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Opinion
Exploring ‘new’ bioactivities of polymers at the nano–bio interface Chunming Wang1 and Lei Dong2 1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau SAR, China 2 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing, 210093 China
A biological system is essentially an elegant assembly of polymeric nanostructures. The polymers in the body, biomacromolecules, are both building blocks and versatile messengers. We propose that non-biologically derived polymers can be potential therapeutic candidates with unique advantages. Emerging findings about polycations, polysaccharides, immobilised multivalent ligands, and biomolecular coronas provide evidence that polymers are activated at the nano–bio interface, while emphasising the current theoretical and practical challenges. Our increasing understanding of the nano–bio interface and evolving approaches to establish the therapeutic potential of polymers enable the development of polymer drugs with high specificities for broad applications. Biological systems: elegant assemblies of polymeric nanostructures If we could see all biological events taking place at the nano scale, we would recognise biological systems as natural assemblies of polymeric nanomaterials. Cellular components consist of biopolymers, such as proteins, polysaccharides, nucleic acids, and their complexes, with sizes ranging from a few to hundreds of nanometres. Cells function by way of various interactions between these nanostructures, typified by protein binding events, such as receptors recognising ligands, integrins adhering to the cell matrix, and antigens binding antibodies. Collectively, biopolymers elegantly combine to orchestrate cell functions and mediate crucial physiological events. Dissecting biological systems into interactions between polymeric nanostructures inspired investigations into whether non-biological polymers can be used as therapeutic agents [1]. Conventionally, most polymers, apart from a few classes of biologically derived macromolecules like collagen, are hardly considered bioactive and rarely top the list of drug candidates. They are useful biomaterials tools to support tissue regeneration, resist pathogens, or deliver drugs, chiefly because of their physical advantages Corresponding author: Dong, L. ([email protected]). Keywords: polymers; nano–bio interface; polycations; polysaccharides; corona; macromolecular drugs. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.11.002
[2,3]. However, increasing evidence has emerged that many polymers, either natural or synthetic, can acquire new, mostly unpredicted bioactivities when they interact with living systems [4,5]. Even the so-called ‘bio-inert’ materials, such as gold or high-molecular-weight polyethylene, gain significant bioactivities when they form nanoparticles [6,7]. Polymers can be ‘activated’ at the nano–bio interface, no matter how active or inert their constituents. The notion that polymers, especially those that are nontissue derived, should be bio-inert is now proving incorrect. Polymers can act as drugs and may offer unique advantages over smaller compounds. For instance, their interaction with the biological interface emulates the natural interplay between biomacromolecular complexes in the body. They may also be more specific than smaller compounds, as the latter usually have more diverse targets [8]. Investigation and active control of the nanoscopic interactions between polymers and native biomolecules may open up enormous possibilities for development of polymer drugs with unique, high, and specific therapeutic activities. Polymers are activated at the nano–bio interface: cases and inspirations Polymers should have several essential features in order to gain various bioactivities at the nano–bio interface. The polymers must be sufficiently large, usually measuring between tens of nanometres and a few micrometres. Additionally, the biological effects exhibited by the polymers should not be present or be much weaker in their monomers or oligomers. The monomers or oligomers may have no activity at all or other activities that disappear after polymerisation. The polymers could interact with cell receptors, antigens, or growth factors, creating or changing an existing nanostructure and thereby activating or blocking cell signalling. Fourth, apart from a few carbohydrates, the polymers do not have specific antigens/receptors, but utilise the antigens/receptors that are specific to other antibodies/ligands (Figure 1A). The cationic polymer polyethyleneimine (PEI), long used as a carrier tool for gene transfection, was recently found to be a promising adjuvant [9,10] and was used as a direct therapeutic agent for immunotherapy [11–13]. As a potent mucosal adjuvant against viral subunit glycoprotein antigens [9,10], PEI co-administrated with viral vaccine antigen gp140 increased the production of antigen-specific IgG Trends in Biotechnology xx (2014) 1–5
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Figure 1. Four major mechanisms through which polymers are activated at the nano–bio interface. (A) A polymer can directly bind and activate a biomolecule, such as an antigen, a receptor, or an intracellular protein, and thereby trigger downstream cellular signals. These biomolecules are not specific to the polymer (and usually have their own specific antibodies or ligands) but are efficiently used by the polymer. (B) Instead of directly binding to a receptor, a polymer can bind a growth factor or cytokine, and form complexes with the receptor for this growth factor, thus enhancing or blocking the function of this growth factor. (C) A polymer can be engineered to conjugate ligands to form a multivalent conjugate, with high specificity and efficacy to activate cellular receptors and control cell behaviour. (D) In the body, a nanoscale polymer can be encapsulated by biomolecular coronas, and its ‘original’ function may be redefined by the corona activities. The corona can be very stable.
