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Langmuir 2007, 23, 7916-7919
Tunable Disassembly of Micelles Using a Redox Trigger Suhrit Ghosh, Katharine Irvin, and S. Thayumanavan* Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003 ReceiVed April 5, 2007. In Final Form: May 20, 2007 A simple design for stimuli-responsive micellar disassembly has been demonstrated on the basis of a surfactant molecule wherein the hydrophilic head group is connected to the hydrophobic tail through a redox-sensitive disulfide linker. The disassembly kinetics is tuned by mixing the stimuli-responsive surfactant with a suitable nonresponsive co-surfactant in various ratios, which helps in gaining control over the release rate of encapsulated dye molecules from such mixed micelles.
Introduction Stimuli-responsive disassembly of an organized supramolecular structure has been a topic of great interest.1 If a disassembly event results in the release of an otherwise encapsulated guest molecule, then the impact of the study becomes greater.2 Among the various stimuli studied for these purposes, redox cleavage of the disulfide bond3 is of particular interest because of the possibility of an elevated glutathione level in specific cell types.2b There are a few examples of redox cleavage of supramolecular assemblies based on disulfide functionality.4 These examples arise mainly from chemically cross-linked polymer nanoparticles and liposomes. In the case of cross-linked polymer nanoparticles, guest molecules are physically entrapped during the cross-linking process and are released during the disulfide cleavage-based uncrosslinking event. In the case of liposomes, water-soluble guest molecules are entrapped within the interiors during the assembly process, which are then released in response to a stimulus. In both cases, the guest molecules are sequestered within the interior in competition with the bulk solvent. This is mainly because the guest molecules here are most often soluble in the solvent in which the assembly is done. Micellar assemblies are quite complementary in this regard. The guest molecules that micelles sequester are hydrophobic. Because micelles are formed in water, the hydrophobic guest molecules that are often insoluble in water can be loaded into the assemblies with high efficiency. To our knowledge, the disassembly of micelles has not been studied in the context of redox-triggered cleavage. Although disassembling micelles using the redox trigger itself is interesting, this process is even more useful, if the kinetics of the response to the stimulus is tunable through molecular * Corresponding author. E-mail:
[email protected]. Fax: +1-413545-4490. (1) (a) Rui, Y.; Wang, S.; Low, P. S.; Thompson, D. H. J. Am. Chem. Soc. 1998, 120, 11213. (b) Goodwin, A. P.; Mynar, J. L.; Ma, Y.; Fleming, G. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2005, 127, 9952. (c) Jaeger, D. A.; Zeng, X. Langmuir 2003, 19, 8721 and references therein. (d) Menger, F. M.; Gabrielson, K. J. Am. Chem. Soc. 1994, 116, 1567. (e) Heskins, M.; Guillet, J. E. J. Macromol. Sci. Chem. 1968, 1441. (f) Jiang, J.; Tong, X.; Zhao, Y. J. Am. Chem. Soc. 2005, 127, 8290. (g) Eastoe, J.; Vesperinas, A. Soft Matter 2005, 1, 338. (2) (a) Heller, J.; Barr, J.; Ng, S. Y.; Abdellauoi, K. S.; Gunny, R. AdV. Drug DeliVery ReV. 2002, 54, 1015. (b) Saito, G.; Swanson, J. A.; Lee, K.-D. AdV. Drug. DeliVery ReV. 2003, 55, 199. (3) Lamoureux, G. V.; Whitesides, G. M. J. Org. Chem. 1993, 58, 633. (4) For disulfide-containing amphiphilic structures, see (a) Nolan, D.; Darcy, R.; Ravoo, B. J. Langmuir 2003, 19, 4469. (b) Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem. Soc. 1999, 121, 11247. (c) Saily, V. M. J.; Ryhanen, S. J.; Lankinen, H.; Luciani, P.; Mancini, G.; Parry, J. Mikko.; Kinnunen, P. K. J. Langmuir 2006, 22, 956. (d) Zhang, J.; Jing, B.; Tokutake, N.; Regen, S. T. Biochemistry 2005, 44, 3598. (e) Jong, L. I.; Abbott, N. L. Langmuir 1998, 14, 2235. (f) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (g) Li, Y.; Lokitz, B. S.; Armes, S. P.; McCormick, C. L. Macromolcecules 2006, 39, 2726.
