Enzyme-responsive polymer assemblies constructed through covalent synthesis and supramolecular strategy.

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Enzyme-Responsive Polymer Assemblies Constructed Through Covalent Synthesis and Supramolecular Strategy Yan Ding, Yuetong Kang and Xi Zhang*

Received 00th January 2014, Accepted 00th January 2014

Enzyme-responsive polymer assemblies have proved to be promising candidates for biomaterials, biomedicine and biosensing. Traditionally, these assemblies are prepared by the self-assembly of polymer building blocks which are convalently conjugated with enzymewww.rsc.org/ responsive moieties. Moreover, a supramolecular strategy has recently been developed for the preparation of enzyme-responsive polymer assemblies where the enzyme-responsive moieties are non-covalently complexed with the polymer building blocks. In addition, kinetic studies have been conducted on the enzyme-responsive behaviour of the polymer assemblies, which paves the way for tuning the response rate of the assemblies in a controlled manner. copolymers, which will consequently induce the formation, Introduction disassociation or morphological transformation of the polymer Polymer assemblies are of increasing interest due to their assemblies. applications in biomaterials, biomedicine and biosensing.1-5 In addition to the above covalent synthesis, there is also a Polymer micelles and vesicles have proved to be promising supramolecular strategy for the preparation of enzymenanocarriers because of their advantages of enhanced stability, responsive polymer assemblies, which features the nonimproved biocompatibility and extended blood circulation covalent integration of enzymatic substrates into the duration.6-9 Furthermore, stimuli-responsiveness has been assemblies.31,32 In this strategy, the enzymatic substrates and introduced to polymer assemblies so that they can respond to the polymer building blocks are bound together through nonspecific triggers in physiological environment, leading to their covalent interactions instead of covalent chemical bonds. This potential applications in drug/gene delivery.10-13 So far, a non-covalent approach, to some extent saves the labor of variety of stimuli have been employed to control the self- organic synthesis and simplifies the introduction of complicated assembly of polymers, including pH,14,15 light,16-18 enzyme-responsive moieties, thus providing a promising temperature,19-21 and redox.22-24 Among them, enzymes are alternative route to achieve enzyme-controlled self-assembly of especially intriguing.25-27 Enzymes play a key role in metabolic polymers. processes,because they catalyze biochemical reactions in a When enzymatic substrates are incorporated into polymer highly selective and efficient way. Meanwhile, the assemblies, the access of the enzymes to the substrates becomes concentration and the activity of the enzymes are closely susceptible to several factors, such as the affinity between the related to the health condition of cells. Certain enzymes are assemblies and the enzymes, and the permeability of the often upregulated in inflamed or tumour tissues,28-30 which assemblies to the enzymes. As a result, the rate of the allows the preferential drug/gene delivery to the diseased areas enzymatic response of the polymer assemblies will be by enzyme-responsive polymer assemblies. influenced. A few pioneering works have been conducted to illustrate how the different factors can affect the kinetics of the The common strategy for imparting enzyme-responsiveness enzyme-responsive behaviour.33,34 More interestingly, it has to polymer assemblies is to covalently link enzymatic substrates also been demonstrated that this “kinetic trouble” can be to amphiphilic copolymers.13,25 Possible candidates of enzyme exploited to achieve the external stimuli-controlled enzymesubstrates include bio-related motifs (peptides, DNA, etc) and responsiveness.35 non-natural moieties as well. In addition, the enzymeIn this feature article, we will first give a brief introduction responsive sites can be fixed either in the main chains or on the of the progress in the enzyme-responsive self-assembly of the side chains of the polymer building blocks. Upon exposure to covalently modified polymer building blocks. Then the noncertain enzymes, the substrate moieties will undergo the covalent strategy for the preparation of enzyme-responsive corresponding enzymatic reactions, which at the same time will polymer assemblies will be highlighted. In addition, detailed greatly alter the structure and the amphiphilicity of the DOI: 10.1039/x0xx00000x

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discussion will be carried out with regard to how the kinetics of the enzyme-responsive behaviour can be controlled for achieving the tunable response rate of polymer assemblies.


