Letter pubs.acs.org/Langmuir
Block Copolymer Micelles with a Dual-Stimuli-Responsive Core for Fast or Slow Degradation Dehui Han, Xia Tong, and Yue Zhao* Département de chimie, Université de Sherbrooke, Sherbrooke, Québec, Canada J1K 2R1 S Supporting Information *
ABSTRACT: We report the design and demonstration of a dualstimuli-responsive block copolymer (BCP) micelle with increased complexity and control. We have synthesized and studied a new amphiphilic ABA-type triblock copolymer whose hydrophobic middle block contains two types of stimuli-sensitive functionalities regularly and repeatedly positioned in the main chain. Using a two-step click chemistry approach, disulfide and o-nitrobenzyle methyl ester groups are inserted into the main chain, which react to reducing agents and light, respectively. With the end blocks being poly(ethylene oxide), micelles formed by this BCP possess a core that can be disintegrated either rapidly via photocleavage of o-nitrobenzyl methyl esters or slowly through cleavage of disulfide groups by a reducing agent in the micellar solution. This feature makes possible either burst release of an encapsulated hydrophobic species from disintegrated micelles by UV light, or slow release by the action of a reducing agent, or release with combined fast-slow rate profiles using the two stimuli.
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methyl ester by photolysis upon light absorption,10,14 we anticipated that micelles of this BCP might be degraded by the two stimuli in different manners. To our knowledge, this is the first study reporting the incorporation of two types of stimuli-cleavable moieties into the main chain via rational BCP design. As schematically illustrated in Figure 1, we found that micelles of this BCP
INTRODUCTION There is much interest in micelles of amphiphilic block copolymers (BCPs) that can undergo a disruption process in response to stimuli such as temperature or pH change,1 light,2 redox species,3 enzymes,4 and ultrasound.5 Among the many BCP designs, incorporating stimuli-responsive functionalities repeatedly into the main chain of the micelle core-forming hydrophobic block is emerging as an interesting strategy.6−8 A straightforward disintegration of micelles can be achieved if the stimuli-responsive groups can lead to a severe main chain breaking. This feature makes this type of BCP micelles different from those either with only one cleavable linkage between the hydrophilic and hydrophobic blocks9 or having many cleavable side groups on the hydrophobic block.10 On the one hand, we reported recently the study of a BCP whose hydrophobic block has ο-nitrobenzyl methyl ester groups in all monomeric units and whose micelles could undergo fast photodegradation resulting in burst release of encapsulated species.6 On the other hand, BCP micelles with pH11 or redox-sensitive main chain cleavage7,8,12 were also known. In the search of stimuli-responsive BCP micelles with a more versatile and complex level of control, it is of interest to insert multi-stimuli-cleavable units in the main chain of the hydrophobic block in a controlled fashion. Although this may be synthetically challenging, the resulting micelles could exhibit distinguished behaviors due to the main chain degradability in response to two or more stimuli. In this paper, we describe a synthetic method with which a new amphiphilic ABA-type triblock copolymer was designed to possess two stimuli-responsive functionalities positioned repeatedly in the main chain of the hydrophobic middle block, namely, disulfide and o-nitrobenzyl methyl ester. Knowing that disulfide can be cleaved by a reducing agent8,13 and o-nitrobenzyl © 2012 American Chemical Society
Figure 1. Schematic illustration of block copolymer micelles undergoing either fast or slow degradation as a result of either fast photoinduced or slow reduction-induced main chain cleavage.
could indeed be disintegrated either quickly by light or slowly by a reducing agent. This feature allows encapsulated hydrophobic species to be released in aqueous micellar solution either quickly by light exposure, or slowly under the effect of reduction, or with a combined fast-slow rate profile using the two stimuli.
