Ye Huanga,
Ping Yonga,
Yan Chena,
Yuting Gaoa,
Weixiong Xua,
Yongkang Lva,
Liming Yanga,
Rui L. Reisb,
Rogério P. Pirracob and
Jie Chen
*a
aDepartment of Chemical Engineering, School of Environmental and Chemical Engineering, Shanghai University, Shangda Road 99, Shanghai 200444, P. R. China. E-mail: jchen@shu.edu.cn; Tel: +86 21 66137482
b3B's Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Portugal
First published on 30th May 2017
The multi-stimuli-responsive amphiphilic ABC triblock copolymer of poly(methyl methacrylate)-block-poly[N,N-(dimethylamino) ethyl methacrylate]-block-poly(N-isopropylacrylamide) (PMMA-b-PDMAEMA-b-PNIPAM) was synthesized by sequential reversible addition–fragmentation chain transfer (RAFT) polymerizations. Due to the pH- and thermo-responsive blocks of PDMAEMA and thermo-responsive blocks of PNIPAM, the copolymer solution properties can be manipulated by changing the parameters such as temperature and pH, and it formed diverse nanostructures and had gel behavior in different conditions. In detail, when the pH was below 7.3, the pKa of DMAEMA, tertiary amino groups were protonated, the polymer micellar solution like weak gel changed into free-standing gel at around 40 °C even at a low concentration of 2 wt%. Further, the gel behavior and morphology of the system were studied by rheology, turbidimetry measurements, transmission electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. When the pH was above the pKa, the triblock copolymer self-assembled into diverse micellar structures including shell–core, corona–shell–core and shell–shell–core nanoparticles as the temperature increased, but no free-standing gelation. The two-step thermo-responsive behavior, corresponding to the different LCSTs of PNIPAM block and DMAEMA block, was evidenced by turbidity analysis. The assembled structures rapidly collapsed due to the enhanced hydrophobic interaction when the temperature further increased. Dynamic light scattering (DLS) was used to confirm the transitions.
Environment-sensitive polymers play an important role in the development of smart hydrogels.11,18,19 Thermo-responsive polymers can become hydrophobic from hydrophilic when the temperature is above a critical point, where a soluble-to-insoluble phase transition occurs, known as lower critical solution temperature (LCST).20 Poly(N-isopropylacrylamide) (PNIPAM) is a water soluble polymer that exhibits phase separation at the LCST of around 32 °C, depending upon the detailed microstructure of the macromolecule.21 Namely, below 32 °C, PNIPAM dissolves well in water, in contrast, changes into hydrophobic aggregation and may deposit from the aqueous because of the breakdown of the hydrogen bonds presented at a lower temperature.4 Because the LCST is between room temperature(25 °C) and physiological temperature (37 °C), it has been widely employed in many biomedical fields especially in on/off drug release and the reversible attachment/detachment of cultured cells.22
Besides the temperature sensitive hydrogels based on PNIPAM, another type of typical stimuli sensitive hydrogels is the pH sensitive hydrogels, which may change their shapes and volumes with the variation of external pH.23 Poly(dimethylaminoethyl methacrylate) (PDMAEMA) is a pH-sensitive polymer with a pKa of 7.3 in water. Moreover, PDMAEMA is thermos-sensitive, and the LCST is hinged upon its molecular weight and pH. In detail, the LCST strongly decreased with increasing pH of the solution.24 It has been widely used for the producing of multi-sensitive materials.11,25
The copolymerization of DMAEMA and PNIPAM is supposed to have some interesting properties since both monomers are thermos-sensitive and only DMAEMA is pH-sensitive. Wang et al.26 prepared hydrogels based on NIPAM and DMAEMA via free radical polymerization, the resulted P(NIPAM-co-DMAEMA) hydrogels have the strong desire to respond to external temperature and pH stimuli. The synthesis of terpyridine end-capped PDMAEMA-block-PNIPAM diblock copolymers via controlled radical copolymerization has been reported by Jeremy Brassinne and his co-workers.27
Over the last few years, hydrogel formation by ABA triblock copolymers containing hydrophilic mid-blocks and hydrophobic end blocks has been extensively investigated.28–31 A triblock polymer (PPO43–PMPC160–PNIPAM81) was synthesized by ATRP and its gelation efficiency in water was evaluated by Li and his co-workers.32 However, almost 20 wt% polymer was required to be gel, the gelation efficiency was sufficiently lower than that observed in the PNIPAM–PMPC–PNIPAM triblock copolymers. Besides high critical gelation concentration, these hydrogels often exhibit broad or slow sol–gel transitions.32,33 Comparing to ABA model, ABC triblock copolymers, which the C block is hydrophobic and the B and A blocks are water-soluble below the sol–gel transition temperature, are more efficient to realize the sol–gel transition.18,19 Because the three blocks are mutually immiscible, they can form diverse nanostructures in aqueous solution in response to the change of the environment.1,32
