Linear–dendritic biodegradable block copolymers: from synthesis to application in bionanotechnology

Abstract

Besides biodegradability and biocompatibility, linear–dendritic biodegradable block copolymers present unique hierarchical self-assembly and multivalent characteristics, making them appealing in stimuli-responsive nanomedicine and hydrogel applications. This minireview highlights the recent progress on linear–dendritic biodegradable block copolymers synthesized via click chemistry, the DNA–/protein–dendritic biohybrids, and their prospects in bionanotechnology.

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Chang-Ming Dong

Prof. Chang-Ming Dong (born 1972) received his Ph.D. in Polymer Chemistry & Physics under the supervision of Prof. Xin-De Feng and Prof. Kun-Yuan Qiu from Peking University in 2001. After about two-years of postdoctoral research with Prof. Elliot L. Chaikof in the School of Medicine of Emory University, he joined the faculty of the Department of Polymer Science & Engineering in Shanghai Jiao Tong University (Shanghai, P. R. China) in January, 2004. He was promoted to full professor in January, 2009, and currently focuses on the design and synthesis of biodegradable and biomimetic polymeric biomaterials and their applications in drug delivery and nanomedicine.

Gang Liu

Gang Liu is currently a PhD student at School of Chemistry & Chemical Engineering, Shanghai Jiao Tong University (P. R. China). After studying leather chemistry in Shaanxi University of Science and Technology, he joined the group of Prof. Shenghua Lv for a masters-thesis. In 2010, he joined the group of Prof. Chang-Ming Dong for a PhD study on photoresponsive biodegradable polymeric biomaterials & nanomedicine.

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1. Introduction

Dendritic polymers, including both dendrimers and hyperbranched polymers, as the fourth type of polymer, have received much attention in scientific and industrial communities over the past four decades.^1–15 They have a highly branched globular architecture with multiple periphery groups, and present peculiar physicochemical, rheological, and hierarchical self-assembly properties in bulk and solution, and multivalent characteristics, making them promising for many applications, such as nanotechnology, nanomedicine, electronics, energy harvesting, nanoreactors, catalysts, etc.^1–15 Owing to their multi-component constitutions, unique self-assembly properties, and multivalent characteristics, linear–dendritic block copolymers, pioneered by Fréchet et al.,¹⁶ which take advantage of chain-entangled linear polymers and densely chain-packed dendritic counterparts, have been increasingly investigated worldwide. Besides their excellent encapsulation characteristics, the dendritic wedges can be modularly functionalized with drugs, fluorescent dyes, targeting and imaging agents, stimuli-responsive compounds, and other bioactive molecules, exhibiting a multivalency effect for both targeted drug delivery and binding with DNA or proteins. These make linear–dendritic block copolymers especially useful for advanced nanomedicine and bionanotechnology. Several excellent reviews on linear–dendritic block copolymers can be found in the literature;^17–19 Fernandez-Megia et al. recently reviewed poly(ethylene oxide) (PEO)–dendritic block copolymers for biomedical applications.¹⁹ Generally, linear–dendritic block copolymers can be mainly synthesized from three strategies: (i) the coupling or conjugation of a dendritic wedge with a linear polymer; (ii) the “chain-first” method: a dendritic wedge is radially grown from a linear polymer; (iii) the “dendron-first” method: a linear polymer is polymerized from the focal point of a dendritic wedge, acting as a dendritic macroinitiator. Since Chapman et al. first synthesized linear–dendritic biodegradable block copolymers (LDBBCs), i.e., PEO-b-dendritic poly(L-lysine) (generation 4 having 15 lysine residues),²⁰ many scientists have focused on LDBBCs, such as the famous PEO–dendritic aliphatic polyesters composed of 2,2-bis(methylol)propionic acid (bis-MPA) or glycerol–succinic acid units.^17–31 In this minireview, we highlight the “click” and orthogonal syntheses of LDBBCs and DNA–/protein–dendritic biohybrids, and the bionanotechnology thereof.

