Time-dependent shrinkage of polymeric micelles of amphiphilic block copolymers containing semirigid oligocholate hydrophobes

Journal of Colloid and Interface Science 353 (2011) 420–425

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Time-dependent shrinkage of polymeric micelles of amphiphilic block copolymers containing semirigid oligocholate hydrophobes Jing Wu, Xingang Pan, Yan Zhao ⇑ Department of Chemistry, Iowa State University, Ames, IA 50011-3111, USA

a r t i c l e

i n f o

Article history: Received 5 August 2010 Accepted 23 September 2010 Available online 20 October 2010 Keywords: Facial amphiphiles Cholic acid Micelles Block copolymers Self-assembly

a b s t r a c t Alkyne-derivatized poly(ethylene glycol) (M.W. 5000) was coupled to several azide-terminated oligocholates by the click reaction to form amphiphilic block copolymers. A copolymer with a cholate hexamer as the hydrophobic block formed polymeric micelles that shrank by 50% over a period of 10 h at 25 °C. Shrinkage was faster and more dramatic at 35 °C. Shortening the oligocholate by two units or inserting a 4-aminobutyroyl spacer in the hexacholate eliminated or diminished the shrinkage. Metastable aggregates were proposed to form when the block copolymers began to aggregate in water. The large hydrophobic surface, awkward shape, rigidity, and facial amphiphilicity of the cholate repeat unit and the long chain made it difficult for the oligocholates to adjust within the micellar core. As the oligocholates rearranged to maximize hydrophobic interactions and hydrogen-bonding while minimizing conformational strain, the polymeric micelles became more compact over time. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Amphiphilic block copolymers represent a tremendously important class of materials with many applications in chemistry, nanoscience, and drug delivery [1–3]. A key feature of these macromolecules is their self-assembling into superstructures such as micelles, rods, and vesicles [4–6]. The assembling is determined by the composition of the block copolymer, solvent polarity, and relative solubility of individual blocks in the solvent. As chemists develop more sophisticated synthesis of block copolymers, other morphologies, e.g., toroids [7,8], nanotubes [9], helices [10], and disks [11], have appeared in the literature. The major driving force for the assembly of amphiphilic block copolymers in water is hydrophobic interactions. Many polymers have been used as the hydrophobic block, including polystyrene, polybutadiene, polyisoprene, polyacrylate, and polycaprolactone. These polymers offer a number of important parameters, such as molecular weights, glass transition temperature, chemical reactivity, and biodegradability, to tune the properties of the block copolymers. Instead of a randomly coiled polymer such as the above, the hydrophobic block may be a rigid rod, often made of multiple aromatic units. Because the size and shape of the hydrophobe, as well as the directionality in the aromatic–aromatic interactions, constrain how the hydrophobic blocks come together to maximize hydrophobic and/or van der Waals interactions while minimizing solvent exposure [12], the change from a random coil ⇑ Corresponding author. Fax: +1 515 294 0105. E-mail address: [email protected] (Y. Zhao). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.09.071

to a rigid rod in the hydrophobic block impacts the self-assembling greatly [13]. Rod–coil amphiphilic block copolymers are known to form diverse superstructures including hollow spherical micelles [14], bundles [15], ribbons [16], tubules [17], and molecular ‘‘mushrooms” [18] and ‘‘donuts” [19]. Researchers have also employed polypeptides [10,20–26] to construct amphiphilic block copolymers. Different from either a random coil or a rigid rod, the size, shape, and distribution of functional groups of a polypeptide are determined by its conformation. With conformational control being the dominant mechanism in the environmental responsiveness of biopolymers, conjugation of synthetic polymers and peptides allows the rational design of hybrid materials with protein-like, signal-specific responsiveness. Because of the biological compatibility of cholic acid and other bile acids, there is great interest in preparing bile acid-derived polymers [27]. Zhu and co-workers, in particular, have developed a number of novel polymers from bile acids [28–31]. We recently synthesized linear oligomers of cholates in an effort to create conformationally tunable synthetic foldamers [32,33]. These oligocholates display predictable conformational changes in mixed solvents, micelles, and bilayers. Whereas folding in organic solvents is controlled mainly by the preferential solvation of the hydrophilic and hydrophobic faces of the cholate units, the conformation in surfactant solution is dominated by the size, shape, and ionic nature of the surfactant micelles [34]. In this paper, we report diblock copolymers with an oligocholate as the hydrophobic block and poly(ethylene glycol) or PEG as the hydrophilic block. We discovered that, depending on the chain length of the oligocholate, the block copolymer self-assembles

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into polymeric micelles displaying time-dependent shrinkage. We propose that the shrinkage occurs as the semirigid oligocholate block adjusts itself in the hydrophobic micellar core to maximize hydrophobic interactions and hydrogen-bonding while minimizing conformational strain.

