pH-Responsive Nanoaggregation of Diblock Phosphorylcholine

9652

J. Phys. Chem. B 2008, 112, 9652–9659

pH-Responsive Nanoaggregation of Diblock Phosphorylcholine Copolymers Q. S. Mu, X. B. Zhao, and J. R. Lu* Biological Physics Group, School of Physics and Astronomy, The UniVersity of Manchester, Schuster Building, Oxford Road, Manchester M13 9PL, United Kingdom

S. P. Armes Department of Chemistry, The UniVersity of Sheffield, Sheffield S3 7HF, United Kingdom

A. L. Lewis Biocompatibles UK Limited, Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey GU9 8QL, United Kingdom

R. K. Thomas Physical and Theoretical Chemistry Laboratory, The UniVersity of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom ReceiVed: October 26, 2007; ReVised Manuscript ReceiVed: May 18, 2008

We have characterized three diblock copolymers bearing zwitterionic phosphorylcholine and weak tertiary amine groups, namely, poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30-block-(2-(dimethylamino)ethyl methacrylate)60] (denoted as MPC30-DMA60, Mn ) 18 000), poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30-block-(2-(diethylamino)ethyl methacrylate)60) (denoted as MPC30-DEA60, Mn ) 20 000), and poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30-block-(2-(diisopropylamino)ethyl methacrylate)60) (denoted as MPC30-DPA60, Mn ) 21 000), by studying their surface tension and solution aggregation through a combined approach of surface tension measurement, dynamic light scattering, and small-angle neutron scattering. Our results show that larger tertiary amine substituents lead to an increasing tendency to form micellar aggregates, which is consistent with the increasing copolymer hydrophobicity. Thus, MPC30-DMA60 did not aggregate under the experimental conditions studied. The free chains exist in the form of thin cylinders, whose length decreases with copolymer concentration and solution temperature but increases with solution pH. The diameters of the MPC30-DMA60 cylinders remained almost constant at around 30 Å under all the conditions studied. At the lower copolymer concentration of 0.5 wt %, the cylindrical lengths correspond to the persistence length of the copolymer backbone and are close to its full length, indicating a rather high rigidity. Further data analysis showed that, at the two higher concentrations of 2 and 4 wt %, the phosphorylcholine and amine blocks associate, inducing bending of the copolymer backbone. One backbone kink was required to satisfy all the constraints, including the dry volume of the copolymer. MPC30-DEA60 showed a similar trend of pH- and concentration-dependent conformational responses for the free copolymer, but in addition micellar aggregation occurred at pH 9. In contrast, MPC30-DPA60 exhibited significantly reduced solubility associated with strong aggregation, which is consistent with it being the most hydrophobic copolymer in the series. Introduction Nanoaggregation of natural and synthetic polymer molecules is playing an increasing role in various domestic and technological applications. The structural and physicochemical properties associated with molecular self-assembly are determined by the connectivity and flexibility of the polymer chains and their interactions with the solvent. The size and shape of the aggregates formed are usually not constant but vary with the molecular architecture, as well as with the solution conditions such as temperature and pH.1 The diversity of nanostructured materials is all the more remarkable given the relative inde* To whom correspondence should be addressed. Phone: 44-161-3063926. E-mail: [email protected].

pendence of the chemical details of the polymer-solvent systems under consideration.2–6 Many techniques, including small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), optical and electron microscopy, and spectroscopic methods such as NMR and IR, have been deployed to characterize the structural features of micellar aggregates formed by different polymers.7–9 In this work, we have explored how an increase in side chain hydrophobicity affects the aggregation behavior in a group of novel diblock water-soluble copolymers. Dynamic light scattering (DLS) was first used to establish the general features of aggregation, and SANS was subsequently used to provide further structural assessment of the size and shape of the free copolymer chains and their aggregates formed at different concentrations, temperatures, and pH.

10.1021/jp710365u CCC: $40.75  2008 American Chemical Society Published on Web 07/19/2008

Nanoaggregates of Phosphorylcholine Copolymers

J. Phys. Chem. B, Vol. 112, No. 32, 2008 9653

Figure 2. Surface tension plots obtained from MPC30-DEA60 at 25 °C and pH 5 (0), 7 ([), and 9 (∆) with continuous lines to guide the eye.

Figure 1. Chemical structures of the three diblock MPC copolymers, with R equal to methyl, ethyl, or isopropyl. The resulting amine block is referred to as DMA, DEA, and DPA, respectively. The three types of copolymers have m ) 30 (MPC block) and n ) 60 (DMA, DEA, or DPA block).

