The effects of polymeric nanostructure shape on drug delivery

Advanced Drug Delivery Reviews 63 (2011) 1228–1246

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

The effects of polymeric nanostructure shape on drug delivery☆ Shrinivas Venkataraman a, James L. Hedrick b, Zhan Yuin Ong a, c, Chuan Yang a, Pui Lai Rachel Ee c, Paula T. Hammond b, Yi Yan Yang a,⁎ a b c

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore

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Article history: Received 15 March 2011 Accepted 21 June 2011 Available online 6 July 2011 Keywords: Drug delivery Polymeric nanostructure Shape control Shape effect Microfabrication Self-assembly Micelles Elongated micelles Worm-like micelles Vesicles Dendrimers Biodistribution Cellular uptake Cytotoxicity

a b s t r a c t Amphiphilic polymeric nanostructures have long been well-recognized as an excellent candidate for drug delivery applications. With the recent advances in the “top-down” and “bottom-up” approaches, development of well-defined polymeric nanostructures of different shapes has been possible. Such a possibility of tailoring the shape of the nanostructures has allowed for the fabrication of model systems with chemically equivalent but topologically different carriers. With these model nanostructures, evaluation of the importance of particle shape in the context of biodistribution, cellular uptake and toxicity has become a major thrust area. Since most of the current polymeric delivery systems are based upon spherical nanostructures, understanding the implications of other shapes will allow for the development of next generation drug delivery vehicles. Herein we will review different approaches to fabricate polymeric nanostructures of various shapes, provide a comprehensive summary on the current understandings of the influence of nanostructures with different shapes on important biological processes in drug delivery, and discuss future perspectives for the development of nanostructures with well-defined shapes for drug delivery. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . Top-down approach . . . . . . . . . . Bottom-up approach . . . . . . . . . . 3.1. Self-assembly . . . . . . . . . . 3.1.1. Spherical micelles . . . . 3.1.2. Elongated micelles . . . 3.1.3. Vesicles . . . . . . . . 3.2. Well-defined macromolecules . . 4. Influence of shape on biological processes 4.1. Biodistribution . . . . . . . . . 4.2. Internalization . . . . . . . . . 4.2.1. Phagocytosis . . . . . . 4.2.2. Endocytosis . . . . . . . 4.3. Cytotoxicity . . . . . . . . . . . 5. Conclusion and future outlook . . . . . Acknowledgments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Hybrid Nanostructures for Diagnostics and Therapeutics”. ⁎ Corresponding author. Tel.: + 65 68247106. E-mail address: [email protected] (Y.Y. Yang). 0169-409X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2011.06.016

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1. Introduction Encapsulation of drugs using polymeric carriers has emerged as the workhorse solution to manage poor biodistribution and stability of bare therapeutics [1]. With the optimized encapsulation technology, therapeutic efficacy can be increased by many folds. Incredible choices in the polymeric designs offer a direct route to optimal carrier design. The physical and chemical attributes of the polymeric carriers play a crucial role in navigating the biological barriers and hence determining the overall success of the therapy. Amongst these attributes, recently the shape of the carrier has been identified as one of the key factors that influence important biological processes, including biodistribution and cellular uptake, in drug delivery applications. The ability to modulate these vital processes via manipulation of carrier shape has opened up new avenues such as engineering of carriers that can evade phagocytosis [2] or development of long-circulating carriers [3]. Realizing the impact of nanostructural shape, in this review we first provide a critical summary of different approaches to fabricate nanostructures with precise shape control, followed by a discussion of the current understanding on the influence of shape on crucial biological aspects of drug delivery. With the recent advances in synthesis of macromolecules and microfabrication approaches, our capabilities to make nanostructures of different shapes have tremendously increased. Still, there are certain shapes and sizes better accessible than the others via certain methodologies. So, in the first part of this review we will highlight different approaches to make nanostructures of different shapes for drug delivery applications. The discussion of the preparation of nanostructures of different shape will be classified into the topdown and bottom-up approaches. The top-down approaches have opened up the possibilities especially in the sub-micron range, providing access to the range of shapes and also having practically mono-disperse and reproducible nanostructures at will. In the last decade, many methodologies originating from the traditional microfabrication tools have been customized for the preparation of soft nanostructures for biomedical applications. Briefly, in Section 2, the impact of these top-down approaches on the developments of drug delivery vehicles will be presented. Bioinspired “bottom-up” approaches offer unprecedented advantages as with these methods one can not only reliably assemble but also enable programmed dis-assembly of nanostructures. With a rationally designed amphiphile, a wide range of nanostructures can be readily accessed via facile self-assembly of these amphiphiles. This chemistry-centered approach is relatively simple and cost-efficient, particularly for nanoparticles of sub-100 nm range as it may not have need for any expensive fabrication tools, etc. Core-shell spheres, vesicles and elongated rod-like micelles, some of the reliably and routinely accessed shapes have demonstrated potential to encapsulate a diverse array of therapeutics with high drug loading capacities. In Section 3, apart from highlighting the ‘paradigm-shifting’ works on self-assembly of block copolymers that came along in the last decade or so, we will focus on the recent breakthroughs that we believe will impact the following decades. Achieving low polydispersity for certain morphologies and similarly, precise presentation of targeting biochemical functionalities via self-assembly approaches have been challenging. To some extent these issues have been addressed by deploying well-defined complex macromolecules of different architectures such as dendrimers and block copolymeric brushes, etc. These classes of sophisticated macromolecules no doubt involve laborious efforts to synthesize. However, the new developments, focused on accelerating and simplifying the synthesis with efficient novel chemistries [4,5], render these materials worth considering for niche applications, where homogeneity and/or spatial control over ligand presentation are important. In Section 3.2, we will highlight the latest developments in this molecular approach.

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With the synthetic access to numerous nanostructures of different shapes, it is prudent to appreciate the roles of particle shape on various biological aspects involved in drug delivery for the development of carriers optimized for specific biomedical applications. As will be seen in this review, differently shaped particles exhibit vastly different pharmacokinetic properties and propensity for phagocytic or endocytic uptake, which may greatly influence their success as drug delivery vehicles. The emergence of several reports demonstrating the superior in vivo anti-tumor effects of drug-loaded biodegradable and non-traditionally shaped carriers such as filamentous micelles [3,6] and polyester dendrimer-poly(ethylene oxide) hybrid [7], for instance, has undoubtedly highlighted the immense potential of particle shape modulations in improving the overall drug delivery outcome. As such, in Section 4, we will provide a comprehensive overview of recent studies that probe the role of particle shape on various drug delivery aspects including biodistribution, cellular internalization and cytotoxicity. Finally in the outlook, Section 5, we will identify key areas wherein we believe that more work needs to be done so that as a community, we can understand the role of nanostructural shape and capitalize on their biological effects to develop practical and effective carriers for optimized drug delivery. 2. Top-down approach Fabrication techniques typically reserved for microelectronics devices have been employed for the generation of nanoparticles of complex three dimensional nanostructures, of defined size and shape. These techniques are generally referred to as “top down” approaches and recent advances in this area have produced nanostructured materials that have dimensions that approach self-assembly methodologies. Ability to exert precise control over numerous critical nanoparticle parameters, ease of scalability and reproducibility, render “top-down” approaches very attractive. Amongst microfabrication techniques, photolithography is the most widely applied technology, wherein geometric patterns, encoded in photo masks, can be transferred onto substrates with the aid of a light sensitive material (photoresist). Majority of the semiconductor industry rely on photolithography for the manufacture of integrated circuits. Considering the nanostructural size and shape requirements for biomedical applications, recently, many innovative fabrication strategies have been developed to complement photolithography. These techniques including soft- and imprint lithography [8–15], step-flash imprint lithography [16], multibeam interference lithography [17,18], probe lithography [19–21], to name a few, generate both two and three dimensional nanostructures. In addition to employing traditional lithographic techniques to generate nanostructures of controlled size and shape, there are a number of alternative “top down” techniques that permit control of particle shape, mechanical properties and surface topology. In this section, we will highlight some of the recent developments in the preparation of drug delivery carriers via “topdown” approaches. Soft lithography is a microfabrication technique that typically employs a silicone-based elastomer with surface features designed to replicate micro- and nanostructures. Silicone-base elastomers have a low modulus and surface energy, facilitating both the release from the master as well as from the replicated micro or nanoscale objects. Recent advances in soft lithography have shown that particulate drug delivery systems can be successfully fabricated using this technique. For example, Hansford and coworkers [22,23] used soft lithography to prepare microparticles from both thermoplastics and thermosets from different precursors, such as poly(lactic-co-glycolic acid) (PLGA), chitosan, and acrylate-based liquid reactive resins. The microparticles fabricated were used to generate uniform structures, including platelike structures for drug delivery. Importantly, this soft lithography technique allowed the formation of single and multiple reservoirs designed for the oral delivery of pharmaceuticals. In another example,