2. Linear–dendritic biodegradable block copolymers (LDBBCs)

2.1 Synthesis of LDBBCs via click chemistry

As a philosophical concept, click chemistry, coined by Sharpless et al., caused a resolution in synthetic organic/polymer chemistry and materials science in the past two decades.^32–47 Among them, the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), thiol–yne/–ene coupling reaction, and the Diels–Alder reaction are representative examples. Owing to the high reaction efficiency and selectivity, the tolerance of multiple functional groups, and the modest reaction conditions, click chemistry has been widely studied for the modular syntheses of multi-topological (co)polymers and dendritic polymers, hydrogels, biomaterials, drug delivery systems, and bionanotechnology.^33–47 Note that the true click reaction in macromolecular synthesis emphasizes the equimolar feature of starting polymer precursors, besides the aforementioned characteristics.⁴⁸ Thus, can click chemistry be utilized to synthesize LDBBCs with high efficiency? By combining CuAAC and the ring-opening polymerization (ROP) of lactone or α-amino acid N-carboxyanhydride (NCA) monomers, Dong et al. reported the controlled synthesis of asymmetrical and symmetrical AB diblock and ABA triblock copolymer LDBBCs, in which the linear chains could be chosen to be biocompatible and hydrophilic PEO or biodegradable and hydrophobic poly(ε-caprolactone) (PCL), and the dendritic wedges were PCLs or biodegradable polypeptides, including poly(γ-benzyl-L-glutamate) (PBLG), poly(L-glutamic acid) (PLG), poly(ε-benzyloxycarbonyl-L-lysine) and poly(L-lysine), as shown in Scheme 1.^49–54 Both “dendron-first” and “chain-first” methods were tested for the synthesis of the LDBBCs PCL-b-PEO and PCL-b-PEO-b-PCL. In the “dendron-first” strategy, the alkyne focal point poly(amido amine) (PAMAM) dendrons (denoted as Dm, i.e., D0, D1, D2, and D3; generation zero to three, m = 0, 1, 2, and 3) initiated the ROP of CL monomer to generate clickable dendritic PCL wedges with 2^m branches in which the primary amine of Dm played a role as initiator species. The dendritic PCL wedges were then click conjugated with azide-terminated PEO (PEO–N₃) to produce the targeted copolymers. In most cases, the excess linear PEO or PCL precursor was 10%. The click efficiency was >90%, but the copolymer yield varied within about 60–90%. In the “chain-first” strategy, the alkyne PAMAM dendrons Dm can be click conjugated with PEO–N₃ to prepare macroinitiators PEO–Dm and Dm–PEO–Dm, but they are not practicable for polymerizing CL to synthesize the targeted copolymers with narrow polydispersities. Using CuAAC and the “dendron-first” method, Oriol and Sánchez et al. synthesized a series of photoaddressable LDBBCs composed of linear PEO and dendritic aliphatic bis-MPA polyesters (generation 1–4) decorated with mesogenic and photochromic cyanazobenzene peripheries, and their morphologies and phototriggered anisotropy were studied.²⁷ Hvilsted et al. synthesized LDBBCs composed of linear cholesteryl-terminated PCL and a second generation L-lysine dendron by sequential CuAAC and thiol–ene click reactions.²⁹ 100% excess of a second generation L-lysine dendron was used in the CuAAC, while 10-fold thiocholesterol was used in the thiol–ene reaction. Hvilsted et al. further extended CuAAC for the preparation of LDBBC macromers, which were polymerized to produce miktoarm core-crosslinked star copolymers.³⁰ Malkoch et al. used the ROP of CL initiated by alkyne/alkene focal point bis-MPA dendrons (G0–4) to prepare a library of dendritic PCL wedges, which was then conjugated with azide-/thiol-terminated PEO to produce LDBBCs via CuAAC or thiol–ene click chemistry.³¹

Scheme 1 (A) Alkyne focal point PAMAM dendrons Dm (m = 0, 1, 2, 3); (B) both asymmetrical and symmetrical LDBBCs can be synthesized by click chemistry.^49–54

Naturally branched proteins (e.g., collagen, glycoprotein, and proteoglycan) are key components of both the connective tissues and the extracellular matrices in mammals and human beings. Therefore, studying mimics of proteins will shed light on their structure–property relationship and de novo designer guidelines for artificial biomaterials. Keeping these in mind, LDBBC polypeptide block copolymers composed of linear PEO or PCL blocks and dendritic polypeptides, were synthesized by combining CuAAC and ROP of NCA via the “chain-first” or “dendron-first” methods (Scheme 1).^50,51 For example, the LDBBC PBLG-b-PEO was successfully synthesized with a high yield of 90–96%, which was then deprotected to generate the LDBBCs PLG–PEO.⁵⁰ Similarly, the LDBBC PBLG-b-PCL was efficiently synthesized by the “chain-first” method, and the PBLG segment gradually changed from β-sheet to an α-helix conformation with increasing chain length.⁵¹

Collectively, all the above examples do not strictly conform to the criteria of polymer click reactions as excess polymer precursor was used during the reaction process.⁴⁸ However, the excess polymer precursor can be removed from the target by selective precipitation or extraction, and both the reaction efficiency and the yield of LDBBCs are high. The CuAAC utilized for LDBBCs can be regarded as a click conjugation reaction because it is superior to the common polymer conjugation reactions. Meanwhile, thiol–ene chemistry provides a useful method to prepare biomaterials without heavy metal contamination, while the side reaction between thiol-containing precursors greatly decreases the click efficiency compared with CuAAC.

