ARTICLE pubs.acs.org/Langmuir
Thermally Triggered Assembly of Cationic Graft Copolymers Containing 2-(2-Methoxyethoxy)ethyl Methacrylate Side Chains Nur Nabilah Shahidan,† Ruixue Liu,†,‡ Francesco Cellesi,§ Cameron Alexander,|| Kevin M. Shakesheff,|| and Brian R. Saunders*,† †
Biomaterials Research Group, The School of Materials, The University of Manchester, Grosvenor Street, M13 9PL, United Kingdom School of Material and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou, 450002, P.R. China § School of Pharmacy, The University of Manchester, Oxford Road, M13 9PT, United Kingdom School of Pharmacy, The University of Nottingham, University Park, Nottingham, NG7 2 RD, United Kingdom
)
‡
bS Supporting Information ABSTRACT: Thermoresponsive copolymers continue to attract a great deal of interest in the literature. In particular, those based on ethylene oxide-containing methacrylates have excellent potential for biomaterial applications. Recently, some of us reported a study of thermoresponsive cationic graft copolymers containing poly(N-isopropylacrylamide), PNIPAm, (Liu et al., Langmuir, 24, 7099). Here, we report an improved version of this new family of copolymers. In the present study, we replaced the PNIPAm side chains with poly(2-(2-methyoxyethoxy)ethylmethacrylate), PMeO2MA. These new, nonacrylamide containing, cationic graft copolymers were prepared using atom transfer radical polymerization (ATRP) and a macroinitiator. They contained poly(trimethylamonium)-aminoethyl methacrylate and PMeO2MA, i.e., PTMA+x-g-(PMeO2MAn)y. They were investigated using variable-temperature turbidity, photon correlation spectroscopy (PCS), electrophoretic mobility, and 1H NMR measurements. For one system, four critical temperatures were measured and used to propose a mechanism for the thermally triggered changes that occur in solution. All of the copolymers existed as unimolecular micelles at 20 °C. They underwent reversible aggregation with heating. The extent of aggregation was controlled by the length of the side chains. TEM showed evidence of micellar aggregates. The thermally responsive behaviors of our new copolymers are compared to those for the cationic PNIPAm graft copolymers reported by Liu et al. Our new cationic copolymers retained their positive charge at all temperatures studied, have high zeta potentials at 37 °C, and are good candidates for conferring thermoresponsiveness to negatively charged biomaterial surfaces.
’ INTRODUCTION Research activity on thermoresponsive copolymers continues to grow rapidly111 because of the ability to control large-scale conformational changes using macromolecular architectural design. There is a growing trend toward preparing complex architectures with the objective of producing greater tuneability of thermoresponsiveness. Examples include multihydrophilic block copolymers,12 triblock copolymers,1315 double thermoresponsive block copolymers,16 and star-shaped copolymers.17 We have a strong interest in using thermoresponsive copolymers to render particles that are thermoresponsive and gel forming,18,19 because this has potential application in terms of injectable biomaterials. Recently, some of us reported a new family of thermally responsive graft copolymers.20 The copolymers contained a cationic backbone and poly(N-isopropylacrylamide), PNIPAm, side chains. They exhibited thermogelation20 and also conveyed thermoresponsiveness to anonic PLGA (poly(lactic acid-co-glycolic acid)) nanoparticles.18 Thermoresponsive r 2011 American Chemical Society
copolymers based on poly(ethylene glycol) (PEG) have some practical advantages compared to PNIPAm4,21 and have shown good potential for biomaterial applications in preliminary in vivo studies.22,23 In this study, we show that the PNIPAm side chains can be replaced with poly(2-(2-methoxyethoxy)ethyl methacrylate), PMeO2MA. The study of PMeO2MA-based thermoresponsive copolymers was pioneered by Lutz et al.,24,25 who demonstrated facile synthesis, controlled molar mass, and highly tunable thermal transitions with these PEG-methacrylate-type materials. The purpose of the present study was to investigate the thermoresponsive properties of dilute dispersions of a new family of PMeO2MA-based cationic copolymers. We hypothesized that both the micellar structure and the cloud point temperature would be tunable through the architecture of these new graft Received: August 16, 2011 Revised: October 1, 2011 Published: October 03, 2011 13868
