Temperature-/CO₂-dual-responsiveness of a zwitterionic “schizophrenic” copolymer

Abstract

A zwitterionic “schizophrenic” copolymer with dual-responsiveness to temperature and carbon dioxide self-assembles to undergo a reversible phase transition in a weakly alkaline borate buffer solution. It can be switched “on” and “off” when sequentially treated with carbon dioxide/nitrogen (CO₂/N₂) due to the protonation–deprotonation of the tertiary amine groups along the polymer skeleton.

---

Due to their intriguing smart behavior, “schizophrenic” block copolymers have received considerable attention and held great promise for controlled drug release, gene delivery, and nanocatalysis, ever since the first example was reported in 1998.^1–3 A schizophrenic block copolymer can self-assemble to form micelles and inverted micelles in dilute solution by simply changing external conditions such as temperature, pH, redox, photo, ionic strength,^4,5 or subtle solvent changes.⁶ However, the previous schizophrenic block copolymers mainly combined two different response parameters such as pH and ionic strength,¹ or pH and temperature.⁷ The schizophrenic block copolymers that can be switched to form micelles to inversed micelles only by changing temperature or pH were reported in 2002.^8,9 Generally, the fully temperature-switchable schizophrenic block copolymer requires combination of lower critical solution temperature (LCST) behavior and upper critical solution temperature (UCST) behavior,^10–16 and realizes switching by simply changing temperature without addition of salts, acids, or bases to the solution.¹⁴ In comparison, the pH-switchable schizophrenic block copolymer can be achieved switching through varying pH by adding acids or bases. It is possible to obtain byproducts and cause contamination of the existing system, imposing potential side effects on biological cells involving incompatibility, membrane decomposition, cytotoxicity, and gene damage.¹⁶ Therefore, the exploration of novel, mild, and environmentally friendly stimuli is emerging and promising.

Carbon oxide (CO₂) is attractive as a trigger for stimuli-responsive materials due to its availability, cost-efficiency, and benign nature.¹⁷ As an endogenous metabolite, CO₂ can freely diffuse through the cytomembrane without any cytotoxic effect and play a crucial role in regulating bio-aggregated constitution. The “green” stimulus only involves introduction and release of inactive gases such as CO₂ and N₂.^17–20 Recently, Yuan's and Zhao's groups mimicked deformable behavior of vesicles in response to CO₂ trigger.^20–23 In addition to green chemistry, the use of CO₂ as a trigger for switching property or function of materials may also hold promise for application.²³ Therefore, to explore CO₂ as a new trigger to drive block copolymer phase transition and to realize the “schizophrenic” behavior remains a great challenge although some nascent effort has been devoted.

In the current work, we report, for the first time, a temperature-/CO₂-dual-responsive zwitterionic “schizophrenic” triblock copolymer composed of poly(methoxyethylene glycol) (MPEG), poly{[2-(methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)-ammonium hydroxide} (PSBMA), and poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA). This schizophrenic block copolymer combines PSBMA with upper critical solution temperature (UCST) and PDMAEMA with lower critical solution temperature (LCST) in weakly alkaline buffer solution, which is different from most fully temperature-responsive schizophrenic block copolymers based on non-ionic/zwitterionic block self-assembly with poly(N-isopropylacylamide) (PNIPAM) as LCST polymer while polysulfobetaine as UCST polymer so far. For the triblock copolymer, the water-soluble MPEG blocks with biocompatible and anti-fouling properties endow stability to the polymer chains or the formed micelles in aqueous solution by creating a hydrophilic corona.²⁴ PSBMA as a zwitterionic polymer is considered to be in collapsed coil in aqueous solution below UCST due to intra- and/or interchain associations, while in the form of non-associations above UCST.²⁵ Moreover, PSBMA also owns excellent biocompatibility and biofouling-resistance. PDMAEMA is a weak base with pK_a about 7.0 and is water-soluble within wide pH range,²⁶ exhibiting a phase transfer between hydrophilic and hydrophobic characteristics below and above pK_a.^27–29 The chains of PDMAEMA are expanded in aqueous solution with tertiary amine groups of PDMAEMA are protonated and hydrated below the pK_a. In contrast, the polymeric chains are collapsed by deprotonation and dehydration of the amine group above pK_a. There are also conformational changes in PDMAEMA below and above its LCST.^30–32 Beside this, tertiary-containing moiety in PDMAEMA can react with CO₂ in solution to form a charged ammonium bicarbonate, which can be recovered upon CO₂ removal (Scheme 1).³² Most importantly, the combination of non-ionic MPEG, cationic PDMAEMA, and zwitterionic PSBMA results in a unique molecular structure, which can be expected to create an advanced biocompatible and biofouling resistant system and to act as nanocarriers for drug uptake, delivery, and release in living organisms.

