Self-assembly and disassembly of stimuli responsive tadpole-like single chain nanoparticles using a switchable hydrophilic/hydrophobic boronic acid cross-linker

Junliang Zhang a, Joji Tanaka a, Pratik Gurnani a, Paul Wilson a, Matthias Hartlieb a and Sébastien Perrier *abc
aDepartment of Chemistry, The University of Warwick, Coventry CV4 7AL, UK
bWarwick Medical School, The University of Warwick, Coventry CV4 7AL, UK
cFaculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. E-mail:; Tel: +44 2476 528 085

Received 18th May 2017 , Accepted 5th June 2017

First published on 6th June 2017

Living systems are driven by molecular machines that are composed of folded polypeptide chains, which are assembled together to form multimeric complexes. Although replicating this type of system is a longstanding goal in polymer science, the complexity the structures impose is synthetically very challenging, and generating synthetic polymers to mimic the process of these assemblies appears to be a more appealing approach. To this end, we report a linear polymer programmable for stepwise folding and assembly to higher order structures. To achieve this, a diblock copolymer composed of 4-acryloylmorpholine and glycerol acrylate was synthesised with high precision via reversible addition fragmentation chain transfer polymerisation (Đ < 1.22). Both intramolecular folding and intermolecular assembly were driven by a pH responsive cross-linker, benzene-1,4-diboronic acid. The resulting intramolecular folded single chain nanoparticles were well defined (Đ < 1.16) and successfully assembled into a multimeric structure (Dh = 245 nm) at neutral pH with no chain entanglement. The assembled multimer was observed with a spherical morphology as confirmed by TEM and AFM. These structures were capable of unfolding and disassembling either at low pH or in the presence of sugar. This work offers a new perspective for the generation of adaptive smart materials.


Nature uses the sophisticated machinery of the cell to confer precision on its biopolymers (e.g. proteins) in one-dimension through their primary sequences, and in three-dimensions (3D) via their subsequent secondary and tertiary structures, as well as their molecular organisation into multimeric complexes, all of which are imperative for the polymers to perform their specific biological functions. The 3D architectures of proteins originate from the controlled dynamic folding process of a single-stranded polypeptide chain and further self-assembling into selectively tailored molecular assemblies and interfaces which interact and respond to their environment.1–4 Folding a single linear polymer chain into a single chain nanoparticle (SCNP) has been utilized as a versatile way of constructing polymeric nanoparticles to copy nature's ability to form well-defined structures and is a rapidly expanding research area in polymer science.5–30 SCNPs can not only mimic the delicate controlled folding process of proteins with controlled size and morphology,31–33 but can also self-assemble into more complexed 3D structures.34 Furthermore stimuli-responsive polymeric nanoparticles, also called “smart” or “intelligent” nanoparticles that are capable of conformational and chemical changes by adapting to the external stimuli,35,36 have increasingly attracted interest due to their diverse range of applications in the delivery and release of drugs,37,38 diagnostics,39 and sensors.40 Dynamic covalent chemistry is a very suitable candidate for building intelligent materials which can be responsive to the environmental changes, such as pH or other stimuli.33,41–44 Boronic acid containing macromolecules have been widely utilized as an effective route toward bioresponsive architectures and a large body of research has been carried out.45–51 Boronic acid derivatives reversibly react with 1,2- and 1,3-diols (i.e. saccharides) to form boronic or boronate esters depending on the environmental pH.52 At high pH, the anionic boronate ester is hydrophilic (Scheme 1a). Upon acidification the boronate moieties are converted to neutral/hydrophobic groups (Scheme 1b).53,54 Sumerlin et al. reported a novel example of boronic acid containing triply-responsive “schizophrenic” diblock copolymers which displayed self-assembly in response to changes in temperature, pH, and the concentration of diol.52
image file: c7py00828g-s1.tif
Scheme 1 (a) Equilibrium formation of boronate esters from 1,2-diols at high pH in water; (b) equilibrium formation of boronic esters from 1,2-diols at neutral pH in water; (c) schematic representation of the synthesis of hydrophilic diblock copolymers of AB1 and AB2 by RAFT polymerization. (d) Schematic representation of the synthesis of tadpole-like SCNPs.

The self-assembly of amphiphilic diblock copolymers has attracted considerable interest to generate stimuli responsive nanoparticles with tailored structures.35,55–57 The structures and properties of superparticles formed by self-assembled SCNPs have been proven to be entirely different from the traditional block copolymer micelles.58 Zhao et al.59 and Chen et al.58 reported the first examples of the self-assembly and disassembly of diblock single chain Janus nanoparticles (SCJNPs). However, these self-assemblies were obtained either in an organic solvent or required the involvement of an organic solvent to assist the solubility of the hydrophobic part, which will limit the application under physiological conditions. Besides, the disassembly was achieved by utilizing ultra-sonication which will also circumvent its wide use due to the destructive effect of sonication.60

Herein, we report a novel synthesis of completely water soluble SCNPs from a 1,2-diol pendant linear precursor polymer, using a boronic acid crosslinker and utilising the aforementioned pH dependency of boronate esters to promote self-assembly. In contrast to the studies of Zhao et al. and Chen et al., we investigated self-assembly without the need for switching solvents and, also new to this field, we investigated the dis-assembly of the SCNPs back to the linear precursor using pH and sugars as chemical stimuli.