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Scheme 1. Method Used to Prepare Thermoresponsive Cationic Copolymersa
The idealized unimolecular micelle is depicted as a cylinder with two hemispheres at each end. It is likely that the side chains would “spill over” at the ends to some extent, which means that Lm(uni) + dm(uni) is the maximum size and that dm(uni) is the minimum size. Note that the positive charge does not sit exactly on the backbone of the graft copolymer. a
copolymers. The overall aim of this study was to explore the effects of graft copolymer architecture (side-chain lengths and grafting density) on the thermally triggered changes in micelle structure. For PMeO2MA copolymers, copolymerization with longerchain EO-containing methacrylates provides a well-established method for systematically tuning the LCST.4,2426 PMeO2MAcontaining copolymers have very good versatility and have recently been prepared as coreshell dendritic particles,27 conjugated with fluorescent probes, and 28 polymerized from salmon calcitonin,29 as well as being formed as co-networks30 and in thermogelling systems.31 The key advantage for the latter systems is the potential for improved thermoreversibility. PNIPAm-based thermogelling systems often suffer from hysteresis in thermal response due to intermolecular hydrogen bonding.32 The study of cationic thermoresponsive graft copolymers is a relatively new research area.20,33,34 Cationic thermoresponsive copolymers have been well-studied.5,34 Furthermore, nonionic thermoresponsive graft copolymers have also received attention.35 An advantage offered by graft copolymers with a cationic backbone and thermoresponsive side chains is the potential to adsorb onto anionic substrates and provide a thermoresponsive coating.36 Here, we used a similar synthetic method to that established in our earlier study20 to prepare thermoresponsive cationic graft copolymers containing a poly(trimethylamonium)-aminoethyl methacrylate backbone and PMeO2MA side chains, i.e., PTMA+x-g-(PMeO2MAn)y. The copolymers studied here are abbreviated as M(x/y)-PMAn and the preparation method is depicted in Scheme 1. The copolymers were prepared using atom transfer radical polymerization (ATRP) and the macroinitiator initially reported by Armes et al.37 We take advantage of ATRP here to obtain graft copolymer architectures that are not obtainable using other routes. The major differences between this study and our earlier report20 is the side-chain type (PMeO2MA, cf., PNIPAm) and also side-chain length. Because the overwhelming majority of the mass of these copolymers is made up of the side chains, this causes major differences in the behavior. In this paper, we focus on the dilute solution properties of these new copolymers. Because the M(x/y)-PMAn copolymers exist as welldefined unimolecular micelles at 20 °C, new structureproperty
insights are obtained for this family of graft copolymers that were not available from the earlier study.20 The results of this study show that the thermally responsive cationic copolymers investigated here for the first time are better model systems and have thermoresponsive properties that are tunable. The new PMeO2MA-based copolymers also have good potential for future biomaterial applications because PMeO2MA copolymers have been shown to be well-tolerated in vitro and in preliminary in vivo experiments.38,39
’ EXPERIMENTAL SECTION Reagents. 2-(2-Methoxyethoxy)ethyl methacrylate (MEO2MA, 95%) was purchased from Sigma Aldrich and purified using an alumina column twice. Copper bromide (CuBr, 99.9999%) and N,N,N0 ,N00 ,N00 -pentamethyldiethylenetriamine (PMDETA, 99%) were purchased from Sigma Aldrich and used as received. Methanol was analytical reagent grade (Fisher Scientific, 99.99%). Water was Milli-Q grade quality. The solvents were degassed using Ar prior to use. Two macroinitiators (M) were used in this work. They were synthesized using the method described in detail earlier20, which was originally devised by Chen et al.37 M1 and M3 had x/y ratios (Scheme 1) of 1 and approximately 3, respectively. The values of x and y for M1 were both 23. The values of x and y for M3 were 37 and 12, respectively.20 Copolymer Synthesis. The cationic copolymers and the macroinitiators were synthesis using ATRP. The composition used to discuss the copolymers is PTMA+x-g-(PMeO2MAn)y, where x, y, and n are the numbers of TMA, esterified HEMA20, and MeO2MA units in the copolymer, respectively. We use the shortened abbreviation of M(x/y)PMAn in this