Letter pubs.acs.org/macroletters
Effect of Micellization on the Thermoresponsive Behavior of Polymeric Assemblies Lewis D. Blackman, Daniel B. Wright, Mathew P. Robin, Matthew I. Gibson,* and Rachel K. O’Reilly* University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K. S Supporting Information *
ABSTRACT: The chain density of polymer micelles, dictated by their aggregation number (Nagg), is an often overlooked parameter that governs the macroscopic behavior of responsive assemblies. Using a combination of variable-temperature light scattering, turbidimetry, and microcalorimetry experiments, the cloud point and thermal collapse of micellar poly(Nisopropylacrylamide) (pNIPAM) corona chains at lower temperatures than the cloud point were found to be largely independent of the micelle’s Nagg. By controlling the core composition, the degree of hysteresis associated with the thermal transition was found to increase as a function of core hydrophobicity. We performed this study on well-characterized micelles with tunable Nagg values, composed of a thermoresponsive corona (pNIPAM) and a nonresponsive core block poly(n-butyl acrylate-co-N,N-dimethylacrylamide) (p(nBA-co-DMA)), which were synthesized using reversible addition−fragmentation chain transfer (RAFT) polymerization. This allowed for a distinction to be made between thermoresponsive behavior at both the molecular and macroscopic level. The study of the subtle differences between these behaviors was made possible using a combination of complementary techniques. These results highlight the critical need for consideration of the effect that self-assembly plays on the responsive behavior of polymer chains when compared with free unimers in solution. “Smart” polymer materials that can respond to various external stimuli are of increasing interest and have the potential to be used in a range of applications. Key stimuli in the literature include light, pH, redox changes, enzymes, glucose, and CO2 to name a few, and there have been many reviews on these materials in recent years.1−9 One of the most commonly studied stimuli is temperature;8,10−18 unlike nonresponsive polymers, thermoresponsive polymers exhibit a change in solubility over a temperature range with either an upper or lower critical solution temperature (UCST or LCST).19 Above the LCST, or below the UCST, the polymer and solvent exist as a two-phase system, usually observable as macroscopic precipitation of the polymer. Thermoresponsive polymers have shown potential for a variety of applications including their use as delivery vehicles for increased cellular uptake11 and controlled drug release,12,13,18 thermoresponsive bioconjugates of DNA17 and proteins,14,20 and thermosensitive hydrogels for tissue engineering.15,16 One of the most studied thermoresponsive polymers is poly(N-isopropylacrylamide) (pNIPAM) which exhibits an LCST close to body temperature,10 making it especially applicable for biological applications. Although many reports on the potential applications of pNIPAM exist, one drawback is that it exhibits a thermal hysteresis,21 contrary to other thermoresponsive polymers.22−26 The slow reversibility of the thermal transition has been attributed to hydrogen bond formation between polymer chains,27 which has led to slow morphology transitions in some reports.28,29 It is important to stress that although pNIPAM and other thermoresponsive polymers exhibit properties that are well © XXXX American Chemical Society
characterized and studied in solution, when these exist as tethered chains, these properties can change drastically, and this leads to seemingly conflicting conclusions in the literature. For instance, Salmaso and co-workers found that gold nanoparticles decorated with thermoresponsive poly(N-isopropylacrylamideco-acrylamide) chains and folic acid showed no significant macroscopic aggregation when heated above the LCST.30 In contrast to this, Li and co-workers found that in their pNIPAMdecorated gold nanoparticle systems aggregation occurred, and the solution became turbid at temperatures above the LCST.31 While these results seem contradictory, especially considering the fact that both sets of gold nanoparticles were prepared by “grafting to” approaches, the colloidal stability of the former example was attributed to the presence of the hydrophilic folic acid, which prevented aggregation as it became exposed on the surface of the nanoparticle once the thermoresponsive chains collapsed. In both cases, the surface chemistry of the nanoparticle was a critical factor that influenced the macroscopic behavior of heating the system above the transition temperature. Additionally, in stark contrast to the work by Li using “grafting to” pNIPAM-decorated gold nanoparticles, Choi et al. showed that no aggregation occurred upon heating similar sized gold nanoparticles prepared using a “grafting from” approach.8,32 As the chain density of particles prepared by Received: August 6, 2015 Accepted: October 12, 2015
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DOI: 10.1021/acsmacrolett.5b00551 ACS Macro Lett. 2015, 4, 1210−1214
Letter
ACS Macro Letters Scheme 1. Synthesis of pNIPAM-b-[nBA-co-DMA] Block Copolymers and Their Subsequent Self-Assembly in Watera
a
Key: x = molar fraction of nBA monomer in the core-forming block.