Enzyme-controlled self-assembly of polymers covalently modified with enzymatic substrates The covalent conjugation of enzymatic substrates with polymers has been a classic approach to prepare enzymeresponsive polymer assemblies. Taking esterases for examples, a common strategy to prepare esterase-responsive polymer assemblies is to covalently synthesize amphiphilic block copolymers with polyesters as the hydrophobic parts.36,37 Normally used polyesters, including poly(lactic acid) (PLA), polycaprolactone (PCL) and poly(trimethylene carbonate) (PTMC), are good substrates for esterases, such as lipase and porcine liver esterase.38-40 The copolymers can self-assemble into micelles or vesicles in aqueous solutions, and enzymatic hydrolysis of the polyester blocks will result in the disassociation of the assemblies. Instead of the cleavage of the backbones of polyesters, the phosphatase is often exploited to function through the removal of phosphate groups attached on polymers.41,42 For example, Ulijn et al. have prepared enzyme- and thermo-responsive polymers by adding a 9-fluorenyl methoxyloxycarbonylphosphorylated tyrosine (Fmoc-pY) group onto one end of a poly(2-isopropyl-2-oxazoline) (PiPrOx) chain.43,44 The polymer could not form stable assemblies due to the negatively charged phosphate moiety. In the presence of phosphatase, the Fmoc-pY was dephosphorylated to form the readily aggregating Fmoc-Y, which drove the polymers to self-assemble into micellar aggregates. Besides, the enzymatic dephosphorylation also resulted in a decrease in the lower critical solution temperature (LCST) of the polymer. Last a few years have also witnessed the increasing utilization of matrix metalloproteinases (MMPs) in the fabrication of enzyme-responsive systems. MMPs are a class of endopeptidase that can selectively cleave specific peptide bonds between nonterminal amino acids. This means that peptide substrates for MMPs can be incorporated into polymer building blocks as enzyme-cleavable sites, which will allow the fine tailoring of the structure of the building blocks and therefore the morphology of the polymer assemblies.45-47 And notably, MMPs are closely related to the pathological state of cells as they are found to be upregulated in inflamed and tumour tissues. Therefore, MMP-responsive nanocarriers are highly desirable because of their potential application in drug delivery.48 As shown in Figure 1, a kind of MMP-responsive polymer micelles, composed of a diblock copolymer and a plasmid DNA was prepared to act as gene vectors.49 The diblock copolymer, PEG227-GPLGVRG-PAsp(DET)64, consists of two hydrophilic segments, which is poly(ethylene glycol) (PEG) and diethylenetriamine modified poly(aspartamide) (PAsp(DET)) , respectively, with a peptide linkage (GPLGVRG) in between. The positively charged PAsp(DET)

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Journal Name DOI: 10.1039/C4CC05878J block was allowed to bind plasmid DNA through electrostatic interaction, leading to the formation of polyplex micelles with PAsp(DET)/DNA cores and PEG shells. In the presence of MMP-2, the peptide linkage could be cleaved so that the PEG shells of the micelles were shed, exposing the positively charged cores which would subsequently be internalized into cells. Notably, the positive charges on the dePEGylated cores can greatly promote the cell uptake and endosomal escape, thus remarkably enhancing the efficiency of the gene transfection.50

Fig. 1 Preparation of the polyplex micelles through the electrostatic complexation of PEG-GPLGVRG-PAsp(DET) and plasmid DNA, and the enhanced cellular uptake and endosomal escape of the micelles in the presence of MMP-2. Reproduced from Ref. 49.

The advances in enzyme-responsive assemblies are often facilitated by the introduction of new enzymes and enzymecatalyzed reactions into this field. It is gratifying that apart from the commonly used enzymes mentioned above, there are still several other enzymes which have been employed to regulate the behaviors of polymer assemblies, such as chitosanase,51 hyaluronidase52 and some reductases. For example, Khan et al. have recently prepared an azoreductase-responsive polymer assemblies,53 which are formed by an amphiphilic block copolymer poly(ethylene glycol)-b-poly(styrene) (PEG-b-PS) with an azobenzene group as the linker. The copolymers could self-assemble into m icellar aggregates in aqueous solutions. Moreover, in the presence of the reduction-state coenzyme NADPH, the azobenzene linkage of the copolymer could be cleaved under the catalysis of azoreductase, leading to the disassembly of the micellar assemblies. This case illustrates the potential use of the non-natural azobenzene motif in the preparation of new enzyme-responsive materials. Additionally, due to the presence of azoreductase in the human intestine, the micellar assemblies are expected to be applied for colonspecific drug delivery. Another example involves the exploitation of methionine sulfoxide reductase (MSR) as the trigger for the disassembly of polymersomes.54 Unlike the enzymes mentioned above, MSR functions by catalysing the reduction of methionine sulfoxide rather than the cleavage of chemical bonds. A double-hydrophobic copolypepetide poly(Lmethionine)65-b-poly(L-leucine0.5-stat-L-phenylalanine0.5)20 (M65(L0.5/F0.5)20) was prepared. Subsequent oxidation of the



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Fig. 2 Redox-controlled interconversion between M65(L0.5/F0.5)20 and MO65(L0.5/F0.5)20, and the structure of the vesicle formed by MO65(L0.5/F0.5)20. Reproduced from Ref. 54 with permission from the American Chemical Society.

Non-covalent integration of enzyme-responsive moieties into polymer assemblies In addition to covalent modification of enzyme-responsive moieties onto synthetic polymers, another strategy has been developed for the preparation of enzyme-responsive assemblies,31-35 in which the enzyme-responsive moieties are non-covalently conjugated onto the polymer building blocks. Compared with covalent modification, the non-covalent incorporation saves the labour of covalent synthesis to some extent. Building blocks are readily available and no solvents and toxic reagents are used in the preparation of the polymer assemblies. So far, the complexation between the enzymeresponsive moieties and the polymers is mainly driven by electrostatic interaction. Charged enzyme-responsive biomolecules have been employed to bind oppositely charged block copolymers to form enzyme-responsive polymer assemblies.


Fig. 3 (a) Phosphatase-controlled self-assembly of the polymeric supraamphiphile composed of ATP and PEG-b-PLKC. (b) Variation of the count rate of the assemblies in the solution with time in the presence of phosphatase. Adapted from Ref. 31 with permission from John Wiley and Sons.