Scheme 1 Schematic representation of the “schizophrenic” aggregation behavior for the triblock copolymer MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ controlled by temperature and CO₂.

The triblock copolymer MPEG-b-PSBMA-b-PDMAEMA was synthesized by sequential atom transfer radical polymerization (ATRP) using an MPEG-based macroinitiator.³³ The detailed synthesis and characterization are shown in the ESI.† After purification, the obtained copolymer MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ was dissolved in borate buffer solution (pH 9.2) at a concentration of 10 mg mL⁻¹.

The stimuli-responsive behavior of MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ was investigated via UV-vis spectroscopy and dynamic light scattering (DLS). Measurements were conducted between 16 to 65 °C, well encompassing the UCST of PSBMA and the LCST of PDMAEMA.^34,35 As shown in Fig. 1a, a sharp change of transmittance at 32 °C indicates that both PSBMA and PDMAEMA blocks are soluble. Upon decreasing the temperature from 32 to 16 °C, a decrease in transmittance was detected and shown in the black curve in Fig. 1a, accompanied by a reasonable dimensional increase as shown in the black curve in Fig. 1b. Similar results were obtained by increasing the temperature from 32 to 40 °C. An individual UCST (28 °C) or LCST (36 °C) determined at 50% transmittance was exhibited.³³ The temperature-dependent assembly change behaviors can be attributed to PSBMA and PDMAEMA. Below the UCST, collapsed PSBMA forms the core of the aggregates while PDMAEMA and MPEG as the shell, which is attributed to mutual electrostatic attraction by ion pairings between ammonium cation and sulfo-anion of the zwitterionic sulfobetaine groups in PSBMA; above the LCST, PDMAEMA chains become hydrophobic and the hydrogen bond with water molecules weakens, collapsed PDMAEMA forms the core of the aggregates while MPEG-b-PSBMA as shell,³³ as shown in Scheme 1.

Fig. 1 Temperature dependence of transmittance (a) and size (b) for MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ in borate buffer solution (pH 9.2) before (black curve) and after (red curve) CO₂ bubbling.

The CO₂-responsiveness was firstly confirmed by monitoring the transmittance and pH during successive CO₂ and N₂ bubbling cycles. Upon CO₂ bubbling, no cloud point can be detected over the range of temperature for the transmittance measurement from 16 to 60 °C (Fig. 1a). This phenomenon derives from the protonation of tertiary amine groups in PDMAEMA blocks. The number of PDMAEMA units decreased due to the formation of carbonic acid upon CO₂ bubbling in water, which resulted in improved solubility of the triblock copolymer. Therefore, the shell of micelles becomes looser as the PDMAEMA segments are stretched. The protonation caused by CO₂ is reversible with assistance of N₂. It can be observed that there is no significant difference but a slight decrease in transmittance after CO₂ bubbling below 40 °C, which can be attributed to the insoluble PSBMA blocks as the core of micelles. However, the phase transitions are distinct under alternative CO₂ and N₂ above 32 °C, resulting in unimers with CO₂ and precipitation without CO₂.

The scanning electron microscopy (SEM) images of triblock copolymer aggregates at 25 °C before and after being treated with CO₂ shown in Fig. 2, display quite different sizes, which is in accordance with the result determined by DLS. The formed vesicles with a diameter of over 500 nm treated with CO₂, show larger size than that without CO₂. In comparison with the DLS measurement, the smaller diameter by SEM can be attributed to the dry state.

Fig. 2 SEM images of MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ self-assembling aggregates in borate buffer solution (pH 9.2) before (a) and after (b) being treated with CO₂ at 25 °C.

