DOI: 10.1002/chem.201003502

A pH-Responsive Superamphiphile Based on Dynamic Covalent Bonds

Chao Wang, Guangtong Wang, Zhiqiang Wang, and Xi Zhang*[a]

Stimuli-responsive polymers have made rapid advances inrecent years because of prospective applications in biotech-nology and drug-delivery systems.[1] Conventionally, thestimuli-responsive groups are linked to polymers by covalentsynthesis. The new concept of “superamphiphiles” hasemerged as an alternative and powerful method for fabricat-ing stimuli-responsive self-assembled structures. Superam-phiphiles refers to amphiphiles that are synthesized by non-covalent interactions.[2] Thus, in superamphiphiles, stimuli-responsive moieties can be linked to amphiphiles on thebasis of noncovalent interactions that greatly reduces theneed for tedious chemical synthesis.[3–4]

A dynamic covalent bond is in many aspects like a nonco-valent interaction due to the dynamic nature and has al-ready led to controlled formation of gels, homopolymers,functional surfaces, and so forth.[5] Recently, van Esch et al.reported, for the first time, a low-molecular-weight amphi-phile on the basis of dynamic covalent bonds.[5g] Among dy-namic covalent bonds, the benzoic imine bond[6] is especiallyattractive, because whereas it is stable under physiologicalconditions, the linker can be hydrolyzed under mildly acidicconditions close to the extracellular pH of solid tumors.[7]

Although dynamic covalent bonding has been used widelyin the controlled formation of various supramolecular sys-tems, the usefulness as a driving force for fabrication ofpolymeric superamphiphiles has not been reported. Hereinwe report the successful synthesis of a toothbrush-type,polymeric superamphiphile based on dynamic benzoic iminebonds by using a double-hydrophilic polymer with aminemoieties and an organic molecule with benzoic aldehydegroups as building blocks. The superamphiphile can self-as-semble into spherical aggregates under physiological condi-tions. Due to the unique pH-responsive nature of the benzo-ic imine bond, the superamphiphile can be formed and dis-sociated reversibly in response to small pH fluctuations inthe physiologically accessible range; this leads to self-assem-bly and disassembly of the aggregates.

The toothbrush-type superamphiphile consists of twocomponents. As shown in Scheme 1, one component is a

double-hydrophilic block copolymer, methoxy-poly(ethy-leneglycol)114–block–poly(l-lysine hydrochloride)200 (PEG–b–PLKC) in which the PLKC segment contains primaryamine groups. The other component is 4-(decyloxy)benzal-dehyde (DBA), which contains an alkyl chain and a benzal-dehyde end group. Under physiological conditions (pH 7.4),the alkyl chain in DBA can be attached to the polylysinesegment by benzoic imine bonds formed between the aminegroups on the polylysine side chain and benzaldehydegroups in DBA; this leads to the formation of a toothbrush-type superamphiphile. When the pH is reduced to 6.5, how-ever, the benzoic imine bond is broken and the PEG–b–PLKC/DBA superamphiphile decomposes into nonamphi-philic components, resulting in disassembly of the aggregatesand release of guest molecules. When the pH changes backto 7.4, the benzoic imine bond is retrieved and the aggrega-tion is recovered.

To prepare the superamphiphile, PEG–b–PLKC andDBA were mixed in a phosphate buffer, pH 7.4, at themolar ratio PEG–b–PLKC:DBA 1:200 to ensure that there

[a] C. Wang, G. Wang, Prof. Z. Wang, Prof. X. ZhangKey Lab of Organic Optoelectronics and Molecular EngineeringDepartment of Chemistry, Tsinghua UniversityBeijing 100084 (P.R. China)Fax: (+86) 10-62771149E-mail : xi@mail.tsinghua.edu.cn

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201003502.

Scheme 1. Schematic representation of the building blocks of the super-amphiphile and the pH-responsive property of the self-assembled aggre-gates. The superamphiphile can self-assemble into spherical aggregates,which disassemble when the pH is decreased to 6.5.