Our research group has developed several enzymeresponsive assemblies on the basis of the concept called polymeric supra-amphipiles.55 For example, phosphataseresponsive spherical assemblies have been prepared with Adenosine 5’-triphosphate (ATP) and methoxy-poly(ethylene glycol)114-block-poly(L-lysine hydrochloride)200 (PEG-bPLKC) (Fig. 3a).31 The ATP molecules bearing four negative charges were allowed to interact with the positively charged PLKC block in PEG-b-PLKC through electrostatic complexation. The complexation neutralized the positive charges on the PLKC block, making the resultant complex amphiphilic which self-assembled into spherical assemblies. Moreover, the assemblies were responsive to phosphatase. Upon the addition of phosphatase, the multiple-charged ATP molecules was hydrolysed into single-charged phosphate and neutral adenine, which disrupted the multivalent electrostatic interaction between ATP and PEG-b-PLKC, thus resulting in the dissociation of the supra-amphiphiles and the consequent disassembly of the spherical assemblies.

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methionine into hydrophilic methionine sulfoxide (MO) gave the desired amphiphilic copolypeptide MO65(L0.5/F0.5)20, which could self-assemble into vesicles in aqueous solutions (Fig. 2). As the MO group served as a good substrate for MSR, enzymatic reduction of MO back to M would disrupt the amphiphilicity of the copolypeptide, thus leading to the disassembly of the vesicles.

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Journal Name DOI: 10.1039/C4CC05878J fluorescein groups were brought into close proximity, which allowed the intermolecular energy transfer between the fluorescein, thus leading to the quenching of its fluorescence. However, when the lipopeptides were phosphorylated by the protein kinase , the net charges of the lipopeptides decreased, resulting in the dissociation of the nanoparticle. As a result, the fluorescence of fluorescein was amplified. Based on the enzyme-induced fluorescence amplification, the polymer nanoparticles were further employed to evaluate the IC50 of protein kinase inhibitors.

Fig. 4 AChE-catalysed hydrolysis of myristoylcholine and the enzyme-responsive self-assembly of the supra-amphiphile composed of PEG-b-PAA and myristoylcholine. Reproduced from Ref. 56 with permission from the American Chemical Society.

Apart from ATP, the positively charged myristoylcholine is also enzyme-responsive. Following the same strategy, we have been able to prepare a polymeric supra-amphiphile through the electrostatic complexation of myristoylcholine and poly(ethylene glycol)-block-poly(acrylic acid) (PEG-b-PAA) (Fig. 4).56 The supra-amphiphile could self-assemble into spherical aggregates in aqueous solutions. In addition, the myristoylcholine in the supra-amphiphile could be hydrolyzed under the catalysis of acetylcholinesterase (AChE), which would disturb the amphiphilicity of the supra-amphiphile, leading to the disassembly of the spherical assemblies. As AChE plays an essential role in neurotransmission, the cholinesterase-responsive system may find applications in the therapy of neurological diseases. In the two systems discussed above, supra-amphiphiles were composed of charged small molecules and oppositely charged block copolymers. Besides, polymeric supraamphiphiles can also be prepared through the complexation of the small molecules and charged homopolymers, namely polyelectrolytes. For instance, electrostatic complexes could be prepared through the non-covalent conjugation of the negatively charged ATP onto chitosan, which was a natural polycation with its amine groups protonated in the neutral or acidic solutions.57 Herein, the ratio of the protonated amine groups to ATP was kept at around 12:1 so that about a third of the positive charges on the chitosan were neutralized by ATP while the others remained, thus imparting amphiphilicity to the formed electrostatic complexes. In this way, polymeric supraamphiphiles were prepared. The supra-amphiphiles could selfassemble into spherical aggregates which would undergo disassembly upon addition of phosphatase. The controlled self-assembly and disassembly of the enzyme-responsive aggregates make them potential candidates for drug delivery and controlled release. Furthermore, additional functions can be achieved if other functional groups are incorporated into the building blocks. Katayama et al. have reported the preparation of polymer nanoparticles through the electrostatic complexation of hydrophobically modified lipopeptides and polyanions (Fig. 5).32 In particular, fluorescein was modified onto the polyanions. Upon complexation, the

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Fig. 5 Formation of the nanoparticles through the electrostatic complexation of the lipopeptides and the polyanions, and the protein kinase triggered dissociation of the nanoparticles accompanied with the amplification of the fluorescence. Reproduced from Ref. 32 with permission from the American Chemical Society.

The systems discussed above are all based on the complexation between polymers and enzyme-responsive small molecules. Additionally, it is also feasible to prepare enzymeresponsive assemblies by the complexation of two oppositely charged polymers. For example, Zhang et al. have prepared a pH/enzyme-responsive assembly on the basis of the electrostatic complexation between DNA and cationic gelatin (Fig. 6).58 Doxorubicin was intercalated into double-stranded DNA fragments, polyGC, to form the negatively charged intercalation DOX-polyGC. The obtained DOX-polyGC was further coupled with enzyme-responsive cationic gelatin, leading to the formation of the electrostatic complex CPX1 bearing excess positive charges on its surface. Moreover, PEGylated histamine-modified alginate (His-alginate-PEG) with a pKa of 6.9 was employed to cover the exterior of the CPX1 at physiological pH (7.2) through electrostatic interaction, giving the nanoparticle CPX2. The CPX2 nanoparticles could be used for the tumour targeted delivery of doxorubicin. In tumour environment, where the pH was below 6.7, the His-alginate-PEG would get positively charged and thus detach from the surface of CPX1. The exposed CPX1 complex could then be biodegraded under the catalysis of gelatinase and deoxyribonuclease (Dnase) in the tumour cells, which would result in the release of the doxorubicin.