work. Thus, M1-PMA29 represents the cationic MeO2MA graft copolymer prepared using M1 and containing n = 29, i.e., PTMA+23-g-(PMeO2MA29)23. See Table 1. The following describes the preparation of M1-PMA52. A Schlenk flask was purged with Ar, and monomer (MeO2MA, 14.2 g, 75.2 mM) was added with stirring to the flask which contained 120 mL methanol and M1 (0.29 g, 0.16 mM) dissolved in 15 mL of deionized water. Next, the flask was pumped and filled with Ar nine times. Then, PMDETA (50.8 mg, 0.29 mM) dissolved in 5 mL of water was added to the flask under Ar and repeatedly evacuated and the flask filled with Ar. Cu(I)Br (62.0 mg, 0.43 mM) was added to start the polymerization while stirring. The flask was again evacuated and filled with Ar. The flask was immersed in a preheated oil bath at 60 °C for 6.5 h. After polymerization, the 13869
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Table 1. Parameters for ATRP and Characterization Data for Copolymers microanalysis
1
H NMR
copolymer code
PTMA+x-g-(PMeO2MAn)y
M1-PMA18
PTMA+23-g-(PMeO2MA18)23
M1-PMA29
PTMA+23-g-(PMeO2MA29)23 PTMA+23-g-(PMeO2MA52)23 PTMA+23-g-(PMeO2MA101)23 PTMA+37-g-(PMeO2MA69)12 PTMA+23-g-(PNIPAm195)23
M1-PMA52 M1-PMA101 M3-PMA69 M1-PNP195
% conva
%C
%N
n(micro)b
n(NMR)c
50:1:1:1
34
57.4
0.38
17.7 [1.2]
-
90 [5]
33.5
100:1:1:1
30
-
-
-
29.4 [1.5]
140 [6]
28.0
200:1:1:1
26
-
-
-
51.5 [4.2]
236 [18]
30.0
400:1:1:1
25
-
-
-
101 [15]
450[66]
27.0
100:1:1:1
66
55.7
171 [13] f
35.0
200:1:1:1
-
515 g
33.3
[M]:[I]:[CuBr]:[PMEDTA]
-
0.30 -
69.2 [5.7] -
Mnd/ (kg/mol)
-
f
195
a
Tcpe/°C
b
Calculated using the experimentally determined molecular weights compared to theoretical values expected from 100% conversion. Calculated from % C/ % N ratio. The equation used was for PTMA+x-g-(PMeO2MAn)y was n = {(1.166x RCN) 9x 10y 6}/(9y), where RCN = (% C/% N). c Based on A1/A2 values obtained from 1H NMR data (See text). d Values for Mn were calculated using eq 2. e Average values measured in water ((1 °C). f Microanalysis values used to calculate Mn (See text). g From ref 20. The numbers in square brackets in the table represent the estimated (() error of the n or Mn values. catalyst and ligand were removed by dialysis against water. The dialysis tubing used had a molecular weight cutoff equal to 3500 g/mol. The purified copolymer was isolated by freezedrying. The other copolymers were prepared and purified using an equivalent method; see Table 1. Inductively coupled plasma optical emission spectroscopy (ICP-OES) showed that the residual Cu present in M1-PMA29 and M3-PMA69 was 0.002 and 0.001 wt %, respectively. Complex formation of the metal by the copolymers was not significant. The compositions of the copolymers were determined using microanalysis and/or 1H NMR. Physical Measurements. 1H NMR spectra were measured using a Bruker 300 and 400 MHz instrument using D2O or acetone-d6 as the solvent. The Bruker 400 MHz instrument was used for variable temperature measurements. Microanalysis (C, H, and N) was conducted at the School of Chemistry, University of Manchester. The cloud point temperature, Tcp, of the copolymers was determined using a thermostatically controlled Perkin-Elmer UVvis spectrometer and a wavelength of 400 nm. The cationic graft copolymers studied here had a pronounced tendency to adhere to anionic substrates and could not be analyzed using GPC. (This was also the case for the NIPAM-based cationic graft copolymers reported earlier.20) For the present copolymers, 1H NMR spectroscopy or microanalysis was used to calculate the number-average molecular weights. Duplicate (or more) microanalytical data were used. These methods did not permit information regarding the molecular weight distributions or polydispersities to be obtained. ICPOES measurements were performed using a Thermo iCap 6300 inductively coupled plasma optical emission spectrometer. A Malvern Zetasizer with variable temperature capability was used to measure the hydrodynamic diameters and zeta potentials for the copolymers. Zeta potential measurements were performed in the presence of 0.001 M NaCl. TEM measurements were carried out using a Philips CM30 instrument. To prepare samples for TEM measurements, a mixture of 1.6 mL of 2 wt % phosphotungstic acid (Sigma-Aldrich) and 0.4 mL copolymer solution (ca. 0.1 wt %), was prepared. After 15 min, a drop of the mixture was placed on a holey carbon grid and left for 5 min before a tissue was used to remove excess liquid. The samples were left to dry overnight in a desiccator.