In addition, the residual copper catalyst has to be removed from the product if the CuAAC-synthesized LDBBCs are intended for biomedical applications. Purification methods such as iterative resolution-precipitation, column separation, and dialysis can be used, however, these procedures are time-consuming and not practicable. In this case, metal-free click chemistry such as strain-promoted alkyne–azide cycloaddition and thermally activated alkyne–azide cycloaddition might resolve this problem for biomedical applications.^55–58 However, the disadvantage is the complex synthesis of the cyclooctyne derivative and the activated alkyne bearing at least one electron-deficient group.

2.2 LDBBCs for stimuli-responsive nanomedicine and hydrogel applications

When nanotechnology meets medicine, nanomedicine appears as an emerging field. In this case, the hierarchical self-assembly of amphiphilic block copolymers can be used for the fabrication of nanoparticles and the related nanomedicine.⁵⁹ Thus, how do both the topology and copolymer composition of LDBBCs affect the morphology of self-assembled nanoparticles? As for the LDBBC PCL-b-PEO, the copolymer composition had a pronounced effect on the morphology of nanoparticles compared with the topology, and both micelle and polymersome (polymeric vesicle) were fabricated in aqueous solution.⁴⁹ As for the LDBBC PCL-b-PEO-b-PCL, only polymeric micelles self-assembled in aqueous solution, and both the copolymer topology and composition hardly affected the morphology of the nanoparticles.⁵² The clinically used anticancer drug doxorubicin (DOX) was encapsulated into the PCL-b-PEO-b-PCL triblock copolymers to form worm-like nanomedicine, which exhibited a higher stability, sustained a longer drug-release period and improved the premature burst release behavior.⁵² Malkoch et al. also fabricated DOX-loaded nanomedicine from LDBBCs composed of PEO and bis-MPA-b-PCL.³¹

Stimuli-responsive nanomedicine can deliver the cargo drugs to the diseased sites or cells on-demand, as it changes or disassembles under external stimuli such as temperature, pH, biomolecules (e.g., glutathione, glucose and enzyme), and light as well.^60–63 So, imparting (multi)stimuli-response to LDBBCs will accelerate their translation into clinical medicine. On the other hand, synthetic polypeptides with variable secondary conformations have been intensively investigated for nanomedicine and injectable hydrogels, and few of them are commercially available and enter into preclinical and/or clinical trials.^64–69 For example, the polypeptide PLG has a pK_a of 4.32 in water and exhibits a coil–helix conformation transition at pH 5.2, which imparts pH-sensitivity to the PLG-based block copolymers. The LDBBC PLG-b-PEO block copolymers presented a pH-sensitive self-assembly behavior and formed a PLG-cored normal micelle surrounded by a PEO corona via hydrogen-bonding interactions among the helical PLG chains.^70,71 Inspired by the reverse micelle concept, Dong et al. fabricated the reverse micelle of PLG-b-PEO by α-cyclodextrin (α-CD) based host–guest chemistry (Scheme 2).^70,71 The reverse micelle had an α-CD-threaded-PEO (i.e., α-CD–PEO polypseudorotaxane) core stabilized by a negatively charged PLG corona. Note that the copolymer architecture of the reverse micelle was inverted in contrast to normal micelles. This work represents the first example of a biocompatible and biodegradable reverse micelle, and establishes a versatile strategy for the fabrication of reverse micelles. The DOX-loaded nanomedicine fabricated from the LDBBCs PLG-b-PEO gave a higher drug-loading capacity of 24%, and sustained a longer drug-release period of about 70 days compared with its linear counterpart.

Scheme 2 (a) Normal micellar hydrogel and (b) reverse micellar hydrogel fabricated via the cooperation of host–guest chemistry and hydrogen-bonding interactions. Reproduced from ref. 71. Copyright 2010 Wiley InterScience.