approximately 100 fold, compared to gp140 alone. Size was crucial for the activity of PEI, because PEI of higher molecular weight (750 kDa) and in branched form acted more powerfully than lower molecular weight PEI and its linear form. PEI and the antigen formed a relatively large complex (750 nm). The complex was internalised by antigen-presenting cells (APCs) that subsequently induced non-proinflammatory cytokine release, which may be a major mechanism for the immune activity of PEI [10]. In addition to its adjuvant role, PEI was also able to induce cytokine expression that recruited APCs [9], inhibit the development of arthritis [12], as well as promote the response of type 1 T 2
helper cells in vivo [11]. None of these diverse, immunemodulating activities of PEI has been found in its monomers. Similarly, another set of polycations, ‘viologen’ (Nalkylated 4,40 -bipyridinium)-based dendrimers, were designed to modulate immune cells [14,15]. Some viologen-based polycation macromolecules, those with 10–90 charges per molecule, can directly bind the chemokine receptor CXCR4 [15] and exert activity against the human immunodeficiency virus type 1 (HIV-1) in primary human cell cultures [14]. Cationic polymers that can interact with antigens or receptors have emerged as promising candidates for therapeutic agents with broad applications.
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Opinion Polymers may gain new activities less directly but in a more synergistic manner (Figure 1B). Many polysaccharides exhibit therapeutic functions through binding to growth factors and forming complexes with growth factor receptors of the growth factors (but not those of the carbohydrates). Of particular interest are heparin and heparan sulfate (HS). When they are large enough – specifically, when the number of their monosaccharide units exceeds 14 – they have considerable binding affinity for vascular endothelial growth factor-165 (VEGF165) [16]. Inspired by this interaction, researchers engineer HS varieties that preferentially bind and enhance various growth factors, including bone morphogenetic protein-2 (BMP-2) [17] and VEGF-A [18], to promote bone healing and blood vessel formation, respectively. Similarly, another polysaccharide, polysialic acid (polySia), forms a large complex (>650 kDa) with fibroblast growth factor 2 (FGF-2) and can enhance FGF-2-mediated cell proliferation when its chain length reaches 17 monosaccharide units [19]. In comparison with exogenous administration of the growth factors, the supply of growth factor-binding polymers successfully harnesses the power of endogenous growth factors while avoiding the side effects of exogenous growth factor degradation and overdosing [20,21]. In the cases of ischemia or wound healing, the levels of growth factors or cytokines significantly increase locally but may easily diffuse away or degrade. The polymers would synergise the growth factors to trigger cellular events, providing a safer and more elegant mode of ‘bioresponsive’ action, which may be a unique advantage of polymer therapeutics. The area for cell–polymer interaction must be sufficiently large, such as when polysaccharides directly bind and activate cell-surface receptors including toll-like receptors (TLRs) and C-type lectins [22,23]. A recent study demonstrated that a series of soluble b-glucan molecules (16– 400 kDa) could bind the dectin-1 receptor on bone marrowderived macrophages but failed to initiate immune signalling; only the large, insoluble, and particulate b-glucan polymers (3 mm in diameter) could both bind and activate dectin-1-mediated antimicrobial signalling, triggering phagocytosis as well as the production of reactive oxygen species (ROS) [24]. The ‘big’ glucan formed a synapse-like complex with dectin-1 without the involvement of CD45 or CD148, which are usually involved in regulating the activity of macrophages [25]. This finding exemplifies how the sizes of polymers dictate their bioactivities, which is a unique feature of polymer drugs. So, is it possible that different polymers demonstrate a common bioactivity because they share a common structural feature? This question can elicit a series of questions (Box 1), although the exact answer remains unknown. However, one study reported that two cationised polymers – PEI and cationised dextran (c-dextran) – demonstrated a remarkable capacity to convert tumour-associated macrophages (TAMs) from a pro-tumour, M2 subtype to an antitumour, M1 subtype, possibly through TLR-4 signalling [13]. Despite their totally different chemical composition, treatment with either PEI or c-dextran successfully inhibited angiogenesis and eventually decreased tumour size. Although it is still premature to conclude that polycations can be new weapons for cancer immunotherapy, this study
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Box 1. Outstanding questions Can a group of polymers with a similar structural feature (e.g., where all are polycations) but completely different chemical units exhibit common bioactivities? For a certain class of polymer, as its degree of polymerisation increases (from monomer to dimer to oligomer to polymer), would it exhibit more than two types of activities? If so, would it be possible to define and fine-tune this spectrum of functions for this particular polymer? What physiochemical properties of a polymer-based nanoparticle could influence the formation of coronas in specific biological environments? How can we determine the bioactivity of a coronaenveloped nanoparticle? As the corona genesis process is extremely complex and dynamic, to what extent can we control the formation and the composition of the coronas formed in biological fluids, and could this be sufficient for the treatment of some diseases?