’ RESULTS AND DISCUSSION Composition of the Copolymers. The copolymers depicted in Scheme 1 do not show a Br group or a PMA chain at the end of the macroinitiator. These groups are only depicted as side arm chains. We have not established that extension from the end of our macroinitiator chain end is possible. In keeping with reaction schemes from related work40,41 involving macroinitiators, we omit this Br group. We focus on side arm growth only. Even if the
Br group in question produced an additional PMA chain at the end of the macroinitiator, this would only increase the molecular weights by less than 8%. Furthermore, it is unlikely that the presence of a PMA chain at the end of the macroinitiator would significantly affect the properties of these new thermally responsive copolymers. For the copolymerizations conducted in this study, a methanol/ water mixture containing a methanol volume fraction (ϕMeOH) of 0.85 gave the best compromise between macroinitiator solubility and minimization of the polymerization rates. High solution viscosities and gelation occurred if ϕMeOH values less than 0.85 were used. The copolymers studied here are cationic and strongly adsorbed to negatively charged surfaces and also polar stationary phases used for GPC analysis. We therefore determined the number-average molecular weight (Mn) of the copolymers using either 1H NMR spectra or microanalysis (see Table 1). For the latter, the ratio of %C/%N was used to calculate Mn. The average errors (() estimated for the Mn values obtained from NMR or microanalysis data are shown in Table 1. For nonlinear copolymers, it is known that 1H NMR spectroscopy provides more representative molecular weights than GPC, because the latter is usually based on linear polymer standards.12 The following describes the 1H NMR method used to determine the molar mass of the copolymers. We used the ratio of the integrals for A1 and A2 (Figure 1) to estimate the value for n for the M(x/y)-PMAn copolymers. The equation used was 11 R n¼ ð1Þ 3 2R where R = A1/A2. The equation used to calculate the value of Mn from n, x, and y is shown in the following and assumes that the side arm chains have a terminal Br group. Mn ¼ 195 þ 299x þ 279y þ 188ny
ð2Þ
The A1 and A2 peaks are mainly due to CH2 groups in the EO units and the CH3 groups in TMA+, respectively. The experimental and theoretical values for A1/A2 are plotted against [M]/[I] in Figure 1d. The experimental A1/A2 values for the M1-PMAn series were all less than the theoretical values for 100% conversion, with the exception of [M]/[I] = 52. However, the experimentally determined A1/A2 value was artificially high because that copolymer was not completely soluble in acetone-d6. This is because of the relative short PMA side chains. We used microanalysis to determine n for that copolymer 13870
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Figure 1. (a) 1H NMR spectra and assignments for M1-PMAn copolymers. (b) 1H NMR spectra and assignments for M3-PMA69 in D2O at 10 °C or acetone-d6. The peaks in (a) labeled with * are solvent peaks. (c) Structure and assignments for M(x/y)-PMAn. (d) Variation of the calculated (100% conversion) and experimental values for the ratio of the A1 and A2 areas with values of [M]/[I] used for preparation of the copolymers. The M1-PMAn spectra were measured using acetone-d6. The experimental data point for the M3-PMA69 was measured using D2O at 10 °C. The error bars are a similar size to the data points.
(M1-PMA18, Table 1). The values for A1/A2 for the other three copolymers are lower than expected (i.e., smaller n) because the polymerizations did not reach high conversions. The M1-PMAn series typically reached about 2535% conversion (Table 1). This low conversion was advantageous because it avoided gelation and gave thermally responsive micellar solutions. The M3-PMA69 copolymer ([M]/[I] = 100, Figure 1d) had an experimental A1/A2 ratio measured in D2O at 10 °C that was much lower than that expected. This copolymer has the highest number of TMA+ groups and was not very soluble in acetone-d6. It was partly soluble in water at 10 °C (Figure 1b). However, the signals for the backbone protons (a, b, and c) are barely visible in the spectrum. This implies poor solvency for that part of the copolymer. The signals for the PMA protons (e00 , f, and g) have merged but are still present. This is why the A1/A2 ratio is particularly low for that system. The spectra obtained in acetone-d6 (Figure 1b) was subject to broadening in all peaks. On the basis of these considerations, we consider the values for n determined by 1H NMR to be reliable for M1-PMAn when [M]/[I] was within the range 100400 (inclusive). For M1-PMA18 and M3-PMA69, n values determined by microanalysis and eq 2 were used (Table 1). A check of consistency between Mn values calculated using 1H NMR and microanalysis was performed for M1-PMA52. A sample was analyzed using microanalysis, and %C and %N values of 56.9% and 0.15%, respectively, were obtained. The calculated value for n from eq 2 is 47.0 [(3.5]. This is reasonably close to the value of
51.5 [(4.2] determined using 1H NMR spectroscopy (Table 1). We used values for n calculated using 1H NMR for M1-PMA29, M1-PMA52, and M1-PMA101 because the samples appeared to be fully soluble in acetone-d6 and the %N values were less than 0.3%. Dilute Dispersion Behavior. The variable temperature transmittance of the M1(x/y)-PMAn copolymers was investigated in both water (Figure 2a) and aqueous phosphate buffered saline (PBS, Figure 2b). The transmittances of the copolymers were less than 100% at room temperature. This is indicative of scattering due to the copolymer. It was observed that the asmade methanol/water solutions were transparent. However, the dispersions became slightly turbid during dialysis. This is an important observation because it indicates that water is a less favorable solvent for the copolymer than the water/methanol cosolvent blend and strongly suggests micelle formation. Figure 2a shows variation of transmittance with temperature for each of the systems studied in water. The data obtained using M1-PMAn show typical LCST behavior in that the transmittance decreases strongly above a critical temperature, which is the cloud point (Tcp). Here, the value for Tcp was taken as the temperature at which the transmittance had reached half of the total change.34 It can be seen from Figure 2a that the transmittance decreases relatively sharply for M1-PMA29, M1-PMA52, and M1-PMA101. However, it decreases more gradually with temperature for the more highly charged copolymers, M1-PMA18 and M3-PMA69. The data for M3-PMA69 showed a modest 13871
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Figure 2. (a) Transmittance as a function of temperature for 1 wt % solutions of M1-PMAn copolymers or M3-PMA69 measured in (a) water or (b) pH = 7.4, 0.15 M PBS. The legend in (a) applies to both (a) and (b). The variations of Tcp with n (side chain length) for the polymers in water or PBS solution are shown in (c). The lines are guides for the eye.