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promising capsule-like structures were generated for intravenous sustained drug release [23]. Hennink and coworkers [24] fabricated particles using methacrylate-functional polyglycerol hyperbranched polymers via soft lithography as well as other lithographic techniques. High yields were obtained with the soft lithography techniques but with poor resolution for the finer feature sizes. The gels were generated with diverse targeted applications including, for example, tissue engineering and drug delivery matrices. A variation to the traditional soft lithography methods for the generation of a top-down approach to nanoparticles for medical application has been developed by DeSimone and coworkers and is denoted as Particle Replication In Non-wetting Templates (PRINT) [25–28]. This platform allows for the fabrication of on demand nanoparticles of controlled sized, shape, modulus, functionality and other important properties associated with the delivery of therapeutic, diagnostic imaging agent and related cargos. The distinguishing attributes of this process over the conventional approaches is the use of a perfluoropolyether elastomer that has a low surface energy to enable filling of nanoscale molds as well as facilitates the easy recovery of the particles and the solvent resistance for materials deposited with solvent or low molecular weight precursors. Moreover, the use of a continuous mold manufacturing process that employs an inexpensive backing to the moldable polymer has brought this technology to a scalable process. This approach allowed a wide variety of materials to be imprinted including highly cross-linked hydrogels, polylactide and therapeutic peptides. For example, the PRINT process was used to fabricate hydrogels containing iron oxide nanoparticles. This work was geared towards targeted nanoparticle therapy for MR imaging agents, delivery of siRNA, DNA, proteins, chemotherapy drugs and biosensor dyes, to name a few [25,29,30]. The ability to tune size and shape has added an important dimension to this work for the navigation of biological barriers, and this aspect will be described in Section 4. Roy and coworkers [31] used Step and flash imprint lithography (S-FIL) to generate nanoparticles as small as 50 nm of defined shape and size, which allowed a unique cargo encapsulation and release strategies. The molded polymer is derived from an enzymatically degradable peptide (GFLG) with acrylate functional groups together with acrylate functional polyethylene oxide macromonomer. This peptide has been widely studied in many polymer drug conjugates as it is sensitive to Cathepsin B, which is overexpressed in many cancers and is present extracellularly in tumor tissues where it can trigger release of encapsulated cargos. S-FIL is a versatile and scalable process that can imprint large wafers, complex and small features and on top of existing features with 10 nm alignment accuracy. The combination of multiple lithographic techniques to generate nanoparticles with defined length and width has allowed the efficacy of shape effects of nanoparticles in nanomedicine to be studied. For example, Hu and coworkers [32] have shown that the combination of nanoimprint lithography with photolithography on a bilayer polymer scaffold allowed the fabrication of nano-worms. This approach allowed ultra-high aspect ratio structures that avoided the typical problems of vertical molding. The highly defined high aspect ratio materials demonstrated unique biological applications. In another example, microfluidics and the lithographic techniques denoted as stop-flow interference lithography provide a synergistic approach to defined nanostructured anisotropic materials [33]. Mitragotri et al. [34–36] have demonstrated the conversion of spherical nanoparticles into complex shapes (~20) with sizes ranging from ~ 50 nm to microns through a stretching process. Spherical particles were dispersed in water in the presence of a water soluble polymer, polyvinyl alcohol, and cast into films that were subsequently processed/stretched using heat or solvent to generate the requisite particle shape. The samples were then cooled or dried to solidify the newly formed particle-film composite and the particles were harvested by dissolution of the polyvinyl alcohol. The particle

shape is controlled by the properties of the film, viscosity, particle– matrix interactions and stretching parameters. This film stretching method leads to a variety of shapes including one, two and threedimensional shapes with ranges in concavity, curvature and aspect ratios from polystyrene and biodegradable materials (e.g. PLGA). The effect of size and shape from this stretching technique was evaluated on various biological processes including phagocytosis and endocytosis (detailed discussion in Section 4). It is anticipated that this technology will have important ramifications in the delivery of pharmaceuticals. Lahann and coworkers [37] demonstrated an unique approach to the fabrication of multifunctional and multicompartmental microcylinders and microdisks from biodegradable materials such as PLGA with controlled size, number of compartments and aspect ratios. This process involves the electro-hydronamic cospinning followed by microsectioning that converts the fibers into cylinders of defined compartments. Fibers were embedded in a gel and cryo-sectioned where the size and shape of the cylinders were controlled by sectioning parameters including speed and thickness to generate the desired aspect ratios. Fibers were typically harvested by dissolving the gel in de-ionized water and suspended into solution with the aid of sonication. With the incorporation of acetylene-functionalized PLGA, selective functionalization of the mutlicompartmental microcylinders was possible. Reaction of acetylenes with biotin-(ethylene oxide)3-azide and subsequent evaluation of their interaction with steptavidin, demonstrated the feasibility of selective surface modification. Such combinations with click chemistry afforded another dimension with designable chemical and mechanical properties. Recently, multicompartmental particles of 3–5 μm in size with excellent shape and size control have been developed via electrohydronamic co-jetting of polymer solutions by carefully optimizing numerous parameters, including the polymer concentration and solvent composition (Fig. 1) [38]. Even though these particles (likewise, in other examples) are not essentially nanoparticles and

Fig. 1. Illustration of the electrohydrodynamic co-jetting process, involving multiple solutions to result in the formation of bicompartmental microparticles. Upon manipulation of parameters including solvent composition and solution concentration, shape control has been achieved [38]. Copyright (2010) Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

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may not be suitable for systemic drug delivery, they can still be used for drug delivery via other administration routes. 3. Bottom-up approach 3.1. Self-assembly Contrast to ‘top-down’ approaches highlighted in Section 2, ‘bottom-up’ approaches primarily rely on non-covalent interactions for the self-assembly of the constituent (macro) molecules, without any or minimal external intervention, for the formation of ordered aggregates [39]. Hence this approach is cost effective, without any need for huge initial investment on microfabrication tools, and energy efficient owing to the spontaneity of the process and mild processing conditions. Molecular self-assembly is ubiquitously applied in Nature, as a means to create hierarchically ordered composite structures [40] and also to dynamically compartmentalize numerous components so as to exert precise control over the regulation of numerous synchronized biochemical processes with temporal and spatial precision [41]. Inspired from the Nature's compartmentalization strategies, preliminary self-assembly systems involving liposomes were developed to deliver the drugs [1,42–44]. These preliminary studies on liposomes, along with the elegant polymer-therapeutic conjugates, played a crucial role in the elucidation of, now well accepted core tenets of elements of drug delivery vehicle design [29]. These fundamental design principles include but are not limited to the aspects like the installation of neutral hydrophilic macromolecules such as PEG as the shell for delaying the opsonization process, control over the size of the particles to achieve the “enhanced permeability and retention” (EPR) effect and the ability of targeting specific tissues/cells through biological ligands. With this array of indispensible fundamental knowledge of design rules, along with recent revolutionary developments in the synthetic polymer chemistry, selfassembled nanomaterials have attracted tremendous attention in the field of drug delivery. Ingenious developments in the synthesis of amphiphilic block copolymers and evaluation of their self-assembly behaviors have allowed for the reliable preparation of discrete functional nanostructures of different shapes. This ability to control the nanostructural shape has shown potential to better tackle some of the existing biological hurdles. In this section, we will highlight, starting from polymer synthesis that has allowed for the precise shape control of the self-assembled nanostructures in the aqueous environment, cascade of key developments, and in combination with the discussions in Section 4, we will summarize the current understandings on the role of nanostructural shape in the cellular uptake, cytotoxicity and biodistribution. Ability of an amphiphile to micro phase separate has allowed for the spontaneous formation of ordered structures [45]. Key to the modulation of phase separation relies on the synthetic ability to vary the relative hydrophobic and hydrophilic components in an amphiphile. Block copolymers constitute one of the most sophisticated and versatile classes of amphiphiles [46]. Compared to small molecular amphiphiles (b103 Da), block copolymers offer numerous possibilities to vary the design parameters such as chemical composition, molecular weight, number of blocks, block sequences, relative sizes of the corresponding blocks, topology, the physical properties (crystallinity, fluidity, etc.), (bio)degradability and even rate of (bio)degradability. This infinite combinatorial possibility of block copolymer design and synthesis has tremendous impact on numerous disciplines, including the development of ideal drug delivery vehicles in the healthcare industry. Even though the first report on synthesis of block copolymers appeared in 1950s, [47] the renaissance of block copolymers did not occur until 1990s, wherein a host of excellent controlled polymerization methodologies were developed, enabling the facile synthesis [48,49]. Impressively, in a short span of less than ten years, several mechanistically distinct radical polymerization methodologies, in-

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cluding nitroxide mediated radical polymerization (NMRP) [50], atom transfer radical polymerization (ATRP) [51,52] and reversible addition-fragmentation chain transfer (RAFT) [53] polymerization, for the commodity monomers such as styrenics and (meth)acrylates, were developed [54]. These techniques, apart from retaining almost all of the advantages of ionic polymerization mechanisms, such as enabling the synthesis of macromolecules with predictable molecular weights and low polydispersity index, but also offered unprecedented ease in handling reagents [55] and functional group tolerance as compared to anionic polymerization. Now, numerous functional components, required to conduct these polymerization reactions are already commercially available, illustrating the widespread applicability of these methodologies [56–58]. Along with these controlled radical polymerization methodologies, developments in the ring opening polymerization techniques expanded the scope and range of applicability of block copolymers. Recent developments in the organo-catalytic ring opening polymerization [59]of numerous important families of monomers like cyclic lactides, lactones, and carbonates will have tremendous impact on the synthesis of heavymetal contamination-free biocompatible and biodegradable materials for drug delivery applications [60]. Moreover, the recent developments on the facile installation of numerous functionalities onto the biodegradable polymeric platform has enabled access to the range of functional compositions that were, in the past, only accessible to the non-degradable (metha)acrylates [61,62]. Judicious combination of mechanistically distinct class of monomers could also be polymerized to result in block copolymers with appropriate functional initiators or versatile catalysts or clever chain transfer agents. All these developments in the synthetic polymer chemistry along with the ‘click’ chemistry strategies [63] have rendered an almost complete functional macromolecular tool box, required for developing nanoparticulate drug delivery carriers. The complexity and the range of possible self-assembled morphologies with block copolymers are astounding, particularly with the increasing complexity of the block composition and architectures [45]. Apart from the most explored spherical micelles, other exciting morphologies [64] like elongated micelles [65], vesicles [66], ‘hamburger’ micelles [67], helical micelles [68], torroids [69], nanotubules [70] and many other complex structures have been formed by the selfassembly of amphiphilic block copolymers [71–76]. Amongst these exotic nanostructures, many of them may not be well suited for drug delivery applications. The reasons might include but not limited to the factors concerning overall size control, stability, toxicity due to the organic co-solvents, and biocompatibility. So far, spherical micelles, elongated micelles, and vesicles constitute the three different classes of self-assembled morphologies that have been routinely explored for encapsulation of therapeutics and imaging agents. Both spherical and elongated micelles lack the hydrophilic interior space that the vesicles possess. This difference, coupled with the emerging facts that the cells' remarkable preference towards certain aspect ratios, provides unique niche for each of the morphologies [2,77]. In the reminder of this section, we will highlight some of the key advances made in each of these morphologies that demonstrate potential in translating into concrete clinical applications. The morphology of the polymer aggregates dictates their properties and consequently their applications. For instance, encapsulation of a hydrophilic compound might not be feasible in a micelle, whereas this is feasible in the aqueous cavity of the vesicle. Hence there has been an ever increasing impetus to have the capability to design block copolymers for a specific morphology, without having the need of tedious experiments to map out the entire-phase diagram [78]. Importance of developing such theoretical models that can reliably predict the resultant morphologies for a given combination and offer explanation for the observed phenomenon has been highlighted in a recent report, identifying the important challenges in macromolecular science [79].