2. Results and discussion 2.1. Design and synthesis of oligocholate–PEG block copolymers Although an oligocholate has multiple hydrophilic amide and hydroxyl groups, the molecule is overall hydrophobic due to the large hydrophobic b-face of the cholate unit. During the amide coupling of carboxyl- and amino-terminated oligocholates, for example, a common work-up procedure is precipitation of the crude product in water from a DMF solution [32]. Oligocholates prefer nonpolar media and fold best in nonpolar solvents (e.g., hexane/ ethyl acetate mixture, acetate, or CCl4) containing a small percentage of a polar solvent [33]. Their hydrophobicity is also evident from their surfactant-assisted solubilization in water. When fluorescently labeled oligocholates are dissolved in aqueous micellar solutions, their emission indicated their inclusion in the hydrophobic core of micelle [34–36]. We thus reasoned that an oligocholate could be used as the hydrophobe in an amphiphilic block copolymer. Unlike a random-coiled hydrophobic polymer (e.g., polystyrene or polybutadiene) or rigid rod, however, an oligocholate consists of rigid, facially amphiphilic repeat units connected by short tethers. These features should influence the packing of oligocholates and affect the self-assembling of the block copolymer. In a recent study, we discovered that the parent, unmodified, oligocholates pack poorly in water due to the awkward shape of the steroidal repeat unit and the short linkages between the fused aliphatic rings [36]. An interesting dichotomy thus will be created if a hydrophilic polymer is attached to an oligocholate: the large hydrophobic surface of the oligocholates demands their close packing to maximize hydrophobic interactions but such packing is difficult with the awkward shape of the repeat unit and the semirigid backbone. How would an oligocholate-containing amphiphilic block copolymer selfassemble under two contradicting forces? This is the main question we set out to answer in this study. The most convenient way to prepare an amphiphilic block copolymer from an oligocholate is through the click reaction [37–39] with alkyne-terminated PEG 2 (Scheme 1), as the oligoch-

N3

HO HO

NH HO HO

O

NH HO HO

O

m

1a−e

O N

N O

O

With the molecular weight of the PEG significantly higher than that of the oligocholates (M.W. 1600–2600), the diblock copolymers are expected to assemble into polymeric micelles in aqueous solution [1–3]. When an aqueous solution (1.0  105 M) of hexacholate-derived 3a was prepared by adding 10 lL of DMSO solution (1.0  103 M) to 1 mL Millipore water, the average hydrodynamic radius of the polymeric micelles was ca. 140 nm for a freshly made solution. Over time, however, the particles showed a significant decrease in size, ca. 90 nm after 3.5 h and 60 nm after 1 d (Fig. 1S, Supplementary material). Tetracholate-derived 3b also aggregated under the same condition, but the aggregation was more difficult. A freshly made solution of 3b showed a bimodal distribution of nanoparticles, with the larger ones centered at 110 nm and the smaller slightly above 10 nm. The smaller particles decreased in population over 2 h and the larger particles remained unchanged in size during 1 d (Fig. 2S, Supplementary material). The behavior suggests that smaller particles were less stable, presumably due to their insufficient shielding of the hydrophobic surface from water. Difference in the stability of the two polymeric micelles was also evident when they were passed through 0.45-micron syringe filters made of hydrophilized Teflon. Filtration did not change the scattering intensity of the micellar solution of 3a very much, suggesting that the majority of the micelles went through the filters. The micelles continued to display the time-dependent shrinkage, although the average particle size decreased by 20–30% (Fig. 3S,