increase in transmittance prior to the onset of a decrease which, although unexpected, does not contribute to the thermally triggered micellar aggregation processes discussed in this work. Lutz and Hoth25 reported a Tcp for linear PMAn (Mn = 16 700 g/mol) of 28 °C. This is reasonably close to the Tcp values for the M1PMA29, M1-PMA52, and M1-PMA101 copolymers (Table 1). The Tcp values for the more highly charged copolymers (M1PMA18 and M3-PMA69) are 57 °C higher. This new finding is attributed to higher intersegment electrostatic repulsion, which opposes thermally triggered contraction. For a thermoresponsive copolymer that has potential application as a biomaterial, the Tcp value measured at physiological pH and ionic strength is important. This was probed for our copolymers (see Figure 2b). The thermally triggered decreases in transmittance became sharper and shifted to lower temperature for the copolymers in the presence of PBS. Figure 2c shows a comparison of the Tcp values measured in water and in the presence of PBS. For the M1-PMAn systems, the Tcp values decreased by 78 °C in the presence of PBS. This decrease is much greater than the 3 °C decrease reported for linear PMAn copolymers by Lutz et al.24 For all of the M(x/y)-PMAn copolymers, all of the Tcp values are greater than 20 °C. This could provide a useful working window for solution storage prior to in vivo injection in future potential biomaterials applications. The decrease of Tcp due to PBS was 14 °C in the case of M3PMA69, the most highly charged system. These data demonstrate that the thermally triggered aggregation of M(x/y)-PMAn solution species is largely controlled by electrostatic interactions. However, it is noted that the decrease in Tcp due to electrolytes will also have a contribution from competitive dehydration of the copolymer chains. This general effect was reported by Saunders and Daly in 2000 for PNIPAm-based microgels42 and more recently by Lutz et al.24 for PMAn copolymers. The remainder of
the measurements reported in this study were performed in pure water unless otherwise stated. Figure 3a shows the variation of the hydrodynamic diameter with temperature for the copolymers. There are three types of behaviors apparent. The first is where the diameter decreases with increasing temperature. This occurs for the M1-PMA29, M1-PMA52, and M3-PMA69 copolymers. The second type is a general increase in particle size with temperature over the whole temperature range. This is apparent for M1-PMA18. The third type is observed for M1-PMA101 and is a combination of the first two, i.e., both an increase and decrease in size are apparent with increasing temperature. This latter type of thermally triggered increase in diameter is commonly observed for thermoresponsive copolymers.34,43 It is the first type of behavior (diameter decreasing with temperature) that is less commonly observed.16 The variation of size at 20 and 45 °C with n is shown in Figure 3b. Interestingly, it seems that this behavior is controlled by n, i.e., the side arm chain length. If n is too low (ca e20) or too high (g100), then the diameter increases with heating. It was suspected that electrostatic interactions played a role in the hydrodynamic diameter changes with temperature. Specifically, a thermally triggered increase in surface charge density could be expected if the cationic backbone charges approached the micelle surface. This could be expected at temperatures approaching Tcp since the hydrophobic PMAn segments would tend to occupy more of the center of the copolymer micelles. It is well-known that an increase in surface charge density increases colloidal stability in the context of surfactant-free emulsion polymerization.44 However, in the present case the polymers were preformed and emulsion polymerization was not involved. If electrostatic stabilization was present at temperatures greater than Tcp, then added electrolyte should cause screening and aggregation. This was tested for M1-PMA101. It was found that 13872
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Figure 3. (a) Variation of hydrodynamic diameter with temperature for a range of copolymer solutions. (b) Variation of hydrodynamic diameter for the M(x/y)PMAn copolymers at 20 and 45 °C as a function of n (side-chain length). The legend shows the copolymer abbreviations and temperatures in brackets. (c) Calculated aggregation number at 45 °C from eq 3—see text.