It is still difficult to obtain reliable information on the size and shape of micellar aggregates formed by aggregating polymers, although scattering models10–19 have been developed to calculate small-angle scattering curves which take into account the influence of particle size, shape, volume fraction, degree of order, and so on. Furthermore, there is no predictive scheme for nanoaggregation for different polymeric systems. For example, many synthetic and natural polymers tend to form wormlike micelles,20–28 and there are studies that have reported chemical and structural characteristics that lead to the formation of wormlike micelles. However, it may be difficult to predict the solution behavior of any given series of diblock copolymers, and this is particularly the case for the series of diblock copolymers bearing phosphorylcholine (PC) groups. The PC groups on these polymers give the copolymers excellent biocompatibility because of their strong hydration and zwitterionic nature.29 The corresponding tunably hydrophobic moieties comprise 2-(dimethylamino)ethyl methacrylate (DMA) groups, 2-(diethylamino)ethyl (DEA) groups, or 2-(diisopropylamino)ethyl (DPA) groups. The molecular structures of these copolymers are shown in Figure 1. Because these amine groups have pKa values around pH 6-7, their aggregation can be tuned across neutral pH. At low pH, the amine groups are highly charged due to protonation, but as the solution pH exceeds their respective pKa values, they become neutral. Thus, these copolymers aggregate (or dissociate) in response to changes in the solution pH. As the pH conditions across subcellular compartments are known to vary, pH-responsive features have the potential to become powerful handles for controlled local drug/gene delivery. Together with the possibility of effects due to local changes in temperature, such behavior makes this series of PC copolymers more attractive than the widely reported thermoresponsive Pluronics, often referred to as PEOnPPOm-PEOn or PPOm-PEOn-PPOm.30 The aim of this work is to rationalize how the aggregation of these zwitterionic diblock PC copolymers responds to the solution pH, temperature, and ionic strength and, more importantly, to the different sizes and hydrophobicities of the tertiary amine groups. Results and Discussion Surface Tension. The surface tension profiles measured from MPC30-DEA60 solutions at pH 5, 7, and 9 and 25 ( 0.1 °C

Figure 3. Surface tension against pH at a 0.1 wt % concentration of MPC30-DMA60 (0), MPC30-DEA60 ([), and MPC30-DPA60 (∆) measured at 25 °C. The continuous lines are drawn to guide the eye.

are shown in Figure 2. The pKa of this copolymer was found to be around 6.9-7.3.31 The results indicate strong pH-dependent surface adsorption of the copolymer, a trend also reported by Ma et al.31 for similar copolymers. Figure 2 also illustrates how the surface tension varies with the pH at a given copolymer concentration. At pH 7 or 9, the surface tension initially decreases with increasing copolymer concentration, but at a certain critical value, a further increase in the copolymer concentration causes a much smaller decrease in the surface tension. The copolymer concentration at such an inflection is usually identified as the critical micelle concentration (cmc), indicating that above this concentration further addition of the copolymer contributes to the formation of micelles in the bulk solution. However, as we will show below, this interpretation is not correct here. As already indicated, the apparent surface tension is sensitive to the solution pH. Its value is lowest at pH 9, but at pH 5, there is no apparent sign of the occurrence of inflection at all and there is in fact a very small lowering of the surface tension. This is broadly consistent with the variation in charge density of the DEA block in response to adjusting the solution pH. A solution pH of around 7 is just at its pKa; thus, 50% of the DEA groups are expected to be dissociated at this pH. An increase in the solution pH further decreases the cationic charge density on the DEA chains, and the block becomes hydrophobic. In contrast, a decrease in the solution pH increases the charge density on the DEA block, and the copolymer chains become hydrophilic and much more water-soluble. The surface tension profiles for the same series of diblock copolymers bearing DMA and DPA blocks have also been measured, and the results are outlined in Figure 3. As observed for MPC30-DEA60, the surface tension also decreases with increasing pH for these two copolymers. The change from methyl to isopropyl groups increases the hydrophobicity and subsequently reduces the solubility of the copolymers in water. It can be seen from Figure 3 that the most soluble copolymer, MPC30-DMA60, has the highest limiting surface tension, while

9654 J. Phys. Chem. B, Vol. 112, No. 32, 2008

Mu et al. TABLE 1: Structural Parameters Obtained from Fitting a Cylinder Model to the Measured SANS Profiles for MPC30-DMA60 at 25 °C pH

Figure 4. DLS measurements of nominal hydrodynamic diameters plotted against pH for MPC30-DMA60 ([), MPC30-DEA60 (9), and MPC30-DPA60 (2) at 0.1 wt % and 25 °C.

Figure 5. SANS scattering profiles from MPC30-DMA60 solutions at pH 7 and concentrations of (a) 0.5 wt % (∆), (b) 2 wt % ([), and (c) 4 wt % (0) at 25 °C. The continuous lines represent the best fits obtained using the structural parameters given in Table 1.