design. Here, we demonstrate a strategy to fine tune the guest molecule release kinetics from these assemblies using a mixed micelles approach. We envisaged the possibility of designing a surfactant (1 in Scheme 1) that could form a micelle but contains a disuflide linkage between the hydrophilic head group and the lipophilic tail. Treatment of 1 with molecules such as dithiothreitol (2), which is a glutathione mimic, should result in 3-thioproprionate (3) and undecanethiol (4), along with the cyclic water-soluble byproduct (2′) as shown in Scheme 1.5 The consequence of such a reaction is that the lipophilic tail and hydrophilic head group get separated. If such a process were to happen above the critical micelle concentration (cmc) of surfactant 1, then it would result in the degradation of the micellar assembly (Scheme 1a). If a hydrophobic guest molecule is encapsulated within the micellar interior, then this molecule will be released and the kinetics of this release should depend on the disassembly kinetics of the micelle itself. To achieve control over this process, it is necessary to identify a generalized strategy. In this letter, we disclose our findings on the behavior of surfactant 1 as well as a strategy to fine tune the guest molecule release profile using mixed micelles (Scheme 1). Experimental Section Materials and Methods. Dithiothreitol and sodium laurate were purchased from Aldrich Chemical Company and used as such without further purification. Other surfactants were prepared using few synthetic steps. (Details can be found in Supporting Information.) Linear absorption and emission spectra were recorded on a Varian EL01125047 spectrometer and a Jasco FP-6500 spectrofluorimeter, respectively. All spectral measurements were made using a quartz cuvette of 1 cm path length. Determination of Critical Micellar Concentration (cmc) of the Surfactant 5. The cmc of sodium laurate (5) was determined by the pyrene fluorescence method. A stock solution of pyrene (10-4 M) was made in acetone. The stock solution (20 µL) was taken in a vial, and the solvent was evaporated using a mild stream of argon. To this vial, a solution of sodium laurate (2 mL, 10 mM) in water was added, and the solution was sonicated for 30 min to encapsulate the pyrene. The solution was filtered and transferred to a cuvette. A measured volume of this solution was replaced by a 10-6 M pyrene solution in water, and the emission spectrum was recorded every time (λex ) 337 nm). The ratio of the intensities of the first (374 nm) and the third (385 nm) peaks (I1/I3) was plotted against the concentration of the surfactant. The cmc was determined by the inflection point observed in such a plot.7 (5) For an interesting study on the reaction of DTT with molecules containing disulfide bonds, see Clelan, W. W. Biochemistry 1964, 3, 480. (6) Fowler, S. D.; Greenspan, P. J. Histochem. Cytochem. 1985, 33, 833. (7) See supporting information for details.
10.1021/la700981z CCC: $37.00 © 2007 American Chemical Society Published on Web 06/13/2007
Letters
Langmuir, Vol. 23, No. 15, 2007 7917 Scheme 1. Design of Surfactantsa
a (a) Schematic representation of stimuli-responsive micellar disassembly and guest release; (b) conversion of amphiphilic surfactant 1 to hydrophilic and lipophilic components in the presence of DTT; and (c) structures of other surfactants studied.
Determination of the cmc of Surfactant 1. A measured amount of the carboxylic acid form of surfactant 1 was dissolved in a 10 mM aqueous NaOH solution so that 1 equiv of NaOH is present in the mixture. A stock solution (concentration 1 mM) of Nile red in dichloromethane was made, from which 0.1 mL was transferred to another vial and the solvent was removed. To this, 2 mL of the surfactant stock solution was added; the mixture was sonicated for 30 min and filtered into a cuvette, and the emission spectra were recorded (λex ) 550 nm). A measured amount of this solution was replaced by water, and every time, the fluorescence intensity was monitored. The emission intensity was then plotted against the concentration of the surfactant, and the cmc was determined from the prominent inflection point in the plot.7 A similar procedure was followed for the determination of the cmc of all mixed micelles formed by mixing various ratios of surfactants 1 and 5.7 For the mixed micelles, individual surfactant stock solutions were mixed in an appropriate ratio, and the resulting mixture was incubated for 1 h before the start of the experiment. General Procedure for DTT-Induced Dye-Release Studies. To 2 mL of a 10 mM micellar solution was added a measured amount of 0.1 M DTT solution to obtain a 1:1 ratio of DTT/surfactant 1. The solution was stirred in a cuvette, and fluorescence emission spectra were recorded at different time intervals until there was no further reduction in emission intensity. The emission intensity was plotted against time to obtain the release profiles for each composition. The observed blue shift in emission spectra with time is attributed to the change in the local dielectric of the fluorophore due to the disruption of the micelle. In all DTT-induced dye release studies, the total surfactant concentration was kept constant at 10 mM, and 1 equiv of DTT (with respect to the disulfide-containing surfactant) was used.
