† Electronic supplementary information (and details of simulation studies. See DO
Cite this: Chem. Sci., 2014, 5, 200
Received 9th August 2013Accepted 17th September 2013
DOI: 10.1039/c3sc52233d
www.rsc.org/chemicalscience
200 | Chem. Sci., 2014, 5, 200–205
Reversibly switchable polymer with cationic/zwitterionic/anionic behavior through synergisticprotonation and deprotonation†
Harihara Subramanian Sundaram, Jean-Rene Ella-Menye, Norman D. Brault,Qing Shao and Shaoyi Jiang*
A polymer capable of fully and reversibly switching throughout the entire charge regime is highly desirable
for many applications such as drug and gene delivery, controlled ion and molecular transport and tunable
filtration membranes. It is essential that for biologically relevant applications the polymer needs to be
nonfouling. However, conventional nonfouling zwitterionic polymers have a permanently positive
quaternary nitrogen center, making it impossible to switch between charges. Here, we present a
rationally designed polymer with a tertiary amine and a carboxylic acid, which is capable of reversibly
switching among three distinct charged states, viz., cationic, zwitterionic and anionic, and importantly
maintaining the zwitterionic state under physiological pH conditions. Oppositely charged proteins
adsorbed on a charged surface selectively can be completely removed by switching the surface to the
zwitterionic state. We have also found that these two moieties (i.e., a tertiary amine and a carboxylate
moiety) stimulate each other synergistically to achieve a strongly zwitterionic state under physiological
conditions and to resist non-specific protein adsorption from undiluted blood plasma and serum when
they are close to each other.
Introduction
Smart polymers exhibit changes in behavior in response toexternal stimuli such as temperature, pH, redox chemistry, etc.,enabling control over properties such as the transport of ionsand molecules, changes in wettability, and the adhesion ofdifferent species among others.1 These functionalities are beingused for many different applications including drug delivery,2
gene delivery,3,4 ion transport,5,6 protein separation7–11 andnonfouling coatings for immensely different biological12–19 andmarine20–24 environments. Most smart polymers reportedexhibit only two reversible or irreversible charged states. Forexample, a permanently charged cationic polymer with ahydrolysable group was shown to have antimicrobial propertiesand upon hydrolysis become zwitterionic and exhibit ultra lowfouling properties.7 Similarly, a cationic polymer brush with aphoto-cleavable group was previously demonstrated to becomeanionic upon exposure to UV radiation and enable the adsorp-tion of oppositely charged dyes and proteins.8 A polymer havingrandomly distributed groups of temperature sensitive NIPAM(N-isopropyl acrylamide) and pH sensitive acrylic acid was
shown to selectively adsorb positively charged proteins abovethe lower critical solution temperature (LCST) of the NIPAMgroups.9 The oxidation and reduction states of a conductingpolymer surface coating have previously enabled control overthe wettability of oil droplets.25 In all of these examples abovethe polymer switches between only two states.
Di-block copolymers consisting of tertiary amine andcarboxylic acid based monomers have been shown to possesstwo different pKa values, one for each corresponding monomer.These polymers enable reversible micelles to form in solutionby adjusting the pH. At the isoelectric point, the two segmentsbecome cationic and anionic and the resulting polymer willbecome charge neutral if the number of units of each block arethe same.26 However, a single polymer capable of fully andreversibly switching throughout the entire charge regime, fromcationic to neutral zwitterionic to anionic, is highly desirable forapplications such as gene and drug delivery, controlled ion andmolecular transport and tunable ltration membranes. Forbiologically relevant applications it is necessary that the poly-mer exhibits excellent nonfouling properties in complex media.Ultra low fouling properties in complex media, an absoluterequirement for biological applications, have been achieved byonly few materials, prominently, zwitterionic carboxybetainepolymers.7,12,27,28 However, conventional nonfouling zwitterionicpolymers are composed of monomers containing a permanentpositively charged quaternary amine and a negatively chargedgroup, to ensure a zwitterionic structure under physiological
conditions. Due to the presence of the permanently chargedgroup, it is impossible to achieve a fully switchable materialamong all possible charged states. An amino acid based poly-mer has previously achieved reversible charge switching amongall three charged states, but with weak zwitterionic behavior,29
for selective ion transport.6 However, this material achieved thezwitterionic state around pH 5, thus not being relevant in mostbiological environments. A polymer capable of reversiblyswitching among distinctive charged states while maintaining astrong zwitterionic state in physiological conditions would beable to achieve selective adsorption of charged proteins from amixture and then their corresponding complete release.
