , at a temperature of 300 K, is commonly expressed by Eq.3. 153

Eq.3

Thus, when is equal to , the minimum radius below which a wrapping process cannot occur is . 147 In this situation, other endocytic pathways for cell entry, such as transmembrane diffusion, are privileged (Figure 1.4).

In the case of specific interactions between ligand-coated spherical or cylindrical particles with mobile receptors in the cell membrane, mathematical modeling revealed that particles