From a clinical viewpoint, the excipients (i.e., polymers and their degradable units) should be fully secreted and/or metabolized by the human body if LDBBCs-based nanomedicine is approved from bench to bedside. Although some commercial nanomedicines such as Doxil™ (doxorubicin-loaded PEGylated liposome) and Abranxane™ (paclitaxel-loaded albumin nanoparticles) improve therapeutic efficacy and enter into clinical trials, discovering and translating the next generation of biocompatible and biodegradable nanomedicines with both stimuli-response and active targeting is still imperative for cancer therapy.^{60–63,72,73}

Supramolecular hydrogels with a soft and network structure that not only mimic the extracellular matrices, but also respond to some external stimuli such as shear and temperature, have been increasingly studied for injectable drug delivery systems and tissue engineering scaffolds.^74–79 Although increasing efforts have been made for protein hydrogels, the disadvantages of both intrinsic immunogenicity and high production costs hamper their applications.⁷¹ So, designing hybrid polypeptide hydrogels is still challenging for polymer therapeutics and regenerative medicine. Inspired by the injectable hydrogels formed by linear biodegradable polyester-b-PEO block copolymers,^77–79 the LDBBCs PLG-b-PEO polypeptide copolymers can be utilized to fabricate injectable supramolecular hydrogels. PLG-b-PEO cannot directly form hydrogels because of its limited solubility in water, however, it spontaneously self-assembles into micelles. It is reasoned that the hybrid polypeptide micelles can be physically cross-linked into hydrogels with the help of α-CD.^75,76 In the work by Dong and co-workers,⁷¹ the supramolecular cooperation of α-CD-based host–guest chemistry and hydrogen-bonding interactions is utilized to fabricate normal micellar hydrogels and reverse micellar hydrogels (Scheme 2). These hydrogels formed through the secondary aggregation of the polypeptide-cored normal micelles or through that of the polypeptide-shelled reverse micelles, presenting both temperature and pH dual-stimuli-responses. In particular, the storage modulus of the hydrogels reaches up to several ten-thousand Pa at a lower copolymer concentration (<7 wt%), which is superior to biodegradable polyesters-b-PEO hydrogel that has a weaker mechanical strength of <1000 Pa at a higher copolymer concentration of 10–25 wt%.^71,80 It is known that the cell–matrix interactions (e.g., adhesion, proliferation and differentiation) are influenced by the physical cues of the extracellular matrices such as hydrogels.⁸¹ For instance, human bone marrow stromal cells exhibited increasing expression of neuronal, myogenic and osteogenic markers when they were cultured on low (<1 kPa), intermediate (8–15 kPa), and high (25–40 kPa) mechanical hydrogels.⁸² As the elastic modulus of the hybrid polypeptide hydrogels can be easily tuned by the copolymer or α-CD concentrations, and the polypeptide composition, they might provide multiple faceted scaffolds for cell culture. In addition, the reverse micellar hydrogels attained a DOX loading capacity of 10% (w/v) compared with commercial poloxamer hydrogel of 1%,⁷¹ and the controlled drug-release profile can be tuned by both the copolymer topology and the polypeptide composition. Last but not least, imparting stimuli-response and dynamical self-healing properties to the hybrid polypeptide hydrogels will broaden their biomedical applications.⁸³

3. DNA–dendritic biohybrids

Biomacromolecules such as DNA and proteins constitute the key information components of living cells, and recently have been used as versatile building blocks to construct hierarchical hybrid nanostructures for biosensors, bioimaging, and drug delivery as well.^84–86 In fact, both single stranded DNA (ssDNA) and some proteins can be viewed as linear polymer chains and can be connected with dendrimers/dendrons to form linear–dendritic biohybrids. Obviously, developing click and orthogonal chemistry is practicable to prepare exact one-to-one DNA–/protein–dendritic biohybrids.^55,56 These biohybrids are expected to not only retain the secondary conformations, tertiary structures and biological activities of biomacromolecules, but also possess the unique characteristics of synthetic dendrimers/dendrons.