MPC30-DPA60 has the lowest. Note that MPC30-DPA60 has a rather low solubility in the aqueous solution and the solubility decreases so much as the pH is increased from 7 to 9 that the surface tension measurements above 0.2 wt % are less reliable. Dynamic Light Scattering. Although the surface tension results described above indicate micelle formation in the bulk solution, it is still unclear how the micelle size changes under different conditions. Solution aggregation can be conveniently revealed by DLS. Figure 4 shows the plots of the effective size of the copolymers in solution measured against the solution pH for MPC30-DMA60, MPC30-DEA60, and MPC30-DPA60. The effective sizes for all three copolymers are around 100 Å between pH 5 and pH 6, equivalent to the hydrodynamic diameters of the free copolymer molecules. Above pH 6, aggregation starts to occur in the case of MPC30-DPA60. In comparison, aggregation does not occur with MPC30-DEA60 until above pH 7.5. However, no increase in aggregate size occurs for MPC30-DMA60. These changes parallel the changes in pKa values for the series and the relative hydrophobicity of the alkyl groups in the hydrophobic blocks; when the copolymer becomes more hydrophobic, it is easier for it to aggregate. To reveal the detailed structure of the copolymers in bulk solution, a more sensitive technique, SANS, has been used. SANS. Figure 5 shows a set of SANS measurements obtained for MPC30-DMA60 at different copolymer concentrations at pH 7. Higher copolymer concentrations increase the level of scattering. Over the high Q range, the scattering profiles look similar in shape, but as Q decreases the differences between the three profiles become more pronounced. Fits of models to the neutron data indicated that the scattering objects have the form of cylinders of finite length with an almost constant diameter of around 30 Å. As summarized by the fitting results in Table 1, an increase in the copolymer concentration

5 5 5 7 7 7 9 9 9

C/wt %

sf/10-5

R/Å

l/Å

fw ( 0.15

L/Å

4 2 0.5 4 2 0.5 4 2 0.5

3.80 1.20 0.25 2.30 0.80 0.20 1.05 0.43 0.09

14 ( 3 14 ( 3 15 ( 3 15 ( 3 15 ( 3 15 ( 3 16 ( 2 16 ( 2 16 ( 3

45 ( 5 96 ( 20 130 ( 30 61 ( 10 100 ( 15 145 ( 20 91 ( 10 145 ( 20 200 ( 25

0.54 0.65 0.68 0.66 0.71 0.72 0.78 0.80 0.82

92 125 128 120 130 140 156 172 204

leads to a reduction in the length of the cylinder from 140 Å at 0.5 wt % to around 50 Å at 4 wt %. These dimensions can only be those of the free copolymer chains. The reduction in length with increasing copolymer concentration probably arises from bending of the copolymer backbones. The two blocks of the copolymer each contain hydrophobic and hydrophilic groups and can function as independent but linked amphiphiles. The two blocks can then interact with each other either via hydrophobic interactions and/or through electrostatic interactions between their hydrophilic components. Thus, the strong dipole on the zwitterionic PC block could interact with the cationic charge on the DMA block when the pH is not too high. An increase in copolymer concentration will affect the electrostatic screening of the two blocks and could therefore be expected to change the strength of the interaction. The two distinct amphiphilic copolymer components may interact either intramolecularly, which will be opposed by the bending rigidity, or intermolecularly, which will be opposed by entropic factors. The former interaction will inevitably shorten the length of the cylindrical structure, while the latter will lead to the formation of large micelles. Evidently only the former occurs for MPC30-DMA60, but the other two copolymers exhibit both types of behavior. We have represented the change in the structure of unimolecular micelles schematically in Figure 6. Other possible shapes such as spheres, disks, or their equivalent core-shell models were found to be inadequate for fitting the observed neutron scattering profiles. In addition to the length and radius of the cylindrical molecules, the model fitting also led to the determination of the scale factor (sf), which can be related to the volume fraction of a water-swollen molecular cylinder (φrod) and its scattering length density (Frod) through the following equation:

sf ) 108φrod(Frod - FD2O)2

(1)

where FD2O denotes the scattering length density of D2O used. Since the position of bending of the copolymer molecule appears to change with concentration and SANS is sensitive only to the persistence length, the swollen volume (V) of the copolymer is taken to be related to the measured length (l) and radius (R) through

V ) πR2l

(2)

This part of the chain contains water, and the volume fraction of water in the cylinder (fw) can be estimated from

fw )

Vw V - Vp ) V V

(3)

where Vw and Vp denote the volume of water and the dry volume of the copolymer chain in the cylinder and Vw ) V - Vp



Figure 6. Schematic structures for free MPC30-DMA60 diblock copolymer chains as fitted from the three copolymer concentrations of (a) 4 wt %, (b) 2 wt %, and (c) 0.5 wt %.

represents the volume of associated water. The scattering length density of the cylinder can be estimated from

Frod ) (1 - fw)Fp + fwFD2O

(4)

The volume fraction of the water-swollen cylinder in the solution is thus given by

( )

φrod ) Cp 1 +

Vw Cp ) Vp 1 - fw

(5)

where Cp is the fraction of dry copolymer in water. Although the equations above can be solved directly, it is easier to vary fw and compare the resulting scale factor with that obtained from fitting the data. The fw values and more directly fitted parameters are given in Table 1. The full volume corresponding to an entire water-filled polymer cylinder (Vt) can be estimated by assuming that fw and its radius remain constant using

Vt )

Vp ° 1 - fw

(6)

where Vp° is the dry volume for the entire copolymer molecule, and the entire length (L) is then given by

L)

Vt πR2

(7)