Table 1. Block Copolymer and Resultant Particle Characterization. polymer
nBA:DMA feed ratio
DP nBAa
DP DMAa
mol % nBAa
Mn,NMR (g mol−1)a
Mnb
Đb
RH (nm)c
Rcore (nm)d
MCTA 1 2 3 4 5
N/A 1:1 7:3 4:1 9:1 1:0
N/A 20 26 27 37 40
N/A 17 11 7 4 N/A
N/A 54 70 79 90 100
8800 12900 12800 12400 13000 13900
10000 13000 12800 12700 11200 10900
1.07 1.10 1.11 1.10 1.11 1.11
N/A 9.3 13.6 15.1 14.9 19.8
N/A 3.5 5.6 6.2 7.3 8.1
Determined by 1H NMR spectroscopy. bDetermined by SEC (DMF) analysis. cRH of resultant micelles determined by DLS analysis at 20 °C. dRcore of resultant micelles determined by SLS analysis at 20 °C. a
“grafting from” is typically much greater than those prepared by “grafting to”, these results demonstrate the effects of altering the chain density of the thermoresponsive chains on the macroscopic behavior of very similar systems. The above findings highlight that the behavior of a particular nanoscale or bulk polymer system does not necessarily reflect the thermoresponsive properties of molecularly dissolved chains in solution. Similarly, although stimuli-responsive self-assemblies have been widely studied, little investigation has been done on the effects the subtle differences in a self-assembled particle’s structure has on the overall thermoresponsive properties. We hypothesized that the aggregation number (Nagg), defined as the number of polymer chains per particle, could be important in dictating the thermoresponsive behavior of self-assembled micelles. Certain self-assemblies comprised of amphiphilic diblock copolymers have tunable Nagg values by varying the solvophobicity of the core-forming block; indeed, Colombani and Chassenieux have demonstrated that more hydrophobic cores lead to higher Nagg values.33,34 Herein, micelles with varying pNIPAM coronal chain densities were prepared by synthesizing poly(N-isopropylacrylamide)75-b-[(nbutyl acrylate)x-co-(N,N-dimethylacrylamide)1−x]40 (pNIPAMb-[nBA-co-DMA]) block copolymers with varying amounts of permanently hydrophobic nBA in the core-forming block. The tunable Nagg of these micelles was shown to increase with increasing nBA composition. By combining microcalorimetry, light scattering, and turbidimetry analyses, the thermoresponsive behavioral properties, such as the coronal chain collapse and the degree of hysteresis of the pNIPAM coronal chains as a function of Nagg, were also studied. A series of core−shell micelles comprised of thermoresponsive, amphiphilic diblock copolymers of (pNIPAM-b-[nBAco-DMA]) were prepared by reversible addition−fragmentation
chain transfer (RAFT) polymerization followed by selfassembly in aqueous solution (1−5, Scheme 1). A pNIPAM macro chain transfer agent (MCTA) was synthesized first to ensure the diblock copolymers had identical corona-forming pNIPAM blocks of DP = 75. Core-forming blocks of total DP = 40 were targeted, and monomer feeds were selected such that the percentage composition of hydrophobic nBA in the coreforming block of each system ranged from 50% to 90% (1−4, Scheme 1). The reactivity ratios of nBA and DMA have also been shown to yield a nearly random copolymer,35 ensuring the DMA units are distributed randomly within the core-forming block such that phase separation of DMA and nBA units in the micellar core is not expected to occur. Additionally, a control diblock copolymer containing purely nBA in the core-forming block was prepared (5). The polymers were achieved with narrow dispersities, implying good control over the RAFT polymerization process (Table 1). Each block copolymer was self-assembled into micelles using a solvent