To this end, we designed a single polymer containing atertiary amine and a carboxylic acid separated by a one carbonspacer (Scheme 1). The fully reversible charged states weregenerated by taking advantage of the two distinct pKa valuesof the tertiary amine and carboxylic acid. The tertiary aminecan exist in both positive and neutral states while thecarboxylic acid can exist in anionic and neutral forms. Theability of both groups to undergo reversible protonation ordeprotonation with pH also provides a means for hidingpositive and negative charges. When the number of carbonspacers between the tertiary amine and the carboxylic acidgroups is small enough, such as one, both sites can stimulateone another and thus affect their protonation and deproto-nation states (i.e., a synergistic effect). Hence, the combina-tion of these two moieties in a single compound presents adistinctly new material with many unique properties. For therst time, we present a single new material capable of full-charge-reversible switching for selective protein adsorptionover a wide range of pH conditions which can also achieveultra low fouling properties to undiluted human plasma andserum under physiological conditions.
Scheme 1 Schematic representation of the stimuli responsiveness ofSelective protein adsorption on a charge-reversible surface under differenstates while the carboxylic acid can exist in anionic and neutral forms. (C
Our studies in this work addressed several questions: (a) inwhich pH range will the polymer switch reversibly from cationicto zwitterionic to anionic, and will the zwitterionic state occurbetween pH 7 and 8 which is necessary for relevant biologicalapplications? (b) Will the zwitterionic state of the new polymerbehave like conventional zwitterions containing quaternaryamines and will this polymer be strong (zwitterionic) enough toresist nonspecic protein adsorption from undiluted humanplasma and serum? (c) What structural features (e.g., spacer)induce the synergism between tertiary amine and carboxylicacid groups. The goals of this work are to demonstrate that thisnew polymer presents unique capabilities, such as i) fullyreversible switching behavior between the selective adsorptionand resistance of charged bodies, via adjusting the pH of themedium, and ii) ultra-low fouling properties in complex media,similar to conventional zwitterionic materials. Scheme 1Aillustrates the expected response of the polymer to proteins ofopposite charges at either end of the charge spectrum and thenonfouling behavior in the zwitterionic state. The core revers-ible chemistry hypothesized and the predicted charged states ofthe monomer are shown in Scheme 1B and C.
Results and discussion
The monomer (CBMA-1-TAM, where CBMA, 1, and TAM standfor carboxybetaine methacrylate, the number of carbon spacers,and tertiary amine, respectively) solution was rst titratedagainst sodium hydroxide to reveal the two expected pKa values.As shown in Fig. 1A, the titration revealed pKa values of �2.6and�8.7 for the carboxylic acid and amine groups, respectively.This indicates that the monomer exists as a cationic (low pH),zwitterionic (moderate pH), and anionic (high pH) formdepending on the environmental conditions.
the polymer and the chemistry behind this reversible switching. (A)t pH values. (B) The tertiary amine can exist in both positive and neutral) The molecule in three charged states under different pH values.
Fig. 1 Titration and NMR studies supporting different charged forms of the monomer. (A) Titration curve of the monomer (CBMA-1-TAM)solution against 0.1 N sodium hydroxide; (B) peak shift changes in 1H-NMR spectra for three different monomer protons with change in pH andthe corresponding monomer structure.
Table 1 Adsorption of different proteins on the polymeric surface as afunction of pH. Surface fouling (ng cm�2) obtained from SPR issummarized for different proteins over a range of pH values. Thecharges of the proteins and the surface are also given
a Due to pepsin denaturation issues at pH 9, BSA (pI � 4.7) was used forthis particular experiment.