The full length values of the straight and bent cylinders are also given in Table 1. It can be seen from Table 1 that, at the lowest copolymer concentration of 0.5%, the full length is equal to the fitted cylinder length within experimental error, indicating that the copolymer chain is straight. At the highest copolymer concentration of 4%, the mean bending position appears to occur in the middle of the full length. There is some deviation from this in the case of the data at pH 9, but the overall agreement is excellent. It is also interesting that, at the intermediate concentration of 2%, the location of the bend is around 30 Å from one end of the backbone of the copolymer and is comparable to the diameter of the cylinder. This coincidence explains why a second length parameter was not required to produce good fits in these three cases. Although we have identified a very specific kink in the copolymer, there must be a range of characteristic lengths in the system, and this is reflected in the range of length values that would fit the data. This range has been included in Table 1. As well as being concentration-dependent, the change in cylinder length was also found to be pH-dependent. Figure 7 shows a set of the SANS scattering profiles obtained from MPC30-DMA60 with the copolymer concentration fixed at 0.5 wt %. The scattering profiles measured at pH 5 and 7 are very

Figure 7. SANS profiles measured from 0.5 wt % MPC30-DMA60 at pH 9 (a, 0), pH 7 (b, [), and pH 5 (c, ∆), 25 °C. The continuous lines are the best fits to the data.

similar, and the only small differences occur over the lowest Q range. However, the profile measured at pH 9 differs significantly, particularly when Q < 0.1 Å-1. The data analysis suggests that, apart from the slight difference in the length of the scattering objects, the parameters for the cylinders obtained at pH 5 and 7 were approximately identical. This observation is expected from the relatively high charge density on this copolymer at the two lower pH values. At pH 9, the copolymer carries very little cationic charge and its DMA blocks become much less hydrophilic. From Table 1 it can be seen that there is little change in the radius, but the length change is significant and shows a large increase from 140 to 200 Å at 0.5 wt % when the pH increases from 5 to 9. At the highest copolymer concentration of 4 wt %, the length increases from 45-50 Å at pH 5 and 7 to 95 Å at pH 9. These results indicate that loss of cationic charge causes the individual copolymer chains to become stiffer and more stretched. Apart from the strong pHdependent change in the chain length, there appears to be no further change in the position of bending caused by pH variation. In addition, there is no indication of the formation of any micellar aggregates under these pH conditions, especially at pH 9 for MPC30-DMA60. The change in the scale factor at a given pH should be proportional to the copolymer concentration. It can be seen from Table 1 that this holds well. However, because of the large errors involved in deriving the scale factors, it is difficult to interpret the absolute values of the scale factor any further. However, the decrease in the scale factor with increasing pH is significant and indicates some interaction or aggregation of the copolymer chains at higher pH. This is important for the following reason. The surface tension curve obtained at pH 7 in Figure 2 suggests that there is a cmc, but neither DLS nor SANS detects any micelle formation for MPC30-DMA60. A possible reconciliation of this apparent disagreement is that there is a pronounced


Mu et al.

Figure 8. SANS scattering profiles from MPC30-DMA60 at pH 7 and 4 wt % at 25 °C (∆), 4 wt % at 37 °C (2), 0.5 wt % at 25 °C (0), and 0.5 wt % at 37 °C (9). Continuous lines represent the best fits.

change in the activity of the copolymer at the “cmc”. The intermolecular interactions that could give rise to the variation of the scale factor could also have a strong effect on the activity. We discuss other possible explanations below. Copolymers such as PEOn-PPOm-PEOn show interesting thermoresponsive reversible aggregation.32–34 Given that PC groups are known to be highly resistant to dehydration in dichain phospholipids and single-chain water-soluble phospholipid surfactants,35 it is useful to explore how the diblock PC copolymers respond to the temperature. Figure 8 shows the SANS scattering profiles from MPC30-DMA60 at two different temperatures and concentrations. These results show that, at each fixed concentration, an increase in temperature affects the scattering in the lower part of the Q range, indicating a structural variation. The data were analyzed as described above, and the parameters obtained at 37 °C are listed in Table 2. The scale factor increases slightly with temperature, indicating possible dehydration of the copolymer chains with increasing temperature. This trend is consistent with the reduction in cylinder length with copolymer concentration. Thus, an increase in both copolymer concentration and temperature leads to a reduction in the persistence length of the free copolymer, but there is little change in its diameter. The persistence length can therefore be changed by varying the copolymer concentration, solution temperature, or solution pH, but the chain diameter remains constant within experimental error. The SANS studies were also extended to the other two copolymers, MPC30-DEA60 and MPC30-DPA60. As an example, we show in Figure 9 the scattering profiles from all three copolymers at 2 wt %, pH 7, and 37 °C. The experimental procedures were the same as used for MPC30-DMA60. It was however noted that, above pH 7 and 9, MPC30-DPA60 suffered from poor solubility, thus hindering the quantitative analysis of the SANS profiles. In contrast, all the solutions prepared from MPC30-DEA60 up to 2 wt % were transparent and thus had no solubility problems. The SANS profiles for MPC30-DEA60 were analyzed similarly to those for MPC30-DMA60, and the results are given in Table 3. Below pH 7, a higher copolymer TABLE 2: Structural Parameters Obtained from the Cylindrical Model Fits to the SANS Profiles at pH 7 and 37 °C from MPC30-DMA60 C/wt %

sf/10-5

R/Å

l/Å

fw

L/Å

4 2 0.5

2.60 0.96 0.22

15 ( 2 15 ( 2 15 ( 2

43 ( 5 71 ( 15 118 ( 25

0.63 0.69 0.70

113 123 130

Figure 9. SANS scattering profiles for 2 wt % MPC30-DMA60 (a), MPC30-DEA60 (b), and MPC30-DPA60 (c) at 37 °C and pH 7. The continuous lines represent the best fits obtained using the cylinder model.