Using 32 base pair oligonucleotides, Tomalia et al. synthesized single-site ssDNA–PAMAM (G2) diblock biohybrids via a Michael-addition reaction, and then constructed triblock PAMAM–dsDNA–PAMAM via the molecular recognition of Watson–Crick base pairing.⁸⁷ Importantly, this work provides a supramolecular combinatorial methodology for the synthesis of DNA–dendritic biohybrids, in which synthetic dendrimers/dendrons have variable size, shape, peripheries, and functions. By utilizing CuAAC, Gothelf et al. click conjugated alkyne-/azide-terminated PAMAM G4 (64 peripheries, ∼6.5 nm) with azide-/alkyne-decorated ssDNA having 20 oligonucleotides (∼6.8 nm) to produce one-to-one DNA–PAMAM biohybrids.⁸⁸ Then, DNA-templated self-assembly helps to generate dendrimer oligomers with definite lengths, the supramolecular long wires of DNA–PAMAM conjugates, and the macromolecular long dendrimer wires with alternating grafted ssDNA (Scheme 3). As more DNA templates, including 2D and 3D origami and dendrimers or inorganic nanoparticles, can be potentially utilized as building units, this work provides a facile method for the construction of more complex polymerized dendrimer patterns and functional multicomponent nanopatterns. In addition, theoretical simulations of DNA–dendritic biohybrids will decipher their complex structures and the interactions between DNA and dendrimers/dendrons, providing more information for designing DNA-based self-assembly and gene therapy.⁸⁹ Tomalia reasoned that dendrimers/dendrons with well-defined architecture and size (1.0–20 nm) can be viewed as quantized soft nano-element like modules, which will play a significant role in the emerging science of nano-synthesis compared with fundamental small molecule chemistry.^90,91 Thereby, DNA–dendritic biohybrids provide an important platform for the precise and programmable self-assembly of functional nanostructures and devices, which deserve to be intensively studied for bionanotechnology in the coming future.⁹²

Scheme 3 (a) The click synthesis of DNA–PAMAM G4; (b) the polymerization of DNA–G4 conjugates on a long linear duplex template. Adapted from ref. 88. Copyright 2010 American Chemical Society.

4. Protein–dendritic biohybrids

To date, hybrid polypeptides with biodegradable polymers (e.g., polylactides and PCL) and biocompatible PEO, and the PEGylation of protein have been widely investigated, and some PEGylated protein drugs have been approved by the US Food and Drug Administration.^64–69 Inspired by protein therapeutics and the multivalent effect of dendritic motifs, Uludağ synthesized a carboxyl focal point tetra(bisphosphonic acid) dendron, and then coupled it with a model protein bovine serum albumin (BSA) to generate a BSA–dendron biohybrid.⁹³ This biohybrid with 4 bisphosphonic acid peripheries demonstrated 4.1- and 4.7-fold higher delivery (relative to the control BSA) at the femora and tibiae, which was attributed to both the multivalency effect and the strong affinity with the bone mineral hydroxyapatite. Based on the poly(propyleneimine) dendrimers (G1–3), Meijer et al. modified them as cysteine decorated dendrimers, then developed a native chemical ligation method to synthesize multivalent peptide/protein–dendrimer biohybrids under neutral aqueous conditions.⁹⁴ This modular approach might allow chemoselective dendrimer conjugation with lots of multivalent peptides and recombinant proteins, which provides a versatile platform for targeted drug delivery and bioimaging. Interestingly, using N-maleimido focal point Newkome's dendrons with multiple spermines and a mesoscale surfactant protein Class II hydrophobin (HFBI) with a free sulfhydryl or cysteine group, Kostiainen and Smith et al. prepared one-to-one protein–dendron biohybrids (G1–2) in a good yield of 80% via the site-selective addition between thiol and N-maleimido.⁹⁵ Provided one free cysteine residue exists in the protein or can be added via site-directed mutagenesis, the synthetic method is convenient and applicable to other large proteins (e.g., BSA) (Scheme 4).^95,96 As natural spermine has an affinity with DNA, the biohybrid HFBI–G2 demonstrates multivalent and strong binding with DNA. This result also suggests that the DNA-binding ability can be imparted from multiple spermine-decorated dendrimers/dendrons to proteins that do not have intrinsic affinity with DNA. The molecular dynamics simulation further demonstrates that the protein core affects the multivalent effect of dendrimers/dendrons, guiding the design for protein–dendritic biohybrids.⁹⁷ In summary, protein–dendritic biohybrids are expected to combine biological activities and the fine-tunable multivalency effect, opening up a new avenue for bionanotechnology.

Scheme 4 The protein–dendron biohybrids. Adapted from ref. 96. Copyright 2007 American Chemical Society.