the presence of 0.05 M NaCl was sufficient to trigger precipitation upon heating (see Supporting Information Figure 1S). It is concluded that steric stabilization was present at temperatures less than Tcp and electrostatic stabilization became the dominate stabilization mechanism for the M(x/y)PMAn copolymers at higher temperatures. However, an aggregation step was also involved (discussed below). An important question concerns the identity of the micelles present for each of the copolymers at the different temperatures probed in Figure 3a. This can be addressed using a simple geometric model based on the idealized unimolecular micelle structure shown in Scheme 1. If it is assumed that the backbone and side chains are linear (fully elongated), then the values for dm(uni) and Lm(uni) (in nm) can be shown to be approximately equal to 0.48n and 0.24(x + y), respectively. As a simple estimate, we calculate the ratios of the hydrodynamic diameters at 20 °C (d20) to the maximum unimolecular micelle size (which is dm(uni) + Lm(uni) from Scheme 1). The calculated values for d20/(dm(uni) + Lm(uni)) are shown in Supporting Information Table 1S. The average value for all of the copolymers is 1.7. The values are reasonably close to 1.0 given the simplicity of these calculations. We can therefore propose that all of the M(x/y)PMAn copolymers are unimolecular micelles at 20 °C. What happens to the unimolecular micelles when the solutions are heated? To address this question, the aggregation number at 45 °C (p45) was calculated using the following equation, which is based on the general approach used by Zhou et al.13 p45 ¼
πFNA d3 6M
! ð3Þ
For this equation, M, F, and d are molecular weight, density, and hydrodynamic diameter, respectively. The values calculated for p45 are shown in Figure 3c. They were calculated using data from Table 1 and a nominal density of 1.0 g/mL. The presence of the polyelectrolyte backbone should prevent complete collapse of the micelles in the absence of added electrolyte. This means that the values calculated for p45 overestimate the true values. Nevertheless, these values are much greater than 1.0 and support the view that micelle aggregation occurs. That is despite the fact that the overall particle sizes decrease with temperature for several of the systems (Figure 3a). The alternative approach of assuming that no aggregation occurs (p45 = 1) results in unrealistically low polymer volume fractions at 45 °C. We report the new finding that p45 has a minimum (of 68.5) when n = 52. This may be due to an optimum combination of low ionic strength (the copolymers are polyelectrolytes) and closer proximity of the positive charges to the surface due to a moderate n value. The reversibility of the thermally triggered heating was investigated using PCS measurements (Supporting Information Figure 2S) for the M1-PMA52 and M1-PMA101 solutions. The data show that the thermally triggered aggregation processes had good reproducibility when the temperature was cycled between 20 and 45 °C. This was best for M1-PMA52, which is probably due to the lower extents of thermally triggered micellar aggregation (Figure 3c). Figure 4a shows the variation of zeta potential (ζ) with temperature for the copolymer micelles. The M1-PMAn copolymer samples show similar trends. In each case, the ζ values are positive and the copolymer micelles are cationic. At 20 °C, the micelles probably have the PTMA+ backbone partially buried, which is why the ζ values are relatively low. When the temperature exceeds Tcp, the ζ values increase. The ζ values at 37 °C 13873
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Figure 4. (a) Variation of zeta potential with temperature for M(x/y)-PMAn samples. (b) Variation of zeta potential at 20 and 45 °C as a function of copolymer side-chain length (n) for M3-PMA69 and M1-PMAn.
Figure 5. (a) 1H NMR spectra recorded for M1-PMA29 in D2O at various temperatures (shown as °C in the figure). (b) Variation of A1/A2 with temperature for the copolymer in D2O. (c) Variation of transmittance, zeta potential (ζ), and hydrodynamic diameter (d) with temperature. The arrows in (b) and (c) show the critical temperatures—see text.
are high, which suggests that these micellar aggregates would be good candidates for adsorption onto negatively charged surfaces or DNA under physiological conditions. The data shown in Figure 4a imply that there is a continual structural rearrangement that proceeds with increasing temperature that brings positive charges closer to the surface. Figure 4b shows the effect of n on the ζ values measured at 20 and 45 °C. These data emphasize the major changes in electrostatic interactions that occur when the temperature is increased. These data reinforce the proposal above that the stabilization mechanism must switch from electrosteric (i.e., electrostatic plus steric) at 20 °C to electrostatic at 45 °C. It is interesting that the extent of the ζ increase is much less significant for M3-PMA69 compared to the M1-PMAn copolymers. This is probably because of the greater extent of occupancy of positive charge at the periphery of the micelles for M3-PMA69 at 20 °C. M3-PMA69 and M1-PMA18 (which had the shortest side chains) gave the highest ζ values at 20 °C, which is expected from their compositions (Table 1). It can be seen from Figure 4b that for the
M1-PMAn series the value for ζ at 20 °C falls to less than 10 mV once n becomes greater than or equal to 29. This is because the shear plane begins to exceed the double layer thickness, which was about 10 nm for the measurement conditions used (0.001 M electrolyte). Variable-temperature 1H NMR measurements have been established as a powerful method for probing structural changes for thermoresponsive copolymer micelles.14,15,45 Here, we probed the thermally triggered conformation changes for M1PMA29 using 1H NMR (see Figure 5a). It can be seen that the EO CH2 signals at 3.63.7 ppm (A1 in Figure 5a) became smaller as the temperature approached the Tcp for this copolymer (28.0 °C in water). The A1/A2 ratio indirectly probes the extent of hydration of the PMA side chains. A critical temperature based on 1H NMR was taken as the midpoint of the change in A1/A2 (Figure 5b). This was about 25.0 °C and is significantly lower than Tcp. We selected the midpoint rather than the onset of the change,45 because we have used the midpoint of the transmittance transition for Tcp. Further, the decrease in A1/A2 occurs 13874
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Figure 6. (ad) TEM images of M1-PMA52 micellar aggregates. These samples were deposited and stained using phosphotungstic acid. See text. The small white particles in (a) to (c) originate from the phosphotungstic acid which acts as a positive stain for the cationic micellar aggregates. (d) TEM image of particles deposited at a higher temperature (see text).