Results and discussion

In the present contribution, 4-acryloylmorpholine (NAM) and glycerol acrylate (GLA, synthesized by adapting the published procedure,61 Scheme S1, Fig. S1 and S2) were used as monomers to fabricate water soluble, 1,2-diol-containing copolymers. Two diblock copolymers were designed with an initial hydrophilic block of poly(NAM) (Block A), comprising 100 units, to impart water solubility for the later self-assembled structure followed by a statistical hydrophilic segment of NAM/GLA (Block B, 100 units in total) able to react with a suitable diboronic acid cross-linker to form tadpole-like SCNPs.

In order to investigate the effect of the relative molar fractions of the hydrophobic block on the self-assembly behaviour of the SCNPs, B block copolymers with two different compositions were synthesized: PolyNAM100-b-Poly(NAM80-stat-GLA20) (AB1) and PolyNAM100-b-Poly(NAM20-stat-GLA80) (AB2). As illustrated in Scheme 1c, optimized RAFT conditions as previously described for the synthesis of water soluble multiblock copolymers (azoinitiator: VA-044 at 70 °C in H2O)62 were applied to provide a fast (within 2 hours) and quantitative monomer conversion while maintaining high control over the molar mass, narrow dispersity, and high theoretical livingness. 2-[(Butylthio-carbonothioyl)thio]propanoic acid [called (propanoic acid)yl butyl trithiocarbonate (PABTC) in this paper] and 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) were used as the chain transfer agent (CTA) and the initiator, respectively. After 2 h of polymerization of each block (see the ESI for a detailed procedure), near quantitative monomer conversion (>99%) was obtained and confirmed by 1H NMR spectroscopy analysis for both diblock copolymers (Fig. S3 and S4). 1H NMR spectroscopy of both diblock copolymers confirmed the presence of the peaks associated with each segment, especially the presence of the diol functional group at 4.81 and 4.64 ppm (Fig. S3 and S4, signals a and a′). Size exclusion chromatography (SEC) in DMF revealed a monomodal distribution and a shift towards a higher molar mass confirming the successful chain extension after polymerization (Fig. S5 and S6). While a narrow dispersity was detected for both copolymers [PNAM100-b-(PNAM80-GLA20), AB1, Đ = 1.14; PNAM100-b-(PNAM20-GLA80), AB2, Đ = 1.22, Table 1], it needs to be noted that, for the AB2 copolymer, a low molar mass tail was observed in the chromatogram (Fig. S6).

Table 1 Characterization of linear copolymers and SCNPs by 1H NMR spectroscopy, DMF-SEC, DLS and DSC
Sample Composition M n,th g mol−1 M p,SEC g mol−1 M n,SEC g mol−1 Đ < G > c D h nm PDId T g °C
a M n,th = [M]0 × p × MM/[CTA]0 + MCTA, p is the monomer conversion determined by 1H NMR spectroscopy. b Determined by SEC in DMF with PMMA used as molecular weight standards, Mp represents the maximum peak value of the size-exclusion chromatogram. c Compaction parameter <G> = Mp,SCNP/Mp,linear, the molecular weight variation caused by the cross-linking reaction (e.g. the increased DBA units) was not taken into account. d Hydrodynamic diameter (Dh) and size distributions were measured by dynamic light scattering (DLS) in H2O. See the ESI for experimental details. e Glass transition temperature: determined by the second heating curve of DSC.
A PNAM100 14[thin space (1/6-em)]400 14[thin space (1/6-em)]800 14[thin space (1/6-em)]100 1.07 159.2
AB 1 PNAM100-b-P(NAM80-stat-GLA20) 28[thin space (1/6-em)]600 27[thin space (1/6-em)]200 23[thin space (1/6-em)]700 1.14 7.7 0.07 147.9
AB 1 SCNP PNAM100-b-[P(NAM80-stat-GLA20)]SCNP 24[thin space (1/6-em)]400 19[thin space (1/6-em)]900 1.17 0.90 6.1 0.05 172.4
AB 2 PNAM100-b-P(NAM20-stat-GLA80) 28[thin space (1/6-em)]900 27[thin space (1/6-em)]700 22[thin space (1/6-em)]100 1.22 6.5 0.08 95.8
AB 2 SCNP PNAM100-b-[P(NAM20-stat-GLA80)]SCNP 23[thin space (1/6-em)]700 20[thin space (1/6-em)]300 1.16 0.86 5.0 0.08 172.6

This is due to the low re-initiation efficiency of a polyacrylamide macroCTA towards the acrylate monomer considering the large amount of the acrylate monomer in the second block.63 The high molecular weight shoulder evident in the SEC trace of the AB2 copolymer (Fig. S6) is likely associated with the copolymerization of the macromonomer formed by the propagating radical undergoing backbiting β-scission during the radical polymerization of acrylates,64,65 which will not affect the following cross-linking reaction.