5. Conclusions

This minireview highlights the recent progress on the click and orthogonal syntheses of LDBBCs and DNA–/protein–dendritic biohybrids. From scientific and clinical viewpoints, to modularly synthesize LDBBCs without heavy metal contamination and to easily fabricate biodegradable and biocompatible nanomedicine with a controllable size of 10–200 nm are vital for targeted drug delivery and cancer therapy. Note that the reproducible large-scale synthesis, fabrication, and in vivo biodegradation and biocompatibility of LDBBCs-based nanomedicine need to be investigated thoroughly before they are potentially translated from bench to bedside. To take advantage of the multiple structures and biological activities of biomacromolecules (e.g., DNA and protein) and synthetic multivalent dendrimers/dendrons, DNA–/protein–dendritic biohybrids are currently in their infant stage, and opening a new avenue for the directional and programmable self-assembly, the DNA-/protein-based delivery and therapeutics, and the bionanotechnology thereof.

Acknowledgements

The authors are grateful for the financial support of the National Natural Science Foundation of China (21274086, 21074068 and 20874058) and the Shanghai Leading Academic Discipline Project (B202).

Notes and references

1. M. A. Mintzer and M. W. Grinstaff, Chem. Soc. Rev., 2011, 40, 173–190.
2. D. Astruc, E. Boisselier and C. Ornelas, Chem. Rev., 2010, 110, 1857–1959.
3. D. Wilms, S. E. Stiriba and H. Frey, Acc. Chem. Res., 2010, 43, 129–141.
4. M. Calderon, M. A. Quadir, S. K. Sharma and R. Haag, Adv. Mater., 2010, 22, 190–218.
5. A. Carlmark, C. J. Hawker, A. Hult and M. Malkoch, Chem. Soc. Rev., 2009, 38, 352–362.
6. B. M. Rosen, C. J. Wilson, D. A. Wilson, M. Peterca, M. R. Imam and V. Percec, Chem. Rev., 2009, 109, 6275–6540.
7. R. K. Tekade, P. V. Kumar and N. K. Jain, Chem. Rev., 2009, 109, 49–87.
8. M. A. Mintzer and E. E. Simanek, Chem. Rev., 2009, 109, 259–302.
9. M. Scholl, Z. Kadlecova and H. A. Klok, Prog. Polym. Sci., 2009, 34, 24–61.
10. J. B. Wolinsky and M. W. Grinstaff, Adv. Drug Delivery Rev., 2008, 60, 1037–1055.
11. D. A. Tomalia, Prog. Polym. Sci., 2005, 30, 294–324.
12. S. Svenson and D. A. Tomalia, Adv. Drug Delivery Rev., 2005, 57, 2106–2129.
13. C. Liang and J. M. J. Fréchet, Prog. Polym. Sci., 2005, 30, 385–402.
14. C. C. Lee, J. A. Mackay, J. M. J. Fréchet and F. C. Szoka, Nat. Biotechnol., 2005, 23, 1517–1526.
15. C. Gao and D. Yan, Prog. Polym. Sci., 2004, 29, 183–275.
16. I. Gitsov, K. L. Wooley and J. M. J. Fréchet, Angew. Chem., Int. Ed. Engl., 1992, 31, 1200–1202.
17. I. Gitsov, J. Polym. Sci., Part A: Polym. Chem., 2008, 46, 5295–5314.
18. F. Wurm and H. Frey, Prog. Polym. Sci., 2011, 36, 1–52.
19. A. Sousa-Herves, R. Riguera and E. Fernandez-Megia, New J. Chem., 2012, 36, 205–210.
20. T. M. Chapman, G. L. Hillyer, E. J. Mahan and K. A. Shaffer, J. Am. Chem. Soc., 1994, 116, 11195–11196.
21. I. Gitsov, P. T. Ivanova and J. M. J. Fréchet, Macromol. Rapid Commun., 1994, 15, 387–393.
22. H. Ihre, O. L. P. De Jesus and J. M. J. Frechet, J. Am. Chem. Soc., 2001, 123, 5908–5917.
23. A. Wursch, M. Moller, T. Glauser, L. S. Lim, S. B. Voytek, J. L. Hedrick, C. W. Frank and J. G. Hilborn, Macromolecules, 2001, 34, 6601–6615.