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RESULTS AND DISCUSSION Scheme 1 describes the two-step click chemistry utilized to obtain the triblock copolymer of PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO. Received: December 14, 2011 Revised: January 13, 2012 Published: January 20, 2012 2327
dx.doi.org/10.1021/la204930n | Langmuir 2012, 28, 2327−2331
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Scheme 1. Two-Step “Click” Synthetic Route to the Amphiphilic ABA-Type Triblock Copolymer with a Dual-Stimuli-Cleavable Main Chain for the Middle Hydrophobic Block
and 12 o-nitrobenzyl methyl ester groups on the main chain of poly(disulfide-alt-nitrobenzene). On the one hand, with the BCP dissolved in THF (10 mg/mL, 3 mL solution), exposure to UV light (300 nm, 60 mW/cm2, 20 min) resulted in degradation of the polymer due to the photocleavage of o-nitrobenzyl mether ester groups. As seen from trace D, the elution peak of PEO was recovered, together with many low-molecular-weight species eluting at longer times; a small fraction of polymers with molecular weights larger than PEO remained, suggesting the presence of a number of PEO linked to a segment of the middle block. On the other hand, by adding a reducing agent, dithiothreitol (DTT), in the same BCP solution (10 mg/mL, molar ratio of DTT and disulfide = 8/1), the SEC measurement after 16 h, trace E, indicates similarly severe degradation of the polymer chain due to the disulfide cleavage, with the elution peak of PEO recovered and almost no species of larger molecular weights. These results show that the hydrophobic middle block of poly(disulfide-alt-nitrobenzene) could be degraded by either exposure to UV light or the presence of a reducing agent in the solution. SEC was also used to monitor the evolution of photo- and DTT-induced polymer degradation and the results are given in the Supporting Information. The micelle formation of this BCP was investigated using a typical method.15 The polymer sample was dissolved in THF that is a good solvent for both blocks; water was then added to induce the aggregation of poly(disulfide-alt-nitrobenzene) forming the micelle core. Figure 3a shows the determination of the critical water content for micellization (CWCM) for two BCP concentrations. Light scattering intensity measured at 90° increases abruptly upon the formation of micellar aggregates. The CWCM was found to be 14 and 8 wt % for a BCP concentration of 2 and 5 mg/mL, respectively. After micelle formation, more water was added to quench the micellar aggregates and THF removed by evaporation. Figure 3b shows an example of AFM height image recorded by casting an aqueous micellar solution on silicon wafer followed by drying under vacuum. BCP micelles with diameters of 25 ± 11 nm are visible. When BCP micelles in aqueous solution are exposed to UV light or DTT, they can be disintegrated as a result of the photo- or reductioninduced main chain degradation of the poly(disulfide-alt-nitrobenzene) block (AFM images in the Supporting Information). The interest of this type of micelles resides in the fact that not only they can respond to two types of stimuli but also their degradation rates under the effect of the two stimuli are very different. This can be observed from the micelle degradation kinetic process monitored by measuring the light scattering intensity of the solution. Figure 4 shows the results obtained
In the first step, the middle hydrophobic block was prepared using a click condensation reaction between a disulfide monomer having two alkyne groups (1) and an o-nitrobenzyl methyl ester monomer bearing two azide functionalities (2). In the end of the polycondensation, a slight excess of 1 was added to obtain the middle block terminated with two alkyne end groups. In the second step, the middle block was reacted with hydrophilic poly(ethylene oxide) (PEO) having one azide end group to give rise to the amphiphilic triblock copolymer after click coupling. In this way, the main chain of the hydrophobic block poly(disulfide-alt-nitrobenzene) is given one disulfide and two o-nitrobenzyl methyl ester groups in the repeating unit. Details on the monomer and polymer syntheses are presented in the Supporting Information. The degradability of this triblock copolymer under the effect of the two different stimuli was first investigated. Figure 2 shows
Figure 2. Size exclusion chromatograph (SEC) traces of (A) poly(disulfide-alt-nitrobenzene); (B) azide-terminated PEO; (C) triblock copolymer PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO; (D) the triblock copolymer in THF (10 mg/mL) after 20 min exposure to UV light (300 nm, 60 mW/cm2); and (E) the triblock copolymer in THF (10 mg/mL) after 16 h reaction with DTT, the molar ratio of DTT and disulfide groups being 8:1.
the results obtained with size exclusion chromatograph (SEC) measurements. Trace A is for the poly(disulfide-alt-nitrobenzene) middle block. The relatively large polydispersity index (PDI) of 1.6 is signature of a polycondensation reaction. Trace B is for the PEO end block that has a molecular weight of 2000. Trace C is for the triblock copolymer appearing at shorter elution times. On the basis of the molecular weight of PEO, 1H NMR spectral analysis yielded an average molecular weight of 4150 g/ mol for the middle block, which corresponds to ∼6 disulfide 2328
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Figure 3. (a) Determination of the critical water content for the micellization of PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO upon addition of water into a THF solution at two different polymer concentrations. The light scattering intensity was measured at 90°. (b) AFM height image of the block copolymer micelles dried on a silicon wafer.