Reversible addition–fragmentation chain transfer (RAFT) polymerization which is an attractive controlled/living radical polymerization method for the synthesis of block copolymers has arouse great interest in the past decade.34,35 Since it is a versatile and facile technique, a variety of chain transfer agents are used without transition metal catalysts.36,37 This process leads to the production of materials with controlled molecular weights and low polydispersity.38
When thermo-responsive segments are contained in a single polymer chain, the copolymers may show multiple LCSTs.39 That the combination of different functions within a single polymer can contribute to improvement of carrier efficiency is reported in many researches.40 Block copolymers containing PDMAEMA block and PNIPAM segment provide a flexible control to the polymer phase behavior, resulting in multifunctional smart materials. The lack of data of ABC triblock copolymers relating to the RAFT copolymerization of DMAEMA and PNIPAM motivated us to investigate this system.
In this study, we synthesized a novel pH- and dual-thermo-sensitive amphiphilic ABC triblock copolymer composed of PMMA, PDMAEMA and PNIPAM by RAFT polymerization. The hydrophilic triblock copolymer molecularly could be dissolved in aqueous solution and was a viscous liquid like weak gel with shell–core micelle at room temperature. In acidic aqueous solution, that the pH was below the pKa of the PDMAEMA block, the free-flowing liquid changed into free-standing gel with the elevated temperatures. While in alkaline solutions, the sturdy gelation structure would be destroyed with doubly thermo-responsive steps, due to the wide difference of the LCSTs of PNIPAM and PDMAEMA chains. Indeed, it formed diverse nanoparticles such as corona–shell–core and shell–shell–core, finally precipitated from aqueous solution as the temperature increased (Fig. 1). The thermo- and pH-responsive copolymer may boast a great potential and value in biomedical areas, such as tissue engineering and controlled drug delivery and release.
FT-IR spectra of the polymers are shown in Fig. 2. There are some strong absorption peaks around 2900 cm−1, which could be attributed to the stretching vibrations of –CH3 and –CH2– groups.42 All spectra exhibit an absorption band at 1730 cm−1 from CO stretching vibration, which is assigned to the carbonyl stretching vibration of the ester group. Furthermore, they show a peak at 1147 cm−1 corresponding to the C–O stretch. The absorption peak at 1063 cm−1 is the typical stretching vibrations of C
S groups in CTA.44 The FT-IR spectrum (B) of the PMMA-b-PDMAEMA contain the characteristic band for C–N stretching of DMAEMA at 1020 cm−1.44 For PMMA-b-PDMAEMA-b-PNIPAM (spectra C), the peak at 1650 cm−1 and 1544 cm−1 are attributed to C
O and N–H vibrational bands from –CONH of NIPAM, respectively.45 Fig. 3 displays the 1H NMR spectrum of PMMA-b-PDMAEMA-b-PNIPAM in CDCl3. The structure signals have already been mentioned in Experimental part.46,47 The 1H NMR spectra of PMMA and PMMA-b-PDMAEMA are also detected (Fig. S1 and S2†).45,48,49 From structure signals, we calculated the ratio of the integral peak area between the methylic protons of PMMA (marked c) and the para-C6H5 protons of CPADB (marked 1), the Dp of PMMA was estimated to be about 82. Similarly, the Dp of other polymers could be also roughly calculated.42 GPC traces of the block copolymers are shown in Fig. 4. In particular, with the addition of the monomers, the clear shift to short elution time of the GPC peak without any other peaks indicates that the chain extension has occurred. GPC was also used to characterize the molecular weight of the polymers, and the results are summarized in Table 1. The molecular weight of polymers synthesized with processing in every step increased significantly. The molecular weight calculated by 1H NMR is approximate with the average molecular mass of polymers characterized by GPC, and the PDI is broadening. A possible explanation is as follows: the overall RAFT process provides insertion of monomer units into the C–S bond of the RAFT agent structure, polymer chains lengthened constantly with the polymerization processing. In order to ensure higher monomer conversion rate, we extended for several hours. While long polymerization time may cause the reactive end-groups of RAFT agents fall off under the synthetic circumstances, leading to chain termination because of the potential lability of the C–S bond in the RAFT end-group. Therefore, the polymer was contaminated, displayed as broadening PDI detected by GPC. In short, all of the results above indicate that the target triblock copolymer has been successfully synthesized.