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Fig. 6 Preparation of CPX2 and its degradation in tumour environment. Reproduced from Ref. 58 with permission from Elsevier.

Layer-by-layer (LbL) assembly, which is also based on the electrostatic complexation of oppositely charged polymers, has proved to be another way for the preparation of enzymeresponsive assemblies. For example, Caruso et al. have prepared enzyme-responsive DNA capsules through the LbL assembly of oligonucleotides.59 Single-stranded DNA, poly-T30 was modified onto amine-coated silica particles through electrostatic interaction so that colloidal substrates were obtained. Then triblock oligonucleotides A15X15G15 and T15X15C15 were alternately deposited onto the substrate, which was driven by the base-pair hybridization between the A15, T15, C15 and G15 blocks. In this way, multilayer films were prepared on the surface of the silica particles (Fig. 7). Moreover, oligonucleotide with three X’15 blocks, which were complementary to the X15 blocks, was employed to cross-link the multilayer films. Subsequent dissolution of the silica core led to the formation of hollow DNA capsules. The prepared capsules contained the palindromic sequence 5’GAATTC3’, which could be recognized and selectively cut by the restriction enzyme EcoRI. Therefore, the capsules was degraded with the existence of EcoRI. Furthermore, when mesoporous silica was used as the substrate, proteins could be incorporated into the substrate before the LbL deposition and thus be encapsulated in the capsules after the silica was dissolved. Based on that, enzyme-triggered release of the proteins was achieved. Similarly, Akashi et al. have also prepared enzyme-responsive hollow capsules by the LbL assembly of chitosan and dextran sulfate on mesoporous silica particles followed by the removal of the silica.51


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Fig. 7 Structure of the hollow capsule prepared by the LbL assembly of A15X15G15 (red-green-aqua) and T15X15C15 (orange-green-blue) and by the subsequent crosslinking with X’15 X’15 X’15 (light green). The EcoRI cut sites are highlighted in yellow. Reproduced from Ref. 59 with permission from John Wiley and Sons.

Kinetic control on the enzyme-responsive behaviour of the polymer assemblies When an enzyme catalyses a reaction, it is imperative that it should bind the substrate in the first place. The accessibility of the substrate to the enzyme has a great effect on the kinetics of the catalysis. This is an especially important point that needs to be considered when it comes to the enzyme-responsiveness of polymer assemblies. In enzyme-responsive polymer assemblies, the enzyme-responsive substrates are usually surrounded by other parts of the assemblies. The accessibility of the substrates can be affected by several factors, such as steric hindrance and affinity between the assemblies and the enzymes.34,60 Therefore, the rate of the enzyme-catalysed reaction in these systems is strongly dependent on the structure and the composition of the assemblies as well as the location of enzyme responsive sites in the assemblies. The concern about the kinetic aspects of enzyme-responsiveness may have brought extra challenges in the preparation of enzyme-responsive polymer assemblies. However, from another point of view, it also inspires us to purposely tune the accessibility of the enzyme-responsive moieties by changing the structure of the polymer assemblies so that additional control on the kinetics of the enzyme-responsive behaviour of the assemblies can be achieved. The enzyme-catalysed degradation of the cores of polymer micelles can be slowed through the cross-linking of their shells. Wooley et al. have prepared a block copolymer, poly(N(acryloyloxy)succinimide-co-N-acryloylmorpholine)-b-poly(Llactic acid) (P(NAS-co-NAM)-b-PLLA), which could selfassemble into micelles in aqueous solutions (Fig. 8).33 Composed of PLLA, the hydrophobic cores of the micelles could be hydrolysed under the catalysis of proteinase K. About 70% of the cores could be degraded within 48 h. Moreover, the

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shells of the micelles contained the NAS groups which could be cross-linked by diamine cross-linkers to give the shell crosslinked knedel-like (SCK) nanoparticles. With a cross-linking density of 20%, the percentage of the PLLA degraded within 48 h dropped to 50%. The rate of the degradation was lowered because the cross-linked shells greatly limited the migration of

Journal Name DOI: 10.1039/C4CC05878J the enzymes to the PLLA cores. In other words, the accessibility of the PLLA cores was reduced. In addition, the extent to which the degradation was slowed was not very large, which might be explained by the fact that proteinase K was also capable of cleaving the amide bonds in the cross-links.

Fig. 8 Preparation of the SCK nanoparticles by the self-assembly of P(NAS-co-NAM)-b-PLLA followed by cross-linking and the hydrolysis of the PLLA cores of the SCK nanoparticles in the presence of proteinase K. Reproduced from Ref. 33 with permission from the American Chemical Society.

