Nanoscale Cage-like Structures Derived from Polyisoprene

Hollowed materials of nanoscopic dimensions offer exciting possibilities in a number of fields such as therapeutics and imaging. The production of nan...
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NANO LETTERS

Nanoscale Cage-like Structures Derived from Polyisoprene-Containing Shell Cross-linked Nanoparticle Templates

2004 Vol. 4, No. 4 683-688

Jeffrey L. Turner and Karen L. Wooley* Center for Materials InnoVation and Department of Chemistry, Washington UniVersity in Saint Louis, One Brookings DriVe, CB 1134, Saint Louis, Missouri 63130-4899 Received February 5, 2004; Revised Manuscript Received March 5, 2004

ABSTRACT Hollowed materials of nanoscopic dimensions offer exciting possibilities in a number of fields such as therapeutics and imaging. The production of nanoscopic cage-like materials is described, based upon the excavation of shell cross-linked nanoparticle (SCK) templates. This report focuses upon extensive characterization of the ozonolysis reaction conditions used to degrade the core material, which demonstrates that thirty minutes of ozone treatment is optimal. The resulting nanocage is also shown to undergo pH tunable hydrodynamic diameter variations and to demonstrate a loss of hydrophobic guest interactions, relative to the SCK precursors.

Self-assembled core-shell structures have received considerable interest relating to the development of methodologies for their preparation1-5 and to the study of their properties6-9 and potential applications.10-13 When assembled via supramolecular interactions between block copolymers in solution and stabilized through covalent bonds in either the core14 or shell,15-17 the degree to which both composition and structure can be further modified is an area of current exploration. For example, the production of entirely hydrophilic6 and zwitterionic nanostructures,7 architectures that are not thermodynamically stable and, therefore, are not obtainable from supramolecular assembly alone, have been described. Our interest is in the physical and chemical manipulation of shell cross-linked (SCK) polymer micelles. In earlier reports, the chemical degradation of the hydrophobic core material allowed for the preparation of nanoscopic cage-like structures. Ozonolytic18 or hydrolytic6,19 degradation chemistry was utilized to convert the amphiphilic core-shell SCKs to hydrophilic shells surrounding a water-filled core, allowing for the potential sequestration of guests and utilization in applications, such as drug delivery. Similar approaches have been employed in the laboratories of Liu, ozonolytically for shell cross-linked nanoparticles containing polyisoprene core material in organic solvent,12,20 and Sakurai, photochemically for those containing polysilane within the cores.21 A variety of other methodologies have also been investigated for the production of hollow cage-like materials of nanoscopic dimensions, including core removal of dendrim* Corresponding author. Tel. (314) 935-7136. Fax (314) 935-9844. E-mail: [email protected]. 10.1021/nl0497981 CCC: $27.50 Published on Web 03/23/2004

© 2004 American Chemical Society

ers,22 self-assembly techniques,23-30 layer-by-layer deposition,31-33 and templating techniques.34-38 However, the use of the SCK as a template material to produce nanocages offers a number of advantages, including their demonstrated ease of production as well-defined and chemically robust discrete nanoobjects. Moreover, when prepared in water, the resulting hydrophilic hydrogel-like nanocage is well designed for biomedical and environmental applications in water, whereby, for example, the use of poly(acrylic acid) (PAA) as the shell material allows for the attachment and surface presentation of ligands for complexation/coordination.10,11 We have been focused on the ozonolytic degradation of polyisoprene chain segments contained within and covalently attached to a poly(acrylic acid-co-acrylamide) (PAA-coPAAm) cross-linked shell, due to the possibility for control over the internal cage chemical reactivity imparted by the reactive carbonyl groups that remain following the ozonolysis. Therefore, given the high reactivity of ozone, the first challenge was to optimize the ozonolysis conditions to remove the core selectively without loss of shell integrity. Herein, we report the optimization of the conditions that allow for the construction of a hydrogel membrane nanocage material composed of PAA-co-PAAm and produced from ozonolytic treatment of a poly(1,4-isoprene) (PI) cored SCK. Utilization of the carbonyl groups remaining after the ozonolytic core degradation for chemical derivatization of the nanocages is part of an ongoing investigation, results from which will be reported in due time. Production of the nanocage began with a diblock copolymer composed of poly(tert-butyl acrylate) and polyisoprene (1,4 isomers predominant) segments which were prepared

Scheme 1. Production of a Nanocage Resulting from the Ozonolytic Degradation of the Poly(isoprene) Core of an SCK Template

Figure 1. Loss of β-carotene reporter molecule from SCK core upon exposure to ozone: 22% cross-linked (dashed line), 72% cross-linked (solid line).