suggests an interesting direction – to explore the bioactivities of a family of polymers instead of an individual polymer. In theory, it is possible for a class of polymers to share functions, because cell receptors for polymers, such as the pattern recognition receptors (PRRs), recognise nanomaterials that do not have the same chemical composition, but have similar structures and trigger similar biological responses [26–28]. Nanomaterials with similar physicochemical features are already found to share activities such as membrane-penetrating ability, cytotoxicity, and immunogenicity [29–31]. Challenges lie ahead for development of polymer therapeutics. From a theoretical perspective, it is crucial to decipher the structure–activity relationship of polymers, which is now unclear and much harder to study than that of small-molecule drugs. The biological activity of polymers is determined not only by their physicochemical nature, but by their nanoscale features, which would interact with the complex biological system. The 3D structure of the polymer is often a more important property than the chemistry of the monomers. Proteins with different amino acid sequences but similar 3D structures may have the same activity [32], while the same protein molecule can function distinctly when it folds into unusual structures (‘misfold’), as in the case of prions [33]. Formation of higher order structures composed of biomacromolecule complexes, such as focal adhesions, enables cells to survive and proliferate, while destruction of such complexes leads to loss of cell function and cell death [34,35]. Non-biological polymers can also form higher order structures. Moreover, in forming higher-order structures, polymers with ‘active’ chemical groups, such as amidogen, may gain new biological activities. These ‘active’ functional groups can drive the polymer to form more complex 3D nanostructures that may endow the polymer with unforeseen biological activities [36]. In response to such pressing demand for comprehensive information about the structure–function relationship of polymers, technologies and methods to study protein biochemistry, in tandem with platforms for high-throughput screening and establishment of polymer libraries, may be utilised to accelerate the identification and development of polymers as drug candidates. Another long-standing objective, from an engineering perspective, is to design polymers with tailor-made activities. Methods such as controlling molecular sizes and 3
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Opinion chemically modifying the polymers have proven effective [37]. Bioconjugation, the modification of polymer backbones with bioactive small molecules, is a mainstream approach but is encountering several key challenges as well. The conjugation changes the nanostructures of both the small molecule and the polymer backbone, making the small molecule lose its activity or specificity [38], or enabling the supposedly bio-inert polymer to gain a biological activity that is neither predicted nor wanted. Meanwhile, some new strategies attempt to redefine the nano–bio interface in other ways. One representative case is the technology platform introduced to engineer growth factor-binding HS polysaccharides. Instead of conjugating small molecular ligands to the polymer, this approach exploits the natural affinity of the polymer to select and enrich the desired polymers with tailor-made activities against various specific growth factors [17,18]. Another elegant example is to provide multivalent ligands that can organise stem cell receptors into clusters and thereby regulate stem cell differentiation. This approach yielded a brand new interface that could precisely ‘emulate the natural process of receptor–ligand assembly’ [39] (Figure 1C). These studies demonstrate attempts to redefine the nano–bio interface from different perspectives. Biomolecular coronas: redefining polymer functions in the body In addition to the ‘intrinsic’ activities of polymers, the impact of biological coronas, which confer ‘extrinsic’ functions to polymers, is emerging as significant in the future development of polymer drugs [40,41]. Once a nanosized polymer particle enters the body, tens or even hundreds of biomolecules are rapidly absorbed onto its surface to form a layer called the ‘corona’. These corona molecules may cover the whole surface of the particle, and their own properties override the ‘intrinsic’ biological activities of the polymer [41,40,42,43]. In so doing, the corona provides new biological properties for the polymers and redefines the nano–bio interface [41,40,44] (Figure 1D). In practice, coronas may fail to have the targeting efficiency of functionalised drug vehicles in the body, resulting in the disparity between the in vitro and in vivo activities of many particles [41]. However, more importantly, the power of such coronas suggests that control of their formation may inspire new strategies to activate polymers at the nano–bio interface. It is certainly challenging to control corona formation, which is rapid, dynamic, and influenced by many factors, such as identities of the biomolecules, the affinity of various biomolecules to