As mentioned above, the affinity between the enzymes and the assemblies can affect the accessibility of the enzymeresponsive sites. Enzymes, which are proteins in essence, usually bear charges in aqueous solutions. Thus, for charged polymer assemblies, their electrostatic interaction with the enzymes, either attractive or repulsive, should have an impact on the rate of their degradation. Wooley et al. have reported the electrostatic interaction-mediated enzymatic-degradation of polymer micelles.60 Two amphiphilic block copolymers, poly(acrylic acid)80-b-poly-(DL-lactide)40 (PAA80-b-PDLLA40) and poly(acrylamidoethylamine)90-b-poly(DL-lactide)40 (PAEA90-b-PDLLA40) were prepared (Fig. 9). The copolymers could self-assemble to form two classes of micelles which contained identical PDLLA cores but oppositely charged shells. Enzyme-catalysed hydrolysis of the PDLLA cores in the two micelles was carried out at physiological pH with proteinase K, which bore net negative charges. Within 24 h, 35% of the cores in the negatively charged micelles were hydrolysed while only 15% degradation was achieved with the positively charged analogues. The higher rate of degradation for the anionic micelles resulted from the electrostatic attraction between proteinase K and the micelles which could facilitate the binding of proteinase K to the PDLLA cores. In contrast, electrostatic repulsion made it more difficult for proteinase K to access the PDLLA in the cationic micelles. Additionally, negatively charged porcine liver esterase was also employed to catalyse

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the hydrolysis of the two micelles. Similarly, preferential degradation could be observed with the oppositely charged enzyme-micelle pair, which also supported the electrostatic attraction-mediated acceleration of the hydrolysis.

Fig. 9 Preparation of the anionic and cationic micelles by the self-assembly of PAA80-b-PDLLA40 and PAEA90-b-PDLLA40, respectively, and their enzymatic degradation by the positively charged proteinase K and the negatively charged porcine liver esterase. Reproduced from Ref. 60.

Similar phenomena have been found with proteinencapsulating hollow capsules prepared by the LbL assembly of cationic chitosan and anionic dextran sulfate.51 Negatively charged capsules with dextran sulfate as the outermost layer would be faster degraded under the catalysis of chitosanase



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Journal Name because the electrostatic attraction between the positively charged chitosanase and negatively charged capsules promoted the absorption of chitosanase onto the surface of the capsules, thus accelerating the degradation of the chitosan. Furthermore, additional stimuli-responsive moieties can be integrated into enzyme-responsive polymer assemblies so that the accessibility of the enzyme-responsive sites in the assemblies can be altered with external stimuli, leading to the stimuli-controlled enzymatic degradation of polymer assemblies. For example, thermo-responsive polymers have been exploited to regulate the enzyme-responsive behaviour of polymer micelles.35 A complex micelle was prepared through the co-assembly of poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) and poly(N-isopropylacrylamide)-b-poly(lactic acid) (PNIPAM-b-PLA) (Fig. 10). When the temperature is below the LCST of the PNIPAM blocks, the PEG/PNIPAM shells of the micelles were permeable for proteinase K, which could migrate inside and catalyse the hydrolysis of the PLA cores. However, at temperatures above the LCST, the PNIPAM blocks collapsed onto the surface of the PLA cores, forming hydrophobic patchy domains which would impede the enzyme from accessing the cores, resulting in the decrease of degradation rate. The hydrolysis above the LCST could be further slowed as the proportion of PNIPAM in the shells was raised until negligible degradation could be observed due to the formation of a dense and continuous layer of PNIPAM that totally isolated the PLA core from the enzyme.

ARTICLE DOI: 10.1039/C4CC05878J finally led to the degradation of the micelles within 4 days. On the contrary, when the PBA block in the copolymer was replaced by polystyrene (PS), the resultant micelles became resistant to the catalysis of elastase. The resistance resulted from the higher glass transition temperature (Tg) of PS than that of PBA, which greatly inhibited the exchange of the copolymers between the micelles and the bulk solution. This postulation could be further supported by the fact that when the temperature was raised to 70 oC, the PS containing micelles could be degraded under the catalysis of thermolysin due to the enhanced mobility of the PS blocks. In addition, the polymer assemblies prepared with the supra-amphiphile of ATP and PEG-b-PLKC (see the previous section) may also follow a similar mechanism during their degradation. In that system, the phosphatase-catalysed hydrolysis mainly happened to the free ATP molecules in the bulk solution. And the exchange of the ATP molecules between the assemblies and the bulk solution led to the final degradation of the assemblies. In particular, the degradation proceeded pretty fast. Most of the spherical assemblies disassociated within 3 h according to the result from dynamic light scattering (Fig. 3b).31 This is much faster than the previous examples where the enzymatic substrates were covalently modified onto the polymer building blocks. The difference in the kinetics between the two systems, to some extent, should be attributed to the faster exchange of the small molecule ATP than the polymers. Therefore, the non-covalent incorporation of enzymatic substrates may also contribute to the acceleration of the enzymatic response of the polymer assemblies.

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Fig. 10 Thermally controlled enzymatic degradation of the PLA cores of the micelles composed of PEG-b-PLA and PNIPAM-b-PLA. Adapted from Ref. 35 with permission from Elsevier.