by sequential nitroxide-mediated radical polymerization (NMRP) using Hawker’s universal initiator under standard conditions.39,40 Formation of an amphiphilic copolymer was afforded through acidolysis of the poly(tert-butyl acrylate) segment to poly(acrylic acid) with methanesulfonic acid catalyst.11 Purification of this material was accomplished through a dialysis step against nanopure water. Self-assembly of the resulting polymer was afforded by dissolution into tetrahydrofuran (THF, 2 mg/mL), followed by slow addition of an equivalent volume of water (18 MΩ/ cm, ∼10 mL/h). Addition of a second equivalent of water over a 1 h period, followed by dialysis for 5 days to remove THF, afforded the micellar assembly. Cross-linking of the micelle structure yielded PI-containing SCKs, as has been described previously.11 Briefly, to a stirred micellar solution was added a diamino cross-linker (2,2′(ethylenedioxy)bis(ethylamine)). After a 1 h equilibration time, the addition of a water soluble carbodiimide (1-[3′(dimethylamino)propyl]-3-ethylcarbodiimide methiodide) facilitated a condensation reaction of the amine functionalities with acrylic acid residues within the shell to produce the SCK. Four samples of nominal cross-linking densities of 22%, 39%, 55%, and 72% were prepared from the same micellar solution composed of a PI97-b-PAA78 diblock copolymer, by control over the stoichiometric balance of amine to acid functionalities. The cross-linking densities refer to the percentage of acrylic acid residues that are nominally consumed during the cross-linking reaction (assuming 100% conversion). Scheme 1 demonstrates production of the nanocage from the SCK precursor. Ozonolytic degradation of the core was accomplished by sparging ozone through each nanoparticle solution for varying amounts of time to allow for reaction with the unsaturated carbon-carbon bonds along the PI chain segments, followed by reduction of the intermediate ozonides 684

upon reaction with sodium sulfite. Ozone was delivered at a rate of 100 mg/h as a mixture with column-dried compressed air (2 L/min) from a Red Sea AquaZone 100 ozone generator. After the allotted period of time (0, 1, 3, 7, 15, 30, 60, 180, 360, 720, or 1440 min), residual dissolved ozone was removed by sparging with ozone-free nitrogen for 10 minutes. Addition of a freshly prepared saturated aqueous solution of sodium sulfite afforded reduction of resultant ozonides.41 Each solution was allowed to stir overnight prior to purification and removal of small-molecule byproducts by extensive dialysis. As a measure of the rate at which ozone can permeate the shell and reach the olefinic core of the SCK, β-carotene, a known hydrophobic antioxidant,42 was used as a reporter molecule. The highly conjugated structure of β-carotene allows for monitoring the degradation of the molecule through simple UV-vis spectroscopy. β-Carotene was loaded into the core of 22% and 72% cross-linked SCKs by drop-deposition from acetone into an aqueous SCK solution. After stirring overnight, excess β-carotene was removed by centrifugation, followed by syringe filtration through a 0.45 µm Teflon membrane, and finally dialysis (6-8 kDa MW cutoff cellulose membrane) against nanopure water for several days. Despite precautionary measures to minimize sample exposure to light, β-carotene was found to have degraded into a number of apocarotenoid structures during the dialysis procedure, as indicated by a loss of UV absorbance at wavelengths greater than 500 nm and appearance of additional absorbance peaks between 300 and 500 nm. Prior to ozone exposure, the nanoparticle solutions were again filtered through a 0.45 µm Teflon membrane filter. A control experiment using these purification steps for a β-carotene dispersion in water showed complete loss of the β-carotene material, demonstrated by lack of absorption between 300 and 500 nm wavelengths. Loaded SCK solutions were dispensed into vials and subjected to varying times of ozone treatment. Both the 22% and 72% cross-linked nanoparticle samples demonstrated a rapid decrease in absorbance at 330 nm (strong apocarotenoid absorbance) as the time of ozone treatment increased (Figure 1). This behavior corresponds to a rapid infiltration of the ozone into the core material, and subsequent reaction with the highly conjugated structure; complete destruction of the conjugated β-carotene reporter molecule occurred within 1030 min. The use of the reporter molecule, however, gives no information concerning the relative rates of degradation of Nano Lett., Vol. 4, No. 4, 2004

Figure 2. First derivative plots of the absorbance values measured at 540 nm, following the Purpald assay, demonstrate the rates of aldehyde formation as a function of ozone treatment time. The data for four SCK samples of varying nominal shell cross-link densities (see legend), each derived from the same polymer micelle sample composed of PI core material, are shown.