the particle, and the duration the polymer stays in the environment [45–47]. However, opportunities exist. For example, one can design methods to fine-tune the surface chemistry of a polymer, to selectively enrich certain proteins onto the polymer surface, and thereby ‘engineer’ a group of coronas that contains desirable components and excludes unwanted ones [41,48–50]. In particular, molecular manipulation techniques, such as molecular imprinting, are proving to be powerful tools for generating polymers with selective affinities for certain proteins – a crucial step for controlling corona formation [49]. Notably, coronas can be divided into ‘hard’ and ‘soft’ varieties [45,51,52]. The approaches to control their formations must be tailor made. The hard corona binds tightly 4
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and directly to the particle surface; it is sufficiently stable and long-lived. By contrast, the soft corona attaches loosely onto the hard corona instead of adsorbing directly to the particle, and is short-lived because it constantly exchanges its components with the environment [41,52,53]. Depending on the specific purposes and the technical feasibility, some target biomolecules may need to be chemically immobilised, as part of the hard corona, to facilitate the long-term function of the polymer particles. Other target biomolecules can be recruited to the soft corona via their moderate-to-low affinities for a substrate or a protein pre-coated onto the particle (thus forming equivalent to the hard corona). This strategy would particularly favour the exchange of various proteins during a multi-stage process of drug delivery or tissue regeneration. Surprisingly, some coronas are strong enough to be well preserved on the particle surface even after cell entry, and are only degraded in the lysosomes – this is usually followed by lysosomal damage and eventual apoptosis [53]. Such an interesting feature provides important clues for designing polymer drugs with intracellular targets. Certain biomolecules that exhibit therapeutic activities by interacting with intracellular organelles or proteins and have difficulty crossing the cell membrane can be adsorbed onto a polymer core and enriched to become a hard corona. Here, the polymer represents a ‘Trojan horse’ [53] to carry the biomolecules into the cells. Conversely, the biomolecules, in the form of corona components, functionalise the polymer without the use of chemical conjugation, which might compromise the activity of the biomolecules. This scenario, which we propose to define as ‘coronalisation’, would allow for the possibility of treating various diseases with active molecules that already existed in the body. Polymers that are tailor-made for recruiting, enriching, and harnessing these molecules may provide a uniquely different mode of therapy for a broad range of applications. Concluding remarks and future perspectives Polymers, in particular the non-tissue-derived ones, can exhibit specific, diverse, and largely underestimated bioactivities at the nano–bio interface. Polymers of appropriate size have proven efficient in triggering biological responses through direct or synergistic interactions with cellular receptors, cytokines, or growth factors. In such processes, both intrinsic characteristics (such as size and chemistry) and extrinsic effects (such as those of the corona) are vital in dictating the type and strength of polymer activities. Future opportunities exist in both theoretical investigations and engineering innovations. In particular, three aspects – (i) elucidating the structural–function relationships of polymers; (ii) developing new chemical tools to activate polymers; and (iii) controlling corona formation – are probably the most exciting directions. In combining these efforts, we envisage polymers evolving from purely artificial molecules to unique, powerful, and specific drugs that integrate and harness the power of the body to combat disease and injury. Acknowledgements C.W. acknowledges funding grants from the Macao Science and Technology Development Fund (FDCT 048/2013/A2) and the University of Macau (MRG 006/WCM/2014/ICMS).
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References
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1 Kiick, K.L. (2007) Materials science. Polymer therapeutics. Science 317, 1182–1183 2 Williams, D.F. (2009) On the nature of biomaterials. Biomaterials 30, 5897–5909 3 Wolf, M.T. et al. (2014) Naturally derived and synthetic scaffolds for skeletal muscle reconstruction. Adv. Drug Deliv. Rev. Published online August 29, 2014. http://dx.doi.org/10.1016/j.addr.2014.08.011 4 Chen, L. et al. (2013) Bioactive dental restorative materials: a review. Am. J. Dent. 26, 219–227 5 Simanek, E.E. et al. (2013) Computational design principles for the discovery of bioactive dendrimers: [s]-triazines and other examples. Exp. Opin. Drug Discov. 8, 1057–1069 6 Krpetic, Z. et al. (2014) Nanomaterials: impact on cells and cell organelles. Adv. Exp. Med. Biol. 811, 135–156 7 Lin, T.H. et al. (2014) Suppression of wear-particle-induced proinflammatory cytokine and chemokine production in macrophages via NF-kappaB decoy oligodeoxynucleotide: a preliminary report. Acta Biomater. 