In the above examples, the enzymes have to migrate to the enzyme-responsive moieties before the degradation takes place within the assemblies. In contrast, enzymatic degradation can also proceed through another mechanism, where the enzymecatalysed reaction mostly occurs outside the assemblies. In this case, the rate of the degradation is highly dependent on the speed of the exchange between the building blocks in the assemblies and those individually dissolved in the solution. For example, polymer micelles formed by poly(L-glutamic acid-coL-alanine)-b-poly(n-butyl acrylate) (P(Glu30-co-Ala30)-b-PBA) could be degraded through the elastase-catalysed cleavage of the Ala-Ala amide bond in the shells of the micelles.34 In particular, the cleavage mostly happened to the copolymers in the bulk solution because of the steric hindrance in the shells of the micelles. And it was the relatively fast exchange between the copolymers in the micelles and those in the solution that


In conclusion, enzyme-responsive polymer assemblies have gained much progress, benefiting both from the introduction of the new enzymes and from the development of the new strategy for their preparation. The kinetic studies have shed some light on the correlation between the rate of the enzymatic response and the structure/composition of the polymer assemblies. However, in spite of the advances in this field, challenges still remain. First of all, the enzymes that have been employed in enzyme-responsive systems are still very limited. Mostly used are proteases, phosphatases and a few other esterases, which can cleave the peptide bonds or ester bonds by hydrolysis. More enzymes, especially those functioning through other mechanisms should be involved. Moreover, the rate of the enzymatic response is still not well controlled. This will become a serious problem when it comes to the drug delivery by the enzyme-responsive polymer assemblies since the rate of the drug release is highly dependent on how fast the assemblies can respond to the enzyme triggers. To address this issue, more studies should be conducted from the point of view of kinetics. The research on the kinetics will not only help us better understand the mechanism of the enzymatic response, but also guide us in the design of new enzyme-responsive polymer assemblies. Furthermore, although many enzyme-responsive polymer assemblies are prepared for biomedical applications,

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many of them have only proved to function in vitro. That does not necessarily guarantee that they can work equally well in the physiological environment. More emphasis should be laid on the in vivo performance of the enzyme-responsive polymer assemblies.


Notes and references Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China. Fax: +86-10-6277-1149; Tel: +86-10-6279-6283; E-mail: [email protected]

References 1. Kelley, E. G.; Albert, J. N. L.; Sullivan, M. O.; Epps, I. I. I. T. H., Chem. Soc. Rev. 2013, 42, 7057. 2. Cheng, R.; Meng, F.; Deng, C.; Klok, H.-A.; Zhong, Z., Biomaterials 2013, 34, 3647. 3. Elsabahy, M.; Wooley, K. L., Chem. Soc. Rev. 2011, 41, 2545. 4. Delplace, V.; Couvreur, P.; Nicolas, J., Polym. Chem. 2014, 5, 1529. 5. Cao, W.; Gu, Y.; Meineck, M.; Xu, H., Chem. Asian J. 2014, 9, 48. 6. Brinkhuis, R. P.; Rutjes, F. P. J. T.; van Hest, J. C. M., Polym. Chem. 2011, 2, 1449. 7. Fuks, G.; Mayap Talom, R.; Gauffre, F., Chem. Soc. Rev. 2011, 40, 2475. 8. Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W., Acc. Chem. Res. 2011, 44, 1039. 9. Wei, H.; Zhuo, R.-X.; Zhang, X.-Z., Prog. Polym. Sci. 2013, 38, 503. 10. Zhang, Q.; Re Ko, N.; Kwon Oh, J., Chem. Commun. 2012, 48, 7542. 11. Zhuang, J.; Gordon, M. R.; Ventura, J.; Li, L.; Thayumanavan, S., Chem. Soc. Rev. 2013, 42, 7421. 12. Ge, Z.; Liu, S., Chem. Soc. Rev. 2013, 42, 7289. 13. Randolph, L. M.; Chien, M.-P.; Gianneschi, N. C., Chem. Sci. 2012, 3, 1363. 14. Loh, X. J.; del Barrio, J.; Toh, P. P. C.; Lee, T.-C.; Jiao, D.; Rauwald, U.; Appel, E. A.; Scherman, O. A., Biomacromolecules 2011, 13, 84. 15. Chen, W.; Zhong, P.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z., J. Control. Release 2013, 169, 171. 16. Zhao, Y., Macromolecules 2012, 45, 3647. 17. Liu, Y.; Yu, C.; Jin, H.; Jiang, B.; Zhu, X.; Zhou, Y.; Lu, Z.; Yan, D., J. Am. Chem. Soc. 2013, 135, 4765. 18. Nomoto, T.; Fukushima, S.; Kumagai, M.; Machitani, K.; Arnida; Matsumoto, Y.; Oba, M.; Miyata, K.; Osada, K.; Nishiyama, N.; Kataoka, K., Nat. Commun. 2014, 5. 19. Hocine, S.; Li, M.-H., Soft Matter 2013, 9, 5839. 20. Pietsch, C.; Mansfeld, U.; Guerrero-Sanchez, C.; Hoeppener, S.; Vollrath, A.; Wagner, M.; Hoogenboom, R.; Saubern, S.; Thang, S. H.; Becer, C. R.; Chiefari, J.; Schubert, U. S., Macromolecules 2012, 45, 9292. 21. Luo, C.; Liu, Y.; Li, Z., Macromolecules 2010, 43, 8101. 22. Eloi, J.-C.; Rider, D. A.; Cambridge, G.; Whittell, G. R.; Winnik, M. A.; Manners, I., J. Am. Chem. Soc. 2011, 133, 8903. 23. Ma, N.; Li, Y.; Xu, H.; Wang, Z.; Zhang, X., J. Am. Chem. Soc. 2009, 132, 442. 24. Xu, H.; Cao, W.; Zhang, X., Acc. Chem. Res. 2013, 46, 1647. 25. Hu, J.; Zhang, G.; Liu, S., Chem. Soc. Rev. 2012, 41, 5933. 26. Ulijn, R. V., J. Mater. Chem. 2006, 16, 2217.