the isoprene core compared to that of the β-carotene. Direct evidence of the breakdown of the olefinic bonds within the core was obtained through use of an assay that employed the Purpald reagent (4-amino-3-hydrazino-5-mercapto-1,2,4triazole). Purpald is designed to undergo reaction specifically with an aldehyde to give, after oxidation, a purple 6-mercapto-3-substituted-s-triazolo[4,3-b]-s-tetrazine, the formation of which can be monitored by UV-vis absorbance at 540 nm.43 Ozone reacts with poly(1,4-isoprene) to give aldehyde and ketone functionalities in high yield after sodium sulfite reduction. The rate at which the core degradation occurs, therefore, is correlated to the rate at which aldehydes are formed. The Purpald assay was applied to SCK-ozone solutions at varying time points of ozone exposure to investigate the rate of degradation of the PI core material and, hence, to optimize the conditions for nanocage production. After 0, 1, 3, 7, 15, 30, 60, 180, 360, 720, or 1440 min, sodium sulfite was added, the solutions were allowed to stir overnight, a standardized amount of Purpald was added (1 mL of a 0.014 M Purpald solution in 0.1 M NaOH), and the sample was allowed to stir for 18 h prior to measurement of the absorbance intensity at 540 nm. A plot of the first derivative of absorbance (540 nm) versus time of ozone exposure illustrates that the rate of aldehyde formation within each sample was rapid initially and appeared to have been completed within thirty minutes of ozone exposure (Figure 2). It is assumed that in this experiment no poly(isoprene) degradation products were lost through evaporation during the ozonolysis process. However, given the moderate boiling points of the two aldehydic reaction products, butanedial (154 °C) and 4-oxo-pentanal (186 °C), it is possible that a gradual loss of these materials may have occurred, introducing a possible element of error into the experiment which may be observed in extended time periods. Insight into the dimensional changes of the nanoparticle was gained through size analyses as the ozonolytic degradation occurred. Upon breakdown of the hydrophobic core, the resulting hydrophilic nanocage structures swelled in solution, the extent to which corresponded to the limit of the crosslinking material. This swelling process was monitored in Nano Lett., Vol. 4, No. 4, 2004

Figure 3. Examination of hydrodynamic diameter values, measured as a function of ozone exposure times for the 22% cross-linked SCK (dashed line) and 72% cross-linked SCK (solid line) samples. The inset displays the early time points.

solution through dynamic light scattering (DLS) measurements of 22% and 72% cross-linked samples (Figure 3). In examination of the early stages of ozone treatment, both samples underwent an initial expansion consistent with loss of the hydrophobic core. However, despite having similarly sized SCK templates, the two samples exhibited differences in the extents of expansion. After 30 min of ozone exposure and sodium sulfite treatment, the 22% cross-linked SCK gave significantly greater expansion than did the 72% cross-linked SCK (Figure 3). The 22% cross-linked SCK, originating with a hydrodynamic diameter of 66 ( 1 nm, produced a nanocage having a hydrodynamic diameter of 93 ( 3 nm, which corresponded to a 40% diameter increase and 180% volume increase. In contrast, the 72% cross-linked SCK, of 63 ( 1 nm hydrodynamic diameter, produced nanocage structures of 82 ( 3 nm hydrodynamic diameter, which corresponded to a 30% increase in diameter and 120% increase in volume. Examination of the later time points demonstrated a significant loss of particle size and indicated that the PAAco-PAAm shells underwent degradation after extensive ozone treatment. It has been reported in the literature that ozone can react with both amide44,45 and ether46,47 functionalities; both of which are integral portions of the shell structure. The 22% cross-linked sample shows a loss in size after 720 min (12 h) in the presence of ozone, indicating that breakdown of a portion of the material had occurred, whereas, the 72% cross-linked sample appeared to retain the nanostructural integrity after 12 h of ozone treatment. However, after 1440 min (24 h) of ozone treatment, both the 22% and 72% cross-linked samples exhibited significant decreases in hydrodynamic diameters. Atomic force microscopy provided visual insight into the particles’ post ozone treatment. SCK nanoparticles composed of soft core materials have been shown to flatten upon adsorption onto a substrate.4,6 This is observed as both a decrease in height and increase in diameter, as compared to the particle dimensions in solution. Upon conversion of the SCK to the hollowed nanocage, this effect is amplified, showing a decrease in average height and increase in average width of the nanostructure. Ultimately, however, as indicated by the DLS data, the particles break down under long ozone treatment times, and as such, few particles are recognizable 685

Figure 4. Tapping-mode atomic force microscopy images of 22% (a-c) and 72% (d-f) cross-linked SCKs after 0 (a, d), 60 (b, e), and 1440 (c, f) min of ozone treatment. For each image, the sample was drop-deposited from aqueous solution onto freshly cleaved mica and allowed to dry under ambient conditions.