over a 10 °C range which indicates a gradual change in PMAn conformation. This is consistent with the PCS and ζ data, and is discussed further below. The change from H2O to D2O (used for the 1H NMR measurements) should only modestly alter the LCST of thermoresponsive polymers.46,47 Yang et al.47 reported that their Tcp values for thermally responsive copolymers containing PEGMA dispersed in D2O were 1.7 °C lower than those in H2O. Therefore, the midpoint for the change of the A1/A2 values (Figure 5b) could be 1 or 2 °C higher than 25.0 °C if these values could be measured using H2O. Unfortunately, those measurements cannot be made using 1H NMR spectroscopy. This value would still be significantly lower than Tcp. To further understand the structural changes that occur for these copolymers with temperature, we compare the four sets of temperature dependent data for M1-PMA29 in Figure 5b,c. The arrows identify critical temperatures corresponding to the midpoints of the transitions. These critical temperatures range from 25 to ca. 30 °C. (This range could be slightly narrower given the likelihood of an isotope shift for the 1H NMR critical temperature discussed above.) We propose the following interpretation based on these data. At a temperature of approximately 20 °C,
segments of the PMAn side chains begin to dehydrate. This corresponds to the onset of the decrease in A1/A2. The segment mobility begins to decrease. This process becomes more pronounced at 25 °C. At that point, the refractive index of the micelles increases (transmittance decreases). This thermally triggered segment association must occur mainly in the inner parts of the micelles (which may have a lower LCST due to higher segment density) and the effect on the hydrodynamic diameter is only slight. The mobile cationic parts at the micelle center begin moving toward the surface (rearranging), and this begins to increase ζ. As the temperature further increases (beyond 25 °C), contraction of all of the PMAn chains throughout the micelle becomes pronounced and is no longer restricted mostly to the micelle core. In this region, micellar aggregation occurs. However, for most systems (except M1-PMA101) the size of the aggregates is less than the initial unimolecular micelle. Hence, the diameter decreases with temperature. Segment rearrangement leads to cationic species being closer (on average) to the periphery. The similarity of the changes in ζ and diameter between 25 and 35 °C implies a strong correlation between PMAn side-chain contraction and cationic backbone rearrangement. These two 13875
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Figure 7. Proposed thermally triggered structural changes for M(x/y)-PMAn micelles with temperature. Temperature increase causes aggregation and this process continues until electrostatic stabilization is sufficient to stabilize the nanoparticles. The proximity of the positive charges to the surface increases when the temperature exceeds Tcp. The counterions are not shown for clarity.
Figure 8. Comparison of (a) variable temperature transmittance, (b) hydrodynamic diameters, and (c) zeta potentials for M1-PMA101 and M1-PNP195. Data for the latter were taken from ref 20. The data are plotted against the temperature relative to the Tcp values.