Mn, NMRa (g mol−1) | Mn, GPCb (g mol−1) | PDIc (Mw/Mn) | |
---|---|---|---|
a The molecular weight by 1H NMR analysis.b The number-average molecular weight by GPC analysis.c The PDI or the Mw/Mn values determined by GPC analysis. | |||
PMMA macro-CTA | 8217 | 8386 | 1.25 |
PMMA-b-PDMAEMA macro-CTA | 31![]() |
32![]() |
1.43 |
PMMA-b-PDMAEMA-b-PNIPAM | 39![]() |
41![]() |
1.67 |
Typical viscosity vs. temperature plots obtained from pH = 1 to pH = 6 aqueous solutions of the PMMA-b-PDMAEMA-b-PNIPAM copolymer are shown in Fig. 5(A). The viscosity of PMMA-b-PDMAEMA-b-PNIPAM copolymer first increased slowly (20–30 °C), then rose sharply (30–50 °C), and decreased gradually (50–75 °C), finally was steady. The maximum rate of change was about 40 °C, it is higher than the LCST of NIPAM (32 °C), because the ionization degree of the PDMAEMA block can influence the phase transition temperature of not only PDMAEMA but also the neighbour PNIPAM block due to the weakened hydrophobic aggregation force.27,50 The impact on the thermos-sensitivity of polymerized hydrogel with the incorporation of pH sensitivity has been widely reported.26 At room temperature, the viscosity of the micellar solution exceeded 100 Pa s, it was a viscoelastic liquid like weak gel, which was attributed to the effect of hydrogen bonds between NIPAM and DMAEMA. When the temperature was increased to 50 °C, PNIPAM turns to hydrophobic, the terpolymer bears at both ends hydrophobic blocks that should associate forming a stronger network because of the increasing bridging, furthermore, the free amino groups in the PMMA-b-PDMAEMA-b-PNIPAM are ionized in acidic solutions, which might generate the electrostatic repulsion among polymer chains. Therefore, the free-standing gel formed.51 The phase transition corresponding to gelation with high concentration mainly attributed to the hydrophobic PMMA and thermo-sensitive PNIPAM moiety.52 Above 50 °C, the viscosity decreased gradually strangely. A tentative explanation for this unexpected effect is as follows, it might be the relaxation time which increases upon heating in this regime.53 Nevertheless, the gel was still free-standing. The same phenomenon was also detected in the rheological experiments (Fig. S3†). The gel was strengthening with the increasing temperature, and exhibiting maximum at around 55 °C, though there is a deviation of 5 °C comparing to the viscosity experiment, the trend of the modulus were in strong agreement with the viscosity in Fig. 5(A). Interestingly, the viscosity of the free-standing gel was different at the same temperature in various pH, which was higher at pH = 4, probably due to the degree of the protonation of the amine functions of the PDMAEMA block, at pH 4, the degree of ionization of the central PDMAEMA chains is about 90%, this is the reason of the viscosity maximum since it favors a 3D network formation.24,54 In detail, when pH > 4, a gradual deprotonation of the monomeric units will decrease the degree of ionization and increase chain flexibility, making bridge to loop transitions and hence viscosity decrease when pH increased. While pH < 4, lowering pH will make the ionic strength of the solution increase, and lead to electrostatic screening effects that result in viscosity decrease. The ABA triblock copolymers (PMMA-b-PDMAEMA-b-PMMA) synthesized by Frederic Bossard and his coworkers have the similar properties.53
The transition from a viscoelastic liquid to a 3-dimensional network gel was also evidenced by the data collected from frequency sweep experiments (Fig. 5(B)). The frequency dependences of G′ and G′′ of the polymer solution at selected temperatures of 25 °C, 50 °C and 75 °C were obtained by frequency sweep tests from 0.016 Hz to 10 Hz. At 25 °C, the solution was a transparent liquid that can flow when tilted. G′ and G′′ were of similar magnitudes in the frequency range of 1–10 Hz, this is the signature of the transition between liquid-like and solid-like behavior.28 Above 50 °C, the sample was a free-standing gel. G′ is significantly greater than G′′ and is nearly independent of frequency in the selected frequency range, which was a characteristic of solid-like behaviour.55 Further, we investigated the mechanical properties of the gel states to explore the gelation at 50 °C.