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Controlling self-assembly to result in well-defined nanostructures in a predictable and reproducible way remains a significant challenge [78]. This challenge is tremendous for situations involving mesophases or kinetically controlled structures. Aqueous self-assembly is widely rationalized using the elegant ‘packing parameter’ concept, proposed by Ninham and co-workers [80]. The concept of packing parameter has been widely invoked in the literature to rationalize molecular self-assembly in solutions. The dimensionless packing parameter p is defined as v/(al), wherein v and l correspond to the volume and the length of the hydrophobic tail respectively, and a corresponds to the interfacial area per molecule. Different shapes of self-assemblies are related to different packing parameters. For instance, spherical micelles will have p values below 1/3, elongated micelles with p between 1/3 and 1/2 and for bilayers, p values are between 1/2 and 1. This concept of invoking molecular packing considerations and general thermodynamic principles is useful to explain the observed shapes, but considerably lacks the predictive power [81]. This limitation primarily arises from the fact that a, the optimal interfacial area per molecule is not a geometrical area based on the chemical structure but a thermodynamic quantity, derived equilibrium considerations [82]. Likewise, particularly for the polymers, one can imagine that the volume and length of the hydrophobic tail can also vary significantly depending upon the specific solution conditions such as concentration, ionic strength, and pH of the solution. Ratio of hydrophilic to hydrophobic components in a given block copolymer has been used as a guide to keep track of changes in the morphologies. However, this approach might not be perfect as it is not uncommon to find surprises to some of the well accepted notions, across different block copolymer compositions, due to the inherent differences in the nature of thermodynamic interactions between different monomer pairs. In spite of the limitations on predictive power as to the shape of the aggregates for a given block copolymer composition, tremendous progress has been made so far in the development of a variety of systems with precise shape control. In many instances, the preliminary designs were often curiosity driven, involving nondegradable materials. However, the invaluable lessons learned from these elegant preliminary designs will eventually be translated, to suit the specific needs in drug delivery applications. 3.1.1. Spherical micelles Amongst all the shapes, block copolymer-based spherical micelles are the simplest and have been thoroughly studied. With the advent of micellar systems, numerous problems in the field of drug delivery such as poor drug loading capacity, low drug efficacy, broad size distribution and non-specific toxicity of the drugs have been greatly mitigated. Currently, there are several micellar drug delivery systems at various stages of clinical trials. Typically these systems are prepared from amphiphilic diblock copolymers, having sizes less than 100 nm in diameter, with PEG (or other hydrophilic) shell. These micellar systems have proven to be particularly effective with hydrophobic drugs by improving their solubility. Targeting moieties can be effectively coupled onto the shell region to achieve targeting towards specific tissues/cells. The drug release can also be engineered from just simple diffusion or dissolution controlled to situations wherein the degradation of the polymers effects different rates of drug release profiles. A variety of stimuli-sensitive approaches have also been explored to control drug release spatially and temporally. Since there have been a number of excellent review articles describing micellar drug delivery systems [44,83], here we will draw attention only to some of the important recent reports. Solution state self-assembly can be modulated by the manipulation of the several solution parameters including concentration, ionic strength, pH and even binding of proteins or ligands. In the biomedical context, this environmental responsiveness brings in additional challenges associated with the stability of the self-assembled structures. For example, drug-loaded micelles, upon systemic appli-

cation would undergo infinite dilution, necessitating the need for these systems to have considerably lower critical aggregation concentration (CAC) so as to withstand such dilutions. In one clever approach, shell or the core regions have been cross-linked to kinetically freeze the ensembles to prevent from disassembly when diluted [84]. However, this approach may also prevent the renal excretion of nanoparticles as the size of the cross-linked polymeric particle is way above the renal threshold of about 40 kDa [85]. Hence, cross-linking chemistries have been designed to be reversible, specific to external stimuli. Recently, acetal-based pH responsive cross-linkers for the preparation of shell cross-linked micelles have been reported [86]. The cross-linker imparted significant stability at the physiological pH, whereas it underwent rapid dissociation at acidic conditions. It has been well documented that compared to healthy cells, tumor tissues have an acidic environment, and that the endosomal environment is acidic. Such acidic environment-triggered disassembly may pave way for modulating drug release selectively. Armes and McCormick's group have demonstrated the use of cystamine as a reversible cross-linking agent to prepare cross-linked nanoparticles [87]. It has been postulated that such disulfide-containing crosslinks will impart the necessary stability to the micelles in the bloodstream and upon internalization, the reductive environment of the cells will readily facilitate the cleavage of the disulfide bonds, allowing for the disassembly of the particles. Such disulfide-bridged cross-linking chemistry has been successfully extended to other systems as well. With the use of nonbiodegradable block copolymers, this approach can only disassemble to unimers as further degradation of these polymers might not be possible. To overcome this limitation, incorporation of at least one degradable block has been accomplished. In a recent report, Xu et al. have regioselectively incorporated lipoic acid at the junction of the hydrophilic mPEG and hydrophobic biodegradable polycaprolactone blocks [88]. With a catalytic amount of dithiothreitol, they have demonstrated that the lipoic acid enabled cross-linking and stabilization of the micellar assembly. Higher concentrations of dithiothreitol solution (10 mM) allowed for the disassembly of the micelles. Triggered release of doxorubicin, an anticancer drug, was also accomplished with such redox-sensitive, reversibly cross-linked micelles [89]. Alternative to the cross-linking chemistries, block copolymers containing hydrogen bonding units, capable of lowering CAC and also enhancing drug loading capability have been developed [90,91]. By initiating from the chain end alcohol of mPEG polymer, a series of urea-functional aliphatic polycarbonate-based amphiphilic block copolymers have been synthesized via metal-free organocatalytic ring opening polymerization methodologies. Strategic placement of urea groups in the hydrophobic core of the resultant micelles significantly lowered the CAC due to the strong hydrogen bonding interactions between the urea groups. Dramatic improvements in the doxorubicin loading content were also observed, presumably due to the hydrogen bonding interactions between the drug and the urea groups (Fig. 2). In addition, urea groups in the core of the micelles enabled controlled release of the entrapped drug. This example demonstrates the importance of the ability to tailor functionalities for specific therapeutics. Ability to tailor the functionalities of polymers to enhance the loading content has opened up numerous avenues particularly for many promising drugs that are limited by their poor bioavailability. Encapsulation of such drugs in a well-designed carrier has been shown to have a positive impact. Immunosuppressive agent Cyclosporin A, due to its extreme low water solubility and its cyclic peptidebased architecture, is one such challenging drug to deliver. Möller and coworkers have developed PEGylated polylactide block copolymer micelles to efficiently encapsulate Cyclosporin A (~ 23 wt.%) [92]. Manipulation of the hydrophobicity of the micellar core via replacing one or both the methyl groups of the lactide with the hexyl group, was found to be crucial for both the loading content and the long term stability of these micelles.

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Fig. 2. Placement of molecular recognition units enhance the drug loading. A. Schematic illustration of enhancement of doxorubicin loading into PEGylated block copolymers containing urea functionalities and control over the size of doxorubicin-loaded micelles through the H-bonding interactions between the drug and urea groups. B. The diameter of the drug loaded micelles decreases and the doxorubicin loading content increases with the increase in the urea content of the polymer [91]. Copyright (2010) Elsevier. Reproduced with permission.

Upon moving from small peptides to macromolecular drugs, such as proteins the challenges are enormous and so are the opportunities. Recently Kataoka's group has developed charge conversional polyionic complex micelles based on reversible conjugation strategies [93]. In their work, they have functionalized the model cationic protein cyotochrome c, with citronic anhydride and cis-acinitic anhydride to effectively convert the protein's overall charge to negative and also increased the charge density. Thus, they were able to incorporate the modified protein into the core of the polyionic micelles, formed from ionic interaction between modified protein and a PEGylated cationic

block copolymer. The protein was consequently released by taking advantage of the fact that citronic amide and cis-aconitic amide rapidly degraded at acidic endosomal pH, to result in charge conversion, thereby enabling the dissociation of the micelles (Fig. 3). In vitro experiments also demonstrated that the charge conversional polyionic complexes enabled endosomal escape, enabling protein delivery to the cytoplasm. Similar to the delivery of macromolecular therapeutics, delivery of small molecules has their own unique challenges. Nitric oxide (NO), is one such small molecular therapeutics, which could have impact in

A

1 ph 5.5

= Cyt C

B

Cyt-Cit or Cyt-Aco

PIC micelle

C

NH3

-amine of lysine, present in the protein

O

citraconic anhydride (or) cis-aconitic anhydride

O

O 271

O

O NH O

O

O

citraconic amide

NH2 68

O

O HN

O

(or)

NH O

N H

cis-aconitic amide

1

NH

NH3

pH 5.5 NH3 native protein Fig. 3. Schematic representation of general strategy for encapsulation of protein cargoes. A. The charge density of the protein is increased by the reaction with citronic anhydride or cisaconitic anhydride, followed by the formation of polyionic complex micelles with the PEGylated cationic block copolymer. Subsequently, the release of protein occurs under acidic conditions (pH 5.5), triggered by rapid degradation of citronic amide and cis-aconitic amide. B. Reaction of ε-amine of lysine with the citronic anhydride or cis-aconitic anhydride to result in the formation of citraconic amide or cis-acinitic amide. This reaction can be readily reversed at acidic conditions to result in formation of original protein. C. Structure of the polymer 1, used to encapsulate the modified protein in the PIC micelles. Adapted from [93]. Copyright (2009) Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