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were equimolar amine and benzaldehyde groups. After soni-cation for 10 min, a transparent aqueous solution was ob-tained. Because the solubility of DBA is rather low inwater, the enhanced solubility is attributed to the formationof benzoic imine bonds. The chemical structure of the super-amphiphile was confirmed by NMR spectroscopy. As shownin Figure 1, the NMR signals for the aldehyde groups disap-

pear. Because the molar ratio between amine groups andbenzoic aldehyde is 1:1, the absence of the aldehyde protonsignal indicates that nearly all of the aldehyde groups wereconverted. A new signal appeared at 6.5 ppm, correspondingto the proton on the benzoic imine conjugate structure. Atthe same time, the signals of the protons on the alkyl chainof DBA merge into two broad signals near 0.5–2 ppm; thisis a typical feature of protons on polymers. Further evidenceof the formation of the superamphiphile is provided byFTIR. As shown in the Supporting Information, the charac-teristic IR band of the imine at 1650 cm�1 is clearly visiblein the spectrum of the PEG–b– PLKC/DBA complex.

The influence of the amine:aldehyde ratio was evaluated.When the concentration of PEG–b–PLKC was kept con-stant at 0.019 mgmL�1, samples of superamphiphiles withdifferent amine:aldehyde ratios were prepared. As shown inFigure S3 in the Supporting Information, when the molarratio is 10:1, 5:1, 3:1, 2:1, the count rates are very low, indi-cating that no aggregates are formed. When the amine:alde-hyde ratio is 1:1 and all the amine groups have been con-verted, the count rate increases to 120 kilocounts per second(kcps) instantly, indicating the formation of micellar aggre-gates. The formation of aggregates can be explained by thegrowth of the hydrophobic part. Upon addition of DBA, thehydrophilic protonated amine groups are gradually convert-ed into hydrophobic alkyl chains. Therefore, the double-hy-drophilic polymer changes into polymeric amphiphiles, in-ducing the formation of aggregates. The increase of the hy-drophobic part can also be confirmed by using pyrene as aflorescence probe. The I1/I3 value of pyrene decreases upon

the increase of aldehyde concentration. As a result, the su-peramphiphile with an amine:aldehyde ratio of 1:1, with acritical micelle concentration (cmc) of 0.012 mg mL�1, waschosen as a model system for investigation.

The concentration of the sample solution of the superam-phiphile was 0.043 mg mL�1, about three times higher thanthe cmc, to ensure the formation of self-assembled polymermicelles. The self-assembly behavior of the toothbrush-likesuperamphiphile was investigated by TEM and dynamiclaser scattering (DLS).

An aqueous solution of the superamphiphile with a PEG–b–PLKC concentration of 0.75 mg mL�1 was prepared inphosphate buffer, pH 7.4. The TEM image in Figure 2 a re-

veals that the superamphiphile self-assembled into sphericalmicelles in water. The average diameter of the aggregatesaccording to the TEM results is about 65 nm (Figure 2 b).The size of the polymer micelles was further studied byDLS, which gave an average diameter of 70 nm. At pH 6.5,the TEM images showed that very few aggregates werepresent in the solution, thus indicating disassembly of theaggregates (Figure 2 d).

NMR and FTIR experiments were performed to confirmthe pH responsiveness of the superamphiphile. Figure 3 ashows at pH was changed to 6.5, the signal at 7.8 ppm corre-sponding to the imine bond disappeared accompanied bythe appearance of a sharp signal at 9.5 ppm, which corre-sponds to the aldehyde proton. Simultaneously, the broadsignals between 0.5 and 2 ppm separated into a few sharpsignals, confirming the decomposition of the superamphi-phile. When the pH was changed back to pH 7.4, the iminesignal was recovered and the broad signals at 0.5–2 ppm re-appeared, confirming that the formation and decompositionof the superamphiphile is reversible. In the FTIR spectra(see the Supporting Information) the absorption band at

Figure 1. The 1H NMR spectrum (D2O) of PEG–b–PLKC, DBA, and themixture of PEG–b–PLKC and DBA in the molar ratio of 1:200 atpH 7.4.

Figure 2. a) TEM images of PEG–b–PLKC/DBA aggregates in a pH 7.4buffer. b) The statistical analysis of the diameter of spherical aggregates.c) DLS of PEG–b–PLKC/DBA complex at pH 7.4. d) TEM image ofPEG–b–PLKC/DBA at pH 6.5.

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1650 cm�1, ascribed to the stretching vibration of carbon–ni-trogen double bond, was significantly weakened when thepH was reduced to 6.5. The intensity of the 1650 cm�1 bandincreased when the pH was changed back to 7.4.