TABLE 3: Structural Parameters Obtained from the Cylindrical Model Fits to the SANS Profiles Obtained from MPC30-DEA60a pH T/°C C/wt % 5 5 5 7 7 7 7 9

25 25 25 25 37 37 37 25

4 2 0.5 0.5 4 2 0.5 0.2

sf/10-5

R/Å

3.00 15 ( 2 1.05 15 ( 2 0.11 16 ( 3 0.10 16 ( 3 1.64 15 ( 2 0.5 16 ( 2 0.15 16 ( 3 sf1 ) 0.5 25 ( 4 sf2 ) 0.3 160 ( 10

l/Å

fw

L/Å

60 ( 5 95 ( 10 195 ( 30 212 ( 30 56 ( 5 91 ( 15 175 ( 30 l1 ) 80 ( 10 l2 ) 110 ( 20

0.61 0.68 0.80 0.81 0.73 0.78 0.77

112 133 195 202 162 186 167

a Aggregation occurred at pH 9, and disk-shaped aggregates were used to reproduce the neutron scattering profiles.

concentration leads to a shorter cylinder length, but again the cross-sectional radius remains constant. Raising the temperature also causes a slight decrease in the cylinder length. However, MPC30-DEA60 forms a slightly longer cylinder than MPC30-DMA60, indicating that the effect arises from the different hydrophobic side chains. A change from methyl to ethyl substituents may increase the stiffness along the copolymer backbone, resulting in a longer cylinder, although the overall phenomenon of backbone bending and shrinking is still observed for MPC30-DEA60. However, the main difference is the formation of micellar aggregates at pH 9 in the case of MPC30-DEA60. This is in contrast to the total absence of any aggregation for MPC30-DMA60. At pH 9, MPC30-DEA60 formed two different types of nano-objects, a cylinder with a radius of 25 Å and length of 80 Å and a disk with a radius of 160 Å and length of 110 Å. The dimensions of the smaller cylinders are equivalent to about 1-2 fully hydrated copolymer chains, while those of the disks correspond to about 100 chains. Thus, the size and shape of the cylinders are indicative of further change in the structural conformation of the copolymer chains at this pH. SANS measurements conducted at 0.2, 0.5, and 2 wt % gave scattering profiles that could be broadly fitted with the two types of nano-objects, suggesting that these copolymer solutions contained both cylinders and disks. In the case of MPC30-DPA60, however, its lack of solubility prevented quantitative SANS data analysis. Nevertheless, the results were sufficient to show the effects of increasing the size of the alkyl chain in the copolymer series. The SANS results showed that MPC30-DPA60 was in the form of free copolymer chains at pH 5, just like the two other copolymers. This observation indicated the strong effect of charge in counterbal-



TABLE 4: Alternative Fitting to the 2 wt % Neutron Data Obtained for MPC30-DMA60 and MPC30-DEA60 MPC30-DMA60