In the absence of any stimulus, hydrophobic PDMAEMA chains and weak hydrogen bonds result in poor solubility of the block copolymer above 40 °C. Upon CO₂ bubbling, the transmittance of the micellar solution rapidly rises from 4.4% to 78.4% and then to the equilibrium value of ∼80% with an increase of temperature, simultaneously, the solution pH drops from 9.2 to 6.1. Upon N₂ bubbling, CO₂ can be released from the solution, and the transmittance decreases to 4%, while the pH recovers to 8.2. These variations are remarkably reversible and can be repeated for three cycles without alternation (Fig. 3A). The reversible transmittance “turn-on” and “turn-off” response can simulate the cellular respiration.¹⁹ Furthermore, this trigger benefits from the easy removal of the unstable bicarbonate salt^36,37 produced by the reaction of CO₂ with the tertiary amine groups in the PDMAEMA blocks, thus making it truly reversible, and therefore superior to the more traditional pH triggers by addition of acid or base,³⁸ where reversibility is affected by the accumulation of by-products.³⁹

Fig. 3 Transmittance (A) variations of reversible adsorption and desorption of CO₂ by purging with N₂, temperature (B) and time (C) dependence of pH in borate buffered solution for MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅.

It is worthy of note that the stimuli-responsive behavior of MPEG₄₃-b-PSBMA₃₀-b-PDMAEMA₄₅ in borate buffer solution is different from in pure water, which deriving from the super buffer capacity and complexity of the buffer system. The borate buffer solution was obtained by the hydrolysis of sodium tetraborate in water, resulting in NaBO₂–H₃BO₃ system (B₄O₇²⁻ + 3H₂O = 2BO₂⁻ + 2H₃BO₃). Upon CO₂ bubbling, the tertiary amine groups in PDMAEMA can be partially protonated, resulting in quaternary ammonium cations (–N(CH₃)₂ + CO₂ + H₂O = –NH⁺(CH₃)₂ + HCO₃⁻). Concurrently, sodium tetraborate can be recovered to its initial state (4BO₂ + CO₂ = B₄O₇²⁻ + CO₃²⁻) due to the stronger acidity of carbonic acid than boric acid. Effect of temperature on the pH demonstrates that no significant difference between 30 and 60 °C can be observed in Fig. 3B. Time dependence of pH indicates that the pH gap between CO₂ and N₂ bubbling narrowed within an identical time interval with time extending, as shown in Fig. 3C.

To fabricate multi-responsive schizophrenic system has attracted increasing attention during recent years. Hoogenboom's group constructed a triple thermoresponsive schizophrenic diblock copolymer based on PDMAEMA and PmOEGMA, which undergoes transitions from conventional micelles via unimers to reverse micelles and finally precipitation upon heating.¹⁵ To date very few examples of such systems have been reported. In this work, a triple-responsive schizophrenic triblock copolymer based on the UCST transition of PSBMA, the LCST transition and CO₂-responsiveness of PDMAEMA was developed. Following the above results, we propose the mechanism on how temperature and CO₂ drive the morphological transition of the aggregate in borate buffer solution, which is schematically presented in Scheme 1. In the absence of CO₂, at the turning point between the UCST and LCST, the polymer chains are totally soluble. Decreasing the temperature of the system, driven by electrostatic attraction between intra- or inter-ion pairs in PSBMA blocks, the small micelles (or vesicles) tend to fuse each other to form large fusions. Upon raising the system temperature, driven by the energy minimization between the collapsed PDMAEMA chains and water, the small micelles (or vesicles) also tends to form large fusions. Upon exposure to CO₂, the PDMAEMA block can be partially protonated and hydrophilic, gradually water-soluble, leading to the unimer formation. On the other hand, protonation degree plays an important role in morphological transition. In our case, pH plays an important role in determining the protonation degree in borate buffer solution. In the absence of CO₂, a low protonation degree hardly affects the hydrophobicity of the PDMAEMA block. However, the PDMAEMA block becomes hydrophilic with a high protonation degree, likely to induce substantial structural transition of the assemblies and changing of PDMAEMA copolymer structures.⁴⁰