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numerous diseases related to cardiovascular and tissue responses. Delivery of NO has been incredibly difficult due to the strict temporal and spatial requirements. In an elegant approach, Hubbell and coworkers have sequestered NO onto the secondary amine groups in the polymer, thereby converting a double hydrophilic block copolymer to an amphiphile and hence enabling their self-assembly into micelles (Fig. 4) [94]. By incorporating NO into the micellar core, they were able to effectively control the NO release rate, and due to the small size of the polymeric micelles (~ 50 nm), access to the complex tissue structures was also made possible. Spherical micelles can be assembled not only from simple amphiphilic diblock copolymers, but also from a wide variety of complex architectures. In a recent work from the groups of Luo and Lam, they have employed amphiphilic telodendrimer based on PEG-bdendritic oligo cholic acid that self-assembles in aqueous environment to effectively encapsulate paclitaxel, a challenging hydrophobic drug used in cancer treatment [95]. From their detailed studies, varying systematically numerous aspects of the molecular design, including the PEG chain length, cholic acid moiety and the specific number of cholic acid, in relation to the PEG molecular weight were all found to be crucial to achieve optimal particle size and drug loading. The recent examples discussed in this section clearly demonstrate that by clever installation of drug-complementary functionalities in the polymer, many types of therapeutic components can be incorporated into micellar formulation. Knowledge acquired from the spherical micelles-based work has proven to be crucial to develop drug delivery vehicles of other morphologies. 3.1.2. Elongated micelles Self-assembly of amphiphiles into elongated, flexible core-shell structures of high aspect ratio has been demonstrated to possess unique visco-elastic and rheological properties [96,97]. Compared to the spherical micelles, access to the worm-like micelles, via block copolymers has been only possible in the last decade [64,98]. This was partly due to the fact that often, non-spherical morphologies were favored only in a very narrow compositional space [71]. Importance of such elongated micelles in drug delivery applications has been realized with the advent of pioneering works from Discher's laboratory [3,99]. In this section, we will highlight the recent progress in the development of biocompatible and/or biodegradable elongated micelles. So far, numerous classes of polymers such as coil–coil, rod–coil, and crystalline–coil block copolymers have been shown to form elongated micelles, in both aqueous and non-aqueous environments [65,100,101]. Recent reviews, particularly the one from Manners and Winnik's groups,

provided a comprehensive picture on the block copolymer chemistry that has been demonstrated to form elongated micelles [65]. By comparing the self-assembly behaviors of block copolymers with different weight ratios of constituent blocks, the specific compositional window to result in elongated micelles has been proposed.[71,99] However, several additional factors have been shown to influence the morphological outcome [102–108]. For instance, depending upon the polymer concentration, long rod-like, short rod-like, or spherical micelles have been formed from the same PEO-b-PCL block copolymer [109]. Recently, inorganic salt-induced sphere-to-rod transitions have been reported in PEO-b-PCL block copolymers [104], although the nature of core-forming block's propensity to crystallize has been identified as an important factor [107]. In the case of mPEG conjugated with amorphous poly(caprolactone-b-D,L-lactide) copolymer, typically spherical micelles were formed, whereas upon conjugating a crystalline poly(caprolactone-b-L-lactide) segment, rod-like micelles were observed [107]. These findings point out that our understanding of the forces governing the solution state self-assembly process is still in the preliminary stages. Under pure aqueous conditions, very first block copolymers studied extensively for their formation of elongated micelles were based upon poly(ethylene oxide)-b-poly(butadiene) (PEO-b-PB) and its hydrogenated counterpart, poly(ethylene oxide)-b-poly(ethylethylene) (PEOb-PEE) [98]. For such PEO-based diblock copolymers, around 45–55 weight fraction of PEO, has been shown to result in the formation of elongated micelles [71]. The double bonds in the PEO-b-PB reside in the micellar core, where they offer the unique possibility of cross-linking. Such core-crosslinked nanostructures of high aspect ratio served as an excellent stiffness-tunable material [98,110]. Even though these preliminary designs lacked the desirable degradable components, they served as a model system to evaluate the fundamental aspects [110– 112]. For instance, it was recognized that the flexible worm-like micelles can be elongated under flow conditions and their flexibility also enabled them to penetrate into nanoscale pores [111]. In addition, targeted delivery, using biological signaling molecule [112] was feasible with such elongated micelles. Enhancement in the loading content of griseofulvin has been observed by Attwood and colleagues for worm-like micelles with poly(oxyalkylene)s block copolymers [113]. The specific chemical compositions of the hydrophobic block were found to have profound influence on the solubilization of the drugs. Compared to the elongated micelles with cores formed from poly(butylene oxide), poly(styrene oxide) hydrophobic blocks demonstrated four-fold higher drug solubilization capabilities [113].

Fig. 4. Sequestration of nitric oxide (NO) and micellization of water soluble block copolymer occurs at high pressure due to the transformation of hydrophilic poly(N-acryloyl-2,5dimethylpiperazine) block to hydrophobic poly(sodium 1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate)] block. Under physiological conditions, controlled release of the sequestered NO occurs over weeks from the core of the micelles. Upon release of NO, the block copolymers, revert back to their water soluble double hydrophilic configuration [94].Copyright (2010) American Chemical Society. Reproduced with permission.

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Realizing the potential of elongated micelles for drug delivery applications, biodegradable blocks such as poly(caprolactone) (PCL) were designed to replace the bio-inert poly(butadiene) or poly (ethylethylene) blocks [99]. This change allowed for the hydrolytic degradability while retaining the morphology and flexibility. Based on the molecular weight, the degradation kinetics of the caprolactone segments of the PEO-b-PCL block copolymer was found to be tunable from a day to over a week. Interestingly the degradation occurred via chain end cleavage to result in 6-hydroxycaproic acid, the constituent monomer at predictable rates. Since the worm-like micelles exist only at very narrow range of amphiphilicity, such degradation-induced increase in hydrophilicity favors worm-to-sphere transition, with release of spherical micelles from the ends of the worm-like micelles. The degradation rates were also shown to be modulated with temperature and pH of the solution [114]. Paclitaxel was successfully loaded into the worm micelles. From the comparison of paclitaxel loading into spherical and elongated micelles (prepared essentially from the same block polymer), it was clear that the latter resulted in much superior loading content (almost twice, in most instances) [114]. In fact, in some of the recent reports from Discher's group demonstrated unprecedented in vivo performance with circulation half-lives of around 5 days for elongated micelles of about 18 μm length [3,6]. These findings clearly point out that not only in terms of enhanced therapeutic payload, but owing to their unique degradation pathway and flexibility, elongated micelles are also emerging as an indispensible carrier option. The studies dealing with the in vivo behavior and the pharmacokinetics of these worm-micelles will be discussed in detail in Section 4. Conceptually, similar to shell-crosslinked spherical particles [84], Wooley's group reported methods for the preparation of elongated micelles of different lengths [115]. Both the block composition and the processing conditions were important factors to control the dimensions of elongated micelles. The short rod-like micelles of about 20 nm in diameter and 180 ± 120 nm in length were prepared via sonication of aqueous solution of poly(acrylic acid)-b-poly(styrene) and subsequent carbodiimide-mediated crosslinking of the acrylic acid residues residing in the shell regions using diamine-based crosslinker. Longer rod-like micelles were formed from a triblock copolymer, poly (acrylic acid)-b-poly(methyl acrylate)-b-poly(styrene) (PAA-b-PMAb-PS), in the presence of diamine crosslinker in water-THF mixture. The diamine crosslinker allowed for the ionic interactions with the PAA shell, and the presence of organic solvent plasticized the PS-core. By using these optimal conditions, the desired morphology (elongated micelles) was kinetically accessed, and the micelles were subsequently stabilized by the covalent crosslinking of the shell region via carbodiimide-based chemistry. Eventually the organic solvent was removed by extensive dialysis against water. The lengths of these micelles were about 970 ± 900 nm with a diameter of 30 nm. By this approach, many of the ‘exotic’ morphologies, which are exclusive to organic solvent-water mixtures, can be effectively formed in aqueous environments [73,116]. The elongated micelles prepared, were labeled with folate as a targeting moiety [117] and cell-penetrating peptides [115], and the shape dependence of cellular internalization of these materials was evaluated, which will be discussed in Section 4. Sophisticated synthetic polymer designs have provided access to complex solution state morphologies. ABC miktoarm star polymers, wherein poly(ethylene oxide), poly(ethylethylene) and poly(perfluoropreopylene oxide), three mutually immiscible blocks are joined together, at specific composition, results in multi-compartment nanostructures including elongated micelles in water [76,118]. There have since been numerous efforts in understanding [67,75] the self-assembly of these complex macromolecules and also on the development of miktoarm star polymers with biodegradable blocks [119–121]. Recently, elongated micelles with multi-compartment phase-separated cores, perpendicular to the cylinder axis have been prepared via kinetic manipulation of two different triblock co-