As shown in Scheme 1d, the intramolecular cross-linking of the linear polymer chains was obtained by the reaction of the pendant diol groups along the polymer backbone with a cross-linker. In order to reduce the competing intermolecular crosslinking, the reaction is usually carried out at high dilutions (∼10−5–10−6 mol L−1).31 However, as even under dilute conditions intermolecular cross linking is unavoidable,66 continuous addition of one reactant dropwise to the solution of the other reactant is preferable.31 In this work, the synthesis of the tadpole-like SCNPs was carried out by the continuous drop-wise addition (i.e. 15 minutes for AB1, 30 minutes for AB2, see the ESI for a detailed procedure) of the solution of the cross-linker benzene-1,4-diboronic acid (DBA, 0.5 equivalents per diol group) to a premade basic aqueous solution (pH = 10) of the linear polymer precursor, in order to fold the second block. To investigate whether the single chain folding was successful, SEC, dynamic light scattering (DLS), and differential scanning calorimetry (DSC) analysis were performed.

SEC is an ideal technique to monitor any changes in the hydrodynamic volume of a polymer chain allowing us to distinguish between linear precursors, SCNP and intermolecular crosslinked species.67–70 Comparing the SEC chromatograms of the obtained materials with their parent linear copolymers, a shift towards a lower molar mass (i.e. smaller hydrodynamic volume, Fig. 1) was observed for both cross-linking reactions, suggesting the successful formation of single chain polymeric nanoparticles AB1SCNP and AB2SCNP. These results are consistent with previous literature about the intramolecular cross linking of a single polymer chain.33,43,66,71–74 The compaction parameter <G> calculated according to the method of Lutz et al.,67 by comparison of the maximum peak values of the linear precursor and the compacted polymer chains, was obtained to be 0.90 and 0.86 for AB1SCNP and AB2SCNP, respectively (Table 1). These values closely match those of tadpole-like (P-shaped) macromolecules reported by Lutz et al.67 The relatively smaller <G> value of AB2SCNP is likely due to the more significant extent of folding of AB2 given the relatively larger amount of cross-linkable units.

image file: c7py00828g-f1.tif
Fig. 1 SEC chromatograms (RI traces) obtained in DMF for: (a) AB1 and AB1SCNP; (b) AB2 and AB2SCNP.

DLS measurements revealed a characteristic decrease in the hydrodynamic diameter (Dh) of AB1SCNP and AB2SCNP compared to the corresponding linear precursor, which further indicates the intramolecular collapse and the formation of SCNPs (Fig. 2). The average hydrodynamic diameter decreased from 7.7 nm for AB1 to 6.1 nm for AB1SCNP and from 6.5 nm for AB2 to 5.0 nm for AB2SCNP (Table 1).

image file: c7py00828g-f2.tif
Fig. 2 Hydrodynamic size distributions obtained by DLS in H2O for (a) AB1 and AB1SCNP (pH = 10.02); (b) AB2 and AB2SCNP (pH = 10.20).

DSC analysis was also conducted to demonstrate the successful formation of SCNPs. Compared to the linear polymer, the chain mobility of SCNPs will decrease, resulting in an increased glass transition temperature (Tg) value.31,75–77 The Tg value of the AB1SCNP increased significantly to 172.4 °C from the initial value of 147.9 °C for the linear polymer AB1 (Table 1, Fig. S7; note that the signal at 90 °C is a measurement artefact, see Fig. S8 and the ESI for a detailed explanation). On the other hand, the linear copolymer AB2 contains a larger fraction of GLA in the second block (B2) which leads to a broader glass transition process and a decreased Tg (95.8 °C, Table 1, Fig. S9) compared to AB1 (147.9 °C). The disappearance of the Tg value at 95.8 °C and the characteristic glass transition process with a Tg value of 172.6 °C (Fig. S9) indicate the successful compaction of AB2 leading to the formation of AB2SCNP. The more dramatic change of Tg for AB2SCNP should be caused by the higher degree of compaction which is consistent with the SEC results.

Due to the wide pH ranges present in biological and physiological systems the application of pH-responsive polymeric nanoparticles for controlled encapsulation and release is of great interest.78 The self-assembly behaviour of the tadpole-like SCNPs was investigated by varying the environmental pH. At high pH, the cross-linker exists as hydrophilic anionic boronate esters (Scheme 1a and d);52,79 therefore both segments of the diblock copolymers are hydrophilic. As the pH is lowered to neutral (pH ≈ 7.5), the majority of the cross-linker will become neutral, i.e. hydrophobic boronic esters, causing the tadpole-like SCNPs to be amphiphilic. This in turn will result in the phase segregation of the hydrophobic cross linked “head” block to form the core of a micellar assembly whereas the hydrophilic “tail” segment of NAM constitutes the shell. If the pH is further lowered to acidic conditions, the boronic esters will be hydrolysed to yield free boronic acids and diols (Scheme 1b).79