24. M. A. Carnahan, C. Middleton, J. Kim, T. Kim and M. W. Grinstaff, J. Am. Chem. Soc., 2002, 124, 5291–5293.
25. E. R. Gillies, T. B. Jonsson and J. M. J. Fréchet, J. Am. Chem. Soc., 2004, 126, 11936–11943.
26. Y. Li, Y. D. Zhu, K. J. Xia, R. L. Sheng, L. Jia, X. D. Hou, Y. H. Xu and A. M. Cao, Biomacromolecules, 2009, 10, 2284–2293.
27. J. Barrio, L. Oriol, R. Alcal and C. Sánchez, Macromolecules, 2009, 42, 5752–5760.
28. X. J. Zhang, J. Cheng, Q. R. Wang, Z. L. Zhong and R. X. Zhuo, Macromolecules, 2010, 43, 6671–6677.
29. I. Javakhishvili, W. H. Binder, S. Tanner and S. Hvilsted, Polym. Chem., 2010, 1, 506–513.
30. I. Javakhishvili and S. Hvilsted, Polym. Chem., 2010, 1, 1650–1661.
31. P. Lundberg, M. V. Walter, M. I. Montanez, D. Hult, A. Hult, A. Nystrom and M. Malkoch, Polym. Chem., 2011, 2, 394–402.
32. V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
33. W. H. Binder and R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15–54.
34. H. Nandivada, X. Jiang and J. Lahann, Adv. Mater., 2007, 19, 2197–2208.
35. J. F. Lutz, Angew. Chem., Int. Ed., 2007, 46, 1018–1025.
36. D. Fournier, R. Hoogenboom and U. S. Schubert, Chem. Soc. Rev., 2007, 36, 1369–1380.
37. M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 2952–3015.
38. J. A. Johnson, M. G. Finn, J. T. Koberstein and N. J. Turro, Macromol. Rapid Commun., 2008, 29, 1052–1072.
39. B. Le Droumaguet and K. Velonia, Macromol. Rapid Commun., 2008, 29, 1073–1089.
40. R. K. Iha, K. L. Wooley, A. M. Nystrom, D. J. Burke, M. J. Kade and C. J. Hawker, Chem. Rev., 2009, 109, 5620–5686.
41. C. E. Hoyle and C. N. Bowman, Angew. Chem., Int. Ed., 2010, 49, 1540–1573.
42. A. B. Lowe, Polym. Chem., 2010, 1, 17–36.
43. A. Qin, J. W. Y. Lam and B. Z. Tang, Chem. Soc. Rev., 2010, 39, 2522–2544.
44. G. Franc and A. K. Kakkar, Chem. Soc. Rev., 2010, 39, 1536–1544.
45. M. A. Tasdelen, Polym. Chem., 2011, 2, 2133–2145.
46. S. Hvilsted, Polym. Int., 2012, 61, 485–494.
47. E. Lallana, A. Sousa-Herves, F. Fernandez-Trillo, R. Riguera and E. Fernandez-Megia, Pharm. Res., 2012, 29, 1–34.
48. C. Barner-Kowollik, F. E. Du Prez, P. Espeel, C. J. Hawker, T. Junkers, H. Schlaad and W. V. Camp, Angew. Chem., Int. Ed., 2011, 50, 60–62.
49. C. Hua, S. M. Peng and C. M. Dong, Macromolecules, 2008, 41, 6686–6695.
50. S. M. Peng, Y. Chen, C. Hua and C. M. Dong, Macromolecules, 2009, 42, 104–113.
51. C. Hua, C. M. Dong and Y. Wei, Biomacromolecules, 2009, 10, 1140–1148.
52. Y. Yang, C. Hua and C. M. Dong, Biomacromolecules, 2009, 10, 2310–2318.
53. Y. You, Y. Chen, C. Hua and C. M. Dong, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 709–718.
54. Y. C. Xu and C. M. Dong, J. Polym. Sci., Part A: Polym. Chem., 2012, 50, 1216–1225.
55. J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272–1279.
56. E. M. Slettenand and C. R. Bertozzi, Acc. Chem. Res., 2011, 44, 666–676.
57. A. Qin, J. W. Y. Lam and B. Z. Tang, Macromolecules, 2010, 43, 8693–8702.
58. M. Arseneault, I. Levesque and J.-F. Morin, Macromolecules, 2012, 45, 3687–3694.
59. M. Elsabahy and K. L. Wooley, Chem. Soc. Rev., 2012, 41, 2545–2561.
60. F. Meng, Z. Zhong and J. Feijen, Biomacromolecules, 2009, 10, 197–209.