MPC30-DEA60

pH

temp/°C

sf1/10-5

R1/Å

l1/Å

L1/Å

sf2/10-5

R2/Å

l2/Å

L2/Å

5 7 9 7 5 7

25 25 25 37 25 37

0.26 0.22 0.12 0.51 0.68 0.26

14 ( 3 15 ( 3 15 ( 2 15 ( 2 15 ( 2 14 ( 3

45 ( 5 51 ( 10 86 ( 10 43 ( 5 60 ( 5 45 ( 15

92 120 148 113 112 159

0.80 0.63 0.31 0.49 0.53 0.36

15 ( 3 15 ( 3 16 ( 3 15 ( 3 16 ( 3 14 ( 3

130 ( 30 145 ( 20 175 ( 20 115 ( 30 195 ( 30 166 ( 20

127 140 197 125 195 159

ancing the increasing hydrophobicity of the side chain groups. However, as the pH was increased to 7 or above, the MPC30-DPA60 solution became turbid, showing that the copolymer solubility worsened as the DPA block became hydrophobic. The fitted SANS profiles at pH 7 (Figure 9) again indicated the formation of two different types of nano-objects represented by a small cylinder and a large disk. The small cylinder had exactly the same dimensions as found for MPC30-DEA60, but the disk was larger, with a radius of 180 Å and length of 150 Å. This is equivalent to the association of 120 fully hydrated copolymer chains. These observations further reinforce the view that the tendency toward aggregation increases with increasing size and hydrophobicity of the side chains. The occurrence of bending or kinking in the copolymer backbone as proposed in Figure 6 could occur in two ways, either as a mean, representing a distribution, or as a mix of two different chain lengths. For this copolymer we attempted to fit the 2 wt % data in terms of a mix of the straight copolymer chains at 0.5 wt % and those bent at the middle as observed at 4 wt %. An increase in copolymer concentration effectively led to an increase in the fraction of the 4 wt % type copolymers. This gave a better fit to the data, and we conclude that the structure is best characterized as consisting of two ranges of length distribution centered at those obtained at 0.5 and 4 wt %. Both of the scale factors obtained from the two cylindrical models decreased with increasing pH (Table 4), which is similar to the observation made for the previous model fitting. Although the two different model analyses are self-consistent in terms of their scale factor behavior, it is not clear which is the more appropriate representation of the true backbone distributions. What is certain, however, is the need to invoke a kink in the copolymer backbone at higher concentrations to account for the neutron data. Conclusions MPC30-DMA60, MPC30-DEA60, and MPC30-DPA60 represent a group of PC diblock copolymers that have good cell and tissue compatibility: their pH-responsive behavior at around pH 7 makes them attractive for a range of potential applications, including tissue engineering and either gene or drug delivery. The aim of this work is to rationalize their physicochemical properties so that their applications such as gene complexing vectors36 can be optimized. The DLS measurements confirm that micelle formation is strongly dependent on the size and hydrophobicity of the side chains on the tertiary amine methacrylate blocks. No aggregation was detected for MPC30-DMA60 between pH 5 and pH 9, but a definite trend in the onset of aggregation was observed when the side chain was changed to either ethyl or isopropyl, which is consistent with a shift in the pKa of the tertiary amine methacrylate block, as reported previously.37,38 These pHdependent transitions from free copolymer chains to micellar aggregates are important for more effective control of the selfassembly (size, shape) and net charge density of the complexes

formed by such copolymers with DNA and RNA, which in turn have implications for gene therapy applications. The surface tension vs pH curves obtained for MPC30DMA60 and MPC30-DEA60 confirm that significantly lower limiting surface tension values are obtained at higher solution pH, demonstrating that the deprotonated copolymer chains become more hydrophobic and more surface-active. The surface tension inflection points are normally interpreted in terms of a critical micelle concentration. While this is certainly the case for MPC30-DEA60 at pH 9 and MPC30-DPA60 at pH 7 and 9, the inflection or break for MPC30-DMA60 at pH 7 and 9 and for MPC30-DEA60 at pH 7 is not associated with detectable micelle formation in the bulk solution. This discrepancy is attributed to nonideal behavior of these copolymers, even though they exist in the form of free copolymer chains. A conformational change in the copolymer at the interface would not, on its own, have such an effect. Instead, the Gibbs equation requires a change in activity of the copolymer in the bulk solution to produce such an effect. Other possibilities are surface depletion and/or polydispersity effects.40 The molar concentration at the “cmc” is about 10-6 M, which is rather high for depletion effects to be significant. In principle, polydispersity effects may arise from a spread in the copolymer composition, rather than just a spread in the weight distribution. SANS analysis has revealed the cylindrical shape of the free copolymer chains in the aqueous solution. At the lowest concentration of 0.5 wt %, such cylinders were found to be completely straight, consistent with the estimate from the volume restriction requirement and indicating a fair degree of backbone rigidity. As the copolymer concentration was increased to 2 wt %, the mean cylinder length was substantially shortened, suggesting a single kink in the copolymer backbone at an average distance of 30 Å from one end, which is identical to the cylinder diameter. As the concentration was increased to 4 wt %, the average location of the kink appeared to occur at around the middle of the backbone. In the case of micelle formation for MPC30-DEA60 at pH 9 and MPC30-DPA60 at pH 7 and 9, two different types of nano-objects were detected. While the small cylindrical objects remained the same size and shape, the large disk-shaped micelles increased in size, showing that as the copolymer became more hydrophobic its micelle aggregation number also increased. Experimental Details SANS experiments were carried out using the instrument LOQ at Rutherford Appleton Laboratory (RAL) near Oxford, U.K. An appropriate sample background was measured for every sample. For each of these samples, both the scattering and the neutron transmission runs were obtained. Each transmission run lasted typically 10 min, while each scattering run lasted 1-2 h. All samples were prepared in D2O, and the pH was controlled using phosphate buffer (PBS) prepared from NaH2PO4 and Na2HPO4 (AR grade from Aldrich). Quartz cells with a 2 mm path length were used, and the copolymer solutions were fed into the cells using a 2 mL syringe with a flattened tip. Care


Mu et al.