The self-assembly behaviour of tadpole-like SCNPs adapting the pH changes was monitored by DLS analysis. When the pH of the aqueous solution of the AB1SCNP was gradually lowered from basic (pH = 10.02) to acidic (pH = 2.36), the particles displayed similar sizes across the whole range and no self-assembly was observed (Table S1 and Fig. S10). On the other hand, when the pH of the aqueous solution of AB2SCNP was lowered from basic to neutral, multimolecular aggregates were observed which indicated the occurrence of self-assembly. The hydrodynamic diameters of AB2SCNP increased from 5.0 nm (at pH 10.20) to 111 nm and 245 nm at pH 8.00 and 7.60, respectively (Table S2, Fig. 3 and S11), revealing that the aggregate size could vary depending on the pH. Upon further lowering the pH to acidic, DLS displayed the dissociation of the aggregates and hydrolysis of the boronic esters leading to the formation of polymers with slightly bigger sizes than AB2SCNP under basic conditions (Table S2, Fig. S11). This phenomenon is consistent with the assumption that assembled micellar structures were formed, composed of a hydrophilic polyNAM shell and a hydrophobic core, the size of which gradually increases when the pH was decreased as the anionic/hydrophilic boronate ester groups were converted to neutral/hydrophobic boronic ester groups. Once the pH-value reached a critical level, the hydrolysis of boronic esters started occurring and led to the dissociation of the micelles. It is noteworthy that under acidic conditions (pH ≈ 2), AB1SCNP still displays a similar size to that under basic conditions, whereas AB2SCNP shows an increased size value. 1H NMR and SEC studies were utilized to investigate the transition further.

image file: c7py00828g-f3.tif
Fig. 3 Hydrodynamic size distributions obtained by DLS in H2O for: AB2, AB2SCNP at pH = 10.20, and AB2SCNP self-assembly at pH = 7.60.

In order to be able to monitor the hydrolysis of boronic esters, DMSO-d6 was used to observe the appearance of OH groups of the GLA unit. 1H NMR spectroscopy investigation of AB1 and AB1SCNP in DMSO-d6 was examined first (Fig. 4, the integral of the peaks between δ = 1.90 and 1.30 ppm was used as an internal reference; see the methods part in the ESI for information of peak integration). The spectrum of AB1SCNP at pH 10.02 revealed the appearance of signals associated with crosslinked DBA (peak b; for a comparison with free DBA mixed with free linear polymer AB1, see the top spectrum in Fig. 4; for a comparison with free DBA and free DBA at pH ≈ 10, see Fig. S12 and S13, respectively). The spectrum displayed the signals of unreacted diol groups (peaks a and a′) which is probably due to the high steric hindrance after the folding of the polymer.70,71 The 1H NMR spectrum of AB1SCNP under acidic conditions (pH = 2.36) revealed that the integral of the signals associated with the free diol (peaks a and a′) increased to 26.01 from 14.09 (for pH = 10.02), indicating 46% [(26.01–14.09) ÷ (40.00–14.09) = 46%, see the ESI for a detailed explanation] hydrolysis of the total number of boronic esters. Similarly the integration of aromatic protons (peaks b + b′) and OH groups (peak c) corresponding to the DBA cross-linker also demonstrates an equivalent value for hydrolysis. This equates to a value between 100% and 53% of the cross-linker still being attached to the polymer backbone depending on the number of DBA units existing as a mono-boronic ester (100%, meaning all the DBA units were attached to the polymer backbone by one side) and di-boronic ester [53%, in this case all the OH groups (peak c) corresponding to the DBA cross-linker belong to free DBA units; therefore the amount of the cross-linker still being attached to the polymer backbone is 28.32–13.45 = 14.87. The percentage of the attached DBA is therefore calculated to be 14.87 ÷ 28.32 = 53%], respectively. It is noteworthy that the signals of aromatic protons (peak b) corresponding to the DBA cross-linker attached to the polymer chain shifted downfield at lower pH. This is consistent with the fact that boronate esters are negatively charged at high pH causing a rich electron environment (low chemical shift) around the aromatic ring and a poor electron environment (high chemical shift) when uncharged at low pH.

image file: c7py00828g-f4.tif
Fig. 4 1H NMR spectra (300 MHz, DMSO-d6) of: (from bottom to top) linear copolymer AB1, folded copolymer AB1SCNP at pH = 10.02, folded copolymer AB1SCNP at pH = 2.36, and linear copolymer AB1 mixed with free DBA cross-linker in DMSO-d6.

We found AB2SCNP to be insoluble in the NMR solvent we used for this investigation, due to the high density of the anionic boronate ester formed (see Fig. S14 for DBA at pH ≈ 10 in DMSO-d6). However, the 1H NMR spectrum of AB2SCNP under acidic conditions (pH = 2.50, Fig. S15) also displays a similar profile to that of AB1SCNP, revealing between 84% and 42% (see the ESI for the detailed calculation) of DBA cross-linker still attached to the polymer backbone.

The SEC analysis of AB1SCNP under acidic conditions (pH = 2.36) displays a slightly smaller hydrodynamic volume compared to the linear precursor AB1 (<G> = 0.96, Fig. S16) but a higher hydrodynamic volume than AB1SCNP at pH = 10.02 which is consistent with the hydrolysis of the boronic esters. This minor shift is likely to be associated with the low amount of residual intramolecular cross-linking. It is noteworthy that the SEC analysis of self-assembled AB2SCNP under around neutral conditions (pH = 7.60) demonstrates the retention of the tadpole-like SCNP structure with no apparent intermolecular exchange of the DBA cross-linker (Fig. S17). Despite the close proximity of the hydrophobic “heads” in solution and the dynamic nature of the boronic ester, intermolecular exchange of the DBA cross-linker was not apparent, otherwise a higher molar mass shoulder would have been observed in the SEC trace. Moreover, a smaller compaction parameter (<G> = 0.78, Table S3) compared to AB2SCNP at pH = 10.20 (<G> = 0.86) was observed. We suspect that the anionic boronate esters are more solvated due to the solvent screening the charge, hence neutralising the charge reduces the swelling. The SEC trace of the AB2SCNP under acidic conditions (pH = 2.50) shows a shift towards a higher molar mass compared to AB2SCNP at pH = 7.60, suggesting the hydrolysis of the boronic esters (Fig. S17). However, compared to the linear precursor, it still displays lower molar mass distribution indicating intramolecular cross-linking (<G> = 0.88, Table S3). These results are consistent with the 1H NMR analysis. The more pronounced compaction displayed by AB2SCNP compared to AB1SCNP under acidic conditions is likely due to the increased amount of the cross-linker in AB2SCNP which caused the de-crosslinking to be less efficient.