8 | J. Name., 2012, 00, 1-3

Journal Name DOI: 10.1039/C4CC05878J 27. Zelzer, M.; Todd, S. J.; Hirst, A. R.; McDonald, T. O.; Ulijn, R. V., Biomater. Sci. 2013, 1, 11. 28. Lam, E. W.; Zwacka, R.; Seftor, E. A.; Nieva, D. R.; Davidsion, B. L.; Engelhardt, J. F.; Hendrix, M. J.; Oberley, L. W., Free Radical Biol. Med. 1999, 27, 572. 29. Lee, S. W.; Reimer, C. L.; Fang, L.; Iruela-Arispe, M. L.; Aaronson, S. A., Mol. Cell. Biochem. 2000, 20, 1723. 30. Wu, E.; Mari, B. P.; Wang, F.; Anderson, I. C.; Sunday, M. E.; Shipp, M. A., J. Cell. Biochem. 2001, 82, 549. 31. Wang, C.; Chen, Q.; Wang, Z.; Zhang, X., Angew. Chem. Int. Ed. 2010, 49, 8612. 32. Koga, H.; Toita, R.; Mori, T.; Tomiyama, T.; Kang, J.-H.; Niidome, T.; Katayama, Y., Bioconjugate Chem. 2011, 22, 1526. 33. Samarajeewa, S.; Shrestha, R.; Li, Y.; Wooley, K. L., J. Am. Chem. Soc. 2011, 134, 1235. 34. Habraken, G. J. M.; Peeters, M.; Thornton, P. D.; Koning, C. E.; Heise, A., Biomacromolecules 2011, 12, 3761. 35. Xu, Y.; Ma, R.; Zhang, Z.; He, H.; Wang, Y.; Qu, A.; An, Y.; Zhu, X. X.; Shi, L., Polymer 2012, 53, 3559. 36. Du, J.-Z.; Chen, D.-P.; Wang, Y.-C.; Xiao, C.-S.; Lu, Y.-J.; Wang, J.; Zhang, G.-Z., Biomacromolecules 2006, 7, 1898. 37. Sanson, C.; Schatz, C.; Le Meins, J.-F. o.; Brûlet, A.; Soum, A.; Lecommandoux, S. b., Langmuir 2010, 26, 2751. 38. Yew, G. H.; Mohd Yusof, A. M.; Mohd Ishak, Z. A.; Ishiaku, U. S., Polym. Degrad. Stab. 2005, 90, 488. 39. Iwamoto, A.; Tokiwa, Y., Polym. Degrad. Stab. 1994, 45, 205. 40. Yang, J.; Liu, F.; Yang, L.; Li, S., Eur. Polym. J. 2010, 46, 783. 41. Amir, R. J.; Zhong, S.; Pochan, D. J.; Hawker, C. J., J. Am. Chem. Soc. 2009, 131, 13949. 42. Kühnle, H.; Börner, H. G., Angew. Chem. Int. Ed. 2009, 48, 6431. 43. Caponi, P.-F.; Winnik, F. M.; Ulijn, R. V., Soft Matter 2012, 8, 5127. 44. Caponi, P.-F.; Qiu, X.-P.; Vilela, F.; Winnik, F. M.; Ulijn, R. V., Polym. Chem. 2011, 2, 306. 45. Chien, M.-P.; Rush, A. M.; Thompson, M. P.; Gianneschi, N. C., Angew. Chem. Int. Ed. 2010, 49, 5076. 46. Chien, M.-P.; Thompson, M. P.; Lin, E. C.; Gianneschi, N. C., Chem. Sci. 2012, 3, 2690. 47. Ku, T.-H.; Chien, M.-P.; Thompson, M. P.; Sinkovits, R. S.; Olson, N. H.; Baker, T. S.; Gianneschi, N. C., J. Am. Chem. Soc. 2011, 133, 8392. 48. de Graaf, A. J.; Mastrobattista, E.; Vermonden, T.; van Nostrum, C. F.; Rijkers, D. T. S.; Liskamp, R. M. J.; Hennink, W. E., Macromolecules 2012, 45, 842. 49. Li, J.; Ge, Z.; Liu, S., Chem. Commun. 2013, 49, 6974. 50. He, C.; Hu, Y.; Yin, L.; Tang, C.; Yin, C., Biomaterials 2010, 31, 3657. 51. Itoh, Y.; Matsusaki, M.; Kida, T.; Akashi, M., Biomacromolecules 2006, 7, 2715. 52. Baier, G.; Cavallaro, A.; Vasilev, K.; Mailänder, V.; Musyanovych, A.; Landfester, K., Biomacromolecules 2013, 14, 1103. 53. Rao, J.; Khan, A., J. Am. Chem. Soc. 2013, 135, 14056. 54. Rodriguez, A. R.; Kramer, J. R.; Deming, T. J., Biomacromolecules 2013, 14, 3610. 55. Wang, C.; Wang, Z.; Zhang, X., Acc. Chem. Res. 2012, 45, 608. 56. Xing, Y.; Wang, C.; Han, P.; Wang, Z.; Zhang, X., Langmuir 2012, 28, 6032.