10, 3747–3755 8 Keiser, M.J. et al. (2009) Predicting new molecular targets for known drugs. Nature 462, 175–181 9 Sheppard, N.C. et al. (2014) Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens. Int. Immunol. 26, 531–538 10 Wegmann, F. et al. (2012) Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat. Biotechnol. 30, 883–888 11 Chen, H. et al. (2010) The promotion of type 1 T helper cell responses to cationic polymers in vivo via toll-like receptor-4 mediated IL-12 secretion. Biomaterials 31, 8172–8180 12 Dong, L. et al. (2010) Anti-arthritis activity of cationic materials. J. Cell. Mol. Med. 14, 2015–2024 13 Huang, Z. et al. (2013) Anti-tumor immune responses of tumorassociated macrophages via toll-like receptor 4 triggered by cationic polymers. Biomaterials 34, 746–755 14 Asaftei, S. and De Clercq, E. (2010) Viologen’’ dendrimers as antiviral agents: the effect of charge number and distance. J. Med. Chem. 53, 3480–3488 15 Asaftei, S. et al. (2012) HIV-1 X4 activities of polycationic ‘‘viologen’’ based dendrimers by interaction with the chemokine receptor CXCR4: study of structure-activity relationship. J. Med. Chem. 55, 10405–10413 16 Zhao, W. et al. (2012) Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides. Biosci. Rep. 32, 71–81 17 Murali, S. et al. (2013) Affinity-selected heparan sulfate for bone repair. Biomaterials 34, 5594–5605 18 Wang, C. et al. (2014) Engineering a vascular endothelial growth factor 165-binding heparan sulfate for vascular therapy. Biomaterials 35, 6776–6786 19 Ono, S. et al. (2012) Novel regulation of fibroblast growth factor 2 (FGF2)-mediated cell growth by polysialic acid. J. Biol. Chem. 287, 3710–3722 20 Karvinen, H. et al. (2011) Long-term VEGF-A expression promotes aberrant angiogenesis and fibrosis in skeletal muscle. Gene Ther. 18, 1166–1172 21 Yla-Herttuala, S. et al. (2007) Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J. Am. Coll. Cardiol. 49, 1015–1026 22 Drummond, R.A. and Brown, G.D. (2013) Signalling C-type lectins in antimicrobial immunity. PLoS Pathog. 9, e1003417 23 Iliev, I.D. et al. (2012) Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science 336, 1314–1317 24 Goodridge, H.S. et al. (2011) Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature 472, 471–475 25 Zhu, J.W. et al. (2011) Receptor-like tyrosine phosphatases CD45 and CD148 have distinct functions in chemoattractant-mediated neutrophil migration and response to S. aureus. Immunity 35, 757–769 26 Fan, Y. et al. (2014) Scavenger receptor-recognized and enzymeresponsive nanoprobe for fluorescent labeling of lysosomes in live cells. Biomaterials 35, 7870–7880 27 Flo, T.H. et al. (2002) Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers. J. Biol. Chem. 277, 35489–35495
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Opinion
Exploring ‘new’ bioactivities of polymers at the nano–bio interface Chunming Wang1 and Lei Dong2 1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau SAR, China 2 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing, 210093 China
A biological system is essentially an elegant assembly of polymeric nanostructures. The polymers in the body, biomacromolecules, are both building blocks and versatile messengers. We propose that non-biologically derived polymers can be potential therapeutic candidates with unique advantages. Emerging findings about polycations, polysaccharides, immobilised multivalent ligands, and biomolecular coronas provide evidence that polymers are activated at the nano–bio interface, while emphasising the current theoretical and practical challenges. Our increasing understanding of the nano–bio interface and evolving approaches to establish the therapeutic potential of polymers enable the development of polymer drugs with high specificities for broad applications. Biological systems: elegant assemblies of polymeric nanostructures If we could see all biological events taking place at the nano scale, we would recognise biological systems as natural assemblies of polymeric nanomaterials. Cellular components consist of biopolymers, such as proteins, polysaccharides, nucleic acids, and their complexes, with sizes ranging from a few to hundreds of nanometres. Cells function by way of various interactions between these nanostructures, typified by protein binding events, such as receptors recognising ligands, integrins adhering to the cell matrix, and antigens binding antibodies. Collectively, biopolymers elegantly combine to orchestrate cell functions and mediate crucial physiological events. Dissecting biological systems into interactions between polymeric nanostructures inspired investigations into whether non-biological polymers can be used as therapeutic agents [1]. Conventionally, most polymers, apart from a few classes of biologically derived macromolecules like collagen, are hardly considered bioactive and rarely top the list of drug candidates. They are useful biomaterials tools to support tissue regeneration, resist pathogens, or deliver drugs, chiefly because of their physical advantages Corresponding author: Dong, L. ([email protected]). Keywords: polymers; nano–bio interface; polycations; polysaccharides; corona; macromolecular drugs. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.11.002