It is interesting to note that while DMF-SEC of AB1SCNP under acidic conditions (pH = 2.36) only shows a minor shift towards a lower molar mass compared to AB1 (Fig. S16) DLS still displays a similar size to AB1SCNP under basic conditions (Table S1, Fig. S10); whereas AB2SCNP under acidic conditions (pH = 2.50) reveals a relatively big shift toward a lower molar mass compared to AB2 by DMF-SEC (Fig. S17) but displays bigger size distribution than AB2SCNP under basic conditions in DLS (Table S2, Fig. S11). This is probably due to the hydrophobicity of the remaining DBA cross-linker attached to AB1SCNP under acidic conditions causing the chains to collapse in H2O leading to a smaller size as reflected by DLS. On the other hand, considering there are still relatively high amounts of DBA cross-linkers in AB2SCNP under acidic conditions as illustrated by DMF-SEC (Fig. S17), these hydrophobic DBA cross-linkers will still cause the aggregation of AB2SCNP to a certain extent which caused bigger sizes than AB2SCNP under basic conditions but are insufficient for self-assembly into bigger particles. Therefore, it is reasonable to assume that AB2SCNP under acidic conditions in H2O is composed of small self-assembled aggregates consisting of amphiphilic tadpole-like SCNPs with a low degree of compaction. The reason why AB1SCNP did not self-assemble into micellar structures was hypothesized to be due to the low amount of the boronate ester compared to AB2SCNP as a result of the low diol content of AB1, and therefore insufficient hydrophobicity to promote self-assembly.

Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging were employed to further explore the morphology of the nanoparticles formed by the self-assembly of AB2SCNP at pH 7.60 in aqueous solution. Spherical nano-objects with diameter sizes of around 38 (±6.6) nm were visualized by TEM (Fig. 5 and S18). AFM also revealed nanoparticles with similar diameter values to TEM (Fig. 6, S19 and S20, samples used for TEM and AFM were diluted 10 times after self-assembly of AB2SCNPat pH = 7.60). The relatively small size compared to the values obtained by DLS analysis could be due to the shrinking of the samples in the dry state, whereas water-swollen structures were observed in aqueous solution using DLS.

image file: c7py00828g-f5.tif
Fig. 5 Representative image of nanoparticles formed by the self-assembly of AB2SCNP obtained by TEM (a) and size distributions of nanoparticles analyzed from TEM results (b).

image file: c7py00828g-f6.tif
Fig. 6 Representative AFM topography image of nanoparticles formed by the self-assembly of AB2SCNP. The red line in the topography image shows the analyzed particles.

In addition to the pH responsive nature, the diol responsiveness of the tadpole-like SCNPs and the self-assembled micelles was also investigated in order to exploit the potential applications in sensors for sugars.80 Due to the reversible nature of the dynamic covalent bond of the cyclic boronate/boronic esters formed by the boronic acid groups with 1,2- and 1,3-diols,52,80 the free diol containing molecules will competitively react with boronic ester via transesterification. Upon the addition of glucose to the aqueous solution of the AB1SCNP and AB2SCNP under basic conditions, decross-linking of the SCNPs was triggered leading to polymers with similar sizes to the respective linear precursor as detected by DLS (Fig. 7). SEC analysis of the SCNP samples treated with sugar also revealed similar molar mass distributions to the corresponding linear copolymers (Fig. S21 and S22). The addition of glucose to the solution of micelles formed by the self-assembly of AB2SCNP at pH 7.60 caused the disruption of the self-assembled structure and led to the formation of unimers as displayed by DLS showing a similar hydrodynamic diameter to the linear AB2 (Fig. 7b). In addition to the DLS results, dissociation was also illustrated by SEC (Fig. S22) analysis which shows similar molar mass distribution to the AB2 precursor for the disassembled sample.

image file: c7py00828g-f7.tif
Fig. 7 Hydrodynamic size distributions obtained by DLS in H2O for: (a) AB1SCNP at pH = 10.02, AB1SCNP with the addition of glucose at pH = 10.02, and the linear copolymer AB1; (b) linear copolymer AB2, AB2SCNP with the addition of glucose at pH = 10.20, AB2SCNP self-assembly with the addition of glucose at pH = 7.60, and AB2SCNP self-assembly at pH = 7.60.