switch technique from acetone into cold water (see SI for details). Variable temperature, multiangle dynamic, and static light scattering (DLS and SLS) were employed as techniques to investigate coronal chain collapse as a function of increasing temperature (see SI for SLS data analysis).36 Hydrodynamic radii, RH, were determined using DLS analysis at 20 °C and are outlined in Table 1. Since Rg is below 20 nm, these values could not be determined by SLS.36 The Nagg values determined from SLS increased with increasing composition of nBA units in the core-forming block which is in agreement with the literature (Figure 1).33,34 The CMCs of all the micelles investigated were below 0.157 μM (see SI for discussion). The controlled variability of Nagg across the series of micelles enabled us to study the effects of coronal chain confinement on the thermoresponsive behavior of the pNIPAM corona chains. 1211
DOI: 10.1021/acsmacrolett.5b00551 ACS Macro Lett. 2015, 4, 1210−1214
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ACS Macro Letters
The ω profile depicted in Figure 2 is independent of the percentage composition of the micellar cores and therefore of Nagg in the grafting regime investigated. This finding suggests that the coronal chain collapse reported by other groups38−40 occurs in micelles of both high and low Nagg. The fact that ω decreases at such low temperatures has implications for a wide range of applications including thermoresponsive surfacegrafted polymer coatings and thermoresponsive self-assemblies. As discussed previously, thermoresponsive polymers such as pNIPAM exhibit a cloud point in aqueous solution when heated above the LCST. Note that this is the optically observable macroscopic effect of the LCST and is defined here as the temperature at which the transmittance is reduced to half the original transmittance observed in the homogeneous regime. In each micellar system, macroscopic precipitation occurred. Turbidimetry analysis of the micelles confirmed that the cloud point upon heating the samples is largely independent of the aggregation number (Figure 3). Therefore,
Figure 1. Plot of Nagg with varying nBA units in the micellar core determined by SLS at 20 °C with 10% error bars.
Similar analyses were performed at temperatures ranging from 10 to 25 °C. Using the Nagg values obtained from SLS at each temperature, the core radii, Rcore, of the micelles were determined as a function of temperature (see SI for more details).36 It can be seen that in each system the RH values decrease with increasing temperature (Figure S6); however, Rcore did not vary significantly across the temperature range (Figure S7). The observed trend in RH is rationalized by the collapse of the coronal chains with increasing temperature as the solvent quality decreases resulting in more hydrogen bonds made between polymer chains and fewer solvent−polymer interactions. By subtracting Rcore from RH, the corona radius, Rcorona, could be calculated. Dividing Rcorona by the maximum chain length of 18.75 nm (assuming a repeat unit length of 0.25 nm)36 allowed calculation of the coronal chain stretching, ω (see SI). Figure 2 shows the normalized variation of ω as a
Figure 3. Left axis: Temperature of phase transitions measured by variable temperature techniques at 1 mg/mL with 10% error bars. Black squares represent cloud points determined by turbidimetry. Red circles represent Tp values determined by microcalorimetry. Right axis: Degree of hysteresis observed by turbidimetry analysis. Error bars represent the standard deviation over three repeats.