[2,3]. However, increasing evidence has emerged that many polymers, either natural or synthetic, can acquire new, mostly unpredicted bioactivities when they interact with living systems [4,5]. Even the so-called ‘bio-inert’ materials, such as gold or high-molecular-weight polyethylene, gain significant bioactivities when they form nanoparticles [6,7]. Polymers can be ‘activated’ at the nano–bio interface, no matter how active or inert their constituents. The notion that polymers, especially those that are nontissue derived, should be bio-inert is now proving incorrect. Polymers can act as drugs and may offer unique advantages over smaller compounds. For instance, their interaction with the biological interface emulates the natural interplay between biomacromolecular complexes in the body. They may also be more specific than smaller compounds, as the latter usually have more diverse targets [8]. Investigation and active control of the nanoscopic interactions between polymers and native biomolecules may open up enormous possibilities for development of polymer drugs with unique, high, and specific therapeutic activities. Polymers are activated at the nano–bio interface: cases and inspirations Polymers should have several essential features in order to gain various bioactivities at the nano–bio interface. The polymers must be sufficiently large, usually measuring between tens of nanometres and a few micrometres. Additionally, the biological effects exhibited by the polymers should not be present or be much weaker in their monomers or oligomers. The monomers or oligomers may have no activity at all or other activities that disappear after polymerisation. The polymers could interact with cell receptors, antigens, or growth factors, creating or changing an existing nanostructure and thereby activating or blocking cell signalling. Fourth, apart from a few carbohydrates, the polymers do not have specific antigens/receptors, but utilise the antigens/receptors that are specific to other antibodies/ligands (Figure 1A). The cationic polymer polyethyleneimine (PEI), long used as a carrier tool for gene transfection, was recently found to be a promising adjuvant [9,10] and was used as a direct therapeutic agent for immunotherapy [11–13]. As a potent mucosal adjuvant against viral subunit glycoprotein antigens [9,10], PEI co-administrated with viral vaccine antigen gp140 increased the production of antigen-specific IgG Trends in Biotechnology xx (2014) 1–5
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New signal
Polymerised receptor
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Theorecal receptor
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Signal B
Biomacromolecules Parcle with corona
Actual receptor TRENDS in Biotechnology
Figure 1. Four major mechanisms through which polymers are activated at the nano–bio interface. (A) A polymer can directly bind and activate a biomolecule, such as an antigen, a receptor, or an intracellular protein, and thereby trigger downstream cellular signals. These biomolecules are not specific to the polymer (and usually have their own specific antibodies or ligands) but are efficiently used by the polymer. (B) Instead of directly binding to a receptor, a polymer can bind a growth factor or cytokine, and form complexes with the receptor for this growth factor, thus enhancing or blocking the function of this growth factor. (C) A polymer can be engineered to conjugate ligands to form a multivalent conjugate, with high specificity and efficacy to activate cellular receptors and control cell behaviour. (D) In the body, a nanoscale polymer can be encapsulated by biomolecular coronas, and its ‘original’ function may be redefined by the corona activities. The corona can be very stable.
approximately 100 fold, compared to gp140 alone. Size was crucial for the activity of PEI, because PEI of higher molecular weight (750 kDa) and in branched form acted more powerfully than lower molecular weight PEI and its linear form. PEI and the antigen formed a relatively large complex (750 nm). The complex was internalised by antigen-presenting cells (APCs) that subsequently induced non-proinflammatory cytokine release, which may be a major mechanism for the immune activity of PEI [10]. In addition to its adjuvant role, PEI was also able to induce cytokine expression that recruited APCs [9], inhibit the development of arthritis [12], as well as promote the response of type 1 T 2
helper cells in vivo [11]. None of these diverse, immunemodulating activities of PEI has been found in its monomers. Similarly, another set of polycations, ‘viologen’ (Nalkylated 4,40 -bipyridinium)-based dendrimers, were designed to modulate immune cells [14,15]. Some viologen-based polycation macromolecules, those with 10–90 charges per molecule, can directly bind the chemokine receptor CXCR4 [15] and exert activity against the human immunodeficiency virus type 1 (HIV-1) in primary human cell cultures [14]. Cationic polymers that can interact with antigens or receptors have emerged as promising candidates for therapeutic agents with broad applications.