R

a, m = 2, R = OMe; b, m= 1, R = OMe

O

O

2

OH

OH HO

HO HO

NH HO HO

NH HO HO

O

N

O

d, m= 1, R = HO NH HO m

O

H N

HN

O

S

S

R O

e, m =1, R =

3a−e O

HN

O n

CuSO4, Cu(metal) CH2Cl2, MeOH, H 2O

O

H N

c, m= 2, R = O

4

N

2.2. DLS and TEM study of oligocholate–PEG block copolymers

O

N HO

HO NH HO

O

olates are terminated with an azide group at the end of the synthetic route [32]. We recently took advantage of this feature and utilized the click reaction to synthesize otherwise synthetically challenging long oligomers of cholate [40]. The parent and fluorescently labeled oligocholates (1a–e) were synthesized according to reported procedures [32]. Commercially available methoxy-terminated PEG (M.W. 5000, Mw/Mn = 1.05) with narrow M.W. distribution was coupled to 4-pentynoic acid by a literature procedure to afford 2 [41]. The dansyl group attached to the chain end of 1c–e is sensitive to environmental polarity [42] and was included to probe the self-assembling in water. Its quenching by stable radical 4 and Cu2+ was used to probe the compactness of the polymeric micelles formed by 3c–e. In compound 3e, a flexible 4-aminobutyroyl spacer was inserted into the oligocholate sequence to understand whether rigidity of the oligocholate was important to the assembling.

O n

N

O N

O

HN

NH

N O

Scheme 1. Synthesis of oligocholate–PEG block copolymers 3a–e.

O

S

O N H

HN

O

HO HO OH OH

O NH

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Supplementary material). The micelles of 3b, on the other hand, did not survive the filtration and the filtrate contained no particulate materials detectable by DLS. Fig. 1 plots the average hydrodynamic radii of the polymeric micelles of 3a and 3b as a function of time. Both the filtered and unfiltered micelles of 3a displayed a decrease in size. The size change at 25 °C over a period of 10 h was about 50%. The shrinkage was faster and more dramatic at 35 °C. The radius decreased over 70% over the same period of time, from 110 to 30 nm. For tetramer 3b, the average size initially was smaller due to the bimodal distribution of nanoparticles (Fig. 2S). Once the small, unstable particles merged into the larger ones, however, the size was fairly constant over time. Why did the two block copolymers behave so differently? The instability of the tetracholate micelles is easy to understand, as a shorter oligocholate has a smaller hydrophobic surface and thus lower hydrophobic driving force for aggregation. What could be the cause for the shrinkage of the hexacholate micelles? The oligocholate consists of rigid building blocks connected by short linkages and each cholate unit only has a few bonds capable of rotation [43]. Cholic acid is known to pack poorly in the solid state due to its awkward shape and the crystal lattice tends to include solvent and guest molecules in the voids [44]. When multiple cholates are joined by short covalent tethers, their close packing will be even more difficult and the initially formed micelles may have significant ‘‘voids” in the hydrophobic core. These voids, of course, would be filled with water and/or residual solvent during micelle formation. The polymeric micelles were prepared by injecting a DMSO solution (10 lL) of the block copolymers into water (1 mL). When 3a first enters water and DMSO molecules are displaced by water in the surrounding, there is a high driving force for aggregation due to the large hydrophobic surface of the hexacholate. For a conventional amphiphilic block copolymer, its self-assembly mainly needs to eliminate solvent exposure of the hydrophobic block. For a long oligocholate, however, many additional parameters have to be optimized to reach the thermodynamically most stable states. In addition to maximal hydrophobic contact of the b-faces, the a-faces of cholates need to be satisfied by hydrogen-bonding. The hydroxyl and amide groups of the oligocholates can be fully hydrogen-bonded if they are exposed to water. During aggregation, these polar groups have to hydrogen-bond with one another (and possibly with and through some residual water/DMSO molecules in the micellar core). It is difficult to imagine that the hexacholate block copolymers can instantaneously and simultaneously have all the parameters optimized when they first come together in aggregation. The packing with maximal hydrophobic contact is not necessarily the one

2.3. Quenching of fluorescently labeled oligocholate–PEG block copolymers in water If the shrinkage of the polymeric micelle of 3a was caused by the slow adjustment of the oligocholates in the micellar core, a freshly prepared micelle should be less tightly packed than an aged one. Block copolymers 3c and 3d are basically 3a and 3b with the methyl ester replaced by the dansyl. Polymer 3e is quite similar to 3c, except that a flexible 4-aminobutyroyl spacer is inserted in between the second and third cholate from the dansyl. To probe the packing of the hydrophobic oligocholates in the micellar core, we studied the quenching of fresh and aged polymeric micelles of these fluorescently labeled block copolymers by Cu(NO3)2 and 4, respectively. For the aged samples, fluorescence