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To further verify the pH range for the different charged statesof CBMA-1-TAM, 1H-NMR was used to monitor the chemicalshis of three sets of protons (labeled 1, 2 and 3 in Fig. 1Band S1†) adjacent to the charged groups. The results indicatedthree distinct regions. From pH 1 to 2, tertiary amine proton-ation made the monomer cationic thereby electronicallydeshielding all three protons. Between pH 2 and 9, the mono-mer became zwitterionic as indicated by only proton 1, near theelectron-rich carboxylate, revealing any signicant shieldingeffects. Beyond pH 9, the monomer became anionic, againresulting in a chemical shi for all three neighboring protons tothe amine as it was deprotonated which leveled out with furtherincrease in pH. This pattern in peak shi conrms the existenceof the three different charged states between pH 1 and pH 11.Noteworthy, the titration and 1H-NMR data collectively indicatea zwitterionic species under relevant biological conditions(pH 6–8). It should be emphasized that the monomer behaviorcould be slightly different from a polymer brush on the surfacewhich has restricted movement due to the denser graing onthe surface as well as effects from overlapping side-chains.However, this provided fundamental information necessary forsubsequently studying the polymer system.
Polymer brushes were prepared using photopoly-merization30 (Fig. S2†) to provide further evidence of theexistence of three charged states via protein fouling experi-ments with surface plasmon resonance (SPR) spectroscopy.Iniferter polymerization is one among the controlled livingradical polymerization (CLRP) methods compatible withmany of the functional monomers.31–33 The polymerizationwas performed in methanol and water. The polymerizationwas carried out at different intervals to vary the thickness. Thesolvent ratio was known to affect the change in gra density ofthe grown polymer brushes. The concentration of the mono-mer used was kept constant throughout all the experiments at160 mM. The polymerization kinetics using 10% water inmethanol is given in Fig. S2.†
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The thickness could be adjusted from 15 nm to 60 nm bychanging the polymerization time. The pH dependent proteinadsorption was studied between pH 3 and pH 9 using lysozyme(pI � 11) and pepsin (pI � 1).10 These proteins were selectedsuch that a set of proteins are oppositely charged at any givenpH between pH 3–9. Hence, lysozyme was the positively chargedprotein while pepsin formed the negatively charged counterpartover the entire pH range. Based on the expected charge of theproteins and the corresponding fouling behavior, combined
with the monomer titration and 1H-NMR data, it was possible tostudy the surface charge-pH effect of the polymer lm madefrom CBMA-1-TAM. These results are summarized in Table 1.SPR sensorgrams showing the surface adsorption of oppositelycharged proteins at pH 3 and pH 9 are shown in Fig. S3.†
The data given in Table 1 show that between pH 4 and pH 8,neither the positively charged protein lysozyme nor the nega-tively charged pepsin adsorbed onto the surface. This indicatescharge neutrality of the brush under these conditions. Based onthe monomer titration curve, the NMR study, and the pHdependent fouling experiments using SPR, this result is due tothe polymer brushes exhibiting zwitterionic structure andshowing protein resistance from pH 4 to pH 8. However, thefouling at pH 3 and pH 9 was very different. At pH 3, thenegatively charged pepsin adsorbed heavily (24 ng cm�2)whereas the positively charged lysozyme showed only negligiblebinding. This clearly shows that the surface is positively chargedat pH 3, attracts the negatively charged protein and repels thepositively charged protein. At pH 9, only positively chargedlysozyme was strongly adsorbed on the surface (26 ng cm�2)while the negatively charged BSA (used due to the denaturationissues with pepsin at pH 9) showed negligible binding (0.2 ngcm�2). This demonstrates that the surface is negatively chargedat pH 9. These protein adsorption experiments illustrate thatthe polymer exists as cationic, zwitterionic and anionic formsover discrete pH ranges. It should be pointed out that one cantune the pH range at which the polymer is positive, negative andzwitterionic by changing its structure.
Noteworthy, especially for protein separation applications,as an example, pepsin which bound to the surface at pH 3 wascompletely removed by changing the pH to 7.4. The SPR sen-sorgram is shown in Fig. 2A along with a scheme (Fig. 2B)showing the reversible switching behavior of the polymer. Abase line was established rst and then pepsin was owed onthe surface at pH 3 which clearly showed a denite adsorptionof pepsin. Then the surface was washed with PBS (pH 7.4) andnow the base line reached the initial established level, revealingcomplete removal of pepsin from the surface. Hence, by
Fig. 2 (A) SPR sensorgram showing pepsin adsorption at pH 3 and release(B) Scheme showing the reversible switching behavior of the polymer.
utilizing the reversible charge switching of the polymer it ispossible to adsorb and then completely remove a given proteinfrom the polymer surface.