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57. Kang, Y.; Wang, C.; Liu, K.; Wang, Z.; Zhang, X., Langmuir 2012, 28, 14562. 58. Dong, L.; Xia, S.; Wu, K.; Huang, Z.; Chen, H.; Chen, J.; Zhang, J., Biomaterials 2010, 31, 6309. 59. Johnston, A. P. R.; Lee, L.; Wang, Y.; Caruso, F., Small 2009, 5, 1418. 60. Samarajeewa, S.; Zentay, R. P.; Jhurry, N. D.; Li, A.; Seetho, K.; Zou, J.; Wooley, K. L., Chem. Commun. 2014, 50, 968.


J. Name., 2012, 00, 1-3 | 9

ChemComm ARTICLE


Journal Name DOI: 10.1039/C4CC05878J

Yan Ding received his B.S. degree from Jilin University in 2009. Since then he has been pursuing his Ph.D. degree in the Department of Chemistry at Tsinghua University under the supervision of Prof. Xi Zhang. His work has been focused on stimuli-responsive supramolecular assemblies, including gasand thermo-responsive assemblies.



School of Science (2013-) at Tsinghua University. Currently, he is vice president of Chinese Chemical Society. His scientific interests include supra-amphilphiles for controlled selfassembly and disassembly, supramolecular polymerization driven by host-enhanced charge transfer or π-π interactions, selenium-containing polymeric materials, interfacial assembly and two-dimensional assemblies, and single molecule force spectroscopy of polymers.

Yuetong Kang got his B.S. from the Department of Chemistry, Tsinghua University. Since 2012, he is a Ph.D. student in Prof. Xi Zhang’s group at the Department of Chemistry, Tsinghua University, Beijing. His research is focused on polymeric supra-amphiphiles for controlled assembly.

Xi Zhang received his Ph.D. degree (1992) in Polymer Chemistry and Physics at Jilin University. He joined the Department of Chemistry at Jilin University as a lecturer in 1992 and was then promoted to be a professor in 1994. He moved to Tsinghua University in late 2003. He serves the Chair of the Department of Chemistry (2008-), and the Dean of

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Table of Contents


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A novel supramolecular polymer gel constructed by crosslinking pillar[5]arene-based supramolecular polymers through metal-ligand interactions.

Protein-polymer supramolecular assemblies: a key combination for multifunctionality.

Natural supramolecular protein assemblies.

A hyperbranched supramolecular polymer constructed by orthogonal triple hydrogen bonding and host-guest interactions.

Supramolecular and dynamic covalent reactivity.

Solid-state hierarchical cyclodextrin-based supramolecular polymer constructed by primary, secondary, and tertiary azido interactions.

guest interactions.

A water-soluble supramolecular polymer constructed by pillar[5]arene-based molecular recognition.

Asymmetric synthesis of tetrahydroquinolines through supramolecular organocatalysis.

Supramolecular proteoglycan aggregate mimics: cyclodextrin-assisted biodegradable polymer assemblies for electrostatic-driven drug delivery.

Exploiting lanthanide luminescence in supramolecular assemblies.

Covalent bond or noncovalent bond: a supramolecular strategy for the construction of chemically synthesized vaccines.

ZnS quantum dots: assemblies and optical properties.

Supramolecular assemblies of nucleoside functionalized carbon nanotubes: synthesis, film preparation, and properties.

Metal-Directed Design of Supramolecular Protein Assemblies.

Supramolecular Assemblies from Poly(phenylacetylene)s.

A chiroptical switch based on supramolecular chirality transfer through alkyl chain entanglement and dynamic covalent bonding.

Discrete stimuli-responsive multirotaxanes with supramolecular cores constructed through a modular approach.

Photomodulated fluorescence of supramolecular assemblies of sulfonatocalixarenes and tetraphenylethene.

Responsive Polymer-Based Assemblies for Sensing Applications.

Supramolecular luminescence from oligofluorenol-based supramolecular polymer semiconductors.

Reversible morphological transformation between polymer nanocapsules and thin films through dynamic covalent self-assembly.

Malleable and Self-Healing Covalent Polymer Networks through Tunable Dynamic Boronic Ester Bonds.

Synthesis of discrete Re(I) di- and tricarbonyl assemblies using a [4 × 1] directional bonding strategy.