Because the PEG–b–PLKC/DBA complex is pH respon-sive, we considered that the pH responsiveness is responsi-ble for the disassembly of the spherical aggregates shown inFigure 2 d. To provide further evidence, the aggregation be-havior at pH 6.5 was also monitored by DLS count rate,which revealed that the aggregates disappeared at pH 6.5.When the pH was changed back to 7.4, TEM imagesshowed that the spherical aggregates were formed again,and DLS gave an average diameter of 70 nm, which indi-cates that the pH-responsive self-assembly and disassemblyprocesses are indeed reversible in accordance with the rever-sible pH responsiveness of the formation and decompositionof the superamphiphile.

The self-assembly and disassembly process in response topH was also monitored by zeta potential measurements. Be-cause the pKa of PLKC is about 9.0, the aggregates re-mained positively charged in the pH region of interest. Asshown in Figure 3 b, above pH 7.4 the zeta potential wasclose to zero as a result of the high conversion of aminogroups and the shielding effect of PEG. When the pH wasreduced to 6.5 a significant increase of the zeta potentialwas observed, indicating decomposition of the superamphi-phile and disassembly of the aggregates. When the pH waschanged back to 7.4 the zeta potential returned to zeroagain (Figure 3 c), providing additional confirmation of thereversible nature of the self-assembly.

The PEG–b–PLKC/DBA aggregate was further studied asa possible medium for encapsulation and release of guestmolecules in response to pH fluctuations under physiologi-cal conditions. Nile Red (NR) as a model guest moleculewas loaded into the spherical aggregates. Figure 4 a shows

the amount of encapsulated NR determined by fluorescenceemission microscopy. Above pH 7.4 the amount of NR en-capsulated was almost constant at a concentration of about0.6 mg mL�1 according to the fluorescence intensity. Howev-er, at a mildly acidic pH value (6.5), the fluorescence inten-sity decreased drastically and the encapsulated concentra-tion dropped to about 0.05 mg mL�1, indicating release of theguest molecules. The release kinetics was studied by time-dependent fluorescence microscopy. Figure 4 b shows thatwhen the pH changed from 7.4 to 6.5, the encapsulated NRwas released in less than 20 min, reflecting the fast-releasefeature of the self-assemblies. At the same time, NR can beused as a hydrophobic probe. As shown in the SupportingInformation, in a solution of PEG–b–PLKC/DBA in phos-phate buffer (pH 7.4), NR emission was observed at 641 nm,that is, emission in a hydrophobic environment. At pH 6.5,the wavelength of the maximum emission gradually shiftedto 661 nm, which is the typical emission wavelength of NRin water. The redshift of the NR emission further confirmsthe disassembly of the aggregates and release of the guestmolecules upon pH stimulus.

In conclusion, we have reported the first example of apolymeric superamphiphile based on dynamic covalentbonds. The superamphiphile can self-assemble in water toform spherical polymer micelles under physiological condi-tions and the aggregates disassemble in response to a pHstimulus, thus providing a new carrier for loading and releas-ing guest molecules. It should be noted that the assemblyand disassembly processes are reversible and that disassem-bly occurs at pH 6.5, near the extracellular pH of tumorcells. Moreover, the loaded guest molecules can be rapidlyreleased. We anticipate that dynamic covalent bonding canbe used as a driving force to fabricate superamphiphileswith different architectures as an approach to new intelli-gent materials.

Acknowledgements

This work was financially supported by the National Basic Research Pro-gram (2007CB808000), NSFC (50973051, 20974059), NSFC–DFG jointgrant (TRR 61) and Tsinghua University Initiative Scientific ResearchProgram (2009THZ02230). We acknowledge Prof. Lidong Li and FuTang at the University of Science and Technology Beijing for help withthe DLS experiments.

Figure 3. a) 1H NMR spectra of PEG–b–PLKC/DBA at different pHvalues: 7.4, 6.5, and again 7.4. b) The zeta potential of the PEG–b–PLKC/DBA aggregates at different pH values. c) The change of the zetapotential in the pH-tuning process.

Figure 4. a) The fluorescence intensity of NR at different pH values.b) The controlled-release capability of PEG–b–PLKC/DBA determinedthrough the time dependence of the fluorescence intensity of NR.

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X. Zhang et al.

Keywords: amphiphiles · benzoic imine · dynamic covalentbonds · pH responsive · self-assembly

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Received: December 5, 2010Published online: February 21, 2011

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