Collectively, the data from Fig. 1 and Table 1 reveal a novelsmart surface which can be switched among cationic, zwitter-ionic and anionic states simply by changing the pH. It should beemphasized here that since the surface was tested between pH 3and 8 using single proteins, fouling behaviors will be quitedifferent for complex media such as undiluted blood plasmaand serum. Thus the nonfouling behaviour was further testedwith undiluted plasma and serum at physiological pH (pH 7.4).
Human plasma and serum are far more complex than indi-vidual proteins and offer a much greater challenge to resistingnonspecic adsorption. Conventional zwitterionic polymers(carboxybetaine and sulfobetaine) have been shown to be ultralow fouling against human serum and plasma which is vital forbiomedical applications.34 To further explore the extent of thezwitterionic behavior of CBMA-1-TAM, the polymer brush wassubjected to fouling experiments with undiluted human plasmaand serum. The surface showed only 5.0 � 2.1 ng cm�2 foulingfor serum and undetectable adsorption for plasma (Fig. 3), thusproviding ultra low fouling properties and demonstratingstrong zwitterionic characteristics, akin to conventionalzwitterions.
We also compared CBMA-TAMs with one and two carbonspacers based on their behavior and performance. Their elec-trostatic potential surfaces are shown in Fig. 4. The resultsindicate a more uniform charge distribution for the singlespacer as expected (Fig. 4A). The gure also indicates highlylocalized charge density, such as dark blue (electron decient)and dark red (electron rich), near the nitrogen proton and thecarboxylate for the two-spacer monomer (Fig. 4B). The radialdistribution function for the intermolecular interaction of thenitrogen proton with that of carboxylate is plotted in Fig. S4.†Unlike CBMA-2 with a quaternary amine,35 this promotesintermolecular interactions among CBMA-2-TAM groups(Fig. 4), leading to aggregation and fouling at pH 7.4 (Fig. S5†).The synergistic interaction between tertiary amine and
by running PBS (pH 7.4) on the polymer brush surface (CBMA-1-TAM).
Fig. 3 Fouling tests on the polymeric surface with undiluted humanplasma and serum. Typical SPR sensorgrams for fouling experimentswith undiluted human plasma and serum.
Fig. 4 Charge distributions of the two monomers are different asshown by their electrostatic potential surfaces. The electrostaticpotential surfaces for the zwitterionic forms of (A) CB-1-TAM(–OOCCH2N
+H(CH3)2) and (B) CB-2-TAM (–OOCCH2CH2N+H(CH3)2).
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carboxylic acid groups is thus evident when they are separatedby one spacer. The charge densities are well distributed whenthe number of carbon spacers is one whereas there is no suchinteraction when the carbon spacer number is two. The twospacer polymer (CBMA-2-TAM) shows a fouling of 51 ng cm�2
and 45 ng cm�2 for brinogen and lysozyme respectively at pH7.4 whereas the one spacer polymer (CBMA-1-TAM) showedultra low fouling even for undiluted plasma and serum.
Conclusions
In conclusion, we present the design, synthesis and character-ization of a novel reversibly switchable material. Due to itspossessing a tertiary amine and a carboxylic acid group separatedby a single carbon spacer, the material reversibly switches amongcationic, zwitterionic and anionic forms depending on pH andthe material behaves as perfectly zwitterionic at physiological pHvia a synergistic mechanism. The monomer clearly indicated asynergistic mechanism between the tertiary amine and thecarboxylic acid groups, thereby offering a convenient approachfor controlling the pH-dependent properties. Subsequentquantum chemical calculations indicated that the stable
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zwitterionic state was enabled by charge transfer. These polymerbrushes showed ultra low fouling properties to undiluted humanserum and plasma under physiological conditions. The chargeswitching ability of the polymer combined with the ultra lowfouling properties at physiological pH make this material desir-able for specialized applications.
Acknowledgements
This work was supported by the Office of Naval Research(N000141210441), National Science Foundation (DMR 1005699),and U.S. Army Natick Soldier Research, Development and Engi-neering Center.
Notes and references
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