reaction mechanisms. In the presence of DTT, the reduction reaction of disulfide groups is likely to take place at the micelle corona−core interface because water-soluble DTT molecules could not diffuse into the rigid micelle core. The reaction is more like a slow erosion of the micelles. With a lower ratio of DTT to disulfide, such as 2/1, the degradation of micelles was even too slow to be observed on the used time scale. By contrast, under UV light exposure, the rigid micelle core can absorb photons quickly, and the photolysis of o-nitrobenzyl methyl esters, which is an intramolecular rearrangement reaction without the need for water molecules, can proceed quickly and result in fast disintegration of the micelles. The fast photoreaction in aqueous micellar solution was also revealed by the absorption spectra recorded over UV exposure time (see the Supporting Information). Conceptually, the feature of dual-stimuli-responsiveness with either fast or slow disintegration of micelles is interesting for potential drug delivery applications. While fast drug release is required for some therapies, a slow and sustained release may be desired in other cases. Combined use of the two stimuli offers more possibilities. These include a slow release using DTT with, in the course, controlled burst release at chosen times with light (Figure 4), or fast partial release by light followed by a slow process with DTT, or vice versa. We further investigated the dual-stimuli-controlled release of Nile Red (NR), a model hydrophobic dye often utilized as a probe of release in aqueous solution.10,14,16 The results obtained using NR-loaded BCP micelles are summarized in Figure 5. In (a), the fluorescence intensity of NR at the maximum emission wavelength is plotted as a function of UV light exposure time for micelles in aqueous solution and in a mixed solvent of water/THF (1/2, v/v). In the mixed solvent, the release of NR molecules as a result of photoinduced degradation of the micelles resulted in no change in the fluorescence intensity because NR is soluble in the solution. By contrast, the fluorescence of NR decreased rapidly in aqueous solution upon degradation of the micelles bringing NR into water in which the dye is insoluble and its fluorescence is quenched. This result shows the fast photoinduced release of encapsulated NR. In (b), the change in fluorescence intensity of NR over time in aqueous solution in the presence of DTT (4/1 for the molar ratio of DTT and disulfide) is shown. Here we also performed a control test to make sure that the fluorescence quenching of NR arises from the micellar disintegration due to
Figure 4. Change in the light scattering intensity over time (measured at 90°) for a micellar solution of PEO-b-poly(disulfide-alt-nitrobenzene)b-PEO (1.0 mg/mL in dioxane/water, 1/1, v/v) both in the absence and in the presence of the reducing agent DTT (molar ratio of DTT and disulfide: 8/1). In the latter case, the micellar solution (3.0 mL) was exposed to UV light twice for 1 min duration at indicated times (300 nm, 60 mW/cm2).
with a micellar solution (in a mixed solvent of dioxane/water to ensure the solubility of all degraded species). In the absence of DTT and without exposure to UV light, the scattering intensity remained constant over time, indicating the micellar stability in the solution. By contrast, in the presence of DTT (8/1 for the molar ratio of DTT and disulfide), the scattering intensity decreased continuously over time indicating the disintegration of micelles. After 4 h of reaction, the solution was exposed to UV light for 1 min (300 nm, 60 mW/cm2), the scattering intensity dropped by an amount similar to that after the first 2 h of reaction with DTT. After the fast photodegradation, the decrease in the scattering under the effect of DTT became very slowly, approaching an apparent equilibrium. However, when UV light was applied for 1 min again, the fast disintegration of the micelles was resumed, as can be noticed from the second drop of the light scattering intensity. These results show that the disintegration of the micelles could proceed either quickly under UV light exposure or relatively much slowly upon reaction with DTT. This is understandable due to the two different 2329
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Figure 5. (a) Normalized fluorescence emission intensity of Nile Red (NR)-loaded in micelles of PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO in water (closed circles) and in a mixed solvent of water/THF (1/2, v/v) (open circles) exposed to UV light (solution volume 1.5 mL, 300 nm, 250 mW/cm2). (b) Normalized fluorescence intensity of NR-loaded in micelles of PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO (solid circles) and in micelles of PEO-b-poly(nitrobenzene)-b-PEO (open circles) in water with added reducing agent DTT (molar ratio of DTT/disulfide: 4/1). (c) Fluorescence emission spectra of NR-loaded in micelles of PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO in water with added DTT (molar ratio of DTT/disulfide: 4/1), the solution being exposed to short-time UV light twice (300 nm, 250 mW/cm2). (d) Change in the normalized fluorescence emission intensity over time for the NR-loaded micellar solution in (c). All fluorescence intensities were measured at the maximum emission wavelength, with the emission spectra obtained at λex = 550 nm.