the degree of chain aggregation, as dictated by Nagg, does not influence the temperature at which the macroscopic effect of phase separation and precipitation occurs. In addition to the macroscopic precipitation, the temperature (Tp) at which the heating endotherm from microcalorimetry occurs is also independent of Nagg. This indicates that the temperature at which the breaking of hydrogen bonds between the polymer and the solvent occurs does not depend on the degree of aggregation at the chain densities investigated. As has also been observed in the literature, for high chain densities of thermoresponsive polymers, two distinct transitions occur upon heating the sample, as observed by microcalorimetry.38,40 This has been attributed to the existence of distinct inner and outer coronal subzones that undergo transitions at different temperatures to one another owing to the higher chain density of the inner subzone, which promotes polymer−polymer interactions at lower temperatures. However, in our work, microcalorimetry of the micelle solutions showed that the heat capacity (Cp) profile of each of the systems shows only one obvious transition. This is rationalized by considering that in our systems the core is more mobile than the gold or dendritic cores of the aforementioned studies, and so the coronal chains in our study do not exhibit this behavior.
Figure 2. Normalized ω as a function of temperature, as determined by a combination of DLS and SLS. Key: Micelles formed from copolymers of 1 (black squares), 2 (red circles), 3 (blue triangles), and 4 (pink triangles).
function of temperature. These results are of great consequence, as it can be seen that for all four systems investigated the onset of thermoresponsive behavior at the molecular level occurs at much lower temperatures than the cloud point of pNIPAM, which is known to be around 32 °C.10,37 The observed coronal chain collapse at temperatures lower than the expected LCST is in agreement with the work on pNIPAM from both Liu and Tenhu, for both micelles comprised of dendritic cores and amphiphilic block copolymers, respectively.38,39 This finding highlights that macroscopic precipitation (cloud point) is distinct from the LCST and that individual chains begin to collapse well before this temperature. 1212
DOI: 10.1021/acsmacrolett.5b00551 ACS Macro Lett. 2015, 4, 1210−1214
Letter
ACS Macro Letters Micelles of polymer 4 have a chain density of 0.28 chains nm−2 at the core−corona interface (see SI for details of this calculation), which is comparable to the work by Tenhu and co-workers,40 so the coronal chains within each micelle are in close enough proximity to interact with one another. The cloud points upon cooling the samples, as determined by turbidimetry, varied drastically across the micelle systems. In the cooling cycle, the micelles are resuspended and the coronal chains rehydrated since the temperature decreases back through the spinodal and coexistence curves of the phase diagram to reform a one-phase system. The degree of hysteresis, defined as the difference in the cloud points upon heating and upon cooling, in each system increased as a function of the molar percentage composition of nBA in the core-forming block (Figure 3). The increase of hysteresis with Nagg provided an opportunity to investigate the origin of hysteresis in these systems. The trend is attributed to two possible phenomena: one being that in loosely packed micelles, with low Nagg, the chains are free to collapse and redissolve with little steric repulsion from intraparticle coronal chains leading to rapid resuspension of the micelles upon cooling. Conversely, in high Nagg micelles, confinement of the chains means that upon aggregation chains within an individual particle become entangled and need to overcome the additional energy barrier of disentanglement in order to redissolve, leading to a higher degree of hysteresis (Figure 4A).
micelles that exhibit a low degree of hysteresis would also have more solvated cores owing to their lower nBA content which makes the core less hydrophobic. This phenomenon leads to the formation of more hydrated precipitates above the cloud point. Upon cooling, less penetration of water molecules into the precipitates is necessary in order to resuspend the micelles leading to a lower degree of hysteresis (Figure 4B). Turbidimetry studies on micelles comprised of a pNIPAM300b-nBA32 diblock copolymer (6) revealed that the hysteresis was reduced upon increasing the molecular weight of the corona. This result disagrees with the notion that chain entanglement is the cause of the hysteresis and indicates that core hydration is the most likely cause of the differences in the degree of hysteresis across the series of micelles of polymers 1−5 (see SI). In summary, we have found that for pNIPAM coronal chains the observed cloud point upon heating micellar solutions is largely independent of Nagg. Collapse of the pNIPAM coronal chains was observed prior to the cloud point; however, the degree of coronal collapse over the temperature range investigated was not dictated by Nagg. The observed hysteresis showed a positive correlation with core hydrophobicity. This hysteresis is attributed to decreased hydration of the micellar cores with increasing core hydrophobicity. We were able to study this by controlling the Nagg of pNIPAM-b-[(nBA-coDMA)] micelles by varying the percentage composition of nBA in the core-forming block. A combination of multiangle variable-temperature DLS and SLS, turbidimetry, and microcalorimetry was used to probe the effects of Nagg on the thermoresponsive behavior of the pNIPAM coronal chains. These findings demonstrate that free unimeric polymer chains in solution do not necessarily exhibit the same responsive behavior when tethered as part of a self-assembled particle and that small changes in the particles’ structure can have a dramatic effect on its physical properties. These results also highlight that consideration of the behavior at both the macroscopic and molecular levels needs to be addressed when designing responsive polymer systems for real-world applications.