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polymers with comparable molecular weights and immiscible coreforming block composition [72]. These micelles provided a unique approach to encapsulate or carry two kinds of payloads in different compartments [122]. Even though some of these new developments in cylindrical micelles are not readily applicable in drug delivery applications due to the concern of non-biodegradability, their design principles may be employed to develop biodegradable nanostructures with similar features. 3.1.3. Vesicles Vesicles, spherical structures formed by bilayers are different from the micelles with respect to the enclosed aqueous compartment. Vesicular structures constitute the very essence of life. Routinely, in a programmed fashion a variety of components are actively compartmentalized into vesicles, transported and delivered with spatial and temporal resolution [41]. It is remarkable to note that in biological systems, vesicles are formed and deformed at will. Compared to nature, such a level of sophisticated synchronization and coordinated manipulation of various components in the synthetic materials, to date remains a distant dream. It has been recognized that the vesicles in nature have evolved for the membrane fluidity. This engineering of membrane fluidity is crucial for the biological processes involving budding out and fusion of vesicles, such as endocytosis, exocytosis, cell division, etc. Lack of membrane fluidity would impede the requisite dynamism. In contrast to the biological processes, in the drug delivery applications more than membrane flexibility, their stability plays an important role. Only the programmed instability (for example to facilitate the release of payload) is desired. Accelerated developments in polymeric vesicles are primarily due to the well established liposome-based technologies that have laid a strong foundation in terms of all the necessary characterization techniques [123]. Some of the early FDA approved liposomes-based drug delivery systems that hit the market in 90s also demonstrated the advantages of encapsulation, thereby installing lots of confidence and conviction to pursue further research and development in this direction. Access to vesicles of macromolecular origin has opened up innumerable possibilities in nanomedicine, primarily due to flexibility in synthetic design of macromolecules [124]. Owing to their higher molecular weight, compared to small molecular amphiphiles (e.g. lipids, ≤1 kDa), block copolymer-based macromolecular amphiphiles offer the essential stability, toughness and versatile functionalities for drug delivery. The first polymeric vesicles were reported by Eisenberg's group, which were based on poly(acrylic acid)-b-poly(styrene) in organic solvent–water mixtures so that the glassy poly(styrene) core can be plasticized enough to obtain the desired morphology. Subsequently, developments have been focused on the biocompatible and/or biodegradable materials that often contain low Tg hydrophobic blocks in pure aqueous conditions [124–129]. Compared to lipid-based amphiphiles, with the block copolymers, the thickness of hydrophobic membrane can be engineered [126,127]. Such tunable membrane thickness imparts tunable stability, toughness and permeability [66,130]. Now, there is a rich and diverse array of block copolymers and other complex macromolecules reported to form vesicles, and these developments have been summarized from time to time in several reviews [66,131–134]. In this section, we will briefly highlight some of the key milestones and the most recent developments in the preparation of polymeric vesicles as drug delivery carriers. Unlike the nanostructures discussed in the earlier sections, vesicles have the unique capability to encapsulate both the hydrophilic components in the aqueous compartment and the hydrophobic components within the membrane, hence offering unprecedented flexibility of being able to encapsulate distinct classes of compounds in the same nanostructure. In a recent report, Chen et al., reported the development of pH-sensitive degradable polymeric vesicles from a block copolymer consisting of PEG and acid-sensitive poly(2,4,4-

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trimethoxybenzylidenepentaerythritol carbonate) (PTMBPEC) segments [135]. Encapsulation of both hydrophobic paclitaxel and hydrophilic doxorubicin hydrochloride anticancer drugs were accomplished in the polymeric vesicles. Likewise, the release of both the encapsulated drugs occurred in a pH-responsive fashion. It is not surprising that in the micellar system resulting from similar block copolymers, only the hydrophobic drug was encapsulated. This comparative study is an excellent example of the potential of polymeric vesicles in the concurrent delivery of multiple drugs. By incorporating the dual drug combination into a polymersome with degradable poly(lactide) as part of the hydrophobic membrane, Ahmed et al. demonstrated that both the drugs can be released and accumulated in the tumor site to induce significantly higher apoptosis and consequently tumor shrinkage than the free drug combination [136]. With astute selection of therapeutics, challenging combinations or drugs that can act via different mechanistic pathways can be delivered simultaneously using a single formulation as well [135,136]. Such combination of drug delivery imparts synergistic effects and improved patience compliance [137]. There has always been a need for the development of effective drug loading methodologies as they directly impact the drug loading content, efficiency of encapsulation, and the important parameters that influence the effective dosage and cost. A simple nanoprecipitation method involving basic conditions (pH = 10.5) has been developed by Sanson et al. for the encapsulation of doxorubicin into poly(trimethylene carbonate)-b-poly(L-glutamic acid)-based block copolymers for high drug loading (~47 wt.%) with acceptable efficiency (~50–70%) [138]. For proteins and other potentially expensive therapeutics, there is a tremendous advantage to focus on improving the encapsulation efficiency. Zhang et al. reported a unique two-phase method via mixing concentrated solutions of poly(ethylene glycol)-b-poly (caprolactone) (PEG-b-PCL) and dextran-b-poly (caprolactone) (DEX-b-PCL) to result in polymersomes that encapsulated proteins with high efficiency (~90%) [139]. In their approach, PEGylated asymmetric bilayer vesicles were prepared with PCL forming the hydrophobic membrane and dextran forming the inner layer (Fig. 5). The dextran-lined interior facilitated loading of erythropoietin with well-preserved bioactivity presumably due to the thermodynamically favored partition. In a different approach, asymmetric vesicles were prepared by using a triblock copolymer poly(ethylene glycol)-b-poly(caprolactone)-b-poly (2-(diethylamino) ethyl methacrylate) (PEG-b-PCL-b-PDEA), which was synthesized by the combination of ROP and RAFT polymerization strategies [140]. The block sequence and their relative sizes were designed such that the PDEA would form the interior leaf of the vesicles, to allow for the enhanced protein encapsulation and assist in the release of the encapsulated proteins into the cytosol due to the ‘proton sponge effect’. A variety of proteins, including bovine serum albumin (BSA), cytochrome C, lysozyme, ovalbumin and immunoglobin G (IgG) were

efficiently encapsulated by these triblock copolymer vesicles. Depending upon the protein and feed ratio, the encapsulation efficiencies were between 53 and 97% with loading content varying from 10 to 56 wt%. By using this system, it was also possible to encapsulate doxorubicin together with the proteins. Clearly, the systems having the enhanced drug loading capability along with the ability to deliver (simultaneously) distinct classes of therapeutics are promising for therapeutic applications. For the encapsulation of sensitive hydrophilic therapeutics such as siRNA, vesicular membrane offers excellent protection from the harsh in vivo conditions (e.g., nucleases) [141], but at the same time if required, the membranes could be designed to selectively allow for the diffusion of small molecules [130]. However, the release of the macromolecular hydrophilic therapeutics from the aqueous interior, might be a challenge because of the low diffusivity, due to the thicker (in comparison to liposomes) hydrophobic membrane. Partly, this issue has also accelerated the development of numerous stimuliresponsive release strategies, which were discussed in recent excellent review articles [142–144]. Judicious incorporation of “smart” drug release mechanisms can greatly influence the therapeutic efficacy. With the incorporation of targeting moieties in the shell region of the vesicles, therapeutic efficacy can be dramatically increased [145]. For instance, vesicles coupled with mouse-anti-rat antibody targeting the receptors linked to the blood–brain barrier, were found to be effective. Encapsulation of peptides was also accomplished in these vesicles. In vivo delivery of the peptides to brain was feasible, and the delivered peptides improved the learning and memory impairments [146]. Concurrent developments in molecular biology can provide knowledge on novel targets for complex diseases. Successful integration of such information, onto the drug-delivery vehicles, would pave way for the development of novel therapeutic formulations. 3.2. Well-defined macromolecules Apart from self-assembly of macromolecules into well-defined nanostructures, tailored control of polymer architectures has also been in continuous and parallel development among the “bottom-up” strategies in recent decades. It is well understood that the polymers with identical chemical compositions but different architecture/topology may show distinctive physical and functional properties. Thus, tremendous effort has been devoted towards the development of precisely controlled polymer architecture and topology such as dendrimers, brush-block copolymers [147–153], dendronized polymers [154–159], to name a few. Among the present emerging polymers of various architectures and topologies, dendrimers not only have the most defined nanostructure constructs but also have been extensively studied in detail for their in vitro and in vivo applications. Hence in this section, briefly we will highlight some of the key developments in dendrimers.

Fig. 5. Phase-guided assembly of polymersomes with asymmetric bilayer membrane. A. Schematic representation of formation of polymersomes by the addition of block copolymers PEG-b-PCL and PCL-b-DEX into two-phase aqueous solution containing PEG as the continuous phase and dextran as the dispersed phase. By this approach, biomolecules are partitioned selectively into the interior of the vesicles. B. Photographs of mixture of PEG and dextran solutions, allowed to stand for 60 min with (i) no block copolymers, (ii) PEG-bPCL, (iii) PCL-b-DEX and (iv) both block copolymers added. Without the two block copolymers, solutions phase-separate and no polymersomes are formed [139]. Copyright (2010) Elsevier. Reproduced with permission.

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Dendrimers are large complex molecules with well defined and symmetrically arranged core-shell structures, which leads to an almost monodisperse three-dimentional spherical and globular shape. They possess three distinct architectural components: (a) an initiator core, (b) an interior layer (generations) consisting of repeating units, radially attached to the initiator core, and (c) terminal functional groups [160–166]. Dendrimers are generally produced in an iterative sequence of reactions. Each step, called a generation, adds a distinct layer to the previous one. Dendrimers are usually synthesized through stepwise chemical approaches by either divergent or convergent branching methods, which differ in their directions of synthesis. Divergent method, where the dendrimer is grown outward from the initiator core, was pioneered by Tomalia and Newkome's groups [167–169]; on the other hand, Fréchet and coworkers developed the convergent method, in which the dendrimer is synthesized from the periphery inward, terminating at the core [170]. The divergent method is currently the preferred commercial approach because the molecular weight of the dendrimers nearly doubles with the increase of each layer (generation) in each reaction step, and so does the number of end groups (or active sites). This method is indispensable for synthesis of dendrimers with many generations. However, minor defects may occur for higher generations as congestion-induced De Gennes dense packing begins to take effect [162]. Moreover, most divergent synthesis methods require excessive monomer, and the product obtained is difficult to purify and lengthy chromatographic separation is necessary, particularly at higher generations, because they are structurally similar to the side products [166,171]. Distinct from the divergent method, the convergent method can produce the most monodisperse dendrimers as this method allows for purification at each step of the synthesis and eliminates cumulative effects due to failed coupling [162,172]. Other advantages include: 1) the molecular weight of dendrimers synthesized through the convergent method can be precisely controlled; 2) the surface functionalities of the dendrimers can be made in precise positions and numbers, and 3) dendrimers can be synthesized in a rapid and relatively cheap way [171]. A significant disadvantage of this method is the difficulty to construct high generation dendrimers due to the presence of steric hindrance when attaching dendrons to the molecular core. Also, this synthesis method has been reported to suffer from low yields [166,171]. In both divergent and convergent methods, the key topological feature, i.e. the fractal structure of the dendrimers, is encoded in the monomers. The requirements of the special design of the monomers, and the tedious, expensive and time-consuming multistep synthetic processes limit the synthesis of novel dendritic polymers. Recently many synthetic approaches including double-stage [173], double exponential [174], orthogonal coupling strategies [175], Lego chemistry [176], click chemistry [177], and self-assembly methodologies [178], etc. have been developed to simplify synthetic procedures or as alternative or complementary approaches. For example, Hawker and coworkers developed a highly orthogonal and time efficient protocol, which yielded a 6th generation dendrimer in a single day. The synthesis of the dendrimer started from the core containing one alkyne and two alkene groups, and the following thiol-ene coupling reaction and copper catalyzed azide alkyne cycloaddition (CuAAC) reaction made the purification process of products facile and straightforward [5]. Fréchet's group also developed a rapid approach for preparation of heterobifunctional biodegradable dendrimers that were derived from 2,2-bis (hydroxymethyl) propanoic acid bearing a cyclic carbonate periphery, and functional amines via ring-opening reaction. This approach is efficient and scalable since no chromatographic purification steps are involved [4]. Over the last two decades, many dendrimers have been developed, including polyamidoamine (PAMAM), poly(propylene imine) (PPI), poly(glycerol-co-succinic acid), poly(L-lysine) (PLL), poly(glycerol),