61. M. C. Moore, W. B. Liechty and N. A. Peppas, Acc. Chem. Res., 2011, 44, 1061–1070.
62. J. J. Shi, Z. Y. Xiao, N. Kamaly and O. C. Farokhzad, Acc. Chem. Res., 2011, 44, 1123–1134.
63. E. Fleige, M. A. Quadir and R. Haag, Adv. Drug Delivery Rev., 2012, 64, 866–884.
64. H. Tian, Z. Tang, X. Zhuang, X. Chen and X. Jing, Prog. Polym. Sci., 2012, 37, 237–280.
65. J. Cheng and T. J. Deming, Top. Curr. Chem., 2012, 310, 1–26.
66. S. N. S. Alconcel, A. S. Baas and H. D. Maynard, Polym. Chem., 2011, 2, 1442–1448.
67. H. Cabral, N. Nishiyama and K. Kataoka, Acc. Chem. Res., 2011, 44, 999–1008.
68. H. Sun, F. Meng, A. A. Dias, M. Hendriks, J. Feijen and Z. Zhong, Biomacromolecules, 2011, 12, 1937–1955.
69. G. J. M. Habraken, A. Heise and P. D. Thornton, Macromol. Rapid Commun., 2011, 33, 272–286.
70. Y. Chen and C. M. Dong, J. Phys. Chem. B, 2010, 114, 7461–7468.
71. Y. Chen, X. H. Pang and C. M. Dong, Adv. Funct. Mater., 2010, 20, 579–586.
72. L. Sun, Y. Yang, C. M. Dong and Y. Wei, Small, 2011, 7, 401–406.
73. G. Liu and C. M. Dong, Biomacromolecules, 2012, 13, 1573–1583.
74. A. Altunbas and D. J. Pochan, Top. Curr. Chem., 2012, 310, 135–168.
75. J. Li, Adv. Polym. Sci., 2009, 222, 79–113.
76. J. Li, NPG Asia Mater., 2010, 2, 112–118.
77. L. Yu and J. D. Ding, Chem. Soc. Rev., 2008, 37, 1473–1481.
78. M. H. Park, M. K. Joo, B. G. Choi and B. Jeong, Acc. Chem. Res., 2012, 45, 424–433.
79. H. J. Moon, D. Y. Ko, M. H. Park, M. K. Joo and B. Jeong, Chem. Soc. Rev., 2012, 41, 4860.
80. K. Nagahama, T. Ouchi and Y. Ohya, Adv. Funct. Mater., 2008, 18, 1220–1231.
81. S. H. Parekh, K. Chatterjee, S. Lin-Gibson, N. M. Moore, M. T. Cicerone, M. F. Young and C. G. Simon Jr, Biomaterials, 2011, 32, 2256–2264.
82. A. J. Engler, S. Sen, H. L. Sweeney and D. E. Discher, Cell, 2006, 126, 677–689.
83. A. B. W. Brochu, S. L. Craig and W. M. Reichert, J. Biomed. Mater. Res., Part A, 2011, 96A, 492–506.
84. N. C. Seeman, Nature, 2003, 421, 427–431.
85. F. A. Aldaye, A. L. Palmer and H. F. Sleiman, Science, 2008, 321, 1795–1799.
86. C. Krejsa, M. Rogge and W. Sadee, Nat. Rev. Drug Discovery, 2006, 5, 507–521.
87. C. R. DeMattei, B. Huang and D. A. Tomalia, Nano Lett., 2004, 4, 771–777.
88. H. Liu, T. Tørring, M. Dong, C. B. Rosen, F. Besenbacher and K. V. Gothelf, J. Am. Chem. Soc., 2010, 132, 18054–18056.
89. M. Venkata, S. Kumar and P. K. Maiti, Soft Matter, 2012, 8, 1893–1900.
90. D. A. Tomalia, Soft Matter, 2010, 6, 456–474.
91. D. A. Tomalia, New J. Chem., 2012, 36, 264–281.
92. S. J. Tan, M. J. Campolongo, D. Luo and W. Cheng, Nat. Nanotechnol., 2011, 6, 268–276.
93. G. Bansal, J. E. I. Wright, C. Kucharski and H. Uludağ, Angew. Chem., Int. Ed., 2005, 44, 3710–3714.
94. I. Baal, H. Malda, S. A. Synowsky, J. L. J. Dongen, T. M. Hackeng, M. Merkx and E. W. Meijer, Angew. Chem., Int. Ed., 2005, 44, 5052–5057.
95. M. A. Kostiainen, G. R. Szilvay, D. K. Smith, M. B. Linder and O. Ikkala, Angew. Chem., Int. Ed., 2006, 45, 3538–3542.
96. M. A. Kostiainen, G. R. Szilvay, J. Lehtinen, D. K. Smith, M. B. Linder, A. Urtti and O. Ikkala, ACS Nano, 2007, 1, 103–113.
97. G. M. Pavan, M. A. Kostiainen and A. Danani, RSC Adv., 2011, 1, 1677–1681.

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