In summary, tadpole-like SCNPs were synthesised using a pH responsive DBA cross-linker and suitable linear polymer precursors, which exhibited self-assembly due to the hydrophobic nature of the cross-linker past its isoelectric point. The assembled SCNPs displayed spherical morphology as characterised by TEM and AFM. The intramolecular folding of individual SCNPs was intact and no chain entanglement occurred after self-assembly according to the SEC. We found that the volume fraction of cross-linkable GLA in the second block played a crucial role in the self-assembly of the SCNP, as sufficient hydrophobicity is required to promote the “head” group to drive self-assembly. The dissociation of assemblies can be triggered by varying the environmental pH or exposing to an external stimulus as demonstrated by the addition of glucose. The use of boronic acid containing polymers for pH dependent self-assembly has been demonstrated elsewhere; however, forming an SCNP with a boronic acid cross-linker and taking advantage of its stimuli-responsive properties to drive self-assembly have not been reported. The present study demonstrates the ability of synthetic polymers to mimic the folding of natural polypeptide chains and assembly into a higher order structure found in natural multiprotein complexes, which also display a stimuli responsive character. We hope this study will encourage more research in this active area and provide more perspective for building more complexed biomimetic self-assembled structures with potential applications in healthcare.


The Royal Society Wolfson Merit Award (WM130055; SP) and the Monash-Warwick Alliance are acknowledged for funding (SP; PG; JZ). JT is funded by the Engineering and Physical Sciences Research Council (EPSRC) under grant EP/F500378/1 through the Molecular Organisation and Assembly in Cells Doctoral Training Centre (MOAC-DTC). MH gratefully acknowledges the German Research Foundation (DFG, GZ: HA 7725/1-1) for funding. PW thanks the Leverhulme Trust for the award of an Early Career Fellowship (ECF/2015-075).