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poly(2,2-bis(hydroxymethyl)propionic acid), and melamine, etc. Commonly studied dendrimers can be classified as PAMAM, polyamine, polyamide, poly(aryl ethers), polyester, carbohydrates and DNA dendrimers [179–181]. Polyester dendrimers are biodegradable, and their hydrolysis rates can vary dramatically depending on the hydrophobicity of the monomer, steric environment, and the reactivity of functional groups located within the dendrimers [182]. In contrast, polyamine and polyamide dendrimers do not degrade as easily in the body, and thus they may be more prone to long-term accumulation in vivo [183,184]. Unique properties of dendrimers including nearly monodisperse and predictable nanoscale dimensions, surface and interior functionalities make them interesting targets for biomedical applications. For example, the terminal surface groups are ideal for bioconjugation of drugs, biological signals, targeting moieties and other biocompatible groups [185,186]. However, the well-defined interior void space within the dendrimer can be used to encapsulate small molecular drugs, metals, and imaging agents. Encapsulation in the internal void space reduces drug toxicity to healthy tissues/cells and facilitates controlled drug release [185–188]. Hence, dendrimers have attracted significant attention as potential drug delivery carriers, where drugs can be either non-covalently encapsulated in the void space or conjugated to the surface to form macromolecular prodrugs. A number of dendrimers have been reported for drug delivery. Since there have been several excellent review articles describing dendrimersbased drug delivery systems [166,187,189], we will focus only on some of the important recent findings. Ideally, dendrimers as drug delivery carriers should exhibit high water-solubility and drug-loading capacity, biodegradability, low toxicity, favorable retention and biodistribution characteristics, specificity, and appropriate bioavailability [179,190]. PAMAM is one of the most commonly reported dendrimers based on its terminal-modifiable amines and tertiary amines and amide linkages, which allow for the binding of numerous targeting and guest molecules. Hence, it has been extensively reported for drug and gene delivery [166,187]. For example, paclitaxel (PTX) was incorporated into PAMAM dendrimers through chemical conjugation or physical incorporation. After the incorporation, the water-solubility of PTX was increased up to 109-fold, and the cytotoxicity of PTX-PAMAM complexes was enhanced in PC-3M human prostate cancer cells due to increased water-solubility and cellular uptake [191]. In addition, PTX was covalently attached to the surface functional groups of PAMAM dendrimer, and the resulting PTXPAMAM conjugate showed significantly increased water-solubility. As compared to free PTX, the cytotoxicity of the dendrimer-conjugated PTX was increased by 10-fold against A2780 human ovarian carcinoma cells [192]. The multiple surface functional groups of dendrimers enable simultaneous incorporation of drugs and biological ligands for active targeting. A number of targeting signals such as folic acid [193], monoclonal antibodies [194], saccharides [195] and peptides [196] have been successfully introduced onto the periphery of dendrimers. One of the key advantages that arise from the dendritic architecture in the context of drug delivery is having access to the spatial-control over the bioactive-ligand presentation such that the targeting capability of the carrier could be optimized. Recently, Hammond's team prepared “patchy” nanoparticles by using poly(benzyl-L-aspartic acid)-b-PEGylated polyester dendron, wherein the carboxylic acid residues at the ω-end of PEG chains were functionalized with controlled amounts of folate functionalities, and by mixing these functional polymers with their counterparts without functionalization (Fig. 6A) [197]. By this approach, micelles containing about 1.5 to 16.5 folate groups per stochastic cluster but with comparable overall number of folate groups presented on the micellar surface (~2000– 2400 groups per micelle) have been prepared (for instance, by using a 60% of 20% folate functionalized polymer along with the nonfunctionalized polymer resulted in 20%F–60% mix sample). Different patterns of ligand-clustering were shown to have different performance in

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Fig. 6. Role of ligand clustering in targeting capability of functional, linear dendritic polymers. (A) Chemical structure of poly(benzyl-L-aspartic acid)-b-PEGylated polyester dendron. Ligands can be readily conjugated onto the carboxylic acid functionalities presented on the ω-end of PEG chains and the extent of folate functionalization can also be readily varied. By judicious mixing of different proportion of unfunctional and folate-functionalized polymers, self-assembled nanostructures containing different patterns of folate clusters and yet presenting statistically similar number of folate groups can be obtained. (B) Normalized tumor fluorescence (VivoTag680(VT680)/AngioSense750(AS750)) of tumors for different formulations, presenting equivalent amounts of folate ligands but in different cluster patterns were evaluated on nude mice bearing two different tumors (KB, folate receptor+ and A375, folate receptor−), demonstrate highest in vivo targeting with 20%F–60%mix micelles. These results indicate that the targeting has been achieved for the KB tumors and also the enhancement in the targeting was dependent on the specific ligand clustering [197]. Copyright (2010) Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

cellular uptake from the in vitro experiments. In vivo evaluation of these nanoparticles in nude mice bearing two different tumors (KB, folate receptor+ and A375, folate receptor−) demonstrated that the specific nature of ligand-clustering was an important parameter in targeting. Optimal targeting was achieved with a folate cluster size of about 3 (20%F–60% mix) (Fig. 6B). This work also clearly demonstrates the potential and importance of tunable dendritic design in the development of drug carriers [197]. In addition to drug delivery, amino-terminated PAMAM and polypropyleneimine (PPI) dendrimers have often been used for gene delivery because of their high affinity to negatively charged genes [198–201]. Similar to other polycationic compounds, PAMAM or PPI dendrimers form complexes with DNA through electrostatic interactions between the negatively charged DNA and the protonated primary amino groups on the dendrimer surface. Apart from the primary amino groups on the dendrimer surface, tertiary amino groups that exist at the branching points of the dendrimer interior provide a buffering capacity for endosomal release of the DNA or dendrimer/DNA complexes [188]. It should be noted that partially degraded dendrimers resulted in higher gene expression than intact symmetrical dendrimers probably due to their flexibility, which allows for the formation of more compact DNA complexes [172,188]. Various surface modifications and introductions of targeting moieties have been performed with cationic dendrimers to improve gene transfection efficiency [190]. Moreover, triazine dendrimers have been used for siRNA delivery [202–204]. It was found that both surface functional groups and dendrimer scaffold manipulations significantly impact dendrimer-mediated siRNA transfection efficiency, and increasing the rigidity of the dendrimer core resulted in increased gene knockdown than the flexible analogs. This finding is different from that with DNA delivery probably because siRNA of smaller size and more rigid structure may require more rigid cationic molecules to condense into nanoparticles. Even though we have greatly focused on the dendritic-based macromolecules in this section, strategies to develop well defined macromolecules via sophisticated synthetic tool box, are practically limited only by our imagination. Recent elegant work by Percec and coworkers on the self-assembly of library of Janus dendrimers is an apt example, demonstrating that judicious design of well-defined macromolecules such as dendrimers, will not only allow for the engineering of the self-assembly behaviors of these materials but could also potentially impact the development of sophisticated drug delivery vehicles [205]. With unique possibility of having chemo- and spatio-selective func-

tionalities, these materials alone or as a component in molecular design for self-assembly, have been demonstrated to be crucial in niche problems involving targeting ligands [206]. Tremendous progress has been made in the last decade in the development of efficient orthogonal chemistries, enabling the transformation of the complex architectures as a subject of mere academic interest to one with practical relevance and importance [63]. 4. Influence of shape on biological processes Although extensive work using spherical particles has yielded many valuable insights on the contributions of particle parameters such as size and surface chemistries to biological processes, information on the biological influence exerted by particle shape are relatively lacking [207]. In recent years, emerging evidence from a limited number of in vivo studies comparing shape effects of synthetic structures (Table 1) have highlighted discrepancies in biological behaviors between conventional spherical particles and their non-spherical counterparts. Therefore, in this section, the effects of the shape of bioengineered structures on important biological processes such as biodistribution, cellular internalization and toxicity will be discussed. Due to the comparative lack of biological data for biodegradable nano-sized polymeric carriers, data for various types of micro-sized carriers, including liposomes and silica particles, will also be discussed. These findings are nevertheless expected to provide important insights on the shape requirements for different pharmacokinetic or cellular functions and could thus have important ramifications on the design of biodegradable and/or biocompatible nanoparticles for future drug delivery and imaging applications. 4.1. Biodistribution Carriers with shapes deviating from the conventional spherical forms have been identified to possess distinct pharmacokinetic properties which may be more favorable towards the therapeutic intent. The potential therapeutic benefits of using non-spherical drug delivery systems were clearly illustrated through the work of Discher and coworkers [3]. In the study, persistent circulation of filamentous micelles (filomicelles) formed by a solvent evaporation self-assembly process using diblock copolymers of PEG and the inert poly (ethylethylene) or biodegradable poly(ε-caprolactone) was observed for up to a week, which was in stark contrast with the spherical PEGylated stealth vesicles that were cleared within 2 days.