plus the two linkages between the three blocks (Scheme 1). Therefore, this BCP may be ultrasound-responsive as well. Should this be the case, the BCP micelles could be disintegrated through main chain degradation induced by three types of stimuli: reduction, UV light, and ultrasound, making them truly multi-stimuli-degradable micelles. We investigated this possibility by performing several experiments, including exposure of the triblock copolymer to ultrasound under conditions similar to those reported (polymer dissolved in acetonitrile at 0 °C, ultrasound frequency 20 kHz, power 750 W), and also exposure of the BCP micelles in aqueous solution to ultrasound. In all tests, no ultrasound-induced cleavage of triazole groups and degradation of micelles were observed, suggesting that the specific conditions for the occurrence of ultrasound cleavage of triazole groups (high polymer molecular weight with one triazole group placed at the middle)17 were not met with the BCP architecture containing many triazole groups, neither in the form of self-assembled micelles. The potential of exploring ultrasound for controlled cleavage of triazole groups in polymer micelles may be limited.
the cleavage of disulfide groups. NR was loaded in micelles of another ABA triblock copolymer composed of PEO end blocks and a hydrophobic middle block bearing no disulfide but only o-nitrobenzyl methyl ester groups.6 As seen, a certain degree of fluorescence quenching was observed for micelles containing no disulfide groups and thus stable in solution, which suggests an effect of the reducing agent DTT on the fluorescence of NR. However, when loaded in the triblock copolymer with the poly(disulfide-alt-nitrobenzene) block, the fluorescence quenching became much more prominent, which is result of the degradation of the micelles bringing NR into an aqueous medium. Comparing the time scales in Figure 5a and b, it is clear that the photoinduced release of NR is much faster than the reduction-induced release. We then carried out an experiment with combined use of DTT and UV light. The reducing agent was added in an aqueous solution of NR-loaded micelles (molar ratio 4/1), and the fluorescence emission spectra of NR were recorded as a function of time. As shown in Figure 5c, the decreasing intensity indicates reduction-induced release of dye molecules due to slow micellar disintegration. After 3 h, UV light exposure was applied for 1 min; the drop in the fluorescence intensity indicates a burst release due to fast photoinduced degradation. This alternating use of the two stimuli was repeated for another cycle. Figure 5d shows the plot of fluorescence intensity of NR vs time under the combined use of the two stimuli. Before concluding, it is worth mentioning that according to a recent report the triazole group resulting from the click reaction between azide and alkyne can be cleaved by ultrasound.17 In our PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO, each repeating unit in the middle block also contains two triazole groups,
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CONCLUSIONS
We have reported a general two-step click chemistry approach for preparing amphiphilic ABA triblock copolymers of which the repeating unit of the hydrophobic middle block contains functionalities in the main chain that are responsive to two different types of stimuli. Using the method, we synthesized PEO-b-poly(disulfide-alt-nitrobenzene)-b-PEO whose hydrophobic block contains one disulfide and two o-nitrobenzyl methyl ester groups in the repeating unit. With this BCP design, micelles can 2330
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be disintegrated under the effect of either a reducing agent that breaks the disulfide bonds or UV light that cuts the o-nitrobenzyl methyl ester groups. We showed that photoinduced degradation of the BCP micelles in aqueous solution is much faster than the reduction-induced degradation. This feature makes it possible to have either burst release of an encapsulated hydrophobic species from disintegrated micelles by UV light exposure, or slow release by the action of a reducing agent in the micellar solution. The two stimuli could also be utilized in combination to generate on-demand release rate profiles. To the best of our knowledge, this is the first example of BCP design that allows two types of stimuli-cleavable moieties to be repeatedly inserted into the main chain of the hydrophobic block, making BCP micelles undergo main chain degradation in response to two different stimuli.
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ASSOCIATED CONTENT
S Supporting Information *
More details on the synthesis and characterization of PEO-bpoly(disulfide-alt-nitrobenzene)-b-PEO triblock copolymers. UV−vis, SEC, and AFM characterization results of triblock copolymer micelle. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and le Fonds québécois de la recherche sur la nature et les technologies of Québec (FQRNT). Y.Z. is a member of the FQRNT-funded Center for Self-Assembled Chemical Structures.
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