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Figure 4. Schematic representation of the two possible proposed causes of the increased degree of hysteresis in more hydrophobic micelles. (A) Higher chain density in high Nagg micelles leads to higher interchain entanglement in the globular state, above the transition temperature. (B) Increased water exclusion in highly hydrophobic micelles above the transition temperature, compared to the less hydrophobic cores which exhibit more hydrated precipitates.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00551. Experimental procedures and characterization data, additional information regarding the light-scattering analysis, additional microcalorimetry, rheology, and CMC data, and a discussion on the effect of particle concentration and corona molecular weight on hysteresis (PDF)
This is a phenomenon proposed by Zhu and Napper who showed by light scattering that this occurs in poly(styrene) latex particles with grafted pNIPAM coronas.41 Briefly, they found that when held at temperatures above the transition temperature, and then quenched at temperatures below the transition temperature, different degrees of hysteresis were observed which were dependent upon the holding temperatures and the amount of time held at the temperature above the transition temperature. This was attributed to an increased degree of entanglement at both higher temperatures and longer heating times. Rheology data of MCTA indicate that the corona chains in our system are able to entangle within the micelle (see SI). In our system, however, the nature of the core-forming block is also a variable so the differences in the degrees of hysteresis could be caused by something other than simply differences in entanglement. One other explanation is that as the cores of the micelles differed in hydrophobicity, low Nagg
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (M. I. Gibson). *E-mail:
[email protected] (R. K. O’Reilly). Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest. 1213
DOI: 10.1021/acsmacrolett.5b00551 ACS Macro Lett. 2015, 4, 1210−1214
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ACS Macro Letters
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(33) Wright, D. B.; Patterson, J. P.; Pitto-Barry, A.; Cotanda, P.; Chassenieux, C.; Colombani, O.; O’Reilly, R. K. Polym. Chem. 2015, 6, 2761−2768. (34) Lejeune, E.; Drechsler, M.; Jestin, J.; Müller, A. H. E.; Chassenieux, C.; Colombani, O. Macromolecules 2010, 43, 2667−2671. (35) Neugebauer, D.; Matyjaszewski, K. Macromolecules 2003, 36, 2598−2603. (36) Patterson, J. P.; Robin, M. P.; Chassenieux, C.; Colombani, O.; O’Reilly, R. K. Chem. Soc. Rev. 2014, 43, 2412−2425. (37) Djokpé, E.; Vogt, W. Macromol. Chem. Phys. 2001, 202, 750− 757. (38) Luo, S.; Xu, J.; Zhu, Z.; Wu, C.; Liu, S. J. Phys. Chem. B 2006, 110, 9132−9139. (39) Nuopponen, M.; Ojala, J.; Tenhu, H. Polymer 2004, 45, 3643− 3650. (40) Shan, J.; Chen, J.; Nuopponen, M.; Tenhu, H. Langmuir 2004, 20, 4671−4676. (41) Zhu, P. W.; Napper, D. H. J. Colloid Interface Sci. 1994, 168, 380−385.
ACKNOWLEDGMENTS We thank EPSRC, BP, the ERC, and the IAS for financial support. We also thank Malvern Instruments Ltd. for use of their microcalorimetry facilities.
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