In conclusion, we have developed a new class of dual-responsive schizophrenic triblock copolymer by using temperature and CO₂ to trigger morphological transition of assemblies. In borate buffer solution, the hydrophilic–hydrophobic transitions of PSBMA and PDMAEMA achieve by varying the system temperature, resulting in the core–shell exchange of assemblies. Furthermore, the aggregates can be reversibly switched “on” and “off” by alternatively bubbling and displacing CO₂, in which the protonation–deprotonation mechanism of the tertiary amine groups along the PDMAEMA block is responsible for this reversible morphological transition. The temperature interval between UCST and LCST is comparatively narrow and mild, completely covering normal body temperature. CO₂ offers many advantages as a trigger, in particular a lack of contaminant accumulation, and thus provides a reversible process over many cycles. To the best of our knowledge, this is the first example of temperature-/CO₂-dual-responsive zwitterionic “schizophrenic” copolymer. It can be anticipated that the unique self-assembly behavior by mild and “green” stimuli my offer new opportunities for selective, well-controlled uptake, delivery and release of more drugs in one nano-vehicle. The biocompatible and bio-fouling resistant property endows the nanomaterial potential and promising application in biomedical area. The system can also provide inspiration for biomimics and simulation by synthetic polymer since the components of the block copolymer are biocompatible, and temperature and CO₂ are crucial for living organisms.

Acknowledgements

The authors gratefully acknowledge financial support from Shandong Provincial Natural Science Foundation of China (Grant No. 2016ZRB01232, ZR2010BM006), Shandong Provincial Science and Technology Development Plan Project of China (2014GGX102013), and Shandong Provincial Overseas Visiting Scholar Project of China (2011).