undecanethiol (4) in aqueous solution. This supposition was independently confirmed by attempting to obtain millimolar solutions of undecanethiol in water. Additional evidence for the release of dye molecules from the micellar interior comes from the observation of the color change with the naked eye. Nile red by itself is not soluble in water and therefore results in a colorless solution (vial 1 in Figure 1b). The dye is taken up in water in the presence of surfactant 1 above the cmc, as evident from the intense color of the solution (vial 2). In the presence of DTT, this color fades away with time, consistent with release from the micelle (vial 3). In the control experiment, wherein no DTT was added, the intensity remained constant in the time frame of the experiment, indicating that the release due to diffusion is insignificant, if present. Although disassembling the micelle and releasing the guest molecule itself is interesting, such a phenomenon would be more useful if there were tunable control over the rate of guest molecule release. It is understood that the rate of reaction will differ with the concentration of DTT. However, it is necessary that there is structure-based control over the rate of release for broader applicability. We envisaged the possibility of forming mixed micelles containing a disulfide-containing surfactant mixed with a non-disulfide surfactant to achieve such control. In choosing such a non-disulfide surfactant, we recognized that it will be important to choose a molecule (i) that exhibits the ability to form a mixed micelle with surfactant 1, (ii) that could form micelles at a cmc higher than that of 1, and (iii) that exhibits a much poorer container property than 1. Sodium laurate (5) satisfied all of the criteria mentioned above. The cmc of 5 was
Results and Discussion We have examined the formation and disassembly of the micelles using the emission spectra of Nile red as the probe. Nile red is a hydrophobic dye that is not soluble in water by itself, as can be discerned from the lack of absorption or emission spectral intensity from the solution. However, this dye can be sequestered inside the hydrophobic pocket generated by micelles.6 The relative emission intensity of Nile red was plotted against the concentration of 1, and a sharp increase in the emission intensity was observed at about 2 mM concentration,7 suggesting this to be the cmc of surfactant 1. To examine the sensitivity of this micellar structure to external stimuli, the Nile red-containing micellar solution was treated with 1 mol equiv of DTT (10 mM concentration of 1; well above the cmc). The change in emission intensity as a function of time is shown in Figure 1a. The decrease in intensity with time is taken to suggest that the micelle is being disassembled. This is supported by the fact that the turbidity of the solution also increases with time, independent of the presence of the fluorophore. This is due to the formation of insoluble
Figure 1. (a) Change in fluorescence emission intensity of Nile red encapsulated in micelles formed by 1 in the presence of DTT. (Inset) relative emission intensity vs time. Wavelengths (nm) at which the intensity is plotted with increasing time are 631.5, 630, 629, 625, 621, 619, 614.5, 614.5, 614, and 615. (b) Color of the Nile redencapsulated solution of 1 before (vial 2) and after (vial 3) treating with DTT for 2 h; vial 1- contains Nile red in water without 1.
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Letters
Table 1. Characterization of Mixed Micelles ratio of 1/5
cmc (mM)
maximum release time (min)a
100:0 65:35 50:50 35:65 25:75 0:100
2.0 2.2 2.5 2.8 3.7 6.5
95 130 170 550 680 b
a Total surfactant concentration is 10 mM (1 equiv of DTT with respect to 1). The maximum release time is the time after which there is no change in the emission intensity of Nile red over an extended period of time. b No dye encapsulation.
Figure 3. Comparison of the DTT-induced release rate of surfactants 1 and 6. Total surfactant concentration ) 10 mM, mole ratio of surfactant 1/DTT ) 1:1 in each experiment.
Figure 2. Time for maximum release (when the change in emission intensity of Nile red reaches saturation) in various micelles. Total surfactant concentration ) 10 mM, mole ratio of surfactant 1/DTT ) 1:1 in each experiment.
determined to be 6.5 mM using pyrene emission spectra,8 and the ability of 5 to induce pyrene uptake was much poorer, relative to that of 1.7 Surfactants 1 and 5 were mixed in different ratios (65:35 to 25:75), and the release rates of Nile red from the resulting mixed micelles were determined. It was gratifying to note that the release rate indeed depends strongly on the composition of the mixed micelle (i.e., as we increase the percentage of nonresponsive surfactant 5 in the mixed micelle, the release rate of the guest molecule slows down significantly, Table 1). The time taken to achieve the maximum release is plotted as a function of the composition of the mixed micelle in Figure 2. For example, it takes nearly twice as much time to release a dye molecule from the mixed micelle containing a 50:50 ratio of 1/5, compared to the time taken for guest release from that of 100% 1. We were interested in achieving a better understanding of the release mechanism. First, we investigated the nature of the micellar container from 1 vs 5. It is intriguing to note that whereas 5 is a poorer host for pyrene and Reichardt’s dye it does not encapsulate Nile red at all, even above its cmc.7 We were, however, delighted to realize that the mixed micelles formed from 1 and 5 (in the ratios studied) are good hosts for Nile red. We were interested in investigating whether the cleavage of the disulfide bond occurs inside the hydrophobic pocket of the micelle or in the free surfactant, which is in dynamic equilibrium with the aggregated form. For this purpose, we synthesized another surfactant, 6 (Scheme 1), that differs only in the location of the cleavable disulfide bond compared to that in 1. The release rates are similar in both cases (Figure 3), which suggests that the cleavage likely happens in the monomeric surfactant. Otherwise, one would have seen a much slower rate of release in the case (8) Kalyansundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039.