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Table 1 In vivo studies comparing shape effects of bioengineered particles. Particle composition

Cargo

Dimensions

Shapes

In vivo effects

Ref.

PEG-b-poly(ethylethylene) or PEG-b-poly(caprolactone) micelles

Paclitaxel

d = 22–60 nm

Filaments vs. spheres

Compared to spherical particles, filomicelles:

[3,6]

l = 2–18 μm (biodistribution); 1 or 8 μm (anti-tumor)

Poly(methylvinylether-co-maleic Doxycycline anhydride) and various lipids hydrochloride

Silicon particles

–

ICAM-1-coated polystyrene

–

Dextran-coated iron oxide nanoparticles

–

• Displayed prolonged blood circulation • Showed enhanced and sustained anti-tumor effects • Increased the MTD and reduced apoptosis in non-tumor organs d = ~ 250–450 nm Regular spheres vs. Preferential accumulation of irregularly shaped irregular shaped nanoparticles in the sinusoidal spleens of rats, dog and rabbits; spheres distributed mainly to the liver Quasi-hemisphere: d = 1.6 μm Quasi-hemisphere vs. Shape-dependent accumulation observed in Disk: d = 1.6 μm, h = 0.3 μm major organs: disk vs. cylinder vs. Cylinder: d = 1 μm, h ≈ 1 μm sphere • Lungs: diskN sphere N cylinder, quasi-hemisphere Sphere: d = 1 μm • Liver: cylinderN sphere, quasi-hemisphereN disk • Heart: disk N sphere, quasi-hemisphere, cylinder • Spleen: disk, quasi-hemisphere N cylinder, sphere Disk: 0.1 × 1 × 3 μm Disk vs. spheres Compared to spherical particles, disk shaped Sphere: d = 0.1–10 μm particles: • Possessed longer blood circulation time • Had higher pulmonary anti-ICAM to IgG ISI Nanoworms: l = 50–80 nm Worm-shaped vs. Nanoworms had significantly higher tumor uptake sphere than spheres Spheres: d = 25–35 nm

[208,212]

[217]

[209]

[210,211]

d: diameter; l: length; h: height; MTD: maximum tolerated dose; ICAM-1: intracellular adhesion molecule 1; IgG: immunoglobulin G; ISI: immunospecificity index.

Interestingly, longer filomicelles corresponded to prolonged circulation times up to an initial length of ~ 8 μm, which is approximately equivalent to the diameter of red blood cells. This feature subsequently translated into significantly greater tumor shrinkage when the drug-loaded filomicelle length was increased from 1 to 8 μm at the same paclitaxel dose (1 mg/kg) in A549 tumor-bearing mice. The superior anti-tumor effects of the paclitaxel-loaded filomicelles were further elaborated in a subsequent study in which the filomicelles were found to accumulate in the xenografted tumor, leading to sustained inhibition of tumor growth while inducing significantly less apoptosis in non-tumor organs of nude mice compared to paclitaxelloaded spherical micelles of the same composition [6]. Apart from prolonging blood circulation times in some instances, non-spherical particles have also been found to display different in vivo distribution profiles from their spherical counterparts, hence providing a means for targeting to specific organs or tissues such as the spleen [208], lungs [209] and tumor tissues [6,210,211]. For instance, the enhanced anti-tumor effects with the long circulating filomicelles described above was associated with enhanced tumor accumulation of paclitaxel delivered by filomicelles [6], presumably via the wellestablished EPR effect. Likewise, worm-like iron oxide nanoparticles also exhibited greater and more prolonged tumor accumulation than nanospheres [210,211]. Additionally, Devarajan et al. demonstrated that irregularly shaped poly(methylvinylether-co-maleic anhydride)/lipid nanoparticles (LIPOMERs) exhibited higher splenic accumulation in rats, rabbit and dog [208,212] and were more effective at evading nonspecific uptake by macrophages than spherical LIPOMERs [208], suggesting potential for the carrier to be used for drug delivery to the spleen. Through extensive investigations comparing the intravascular behaviors of spherical and various non-spherically shaped silica particles fabricated using the top-down standard microlithography approach in combination with wet-etching and/or reactive ion etching techniques, Decuzzi and colleagues have provided some insights on the distinct biodistribution profiles observed with non-spherical particles [213]. Using theoretical models and in vitro flow chamber experiments, the authors have shown that discoidal particles exhibit a greater degree of lateral drifting towards blood vessel walls [214,215] and can potentially adhere more strongly to the endothelial cell wall via multivalent bonding as a function of particle aspect ratios (ARs)

[214,216] in comparison with quasi-hemispherical and spherical particles under flow conditions. In their subsequent biodistribution study, uncoated discoidal silica particles were found to be preferentially distributed to the lungs, heart and spleen, with markedly reduced accumulation in the liver, compared to spherical, quasi-hemispherical and cylindrical particles of similar volumes in tumor bearing mice (Table 1) [217]. The authors thus speculated that the reduced propensity for the phagocytic uptake of elongated particles as described elsewhere [2,218] (also to be discussed in the next section), reduced the amount sequestered by Kupffer cells in the liver, and permited the accumulation of a larger amount of discoidal particles near the vessel walls where they eventually exited the larger blood vessels to gain entry into various organs [217]. These results were further corroborated with important findings from Muro et al. [209]. In the study, anti-intercellular adhesion molecule 1 (ICAM-1)-coated polystyrene elliptical disks (0.1 × 1 × 3 μm), which were prepared from 2 μm diameter polystyrene spheres using the top-down stretching technique described in Section 2, when compared with the corresponding 0.1 μm spheres, displayed longer circulation blood times, distributed less to the liver and were specifically targeted to the lungs where they were internalized by pulmonary endothelial cells via cell adhesion molecule-mediated endocytosis [209]. Taken together, these findings strongly suggest that elongated particles possess an inherent advantage over their spherical counterparts for drug delivery applications that require prolonged blood circulation and/or distribution to specific organs or tumor sites. Szoka et al. have proposed that the entry and passage of polymeric drug carriers with approximately the same hydrodynamic volumes through pores are greatly influenced by the particle shape and degree of flexibility conferred by various molecular architectures (Fig. 7) [219]. As demonstrated through the authors' research, this hypothesis has significant implications on the rate of glomerular filtration or renal clearance, thereby influencing the blood circulation time and biodistribution of the polymers. For instance, linear polymers adopt loose random coil conformations in solution and are expected to readily enter and reptate through glomerular pores via one end of the chain. Cyclic polymers, on the other hand, are required to deform to enter and pass through pores due to the lack of chain ends, making the process more difficult. Recent studies comparing linear and cyclic forms of PEGylated poly(ε-caprolactone) [220] and PEGylated poly (acrylic acid) [221] with molecular weights above the renal threshold

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structure through the pores in the glomerular wall [224]. The enhanced blood circulation time and bioavailability of the higher generation polymers gave rise to high tumor accumulation. The applicability of the polyester dendrimer-PEO hybrid for drug delivery was evidenced in a subsequent study, in which the tumor concentration of doxorubicin delivered by the carrier was 9-fold higher than the free drug following a single dose [7]. The contributions of particle shape and mechanical stiffness associated with different molecular architectures to important pharmacokinetic parameters have definitely provided added imperative for researchers working with nanostructures at the molecular level to explore alternative polymer topologies such as cyclic or dendronized polymers to improve drug delivery outcomes. 4.2. Internalization

Fig. 7. Possible molecular conformations of polymers or dendrimers in solution and the effects of particle shape and flexibility on pore penetration. (A) Loose random coil conformation of a linear polymer facilitates entry and reptation through a pore. (B) A polymer or dendrimer with rigid globular conformation has to undergo substantial deformation to enter and pass through a pore, making the process challenging. (C) A cyclic polymer lacks a chain end; hence passage through a pore requires deformation. (D) A rigid rod-like extended linear polymer readily enters and passes through the pore. (E) Star-like or hyperbranched flexible polymers are more likely to enter a pore via one chain-end (top), however, passage through the pore is sterically hindered due to the need for deformation of the remaining arms; entry of the polymer via multiple chain ends (bottom) is less likely, although the more symmetrical conformation favors passage through the pore. Reproduced with permission from ref. [219]. Copyright (2009) Elsevier.

of 30–40 kDa have positively illustrated the drastically reduced renal elimination of cyclic polymers, giving rise to longer elimination halflives and greater bioavailability than the linear polymers with similar molecular weights. This eventually culminated in the enhanced tumor accumulation of the water soluble cyclic PEGylated poly(acrylic acid) comb polymers over its corresponding linear counterparts in C26 colon carcinoma-bearing BALB/c mice [221]. Inspired by previous imaging studies demonstrating higher tumor penetration of helical secondary structures possessing extended linear conformations of gadolinium-diethylenetriamine penta-acetic acid-poly(lysine) over predominantly coiled or globular constructs [222,223], the authors subsequently fabricated rigid rod-like dendronized linear polymers from poly(4-hydroxystyrene) backbones bearing fourth generation polyester dendrons at each repeat unit and showed that blood circulation times correlated with an increase in the molecular weight, giving rise to high tumor accumulations [155]. Apart from the molecular conformation in solution, the degree of branching of polymeric carriers has also been found to significantly alter the blood circulation time and degree of tumor accumulation [224]. For instance, a study evaluating biodegradable hybrid structures of two covalently attached polyester dendrons with poly(ethylene oxide) (PEO) moieties selectively coupled to one dendron showed that the higher generation number of the PEO conjugated dendron, and hence the greater degree of branching, results in lower renal excretion, which is likely due to decreased flexibility and deformability of the hybrid