Notes and references

1. A. Butun, N. Billingham and S. P. Arms, J. Am. Chem. Soc., 1998, 120, 11818.
2. X. Ji, S. Dong, P. Wei, D. Xia and F. Huang, Adv. Mater., 2013, 25, 5725.
3. (a) Z. Tyrrell, Y. Shen and M. Radosz, Prog. Polym. Sci., 2010, 35, 1128; (b) A. Darabi, P. G. Jessop and M. F. Cunningham, Chem. Soc. Rev., 2016, 45, 4391; (c) V. A. Vasantha, S. Jana, S. S.-C. Lee, C.-S. Lim, S. L.-M. Teo, A. Parthiban and J. G. Vancso, Polym. Chem., 2015, 6, 599.
4. C. Feng, Y. Li, D. Yang, J. Hu, X. Zhang and F. Huang, Chem. Soc. Rev., 2011, 40, 1282.
5. Y. Zhou, Q. Zhang and Z. Luo, Langmuir, 2014, 30, 1489.
6. R. Hoogenboom, H. M. L. Lambermont-Thijs, M. J. H. C. Jochems, S. Hoeppener, C. Guerlain, C.-A. Fustin, J.-F. Gohy and U. S. Schubert, Soft Matter, 2009, 5, 3590.
7. S. Liu, N. C. Billingham and S. P. Arms, Angew. Chem., Int. Ed., 2001, 40, 2328.
8. S. Liu and S. P. Arms, Angew. Chem., Int. Ed., 2002, 41, 1413.
9. M. Arotcarena, B. Heise, S. Ishaya and A. Laschewsky, J. Am. Chem. Soc., 2002, 124, 3787.
10. F. A. Plamper, A. Schmalz and A. H. E. Muller, J. Am. Chem. Soc., 2007, 129, 14538.
11. J. Seuring and S. Agarwal, Macromolecules, 2012, 45, 3910.
12. J. Seuring and S. Agarwal, Macromol. Rapid Commun., 2012, 33, 1898.
13. C. Weber, R. Hoogenboom and U. S. Schubert, Prog. Polym. Sci., 2012, 37, 686.
14. Q. Zhang, J.-D. Hong and R. Hoogenboom, Polym. Chem., 2013, 4, 4322.
15. R. Hoogenboom, H. M. L. Lambermont-Thijs, D. Wouters, S. Hoeppener and U. S. Schubert, Soft Matter, 2008, 4, 103.
16. Y. J. Shih, Y. Chang, A. Deratani and D. Quemener, Biomacromolecules, 2012, 13, 2849.
17. (a) A. Feng, C. Zhao, Q. Yan, B. Liu and J. Yuan, Chem. Commun., 2014, 50, 8958; (b) H. Chen and J. C. M. van Hest, J. Mater. Chem. B, 2016, 4, 4362; (c) K. Jie, Y. Zhou, Y. Yao, B. Shi and F. Huang, J. Am. Chem. Soc., 2015, 137, 10472; (d) B. A. Abel, M. B. Sims and C. L. McCormick, Macromolecules, 2015, 48, 5487.
18. X. Su, M. F. Cunningham and P. G. Jessop, Polym. Chem., 2014, 5, 940.
19. Q. Yan, R. Zhou, C. Fu, H. Zhang, Y. Yin and J. Yuan, Angew. Chem., Int. Ed., 2011, 50, 4923.
20. (a) Q. Yan, J. Wang, Y. Yin and J. Yuan, Angew. Chem., Int. Ed., 2013, 52, 5070; (b) H. Che, M. Huo, L. Peng, T. Fang, N. Liu, L. Feng, Y. Wei and J. Yuan, Angew. Chem., Int. Ed., 2015, 54, 8934.
21. Q. Yan and Y. Zhao, Angew. Chem., Int. Ed., 2013, 52, 9948.
22. Q. Yan and Y. Zhao, J. Am. Chem. Soc., 2013, 135, 16300.
23. Z. Li, E. Kesselman, Y. Talmon, M. A. Hillmyer and T. P. Lodge, Science, 2004, 306, 98.
24. M. Tian, J. Wang, E. Zhang, J. Li, C. Duan and F. Yao, Langmuir, 2013, 29, 8076.
25. Y. Xu, S. Bolisetty, M. Drechsler, B. Fang, Y. Yuan and M. Ballauff, Polymer, 2008, 49, 3957.
26. O. Schepelina and I. Zharov, Langmuir, 2008, 24, 14188.
27. N. Nordgren and M. W. Rutland, Nano Lett., 2009, 9, 2984.
28. J. I. Amalvy, E. J. Wanless, Y. Li, V. Michailidou and S. P. Armes, Langmuir, 2004, 20, 8992.
29. S. H. Cho, M. S. Jhon, S. H. Yuk and H. B. Lee, J. Polym. Sci., Part B: Polym. Phys., 1997, 35, 595.
30. D. Fournier, R. Hoogenboom, H. M. L. Thijs, R. M. Paulus and U. S. Schubert, Macromolecules, 2007, 40, 915.
31. F. A. Plamper, M. Ruppel, A. Schmalz, O. Borisov, M. Ballauff and A. H. E. A. Müller, Macromolecules, 2007, 40, 8361.
32. D. Han, X. Tong, O. Boissire and Y. Zhao, ACS Macro Lett., 2012, 1, 57.
33. Q. Zhang, X. Tang, T. Wang, F. Yu, W. Guo and M. Pei, RSC Adv., 2014, 4, 24240.
34. S. Nayak, D. Gan, M. Serpe and L. Lyon, Small, 2005, 1, 416.
35. C. Sorrell, M. Carter and M. Serpe, Adv. Funct. Mater., 2011, 21, 425.
36. Y. Zhang, Y. Feng, J. Wang, S. He, Z. Guo, Z. Chu and C. A. Dreiss, Chem. Commun., 2013, 49, 4902.
37. Y. Zhang, Z. Chu, C. A. Dreiss, Y. Wang, C. Fei and Y. Feng, Soft Matter, 2013, 9, 6217.
38. S. Kumar, X. Tong, Y. Dory, M. Chaker, Y. Zhao and D. Ma, Chem. Commun., 2013, 49, 90.
39. P. G. Jessop, S. M. Mercer and J. Heldebrant, Energy Environ. Sci., 2012, 5, 7240.
40. D. E. Discher and A. Eisenberg, Science, 2002, 297, 967.

---

Footnote

† Electronic supplementary information (ESI) available: Detailed synthesis and characterization. See DOI: 10.1039/c6ra28018h

---