of surfactant 6. This is conceivable because of the fact that the DTT reagent is very water-soluble and is thus unlikely to be sequestered inside the hydrophobic pocket of the micelle. Note that it is also possible that the cleavage does occur within the aggregate but the diffusion is rapid and the disulfide bond scission is the rate-determining step. We could not unambiguously rule out this alternate possibility. There are two limiting mechanistic possibilities for release: (i) The dye molecule is released because the micelle is completely disrupted and no more micellar containers are available to sequester the hydrophobic dye. (ii) Cleavage of the disulfide bond continuously alters the relative ratio of the two surfactants and finally leaves only non-disulfide surfactant 5, which has the opportunity to reorganize to form a new micelle. The dye molecules subsequently leach out from the reorganized micelle because 5 is an inherently poorer host. It is likely that mechanism i dominates in the case of high 1/5 ratios because the micelle will be completely disrupted. Similarly, in the case of low 1/5 ratios, the latter mechanism (ii) is likely to be operational.9 It is then interesting to ask which of these two mechanisms is favored at intermediate ratios. To address this question, we carried out the following experiments. We measured the release profile of the 65:35 ratio of mixed micelles at two different concentrations (10 and 20 mM). In the first case, the concentration of 5 that would be left behind in solution after the cleavage of 1 is 3.5 mM. In the latter case, it is 7.0 mM. Thus, the remaining concentration of 5 after the cleavage of 1 is below the cmc in the first case and above the cmc in the second case. The guest release profiles in both cases were found to be remarkably similar.7 Thus, mechanism ii alone cannot explain the observations for the intermediate ratios (see below for an alternate possibility). We were interested in identifying the fate of the cleaved surfactant. More precisely, we were interested in identifying whether the hydrophobic 1-undecanethiol (4) byproduct is capable of acting as a competing guest molecule for the micelles. The reason for this interest is that if this were the case then this provides a unique, additional mechanistic pathway for guest molecule release (guest competition-based displacement mechanism). To identify this, we carried out an experiment with mixed micelles containing a 30:70 ratio of 1/5 and compared the turbidity arising from this solution with 100% 1 when treated with DTT. In both of these solutions, the concentrations were chosen in (9) It is important to note that in the initial stage the release is triggered by the cleavage of the disulfide bond for all compositions. Thus in all mixed micelle compositions, the initial rate of release is faster and almost the same (see Supporting Information for details), and the composition-dependent difference in the rate of release arises only in the later stage.
Letters
Figure 4. Comparison of the DTT-induced change in turbidity of the medium as (% transmittance) in a micellar solution. Ratio of surfactant 1:/DTT ) 1:1 in each experiment.
such a manner that the amounts of byproduct 4 generated are identical. In the former case, the remaining amount of 5 is above its cmc and therefore could partly or completely sequester released byproduct 4. In the latter case, this possibility does not exist. When the turbidity is measured with time, the solution from 100% 1 indeed exhibited much more turbidity than did the mixed micelle combination (Figure 4). This suggests that the micelle from 5 can indeed act as the host for byproduct 4 and therefore could potentially contribute to the release of the guest molecule. This also explains the similar release rates from the 65:35 ratio of the mixed micelles at 10 and 20 mM concentrations because the relative amounts of 1-undecanethiol and surfactant 5 are equal in both the cases.
Langmuir, Vol. 23, No. 15, 2007 7919
In summary, we have shown that disulfide-containing surfactants could be used as redox-responsive micelles that result in the release of hydrophobic guest molecules. The release kinetics of the guest molecule can be tuned by forming a mixed micelle with a co-surfactant that is not stimulus-responsive. On the basis of the mechanistic studies carried out here, the model for guest molecule release involves (i) the fact that the co-surfactant is an inherently poor host for the guest molecule and (ii) the participation of the hydrophobic byproducts of the stimulusresponsive cleavage as a competing guest molecule for the micelle. The studies outlined here could have implications in biomedical applications such as drug delivery.10 From a fundamental standpoint, it is also necessary for us to understand the reasons for the differences in container properties of surfactants such as 1 and 5; these have a significant impact on the work outlined here. Experiments to develop such an understanding through further characterization of the micellar assemblies are underway in our laboratories. Acknowledgment. We thank the Army Research Office for financial support. Supporting Information Available: Synthesis of the surfactant molecules, structural characterization data, and spectral data for all of the experiments performed. This material is available free of charge via the Internet at http://pubs.acs.org. LA700981Z (10) For the biological relevance of the present study, it is important to ensure that such cleavage can also be executed with glutathione. Indeed, we found this to be the case with the surfactant 1.