TABLE 5: Calculated Scattering Length Densities (SLDs) and Volumes of the Dried Copolymers and D2O MPC30-DMA60 MPC30-DEA60 MPC30-DPA60 D2O

SLD/10-6 Å-2

volume/Å3

0.72 0.62 0.53 6.35

31 314 34 975 38 716 30

was taken to check that no air bubbles were trapped in the beamilluminated area. The resulting scattering profiles after calibration, normalization, and background subtraction were modeled using a program called FISH2, developed by Dr. R. K. Heenan at RAL.39 DLS was used to measure the intensity correlation function of light scattered from a copolymer solution. Analysis of the intensity correlation profiles provided the decay rate distribution from which the diffusion coefficient was determined. The Stokes-Einstein equation was then used to calculate the hydrodynamic radii of the pure copolymer and the micelles.41 DLS measurements were performed using a Malvern Instruments Nano-S Nanosizer. The instrument was fitted with a helium-neon laser (633 nm) with a size detection range of 0.6 nm to 6 µm. The detection angle was 173° with respect to the incoming beam. Samples were contained in a 1 cm path length quartz cell, and the data were analyzed using Malvern Instruments dispersion technology software. The polymer refractive index was taken to be 1.45 with an absorbance of 0.001. The viscosity and refractive index of water were taken to be 0.8872 cPa and 1.330, respectively. The surface tension of aqueous copolymer solutions was determined using the du Nou¨y ring (Pt/Ir) method with a Kru¨ss K11HRX MK1 tensiometer (Kru¨ss GmbH, Germany). Like many other copolymer solutions, the surface tension showed time-dependent variation. All the measurements were therefore started after the solution had been left in the measuring glass vessel for 30 min. Before each measurement, the ring was rinsed with pure water and flamed to remove possible contaminants. All surface tension experiments were performed at 25 °C under the control of a HAAKE K20 water circulator (HAAKE, Germany). The diblock MPC copolymers used in this work were synthesized via sequential monomer addition using atom transfer radical polymerization as described in the literature.31 Generally, MPC was polymerized first in methanol at 20 °C, and DMA, DEA, or DPA was added once the MPC conversion had reached at least 95%. The target degree of polymerization of the MPC block was 30 in each case, the molar ratio of MPC to DMA, DEA, or DPA was held constant at 1:2, and the three resulting copolymers were denoted as MPC30-DMA60, MPC30-DEA60, and MPC30-DPA60. The molecular weights of MPC30-DMA60, MPC30-DEA60, and MPC30-DPA60 were 18 000, 20 000, and 21 000, respectively. Copolymer purities exceed 99% with relatively low polydispersities (Mw/Mn ) 1.20-1.30). Detailed information about these copolymers, such as their GPC data and 1H NMR spectra, can be found in ref 31. The main physical constants used in neutron data analysis were the volume and scattering length density for each copolymer. These parameters were estimated from relevant physical constants obtained from the literature38 and are listed in Table 5. All the glassware and PTFE troughs were cleaned by soaking in 5% Decon solution (Decon Laboratory, U.K.) overnight and rinsed with tap water and then several times with ultra-highquality (UHQ) water (Elgastat PS, Elga, U.K.). The copolymer solutions were made by dissolving the copolymer first in PBS