Besides dramatically affecting biodistribution, shape also plays an important role in the cellular internalization of macromolecules via endocytosis. Important endocytic pathways relevant in the cellular transport of macromolecules include phagocytosis, macropinocytosis, clarthrin-mediated endocytosis, caveolae-mediated endocytosis, and clarthrin- and caveolae-independent endocytosis; of which the pathway employed is largely dependent on the size of the endocytic vesicle, nature of the cargo and the mechanism of vesicle formation [225]. Shape effects on cellular internalization is a pertinent theme in drug delivery as therapeutic-carrying nanoparticles, particularly those loaded with DNA or siRNA, require internalization into cells where they release their cargoes for activity. Also, in many applications, it may be desirable for nanoparticles to evade phagocytosis by macrophages of the reticuloendothelial system (RES) so as to augment the concentration of particles available for uptake by diseased mammalian cells. For the treatment of various inflammatory diseases as well as bacterial and viral infections, however, phagocytosis of the drug-loaded carriers by macrophages is preferred [226]. In view of these considerations, we will examine the impact of particle shapes on phagocytosis and the other modes of endocytosis in this section. 4.2.1. Phagocytosis As highlighted in Section 2, the successful stretching of polystyrene spheres into particles with a wide array of geometries has enabled Champion and Mitragotri [2] to perform their pioneering work, which was instrumental in demonstrating that the local particle shape from a macrophage's perspective is the major deciding factor on whether phagocytosis or simply spreading will occur. By observing the interaction of diversely shaped micro-sized polystyrene particles, including spheres, oblate and prolate ellipsoids, elliptical and rectangular disks as well as UFOs, with macrophages over time, a dimensionless shape-dependent parameter related to the length normalized curvature, Ω, was defined (see diagram in Fig. 8A). The predisposition to phagocytosis and the internalization velocity of the particles as a function of Ω was also reported (graph in Fig. 8A). In essence, particles were found to be internalized successfully when Ω ≤ 45° via actin-cup and ring formation, with phagocytosis velocity being inversely correlated to Ω (up to 45°); on the other hand, when Ω N 45°, cell spreading, but not internalization occurs (Fig. 8A and B). In contrast, the contribution of particle size or volume to the phagocytotic process was evidently lower compared to particle shape, affecting the completion of particle internalization only when the particle volume is greater than that of the macrophage at Ω of ≤45°. Based on the understanding of the local particle shape on the internalization propensity of macrophages, the authors subsequently fabricated high aspect ratio particles in the form of worm-like polysytrene particles and showed that they were phagocytosed to a significantly less extent by alveolar rat macrophages than spherical particles of equal volumes [218]. The success of the high aspect ratio particles in avoiding phagocytosis was attributed to the

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– Fig. 8. Effects of local particle shape on phagocytosis. (A) Definition of Ω (diagram) and its relationship with internalization velocity (graph). T represents the average of tangential – – angles near the point of cell contact. Ω is the angle between T and the membrane normal at the site of attachment, N. (B) Scanning electron micrographs A–C show cells and particles colored brown and purple, respectively. Images D–F are overlays of brightfield and fluorescent images after staining fixed cells for polymerized actin using rhodamine phalloidin. A and C: The membrane has progressed over half the ellipsoid disk and sphere, respectively. B: The macrophage has spread over the flat side of an ellipsoid disk. D and F: Actin ring and cup, respectively, were formed as internalization proceeded. E: Actin polymerization occurred at the site of attachment to the flat side of an opsonized disk, but no actin ring or cup was visible. Adapted from ref. [2]. Copyright (2006) National Academy of Sciences, USA.

predominance of low curvature regions on the flat sides (Ω = 87.5°) over the high curvature regions (Ω = 2.5°), which were only present at the two discrete ends of the worm-like particles. Similar observations with filomicelles have also been made under simulated splenic flow conditions, in which long filomicelles were extended by flow, resulting in reduced uptake by the macrophages [3]. These results have definitely opened up exciting possibilities for the employment of shape-based approaches, in addition to the existing size and surface modulations [227] established to aid in avoiding the premature loss of nanocarriers via phagocytosis in vivo. 4.2.2. Endocytosis Particle shape, together with size, was also found to affect their internalization by non-phagocytic cells. In one systematic study, Gratton et al. fabricated a series of cationic cross-linked PEG-based hydrogels of varying sizes and shapes via the top-down lithographic PRINT technique and examined the extent, rate and mode of cellular internalization of the particles using HeLa cells [77]. Under comparison, nanometer-sized cylinders were found to be internalized to the greatest extent, followed by the larger micrometer-sized cylinders and lastly, the cubic-shaped particles (Fig. 9). It was further demonstrated that in addition to shape, the internalization kinetics of the nanometer-sized

cylinders in HeLa cells were affected by the particle aspect ratio (AR) and volume: cylinders with 1) higher AR, but similar volume or 2) larger volume, but same aspect ratio were found to have a greater rate of internalization. The greater internalization of the cylindrical particles is speculated to be due to their larger surface area, which allows for more multivalent ionic interactions with the cell membranes to undergo clarthrin- and caveolae-mediated endocytosis as well as to a lesser extent, macropinocytosis. Other similar findings that demonstrate the enhanced propensity for elongated particles to be internalized over their spherical counterparts were also observed with nanosized rod-like biodegradable mesoporous silica nanoparticles [228] and iron oxide nanoworms [211]. Contrary to the observations described above for elongated nanostructures, several other studies have instead found that the spherical forms of gold nanoparticles [229] and shell cross-linked polymeric nanoparticles assembled from block copolymers of poly (acrylic acid) and polystyrene [115] were internalized to a greater extent than their corresponding rod-shaped or cylindrical particles. The opposing findings of these studies thus suggest a need to carefully consider and evaluate the contribution of particle shapes towards cellular internalization in the context of the nanostructure concentration, chemical nature, surface properties and size of the systems

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Fig. 9. Distinct internalization profiles of cylindrical and cubic-shaped PRINT particles of various sizes in HeLa cells over a 4 h incubation period at 37 °C [77]. Copyright (2008) National Academy of Sciences, USA.

investigated. Nonetheless, the majority of the evidence available suggests that elongated nanoparticles are more favorable for therapeutic applications based on their collectively advantageous biodistribution and cellular internalization profiles. Theoretical models for the size effects for receptor-mediated endocytosis of non-spherical particles [230,231] can be useful to explain and predict the likelihood for internalization in the design of nanocarriers for therapeutic delivery applications. For example, Gao et al. [230] developed a mechanic model for the wrapping of a cell membrane with diffusive mobile receptors around a cylindrical or spherical particle covered with compatible ligands and proposed that receptor-mediated endocytosis is unfavorable below a critical radius. Similarly, Decuzzi and Ferrari [231] described a characteristic aspect ratio which affects the endocytic propensity and kinetics of elliptical cylindrical particles [231]. 4.3. Cytotoxicity Recently, it was shown that differently shaped poly(3,4-ethylenedioxythiophene) (PEDT) nanoparticles possessing similar diameters, which were fabricated by chemical oxidization polymerization using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles as the template, affected cell viabilities and inflammatory responses to different extents [232]. Among the three differently shaped nanoparticles tested, PEDT-1 (with lowest aspect ratio) induced a greater degree of cytotoxicity, apoptosis, and reactive oxygen species production in human lung fibroblast IMR90 and mouse alveolar macrophage J774A.1 cell lines. Although these results suggest that particle shape may play a role in affecting cell viabilities possibly through biological interactions, more systemic studies on the effects of nanoparticle shapes for cell viability are required to verify if the findings can indeed be broadly extrapolated to other materials and particle shapes.

the past decade, however, tremendous progress made in the various synthetic methodologies, such as the top-down and bottom-up approaches, for attaining diversely shaped particles, has gradually paved the way for the evaluation of the effects of particle shape on various biological processes important in drug delivery. As can be seen in this review, emerging studies have clearly shown that non-spherical particles possess drug loading capacities [114] and biological behaviors that frequently deviate from their historically well-studied spherical counterparts, which in various instances, have proven to be advantageous for improving blood circulation time [3], organ distribution [6,208– 211] and evading premature clearance by phagocytosis [114,208]. Despite the significant gain in knowledge in recent times, numerous challenges on the biological levels remain to be addressed before the complex relationship between particle shape and biological effects in the context of other parameters such as size, zeta potential, surface chemistry, mechanical property, etc. can be properly delineated and rationally applied to drug carrier designs. On the synthetic level, the different approaches to make nanostructures with precise shape control have their own associated advantages, challenges and limitations. Choice of a particular approach needs to be made after carefully considering the requirements. For instance, on a large scale, if precise reproducibility is required as with a complex shape, top-down approaches may be required. Judicious combination of techniques may also offer new opportunities to fabricate nanostructures that cannot be precisely accessed via any single approach and also transcend size limitations of an individual technique [233]. As with self-assembled structures, morphological dynamics (e.g. potential changes in morphology with variations in amphiphilic balance that may arise due to the degradation of hydrophobic block [99]) needs to be more extensively studied. A sound understanding of the morphological dynamics will be especially useful in predicting and to ensure consistency in the drug delivery performance of materials consisting of biodegradable and/or stimuliresponsive functionalities in vivo. From this review, it is evident that compositional and constitutional variations, which could be trivial or insignificant in different contexts, could lead to great consequences in biological behaviors and eventually impact drug delivery efficacies (e.g., cyclic vs. linear analog [220], unimer vs. aggregate [234], spherical micelle vs. elongated micelle [3], aspect ratio [2,77]). To date, most investigations into shape effects are largely limited to biodistribution studies and there exists a lack of general consensus on shape effects on cellular internalization and cytotoxicity particularly for polymeric nanoparticles synthesized via the established and frequently adopted bottom-up approaches. Therefore, more extensive and systematic bio-evaluations of drug carrier candidates are required to yield greater insights on the shape requirements for specific drug delivery applications. Coupled with the rapid development of synthetic tools that can yield morphologically well-defined biodegradable and/or biocompatible polymeric nanostructures, an improved understanding of the shape effects on biological processes is expected to give rise to a new generation of innovative drug carriers with significantly enhanced drug delivery outcomes for the clinics.

Acknowledgments This work was funded by the Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, Singapore.

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