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Opinion Polymers may gain new activities less directly but in a more synergistic manner (Figure 1B). Many polysaccharides exhibit therapeutic functions through binding to growth factors and forming complexes with growth factor receptors of the growth factors (but not those of the carbohydrates). Of particular interest are heparin and heparan sulfate (HS). When they are large enough – specifically, when the number of their monosaccharide units exceeds 14 – they have considerable binding affinity for vascular endothelial growth factor-165 (VEGF165) [16]. Inspired by this interaction, researchers engineer HS varieties that preferentially bind and enhance various growth factors, including bone morphogenetic protein-2 (BMP-2) [17] and VEGF-A [18], to promote bone healing and blood vessel formation, respectively. Similarly, another polysaccharide, polysialic acid (polySia), forms a large complex (>650 kDa) with fibroblast growth factor 2 (FGF-2) and can enhance FGF-2-mediated cell proliferation when its chain length reaches 17 monosaccharide units [19]. In comparison with exogenous administration of the growth factors, the supply of growth factor-binding polymers successfully harnesses the power of endogenous growth factors while avoiding the side effects of exogenous growth factor degradation and overdosing [20,21]. In the cases of ischemia or wound healing, the levels of growth factors or cytokines significantly increase locally but may easily diffuse away or degrade. The polymers would synergise the growth factors to trigger cellular events, providing a safer and more elegant mode of ‘bioresponsive’ action, which may be a unique advantage of polymer therapeutics. The area for cell–polymer interaction must be sufficiently large, such as when polysaccharides directly bind and activate cell-surface receptors including toll-like receptors (TLRs) and C-type lectins [22,23]. A recent study demonstrated that a series of soluble b-glucan molecules (16– 400 kDa) could bind the dectin-1 receptor on bone marrowderived macrophages but failed to initiate immune signalling; only the large, insoluble, and particulate b-glucan polymers (3 mm in diameter) could both bind and activate dectin-1-mediated antimicrobial signalling, triggering phagocytosis as well as the production of reactive oxygen species (ROS) [24]. The ‘big’ glucan formed a synapse-like complex with dectin-1 without the involvement of CD45 or CD148, which are usually involved in regulating the activity of macrophages [25]. This finding exemplifies how the sizes of polymers dictate their bioactivities, which is a unique feature of polymer drugs. So, is it possible that different polymers demonstrate a common bioactivity because they share a common structural feature? This question can elicit a series of questions (Box 1), although the exact answer remains unknown. However, one study reported that two cationised polymers – PEI and cationised dextran (c-dextran) – demonstrated a remarkable capacity to convert tumour-associated macrophages (TAMs) from a pro-tumour, M2 subtype to an antitumour, M1 subtype, possibly through TLR-4 signalling [13]. Despite their totally different chemical composition, treatment with either PEI or c-dextran successfully inhibited angiogenesis and eventually decreased tumour size. Although it is still premature to conclude that polycations can be new weapons for cancer immunotherapy, this study
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Box 1. Outstanding questions Can a group of polymers with a similar structural feature (e.g., where all are polycations) but completely different chemical units exhibit common bioactivities? For a certain class of polymer, as its degree of polymerisation increases (from monomer to dimer to oligomer to polymer), would it exhibit more than two types of activities? If so, would it be possible to define and fine-tune this spectrum of functions for this particular polymer? What physiochemical properties of a polymer-based nanoparticle could influence the formation of coronas in specific biological environments? How can we determine the bioactivity of a coronaenveloped nanoparticle? As the corona genesis process is extremely complex and dynamic, to what extent can we control the formation and the composition of the coronas formed in biological fluids, and could this be sufficient for the treatment of some diseases?
suggests an interesting direction – to explore the bioactivities of a family of polymers instead of an individual polymer. In theory, it is possible for a class of polymers to share functions, because cell receptors for polymers, such as the pattern recognition receptors (PRRs), recognise nanomaterials that do not have the same chemical composition, but have similar structures and trigger similar biological responses [26–28]. Nanomaterials with similar physicochemical features are already found to share activities such as membrane-penetrating ability, cytotoxicity, and immunogenicity [29–31]. Challenges lie ahead for development of polymer therapeutics. From a theoretical perspective, it is crucial to decipher the structure–activity relationship of polymers, which is now unclear and much harder to study than that of small-molecule drugs. The biological activity of polymers is determined not only by their physicochemical nature, but by their nanoscale features, which would interact with the complex biological system. The 3D structure of the polymer is often a more important property than the chemistry of the monomers. Proteins with different amino acid sequences but similar 3D structures may have the same activity [32], while the same protein molecule can function distinctly when it folds into unusual structures (‘misfold’), as in the case of prions [33]. Formation of higher order structures composed of biomacromolecule complexes, such as focal adhesions, enables cells to survive and proliferate, while destruction of such complexes leads to loss of cell function and cell death [34,35]. Non-biological polymers can also form higher order structures. Moreover, in forming higher-order structures, polymers with ‘active’ chemical groups, such as amidogen, may gain new biological activities. These ‘active’ functional groups can drive the polymer to form more complex 3D nanostructures that may endow the polymer with unforeseen biological activities [36]. In response to such pressing demand for comprehensive information about the structure–function relationship of polymers, technologies and methods to study protein biochemistry, in tandem with platforms for high-throughput screening and establishment of polymer libraries, may be utilised to accelerate the identification and development of polymers as drug candidates. Another long-standing objective, from an engineering perspective, is to design polymers with tailor-made activities. Methods such as controlling molecular sizes and 3