buffer and then diluted to lower concentrations at ambient temperature as desired. D2O was purchased from Aldrich and contained over 99% D. Its surface tension was 71.5 mN m-1 at 25 °C and was close to that obtained for the pure UHQ water. Acknowledgment. We thank Dr. Richard Heenan and Professor Jeff Penfold for insightful discussions, EPSRC for a grant support, and CCLRC for the provision of neutron beam access at the ISIS neutron facility. Drs. Mu and Zhao express their gratitude to Biocompatibles UK Ltd. for studentships. Prof. Armes is the recipient of a five-year Royal Society-Wolfson Research Merit Award. Dr. Y. Ma is thanked for synthesizing the three diblock copolymers used in this study. References and Notes (1) Cates, M. E.; Candau, J. S. Statics and dynamics of worm-like surfactant micelles. J. Phys.: Condens. Matter 1990, 2, 6869–6892. (2) Fo¨rster, S.; Plantenberg, T. From self-organising polymers to nanohybrid and biomaterials. Angew. Chem., Int. Ed. 2002, 41, 689–714. (3) Ying, J. Y.; Mehnet, C. P.; Wong, M. S. Synthesis and applications of supramolecular-templated mesoporous materials. Angew. Chem., Int. Ed. 1999, 38, 56. (4) Seddon, J. M. Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochim. Biophys. Acta 1990, 1031, 1–69. (5) Bleasdale, T. A.; Tiddy, G. J. T; Bloor, D. M. Wyn-Jones, E. The Structure, Dynamics and Equilibrium Properties of Colloidal Systems; Kluwer Academic: New York, 1990. (6) Fo¨rster, S.; Timmann, A.; Konrad, M.; Schellbach, C.; Meyer, A.; Funari, S. S.; Mulvaney, P.; Knott, R. Scattering curves of ordered mesoscopic materials. J. Phys. Chem. B 2005, 109, 1347–1360. (7) Liu, Y.; Chen, S. -H; Huang, J. S. Light-scattering studies of concentrated copolymer micellar solutions. Marcromolecules 1998, 31 (18), 6226–6233. (8) Kriz, J.; Plestil, J.; Pospisil, H.; Kadlec, P.; Konak, C.; Almasy, L,; Kuklin, A. I. 1H NMR and small-angle neutron scattering investigation of the structure and solubilization behaviour of three-layer nanoparticles. Langmuir 2004, 2025, 11255–11263. (9) Jack, K. S.; Wang, J.; Natansohn, A.; Register, R. A. Characterisation of the microdomain structure in polystyrene-polyisoprene block copolymers by 1H spin diffusion and small-angle X-ray scattering methods. Marcromolecules 1998, 3110, 3282–3291. (10) Percus, J. K.; Yevick, G. J. Analysis of classical mechanics by means of collective coordinates. Phys. ReV. 1958, 110, 1–13. (11) Hayter, J. B.; Penfold, J. An analytic structure factor for macroion solutions. Mol. Phys. 1981, 42, 109–118. (12) Hansen, J. P.; Hayter, J. B. Factors related to an effective referral and consultation process. Mol. Phys. 1982, 46, 651–656. (13) Baxter, R. J. Percus-yevick equation for hard spheres with surface adhesion. J. Chem. Phys. 1968, 49, 2770. (14) Matsuoka, H.; Tannaka, H.; Hashimoto, T.; Ise, N. Elastic-scattering from cubic lattice systems with paracrystalline distortion. Phys. ReV. B 1987, 36, 1754–1765. (15) Matsuoka, H.; Tannaka, H.; Iizuka, N.; Hashimoto, T.; Ise, N. Elastic-scattering from cubic lattice systems with paracrystalline distortion 2. Phys. ReV. B 1990, 41, 3854–3856. (16) Stribeck, N.; Ruland, W. Determination of interface distribution function of lamellar 2-phase systems. J. Appl. Crystallogr. 1978, 11, 535– 539. (17) Nallet, F.; Laversanne, R.; Roux, D. Modeling X-ray or neutron scattering spectra of lyotropic lamellar phase interplay between form and structure factors. J. Phys. II 1993, 3, 487–502. (18) Fro¨ba, G.; Kalus, J. Structure of the isotropic, nematic, and lamellar phase of a solution of tetramethylammonium perfluorononanoate in D2O. J. Phys. Chem. 1995, 99, 14450–14467. (19) Pabst, G.; Rappolt, M.; Amenitsch, H.; Laggner, P. Structural information from multilamellar liposomes at full hydration: full q-range fitting with high quality X-ray data. Phys. ReV. E 2000, 62, 4000–4009. (20) Pedersen, J. S.; Schurtenberger, P. Scattering functions of semidilute solutions of polymers in a good solvent. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 3081–3094. (21) Croce, V.; Cosgrove, T.; Maitland, G.; Hughes, T.; Karlsson, G. Rheology, cryogenic transmission electron spectroscopy, and small angle neutron scattering of highly viscoelastic wormlike micellar solutions. Langmuir 2003, 19, 8536–8541. (22) Lee, J. H.; Gustin, J. P.; Chen, T. H.; Payne, G. F.; Raghavan, S. R. Vesicle-biopolymer gels: networks of surfactant vesicle connected by associating biopolymers. Langmuir 2005, 21, 26–33.

Nanoaggregates of Phosphorylcholine Copolymers (23) Garamus, V. M.; Pedersen, J. S.; Maeda, H.; Schurtenberger, P. Scattering from short stiff cylindrical micelles formed by fully ionized TDAO in NaCl/water solutions. Langmuir 2003, 19, 3656–3665. (24) Truong, M. T.; Walker, L. M. Controlling the shear-induced structural transition of rodlike micelles using nonionic polymer. Langmuir 2000, 16, 7991–7998. (25) Espert, A.; Klitzing, R. V.; Poulin, P.; Colin, A.; Zana, R.; Langevin, D. Behavior of soap films stabilized by a cationic dimeric surfactant. Langmuir 1998, 14, 4251–4260. (26) Angelico, R.; Olsson, U.; Mortensen, K.; Ambrosone, L.; Palazzo, G.; Ceglie, A. Relaxation of shear-aligned wormlike micelles. J. Phys. Chem. B 2002, 106, 2426–2428. (27) Fo¨rster, S.; Konrad, M.; Lindner, P. Shearing thinning and orientational ordering of wormlike micelles. Phys. ReV. Lett. 2005, 94, 192303. (28) Koehler, R. D.l; Raghavan, S. R.; Kaler, E. W. Microstructure and dynamics of wormlike micellar solutions formed by mixing cationic and anionic surfactants. J. Phys. Chem. B 2000, 104, 11035–11044. (29) Landin, J.; Pascher, I.; Cremer, D. Effect of a polar environment on the conformation of phospholipid head groups analyzed with the onsager continuum salvation model. J. Phys. Chem. A 1997, 101 (16), 2996–3004. (30) Bu¨tu¨n, V.; Bennett, C. E.; Vamvakaki, M.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. Selective betainisation of tertiary amine methacrylate block copolymers. J. Mater. Chem. 1997, 7, 1693–1695. (31) Ma, Y. H.; Tang, Y. Q; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salavage, J. P. Well-defined biocompatible block copolymers via atom transfer radical polymerization of 2-methacryloyloxyethyl phosphorylcholine in protic media. Macromolecules 2003, 36, 3475– 3484.

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pH-Responsive Nanoaggregation of Diblock Phosphorylcholine

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