The concentration of polymer has a significant impact on gelation property. Therefore, we studied the gelation behaviour in different concentrations at the selected of pH = 4, which afforded total protonation of the amine functions (Fig. 6). The viscosity of triblock copolymer system increased gradually with the increasing of the concentration of polymer, indicating stronger interaction among polymer chains at higher concentrations. The sticky triblock copolymer solution could form free-standing gel at around 40 °C even at a quite low concentration (2 wt% in water). Generally, the gelation of such systems is efficient in the sense of the minimum gelation concentrations.1 The tube was inverted to determine if a physical gel was formed, three times repeated heating and cooling experiments indicated that the transition is fully reversible (Fig. S4†), there was a deviation about 5 °C in each cycle, which may be caused by a lower rate of heat transfer of gel than sol. Fig. 7 shows the strain dependence of G′ and G′′. The polymer solution exhibits a linear viscoelastic response below a critical shear strain 1.2%, where G′ > G′′. Above this linear viscoelastic regime, G′ decreases as shear strain increases whereas G′′ increases, then they reach a equivalence at 18%, that means the system had lower mechanical strength when the strains increased and began flow at strains of 18%.
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Fig. 6 Viscosity curves of PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution at different concentrations at pH = 4. |
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Fig. 7 Strain dependence of G′ and G′′ for 3 wt% PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer at 50 °C. The frequency was held constant at 1 Hz. |
Moreover, the 3-dimensional network gels were evidenced by SEM images. The formation of 3-dimensional network structure of the gel is shown in Fig. 8 and S5.† With the increasing temperature, PNIPAM becomes hydrophobic, the net points which contained PMMA and PNIPAM cores were bridged by ionized PDMAEMA chains (Fig. 1), and more triblock polymer chains associated forming a stronger network structure. In addition, with the help of electrostatic interaction in acid media, the 3-dimensional network became regular and dense.
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Fig. 9 Turbidity curves of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution (2 mg mL−1) at various temperatures in acidic condition. |
In addition, DLS is a powerful tool to get detailed information about the size distribution of the aggregates. Fig. 10 shows the hydrodynamic diameter of PMMA-b-PDMAEMA-b-PNIPAM in 0.15 mg mL−1 aqueous solutions. The hydrodynamic diameter distribution of the triblock copolymer nanoparticles is narrowly dispersed. It can be clearly monitored that the peak of intensity spectra increases and shifts to higher aggregate size, for 79 nm at 25 °C with the polydispersity index (PDI) of 0.328, 122 nm at 50 °C with the PDI of 0.217 and 142 nm at 75 °C with the PDI of 0.174. That means the sizes of micelles become much bigger when temperature increased, especially from 25 °C to 50 °C. It is because of the progressive hydrophobic transformation of PNIPAM blocks in the micellar corona, and some NIPAM blocks may also shrink to form new cores when the temperature increased above the LCST, many micelles aggregated, leading to clusters of micelles at low concentrations and strengthening the gel at high concentrations.