150

(b) 150

130

130

110

110

R (nm)

R (nm)

(a)

that gives maximal hydrogen-bonding and minimal conformational strain in the backbone. Hydrogen-bonds within the hydrophobic micellar core can be quite strong. Reorganization of the oligocholates within the micellar core is thus expected to be slow because multiple hydrogen-bonds will break and re-form during the process. The rigidity and awkward shape of the repeat unit, as well as the long chain, also make the reorganization difficult. The micelles of 3a after 10 h clearly did not represent the global minimum, as the micelles shrank even further at 35 °C. Note that, as solvents got squeezed out over time, the size change slowed down (Fig. 1), possibly because the rearrangement of the oligocholates inside the micellar core became more challenging. Although hexamers 3c and 3e were mainly synthesized to probe the self-assembling by fluorescence quenching (vide infra), we studied their micelles by DLS as well, interested in how the dansyl appendage and the flexible 4-aminobutyroyl spacer affected the micellar size (Figs. 3S and 4S, Supplementary material). These micelles differed in size, with the average hydrodynamic radii somewhat larger than those of 3a. As shown by Fig. 1b, all three hexacholate micelles displayed shrinkage over time, but the extent of shrinkage was the largest in 3a. It seems that the packing of the oligocholates (which was the fundamental reason for the shrinkage) was sensitive to not only the 4-aminobutyroyl spacer but also the dansyl appendage. We could visualize the polymeric micelles directly by transmission electron microscopy (TEM). The samples (aged for 3 d) were stained by 2% phosphotungstic acid (pH 6.2). The hexacholate derivative (3a) formed stable polymeric micelles that showed up mostly as spherical particles with averaging radii about 50– 80 nm (Fig. 2a), in agreement with the DLS sizes. Tetracholate 3b was once again found to be unstable. The micelles did not survive the same sample preparation and only small, irregular particles were found in the TEM micrograph (Fig. 2b).

90

90

70

70

50

50

30

0

2

4

6

t (h)

8

10

12

30

0

2

4

6

8

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12

t (h)

Fig. 1. Average hydrodynamic radius of the micelle of (a) 3a (h, filtered, 25 °C), 3a (e, unfiltered, 25 °C), 3a (s, filtered, 35 °C), and 3b (4, unfiltered, 25 °C) and (b) filtered 3a (h), 3c (4), and 3e (e) in water over time.

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Fig. 2. TEM micrographs of the polymeric micelles of (a) 3a and (b) 3b.

quenching was generally performed generally 48 h after the micelles were prepared. Figs. 3–5 show the representative Stern–Volmer plots for micellar 3c–e. The plots are linear in all cases with the correlation coefficients >0.99. The Stern–Volmer quenching constants (KSV) are summarized in Table 1. The relative standard deviations in KSV ranged from 0% to 6% in repeated measurements. Because of its ionic nature, Cu2+ probably could only quench dansyl located on the surface of the micellar core. Quenching was understandably weak and the I0/I was only slightly above 2 for all three block copolymers even with 200 mM Cu(NO3)2. The Stern–Volmer quenching constants (KSV) ranged from 5 to 9 M1 (Table 1). Interestingly, despite the small differences among the KSV, there seemed to be two consistent trends. First, tetramer 3d was quenched more strongly than either hexamer 3c or 3e, and flexible hexamer 3e more than rigid hexamer 3c. The results were reasonable considering the chain length and the aggregation of the hydrophobic block. A longer oligocholate has a larger hydrophobic driving force for the micelle formation and can better shield the dansyl group from the aqueous phase, where copper ions are located. Assuming dansyl is evenly distributed within the micellar core, a longer oligocholate means that a smaller percentage of dansyl will be on the surface, where it is accessible to the quencher. A recent work of ours indicates that flexible linkers in between the oligocholates make the compounds pack more tightly in water than the more rigid parent compounds [36]. The lower quenching of 3e compared to 3c was thus a reasonable result. It is quite remarkable that a single 4-carbon spacer in a block copolymer nearly 8000 dalton in M.W. could cause detectable differences in the aggregates. Second, quenching was more efficient in the aged than the fresh micelles in all three cases. The KSV values increased by 10–30%,

(a)