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Opinion chemically modifying the polymers have proven effective [37]. Bioconjugation, the modification of polymer backbones with bioactive small molecules, is a mainstream approach but is encountering several key challenges as well. The conjugation changes the nanostructures of both the small molecule and the polymer backbone, making the small molecule lose its activity or specificity [38], or enabling the supposedly bio-inert polymer to gain a biological activity that is neither predicted nor wanted. Meanwhile, some new strategies attempt to redefine the nano–bio interface in other ways. One representative case is the technology platform introduced to engineer growth factor-binding HS polysaccharides. Instead of conjugating small molecular ligands to the polymer, this approach exploits the natural affinity of the polymer to select and enrich the desired polymers with tailor-made activities against various specific growth factors [17,18]. Another elegant example is to provide multivalent ligands that can organise stem cell receptors into clusters and thereby regulate stem cell differentiation. This approach yielded a brand new interface that could precisely ‘emulate the natural process of receptor–ligand assembly’ [39] (Figure 1C). These studies demonstrate attempts to redefine the nano–bio interface from different perspectives. Biomolecular coronas: redefining polymer functions in the body In addition to the ‘intrinsic’ activities of polymers, the impact of biological coronas, which confer ‘extrinsic’ functions to polymers, is emerging as significant in the future development of polymer drugs [40,41]. Once a nanosized polymer particle enters the body, tens or even hundreds of biomolecules are rapidly absorbed onto its surface to form a layer called the ‘corona’. These corona molecules may cover the whole surface of the particle, and their own properties override the ‘intrinsic’ biological activities of the polymer [41,40,42,43]. In so doing, the corona provides new biological properties for the polymers and redefines the nano–bio interface [41,40,44] (Figure 1D). In practice, coronas may fail to have the targeting efficiency of functionalised drug vehicles in the body, resulting in the disparity between the in vitro and in vivo activities of many particles [41]. However, more importantly, the power of such coronas suggests that control of their formation may inspire new strategies to activate polymers at the nano–bio interface. It is certainly challenging to control corona formation, which is rapid, dynamic, and influenced by many factors, such as identities of the biomolecules, the affinity of various biomolecules to the particle, and the duration the polymer stays in the environment [45–47]. However, opportunities exist. For example, one can design methods to fine-tune the surface chemistry of a polymer, to selectively enrich certain proteins onto the polymer surface, and thereby ‘engineer’ a group of coronas that contains desirable components and excludes unwanted ones [41,48–50]. In particular, molecular manipulation techniques, such as molecular imprinting, are proving to be powerful tools for generating polymers with selective affinities for certain proteins – a crucial step for controlling corona formation [49]. Notably, coronas can be divided into ‘hard’ and ‘soft’ varieties [45,51,52]. The approaches to control their formations must be tailor made. The hard corona binds tightly 4
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and directly to the particle surface; it is sufficiently stable and long-lived. By contrast, the soft corona attaches loosely onto the hard corona instead of adsorbing directly to the particle, and is short-lived because it constantly exchanges its components with the environment [41,52,53]. Depending on the specific purposes and the technical feasibility, some target biomolecules may need to be chemically immobilised, as part of the hard corona, to facilitate the long-term function of the polymer particles. Other target biomolecules can be recruited to the soft corona via their moderate-to-low affinities for a substrate or a protein pre-coated onto the particle (thus forming equivalent to the hard corona). This strategy would particularly favour the exchange of various proteins during a multi-stage process of drug delivery or tissue regeneration. Surprisingly, some coronas are strong enough to be well preserved on the particle surface even after cell entry, and are only degraded in the lysosomes – this is usually followed by lysosomal damage and eventual apoptosis [53]. Such an interesting feature provides important clues for designing polymer drugs with intracellular targets. Certain biomolecules that exhibit therapeutic activities by interacting with intracellular organelles or proteins and have difficulty crossing the cell membrane can be adsorbed onto a polymer core and enriched to become a hard corona. Here, the polymer represents a ‘Trojan horse’ [53] to carry the biomolecules into the cells. Conversely, the biomolecules, in the form of corona components, functionalise the polymer without the use of chemical conjugation, which might compromise the activity of the biomolecules. This scenario, which we propose to define as ‘coronalisation’, would allow for the possibility of treating various diseases with active molecules that already existed in the body. Polymers that are tailor-made for recruiting, enriching, and harnessing these molecules may provide a uniquely different mode of therapy for a broad range of applications. Concluding remarks and future perspectives Polymers, in particular the non-tissue-derived ones, can exhibit specific, diverse, and largely underestimated bioactivities at the nano–bio interface. Polymers of appropriate size have proven efficient in triggering biological responses through direct or synergistic interactions with cellular receptors, cytokines, or growth factors. In such processes, both intrinsic characteristics (such as size and chemistry) and extrinsic effects (such as those of the corona) are vital in dictating the type and strength of polymer activities. Future opportunities exist in both theoretical investigations and engineering innovations. In particular, three aspects – (i) elucidating the structural–function relationships of polymers; (ii) developing new chemical tools to activate polymers; and (iii) controlling corona formation – are probably the most exciting directions. In combining these efforts, we envisage polymers evolving from purely artificial molecules to unique, powerful, and specific drugs that integrate and harness the power of the body to combat disease and injury. Acknowledgements C.W. acknowledges funding grants from the Macao Science and Technology Development Fund (FDCT 048/2013/A2) and the University of Macau (MRG 006/WCM/2014/ICMS).
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