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Fig. 10 Intensity distribution of hydrodynamic diameter of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution (0.15 mg mL−1) at pH = 4 at 25 °C, 50 °C and 75 °C, respectively. |
To study the trend of Z-average diameter as the temperature increased, 0.5 mg mL−1 of PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution at pH = 4 was used for the demonstration (Fig. 11). It was observed that Z-average diameter started to increase at around 30 °C and stabilized at around 50 °C. Moreover, the derived count rate gradually increased. It is a measurement of light scattering intensity, which varied directly with the production of particle size and concentration.56 The increasing light scattering intensity until 50 °C suggested that the aggregation of shell–core nanoparticles and tight structure. The Z-average diameter of other research groups were also measured (Fig. S6†). Interestingly, we found that at the pH of 3–5, the Z-average diameters were bigger than that of other conditions at one temperature, the same tendency was observed in the viscosity test and the specific reason has already been elucidated before.
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Fig. 11 (A) The Z-average diameter and (B) light scattering intensity as a function of temperature in a DLS study of 0.5 mg mL−1 PMMA–PDMAEMA–PNIPAM solution at pH 4. |
The morphology of the core–shell micelles is further confirmed by TEM observation. TEM images (Fig. 12) show the micelles were well dispersed in the solution at room temperature, the structure has a change from loose spherical micelle to tight aggregation upon increasing temperature, and the trends of size and distribution are similar with DLS. On the TEM images, we found that the values of the hydrodynamic diameter of the micelles observed by TEM are smaller than those measured by DLS. Firstly, this is because TEM observation shows the diameter of the dried aggregates, whereas the DLS analysis detects the solvated nanoparticles. Moreover, the electrostatic repulsion formed by ionized PDMAEMA in dried micelles will disappear.12,42 Secondly, the intensity-weighted diameter analyzed by DLS is always oversized to the number-averaged diameter observed by TEM.46
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Fig. 12 TEM images of triblock copolymer nanoparticles of PMMA-b-PDMAEMA-b-PNIPAM (0.2 mg mL−1) at different temperatures at pH = 4. |
Fig. 13(B) shows that the storage modulus (G′) is greater than storage modulus (G′′), since the micellar solution was a viscous liquid like soft gel at room temperature. It was noteworthy that the gap between G′ and G′′ became smaller with the increasing of temperature, indicating that the viscosity decreased and became weak gel with increasing temperature. The loss modulus has a slight fluctuation with the increasing temperature, probably caused by the hydrophobic effect of NIPAM and DMAEMA when the temperature is above their LCSTs, respectively. Physical network was formed through an association mechanism between hydrophobic end groups, the sticky property results in the little effect on loss modulus, while the hydrophobic interaction among the three blocks upon increasing temperature leads to the decreasing elastic modulus. The modulus increased when the temperature was above 70 °C, which means that both PNIPAM and PDMAEMA chains could completely reswollen.57 SEM images (Fig. 14 and S7†) were measured at 30 mg mL−1. The loose 3D net structure was found at 25 °C since the polymer solution was a viscous liquid, and the structure was collapsed gradually with the increasing temperature. These observations are highly consistent with the conclusion obtained from rheology measurement previously.