3

which was significant compared to the 0–6% error of the measurements. The result initially was a surprise, since we expected that tighter packing in the aged micelles might shield the dansyl better. However, such expectation assumed that the distribution of dansyl in the polymeric micelles stayed constant during the ageing and enhanced hydrophobic contact was the only change that occurred over time. Aromatic hydrocarbons are considerably less hydrophobic than aliphatic ones [45]. With the amino and sulfamide group, dansyl should be substantially less hydrophobic than cholates (at least the b-faces). It is highly likely the locations of dansyl groups were changing as well during ageing. As the oligocholates reorganized within the core, more dansyl groups probably migrated toward the surface of the micellar core and became accessible to Cu(II). The Stern–Volmer quenching constants of 4 were much larger than those of the copper(II). Different KSV for Cu(NO3)2 and 4 simply means that the two have different quenching efficiencies, but the different trends observed with the organic quencher warrants special attention. A quick glance of Figs. 3b, 4b, and 5b reveals that 80–85% of the initial emission was quenched by 4 for the freshly prepared micelles. If we assume that a significant population of the dansyl remained inside the hydrophobic core of the micelle [46], then its strong quenching by 4 suggests deep penetration of the organic quencher into the micellar core. Significant hydrogen-bonds exist in the hydrophobic core of an oligocholate micelle. As discussed earlier, bile acids, including cholic acid and derivatives, pack poorly in the solid and tend to include guest molecules in the lattice [44]. Similar voids must be present within the micellar core of these oligocholate–PEG block copolymers. In the absence of guest molecules, these voids are filled with the solvent. Although 4 is completely soluble in water, its hydrophobic

(b)

5 4

I0 /I

I0 /I

2 3 2

1

1

0

0

0

50

100

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[Cu(NO3)2] (mM)

200

0

5

10

15

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30

[Quencher 4 ] (mM)

Fig. 3. Stern–Volmer plots for the quenching of freshly made (h) and aged (4) micellar solution of 3c by (a) Cu(NO3)2 and (b) TEMPO-derivative 4 at ambient temperature. [Oligomer] = 2.0  106 M.

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(a)

3

(b)

8 7 6

2

I0 /I

I0 /I

5 4 3

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2 1

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0

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[Cu(NO3)2] (mM)

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[Quencher 4 ] (mM)

Fig. 4. Stern–Volmer plots for the quenching of freshly made (h) and aged (4) micellar solution of 3d by (a) Cu(NO3)2 and (b) TEMPO-derivative 4 at ambient temperature. [Oligomer] = 2.0  106 M.

(a)

3

(b)

5 4

I0 / I

I 0 /I

2 3 2

1

1

0

0

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100

150

200

[Cu(NO3)2] (mM)

0

0

5

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15

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25

30

[Quencher 4 ] (mM)

Fig. 5. Stern–Volmer plots for the quenching of freshly made (h) and aged (4) micellar solution of 3e by (a) Cu(NO3)2 and (b) TEMPO-derivative 4 at ambient temperature. [Oligomer] = 2.0  106 M.

Fresh

Aged

Fresh

Aged

being less tightly packed. The result agrees well with all the previous data on the low stability of the tetracholate micelles. Lastly, 3e, which contained the 4-aminobutyroyl spacer, was quenched less strongly than 3c (Table 1); the flexible spacer thus once again was found to help the packing of the oligocholates [36].

6.34 (0.93) 6.97 (1.02) 5.40 (0.79)

6.81 (1.00) 8.85 (1.30) 6.46 (0.95)

126.9 (1.72) 189.6 (2.57) 112.7 (1.53)

73.9 (1.00) 180.0 (2.44) 70.9 (0.96)

3. Conclusions

Table 1 The Stern–Volmer quenching constants (M1) of block copolymers 3c–e by Cu(NO3)2 and 4 at ambient temperature.a Polymer

3c 3d 3e a

Cu(NO3)2

Quencher 4

Numbers in parentheses are quenching constants relative to that of the aged 3c.