Notes and references

  1. C. B. Anfinsen, Science, 1973, 181, 223–230 CAS.
  2. C. M. Dobson, Nature, 2003, 426, 884–890 CrossRef CAS PubMed.
  3. C. S. Mahon and D. A. Fulton, Nat. Chem., 2014, 6, 665–672 CrossRef CAS PubMed.
  4. M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Muller, C. Ober, M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, F. Winnik, S. Zauscher, I. Luzinov and S. Minko, Nat. Mater., 2010, 9, 101–113 CrossRef PubMed.
  5. A. M. Hanlon, C. K. Lyon and E. B. Berda, Macromolecules, 2016, 49, 2–14 CrossRef CAS.
  6. C. K. Lyon, A. Prasher, A. M. Hanlon, B. T. Tuten, C. A. Tooley, P. G. Frank and E. B. Berda, Polym. Chem., 2015, 6, 181–197 RSC.
  7. M. Ouchi, N. Badi, J. F. Lutz and M. Sawamoto, Nat. Chem., 2011, 3, 917–924 CrossRef CAS PubMed.
  8. O. Altintas and C. Barner-Kowollik, Macromol. Rapid Commun., 2016, 37, 29–46 CrossRef CAS PubMed.
  9. O. Altintas and C. Barner-Kowollik, Macromol. Rapid Commun., 2012, 33, 958–971 CrossRef CAS PubMed.
  10. M. Gonzalez-Burgos, A. Latorre-Sanchez and J. A. Pomposo, Chem. Soc. Rev., 2015, 44, 6122–6142 RSC.
  11. J. A. Pomposo, Polym. Int., 2014, 63, 589–592 CrossRef CAS.
  12. A. Sanchez-Sanchez, I. Perez-Baena and J. A. Pomposo, Molecules, 2013, 18, 3339–3355 CrossRef CAS PubMed.
  13. S. Mavila, O. Eivgi, I. Berkovich and N. G. Lemcoff, Chem. Rev., 2016, 116, 878–961 CrossRef CAS PubMed.
  14. M. Huo, N. Wang, T. Fang, M. Sun, Y. Wei and J. Yuan, Polymer, 2015, 66, A11–A21 CrossRef CAS.
  15. J. Zhang, G. Gody, M. Hartlieb, S. Catrouillet, J. Moffat and S. Perrier, Macromolecules, 2016, 49, 8933–8942 CrossRef CAS.
  16. N. Hosono, A. M. Kushner, J. Chung, A. R. A. Palmans, Z. Guan and E. W. Meijer, J. Am. Chem. Soc., 2015, 137, 6880–6888 CrossRef CAS PubMed.
  17. T. Mes, R. van der Weegen, A. R. Palmans and E. W. Meijer, Angew. Chem., Int. Ed., 2011, 50, 5085–5089 CrossRef CAS PubMed.
  18. N. Hosono, A. R. A. Palmans and E. W. Meijer, Chem. Commun., 2014, 50, 7990–7993 RSC.
  19. O. Altintas, P. Krolla-Sidenstein, H. Gliemann and C. Barner-Kowollik, Macromolecules, 2014, 47, 5877–5888 CrossRef CAS.
  20. O. Altintas, E. Lejeune, P. Gerstel and C. Barner-Kowollik, Polym. Chem., 2012, 3, 640–651 RSC.
  21. E. H. H. Wong, S. J. Lam, E. Nam and G. G. Qiao, ACS Macro Lett., 2014, 3, 524–528 CrossRef CAS.
  22. J. B. Beck, K. L. Killops, T. Kang, K. Sivanandan, A. Bayles, M. E. Mackay, K. L. Wooley and C. J. Hawker, Macromolecules, 2009, 42, 5629–5635 CrossRef CAS PubMed.
  23. O. Shishkan, M. Zamfir, M. A. Gauthier, H. G. Borner and J.-F. Lutz, Chem. Commun., 2014, 50, 1570–1572 RSC.
  24. O. Altintas, J. Willenbacher, K. N. R. Wuest, K. K. Oehlenschlaeger, P. Krolla-Sidenstein, H. Gliemann and C. Barner-Kowollik, Macromolecules, 2013, 46, 8092–8101 CrossRef CAS.
  25. A. M. Hanlon, I. Martin, E. R. Bright, J. Chouinard, K. J. Rodriguez, G. E. Patenotte and E. B. Berda, Polym. Chem., 2017 10.1039/C7PY00320J.
  26. R. Lambert, A.-L. Wirotius and D. Taton, ACS Macro Lett., 2017, 489–494,  DOI:10.1021/acsmacrolett.7b00161.
  27. T.-K. Nguyen, S. J. Lam, K. K. K. Ho, N. Kumar, G. G. Qiao, S. Egan, C. Boyer and E. H. H. Wong, ACS Infect. Dis., 2017, 3, 237–248 CrossRef CAS PubMed.
  28. T. S. Fischer, D. Schulze-Sünninghausen, B. Luy, O. Altintas and C. Barner-Kowollik, Angew. Chem., Int. Ed., 2016, 55, 11276–11280 CrossRef CAS PubMed.
  29. N. D. Knöfel, H. Rothfuss, J. Willenbacher, C. Barner-Kowollik and P. W. Roesky, Angew. Chem., Int. Ed., 2017, 56, 4950–4954 CrossRef PubMed.
  30. M. Aiertza, I. Odriozola, G. Cabañero, H.-J. Grande and I. Loinaz, Cell. Mol. Life Sci., 2012, 69, 337–346 CrossRef CAS PubMed.
  31. E. Harth, B. V. Horn, V. Y. Lee, D. S. Germack, C. P. Gonzales, R. D. Miller and C. J. Hawker, J. Am. Chem. Soc., 2002, 124, 8653–8660 CrossRef CAS PubMed.
  32. E. B. Berda, E. J. Foster and E. W. Meijer, Macromolecules, 2010, 43, 1430–1437 CrossRef CAS.
  33. B. S. Murray and D. A. Fulton, Macromolecules, 2011, 44, 7242–7252 CrossRef CAS.
  34. N. Hosono, M. A. J. Gillissen, Y. Li, S. S. Sheiko, A. R. A. Palmans and E. W. Meijer, J. Am. Chem. Soc., 2013, 135, 501–510 CrossRef CAS PubMed.
  35. N. J. W. Penfold, J. R. Lovett, P. Verstraete, J. Smets and S. P. Armes, Polym. Chem., 2017, 8, 272–282 RSC.
  36. I. Cobo, M. Li, B. S. Sumerlin and S. Perrier, Nat. Mater., 2015, 14, 143–159 CrossRef CAS PubMed.
  37. B. Surnar and M. Jayakannan, Biomacromolecules, 2013, 14, 4377–4387 CrossRef CAS PubMed.
  38. J. Du, L. Fan and Q. Liu, Macromolecules, 2012, 45, 8275–8283 CrossRef CAS.
  39. X. Sun and T. D. James, Chem. Rev., 2015, 115, 8001–8037 CrossRef CAS PubMed.
  40. M. A. J. Gillissen, I. K. Voets, E. W. Meijer and A. R. A. Palmans, Polym. Chem., 2012, 3, 3166–3174 RSC.