processes must occur simultaneously during the later stages of the aggregation process. We used TEM to further probe micellar aggregation. Representative images are shown for M1-PMA52 in Figure 6. For the TEM images (Figure 6ac), we used phosphotungstic acid staining. A similar method was used by Zhu et al. for their fourarm star copolymers.48 Phosphotungstic acid (12WO3 3 H3PO4) is negatively charged at neutral pH and is a positive stain for our cationic copolymer micelles. When a copolymer concentration of 0.1 wt % and temperature of ca. 15 °C were used for deposition, relatively large, loose micellar aggregates were present (Figure 6a,b). A larger aggregate can be seen in the top righthand corner of Figure 6c. The loose aggregate structure was also observed for M1-PMA29 and M1-PMA101 (see Supporting Information Figure 3Sa,b). These images provide strong support for micellar aggregation. During the drying process, dehydration, an increased ionic strength from phosphotungstic acid and increased local concentration are proposed to trigger micellar aggregation. The unimolecular micelles have a strong tendency to aggregate
when the concentration is increased and partial dehydration occurs. When a higher deposition temperature was used (ca. 20 °C), polydisperse nanoparticles with sizes in the range of 40 to 90 nm were obtained (see Figure 6d). We propose that these nanoparticles are irregular, coalesced versions of the loose micellar aggregates shown in Figure 6ac. The micellar aggregates that formed at ca. 15 °C had a tendency to form a regular size. This is evident from Figure 3Sc where 10 micellar aggregates for M1-PMA52 can be seen. From all the results obtained above, we can suggest a mechanism for the thermally triggered structural changes that occur for M(x/y)-PMAn micelles in aqueous solution (see Figure 7). The unimolecular micelles relinquish steric stabilization as the temperature increases beyond Tcp and undergo aggregation. This results in an increased surface charge-tovolume ratio and electrostatic stabilization. Continued temperature increase results in a decreased distance between the positive charges from the backbones and the surface, which increases ζ. The micellar aggregates become more compact at higher 13876
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Langmuir temperature due to interpenetration of the peripheries of the partially collapsed micelles. They coalesce due to the relatively low Tg of the PMA homopolymer (35 °C).49 This interpretation is consistent with the critical temperatures from PCS and ζ data being larger than those from 1H NMR and turbidity (Figure 5). If this interpretation is correct, the final nanoparticles will consist of approximately linear chains of fixed positive charge distributed within a PEO-based matrix. The negatively charged, mobile counterions must also reside near the cationic backbones and linear chains of ionic conductivity may percolate the coalesced nanoparticles. This could enable interesting potential applications for future nanometer-scale ionically conducting devices. Comparison with Cationic Graft Copolymers Containing PNIPAm Side Chains. Finally, we briefly compare the results for M1-PMA101 from this study to those for M1-PNP195 (Table 1) reported earlier. Variable-temperature transmittance, hydrodynamic diameter, and zeta potential data for each system are shown in Figure 8. The data are plotted as a function of the temperature relative to the respective Tcp values (i.e., T Tcp) to facilitate comparison. (The Tcp values are shown in Table 1.) The M1-PNP195 copolymer solution did not scatter light sufficiently at temperature less than Tcp for hydrodynamic diameter or ζ values to be measured.20 Nevertheless, it can be seen from the data that the thermally triggered transitions for M1-PMA101 are more broad compared to M1-PNP195. This must be due to the ability of the PMAn chains to rearrange as a consequence of their low Tg values. This ability to rearrange, combined with the absence of extensive intersegment hydrogen bonding, may also contribute to the higher ζ values achieved for M1-PMA101 at higher temperatures. Considering all the data shown in Figure 8 (and above), the M1-PMA101 copolymer shows generally similar thermally triggered behavior to M1-PNP195. The differences are that M1-PMA101 exists as well-defined unimolecular micelles at temperatures less than Tcp, is able to rearrange its structure at temperatures greater than Tcp, and gives more highly charged micellar aggregates as judged by the zeta potentials.
’ CONCLUSIONS In this study, a new family of thermally responsive cationic graft copolymers has been introduced. The M(x/y)-PMAn copolymers formed unimolecular micelles at temperatures below their Tcp values. The Tcp values measured for these copolymers were strongly dependent on electrostatic interactions. The Tcp values measured in the presence of 0.15 M PBS (pH = 7.4) were decreased by 7 to 8 °C compared to values measured in pure water. The unimolecular micelles aggregated upon heating, and the extent of aggregation was controlled by the side-chain length and was lowest for M1-PMA52. The thermally triggered micelle aggregation was also strongly dependent on electrolyte. The reversibility of the thermally triggered aggregation was good for these copolymers. For the M1-PMA52 system, we identified four distinct critical temperatures. It was argued that the low Tg of the copolymers enabled structural rearrangements to occur at temperatures greater than Tcp. Additional evidence for micellar aggregation was found using phosphotungstic acid staining. A new type of nanoparticle morphology was proposed for these systems consisting of ionic channels through an EO-rich matrix. These could, in principle, offer potential for one-dimensional ionic conduction. The M1-PMAn copolymers have similar thermoresponsive behavior to the M1-PNP195 graft copolymer
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studied earlier;20 however, they exist as unimolecular micelles at low temperature and have higher zeta potentials at temperatures greater than the cloud point. On the basis of our related work,18 it is likely that these new thermoresponsive copolymers have improved potential for conferring thermal responsiveness to anionic biomaterial surfaces under physiological conditions, and a study of this aspect is currently in progress.
’ ASSOCIATED CONTENT
bS
Supporting Information. Variation of hydrodynamic diameter with temperature for the copolymers in the presence of added electrolyte and also reversibility, TEM images for copolymers, and a table showing measured and calculated sizes for the unimolecular micelles. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail
[email protected].
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