framework suggests that its transfer from water to the hydrophobic core of a micelle is energetically favorable. Thus, quenching by 4 most likely was a static process in which the organic quencher penetrated into the micellar core and quenched the dansyl in its proximity. It is important to note that the fresh hexamers (3c and 3e) were quenched much more strongly than the aged ones (Table 1). The 60–70% increase in KSV suggests that the fresh micelles were significantly looser than the aged ones, in agreement with our DLS data. Consistent with the above interpretation, the difference between the fresh and the aged tetramer 3d was small. This is completely in line with the micellar shrinkage, which was only observed in the hexacholate micelles. When short oligocholates aggregate, there are fewer hydrogen-bonds formed through the a-faces and these bonds are weaker in a less hydrophobic core. The oligocholates thus have fewer hurdles to cross in getting to the optimized aggregational states. The quenching constants were larger in the tetramer than the hexamers, suggesting the former

The oligocholate indeed is a unique hydrophobe. The large, awkwardly shaped, facially amphiphilic steroidal ring makes it difficult to pack tightly in the solid state. This is the reason why cholate derivatives, as well as other bile acids, tend to include solvent and guest molecules in the crystal lattice [47]. When multiple cholate units are connected through short amide linkages, close packing becomes even more difficult. Our previous work indicates that poor packing was responsible for the parent oligocholates adopting folded conformations instead of aggregating when dissolved in surfactant solutions [36]. This works reveals that poor packing in the hydrophobic core could create unusual polymeric micelles with time-dependent shrinkage. A size change of 50–70% is significant, as the initially formed polymeric micelles are expected to have significant ‘‘void volume” inside the micellar core. An important application for polymeric micelles is their solubilization of hydrophobic drugs and controlled release [48–50]. As shown by the quenching of 4, the polymeric micelles can bind amphiphilic and/or hydrophobic guests inside the core. If a rational strategy can be developed to modulate the void volume, well-controlled release could be easily engineered in such a system.

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4. Experimental

References

4.1. Materials and instruments used

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4.1.1. General All reagents and solvents were of A.C.S. certified grade or higher, and were used as received from commercial suppliers. Routine 1H and 13C NMR spectra were recorded on a Varian VXR-400 and Bruker DRX-400 spectrometer. MALDI-TOF mass was recorded on a Thermobioanalysis Dynamo mass spectrometer. DLS studies were performed on a PDDLS/ Cool Batch 90 T Dynamic Light Scattering Detector at 25.0 °C. The data was analyzed with the PRECISION DECONVOLVE program and the size measurement was based on five replicates. Transmission electron microscopy (TEM) studies were carried out on a PHILIPS CM 30 instrument, operating at 200 kV. Fluorescence spectra were recorded at ambient temperature on a Varian Cary Eclipse Fluorescence spectrophotometer. The syntheses of compounds 1a [32], 1b [32], 2 [41], and 4 [32] were reported previously. The synthetic procedures for 3a–e are reported in the Supporting material. 4.2. DLS measurements A typical procedure is as follows. An aliquot of 10 lL of a DMSO solution (2.0  103 M) of 4a was added to 1.00 mL of Millipore water in a quartz cuvette. After the sample was gently shaken by hand for 10 s, DLS measurements were taken. Selected samples were filtered through 0.45-micron syringe filters made of hydrophilized Teflon. Intensity data from each sample were collected in five replicates and analyzed by the Precision Deconvolve software. 4.3. TEM imaging The micellar solutions (2.0  105 M) of 3a and 3b were prepared in Millipore water and aged at room temperature for 3 d. A drop of the solution was gently placed on a carbon-coated copper grid (Carbon Type-A, 300 mesh). The TEM grid was stained with 2% phosphotungstic acid (pH = 6.2), dried for 2 h at room temperature, and subjected to TEM observation. 4.4. Fluorescence quenching A typical procedure is as follows. An aliquot of 20 lL of a DMSO solution (2.0  104 M) of 3c was added to 2.00 mL of Millipore water in a quartz cuvette. Stock solutions of 4 (4.0 M) and Cu(NO3)2 (0.6 M) in Millipore water (10.0 lL  10) were prepared. The appropriate amounts of the quencher solution were added to the cuvette via a syringe. After each addition, the sample was vortexed gently for 30 s before the fluorescence spectrum was recorded. For aged samples, the micellar solution was prepared 48 h prior to the fluorescence experiments. The excitation wavelength was 336 nm. Acknowledgments We thank NSF (CHE-0748616) and the Roy J. Carver Charitable Trust for the support of this research. We thank Hongkwan Cho for performing DLS of 3a at 35 °C and Dr. Shiyong Zhang for the TEM images of 3a and 3b. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2010.09.071.