Furthermore, the thermo-response of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer nanoparticles dispersed in water was checked by UV-vis absorbance spectroscopy and dynamic light scattering (DLS). As shown in Fig. 15, the triblock copolymer exhibits two-stage thermally induced collapse, corresponding to the LCST of PNIPAM and PDMAEMA blocks, respectively. The first-stage collapse, which can be ascribed to the shrinking of the PNIPAM chains, the second-stage collapse, is attributed to the shrinking of PDMAEMA chains. At pH = 8, the first-stage collapse ranges from 29 °C to 39 °C, with a middle point at 34 °C, and the second-stage collapse ranges from 52 °C to 60 °C, with a middle point at 56 °C. While at pH = 9, the first-stage collapse ranges from 34 °C to 40 °C, with a middle point at 37 °C, and the second-stage collapse ranges from 44 °C to 60 °C, with a middle point at 52 °C. At pH = 7, there is a broadening transition ranging from 34 °C to 62 °C, with a middle point at 48 °C. This phenomenon is probably caused by the following two factors: firstly, DMAEMA is a cationic hydrophilic polymer as its amine groups are protonated below the pKa value of 7.3, which is the reason why the turbidity curve is similar to that in acidic conditions.54 Moreover, PDMEMA is water-soluble over the whole pH range below the LCST, but the LCST of PDMAEMA decreases with the pH increasing. Secondly, the strong inter-chain interactions are presented among the polymer chains.58,59 This predication have already been confirmed by some experimental results.60 Nevertheless, the results were lower than the triblock copolymers synthesized by Li probably because of the different molecular weight.46
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Fig. 15 Turbidity curves of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution (2 mg mL−1) at various temperatures. |
Fig. 16 shows the evolution of the size distribution of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution as the temperature changed. When the temperature is increased from 20 °C to 50 °C, the evolution of the size distribution is similar to that in acidic conditions. However, the difference occurs when the temperature increases from 50 °C to 75 °C, which means, above the second LCST of the triblock copolymer, the peak of the intensity decreased as the temperature increased.
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Fig. 16 Intensity distribution of hydrodynamic diameter of the PMMA-b-PDMAEMA-b-PNIPAM triblock copolymer solution (0.15 mg mL−1) at pH = 9 at 25 °C, 50 °C and 75 °C, respectively. |
As shown in Fig. 17(A), there is a significant rising of the Z-average diameter when the temperature is increased from 30 °C to 50 °C. Above 32 °C, some of NIPAM blocks may collapse on the PMMA core and shrink to form corona–shell–core structures, the hydrodynamic diameter of the micelles would decrease. However, many more NIPAM blocks may form new cores to be shell–core nanoparticles. Besides shell–core nanoparticles with PMMA as cores, aggregation occurs due to the hydrophobic interactions among hydrophobic groups, which could enlarge the diameter.57,61 When the temperature is above 50 °C, the Z-average diameter starts to decrease. This is because the polymer precipitates from the solution when the temperature is raised sequentially which could be further attributed to the hydrophobic interaction among the three blocks above the second LCST.46 As we can see in Fig. 17(B), the intensity of scattered light, which depends on the concentration of light scatters, starts to decrease gradually at around 35 °C due to the hydrophobic block of NIPAM. Although aggregation occurred as a result of hydrophobic interaction, the high temperature would make the structure loose and viscosity low till precipitation.
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Fig. 17 The Z-average diameter (A) and light scattering intensity (B) as a function of temperature in a DLS study of 0.5 mg mL−1 PMMA-b-PDMAEMA-b-PNIPAM solution at pH 9. |
The TEM images (Fig. 18) confirm the formation of micelles and the temperature-induced collapse of the triblock copolymer system, which were formed by three hydrophobic blocks. It is shown in TEM images that micelles formed were well dispersed as nanoscale micelles at room temperature, and the transition of the aggregating micellar nanoparticles with the increasing of the temperature can be found, and finally precipitation occurred at 75 °C. However, it is very difficult to identify the core layer, shell layer and corona layer of core–shell–corona micelles, the nanoscale micelles is usually observed to be a sphere by TEM, which is just like a core–shell one.38
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Fig. 18 TEM images of triblock copolymer nanoparticles of PMMA-b-PDMAEMA-b-PNIPAM (0.2 mg mL−1) at different temperatures at pH = 9. |
Footnote |
† Electronic supplementary information (ESI) available: Supplementary Fig. S1–S7: 1H NMR spectra, rheology, SEM image of triblock copolymer. See DOI: 10.1039/c7ra04351a |
This journal is © The Royal Society of Chemistry 2017 |