  41. S. J. Rowan, S. J. Cantrill, G. R. L. Cousins, J. K. M. Sanders and J. F. Stoddart, Angew. Chem., Int. Ed., 2002, 41, 898–952 CrossRef PubMed.
  42. D. E. Whitaker, C. S. Mahon and D. A. Fulton, Angew. Chem., Int. Ed., 2013, 52, 956–959 CrossRef CAS PubMed.
  43. A. Sanchez-Sanchez, D. A. Fulton and J. A. Pomposo, Chem. Commun., 2014, 50, 1871–1874 RSC.
  44. A. Sanchez-Sanchez and J. A. Pomposo, Part. Part. Syst. Charact., 2014, 31, 11–23 CrossRef CAS.
  45. W. L. A. Brooks and B. S. Sumerlin, Chem. Rev., 2016, 116, 1375–1397 CrossRef CAS PubMed.
  46. J. Ren, Y. Zhang, J. Zhang, H. Gao, G. Liu, R. Ma, Y. An, D. Kong and L. Shi, Biomacromolecules, 2013, 14, 3434–3443 CrossRef CAS PubMed.
  47. F. Coumes, P. Woisel and D. Fournier, Macromolecules, 2016, 49, 8925–8932 CrossRef CAS.
  48. J. N. Cambre and B. S. Sumerlin, Polymer, 2011, 52, 4631–4643 CrossRef CAS.
  49. C. C. Deng, W. L. A. Brooks, K. A. Abboud and B. S. Sumerlin, ACS Macro Lett., 2015, 4, 220–224 CrossRef CAS.
  50. P. De, S. R. Gondi, D. Roy and B. S. Sumerlin, Macromolecules, 2009, 42, 5614–5621 CrossRef CAS.
  51. A. P. Bapat, D. Roy, J. G. Ray, D. A. Savin and B. S. Sumerlin, J. Am. Chem. Soc., 2011, 133, 19832–19838 CrossRef CAS PubMed.
  52. D. Roy, J. N. Cambre and B. S. Sumerlin, Chem. Commun., 2009, 2106–2108,  10.1039/B900374F.
  53. J. O. Edwards, G. C. Morrison, V. F. Ross and J. W. Schultz, J. Am. Chem. Soc., 1955, 77, 266–268 CrossRef CAS.
  54. J. P. Lorand and J. O. Edwards, J. Org. Chem., 1959, 24, 769–774 CrossRef CAS.
  55. A. Blanazs, S. P. Armes and A. J. Ryan, Macromol. Rapid Commun., 2009, 30, 267–277 CrossRef CAS PubMed.
  56. Y. Mai and A. Eisenberg, Chem. Soc. Rev., 2012, 41, 5969–5985 RSC.
  57. U. Haldar, M. Nandi, B. Ruidas and P. De, Eur. Polym. J., 2015, 67, 274–283 CrossRef CAS.
  58. F. Zhou, M. Xie and D. Chen, Macromolecules, 2014, 47, 365–372 CrossRef CAS.
  59. J. Wen, L. Yuan, Y. Yang, L. Liu and H. Zhao, ACS Macro Lett., 2013, 2, 100–106 CrossRef CAS.
  60. B. Smagowska and M. Pawlaczyk-Łuszczyńska, Int. J. Occup. Saf. Ergon., 2013, 19, 195–202 CrossRef PubMed.
  61. S. C. O. Sousa, C. G. L. Junior, F. P. L. Silva, N. G. Andrade, T. P. Barbosa and M. L. A. A. Vasconcellos, J. Braz. Chem. Soc., 2011, 22, 1634–1643 CrossRef CAS.
  62. G. Gody, T. Maschmeyer, P. B. Zetterlund and S. Perrier, Macromolecules, 2014, 47, 3451–3460 CrossRef CAS.
  63. L. Martin, G. Gody and S. Perrier, Polym. Chem., 2015, 6, 4875–4886 RSC.
  64. A. Postma, T. P. Davis, G. Li, G. Moad and M. S. O'Shea, Macromolecules, 2006, 39, 5307–5318 CrossRef CAS.
  65. G. Moad and C. Barner-Kowollik, in Handbook of RAFT Polymerization, Wiley-VCH Verlag GmbH & Co. KGaA, 2008, ch. 3, pp. 51–104,  DOI:10.1002/9783527622757.
  66. R. K. Roy and J. F. Lutz, J. Am. Chem. Soc., 2014, 136, 12888–12891 CrossRef CAS PubMed.
  67. B. V. Schmidt, N. Fechler, J. Falkenhagen and J. F. Lutz, Nat. Chem., 2011, 3, 234–238 CrossRef CAS PubMed.
  68. J. A. Pomposo, I. Perez-Baena, L. Buruaga, A. Alegría, A. J. Moreno and J. Colmenero, Macromolecules, 2011, 44, 8644–8649 CrossRef CAS.
  69. E. J. Foster, E. B. Berda and E. W. Meijer, J. Am. Chem. Soc., 2009, 131, 6964–6966 CrossRef CAS PubMed.
  70. B. T. Tuten, D. Chao, C. K. Lyon and E. B. Berda, Polym. Chem., 2012, 3, 3068–3071 RSC.
  71. J. B. Beck, K. L. Killops, T. Kang, K. Sivanandan, A. Bayles, M. E. Mackay, K. L. Wooley and C. J. Hawker, Macromolecules, 2009, 42, 5629–5635 CrossRef CAS PubMed.
  72. C. Heiler, J. T. Offenloch, E. Blasco and C. Barner-Kowollik, ACS Macro Lett., 2017, 6, 56–61 CrossRef CAS.
  73. A. M. Hanlon, R. Chen, K. J. Rodriguez, C. Willis, J. G. Dickinson, M. Cashman and E. B. Berda, Macromolecules, 2017, 50, 2996–3003 CrossRef CAS.
  74. J. A. Pomposo, J. Rubio-Cervilla, A. J. Moreno, F. Lo Verso, P. Bacova, A. Arbe and J. Colmenero, Macromolecules, 2017, 50, 1732–1739 CrossRef CAS.
  75. D. Mecerreyes, V. Lee, C. J. Hawker, J. L. Hedrick, A. Wursch, W. Volksen, T. Magbitang, E. Huang and R. D. Miller, Adv. Mater., 2001, 13, 204–208 CrossRef CAS.
  76. A. E. Cherian, F. C. Sun, S. S. Sheiko and G. W. Coates, J. Am. Chem. Soc., 2007, 129, 11350–11351 CrossRef CAS PubMed.
  77. C. Song, L. Li, L. Dai and S. Thayumanavan, Polym. Chem., 2015, 6, 4828–4834 RSC.
  78. J. Du and R. K. O'Reilly, Soft Matter, 2009, 5, 3544–3561 RSC.
  79. D. Roy and B. S. Sumerlin, ACS Macro Lett., 2012, 1, 529–532 CrossRef CAS.
  80. H. Fang, G. Kaur and B. Wang, J. Fluoresc., 2004, 14, 481–489 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available: Experimental details, 1H NMR spectra, GPC traces, additional tables, and figures not depicted in the manuscript. See DOI: 10.1039/c7py00828g

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