after extensive ozone treatment. Representative atomic force microscopy images for both 22% and 72% cross-linked samples are shown in Figure 4. These images demonstrate reduction of the nanostructure height upon transformation from the amphiphilic, PI-filled SCK to the hydrophilic, hollow nanocage structure, followed by destruction of the nanoscale materials upon long-term ozone exposure. The cross-links within the shells of the micelles serve to stabilize the structures as the core breaks down and provide structural support upon complete loss of the core material. As demonstrated previously,18 ozonolytic degradation of a PI-b-PAA micelle has been shown to yield complete loss of the self-assembled nanostructure, due to a loss of the hydrophobic core, which serves as the anchor of the polymer micelle particle. Given the necessity of the cross-linking material to the stability of the nanostructure, the loss of nanostructural integrity, as revealed by DLS measurements initially and confirmed by AFM imaging, is most likely due to either the amide or ether functionalities of the cross-linker undergoing cleavage reactions upon long ozonolysis times. To investigate the chemical stability of the cross-linker, a model compound, designed to emulate the backbone and cross-linker within the shell layer, was synthesized. Exposure of this model cross-linker to ozone under conditions similar to those used for the nanoparticle showed no degradation for up to 4 h, as indicated by 1H NMR spectroscopy (Figure 5a and Figure 5b). After 24 h, additional resonances within the 1H NMR spectrum (Figure 5c) indicated that degradation had occurred to result in a mixture of products. The exact structures of these products were not determined, as there are several side reactions that are possible for both the amide and the ether functionalities.44-47 Although this model compound is a simplistic structure, relative to the nanocage, the demonstration of chemical instability serves to support degradation of the cross-linking units as the mechanism by which the nanocage fails at long exposure times to ozone. Taking into account the data obtained for core degradation and maintenance of shell integrity, thirty minutes of ozone treatment under the conditions described herein was determined to be the optimal time for maximizing core degrada686

Figure 5. 1H NMR spectra (300 MHz, D2O) of solutions containing the model compound for the cross-links present within the shell of the SCKs and nanocages and byproducts that resulted from ozone exposure: (a) 0 min; (b) 240 min, and; (c) 1440 min of ozone treatment. The chemical structure of the model compound and appropriate proton resonance assignments are shown.

tion while minimizing cross-linker degradation for all nanostructures tested. Loss of the hydrophobic core material of the SCK upon conversion to the nanocage has profound effects on the properties of the nanostructures. A large scale production of nanocages (1 L aqueous solution of SCKs at 0.5 mg/mL) was conducted from a 50% cross-linked SCK sample, and these were subjected to further evaluation, following isolation and purification via dialysis against water for several days. One expected difference between the SCK and nanocage was the degrees to which they could undergo structural expansion upon swelling of the nanostructures, a property which was explored through variation of the pH of their solutions. Ionization of the acrylic acid units within the shell induces intramolecular electrostatic repulsions and, thus, causes an expansion of the macromolecule (in these cases an SCK or a nanocage, which because of the cross-linking Nano Lett., Vol. 4, No. 4, 2004

Figure 6. Hydrodynamic diameter of 50% cross-linked SCKs (solid line) and nanocages (dashed line) produced under optimized conditions were monitored in response to pH, as determined by 90° DLS measurements in aqueous solutions under equivalent ionic strengths.

Figure 7. UV-vis spectra demonstrating the capabilities of SCKs (solid line) and nanocages (dashed line) for the sequestration of BODIPY dye (structure shown in upper right).

are nanoscopic single macromolecules), a phenomenon previously observed in similar poly(acrylic acid) systems.28 SCKs and nanocages produced under optimized conditions, were dialyzed into 10 mM phosphate buffer at varying pH values, and the resultant hydrodynamic diameters were measured by DLS. At low pH (pH ) 5), in which a large fraction of the acrylic acid residues were protonated, both the SCK and nanocage showed similar size profiles as measured by DLS (Figure 6). However, as the acid residues became deprotonated with increasing pH, both the SCK and nanocage underwent significant volume expansions (70% and 180%, respectively, at pH ) 9). The extent of the SCK swelling was withheld by the presence of the core material and possibly some influence by the cross-linking, whereas the extent of the nanocage swelling was limited by the crosslinker. The effect of this swelling process on the permeability of the shell is unknown to this point. Studies are currently underway to probe this phenomenon. The loss of the amphiphilic character of the SCK upon conversion to the nanocage was expected to have a profound effect on the uptake of hydrophobic guest molecules as well. Equivalent molar amounts of SCK and nanocage were incubated with a large excess of 4,4-difluoro-4-bora-3a,4adiaza-s-indacene (BODIPY), a hydrophobic fluorescent dye. The dye was dispersed from methanol into a stirred aqueous solution of the nanostructures and allowed to stir overnight. Residual dye was removed by filtration through a 0.45 µm Teflon membrane and the UV-vis absorbance spectrum was recorded for each solution (Figure 7). A control experiment using only water showed no significant absorbance after filtration (spectrum not shown), indicating that the membrane Nano Lett., Vol. 4, No. 4, 2004

had successfully removed insoluble large aggregates of BODIPY. The resulting absorbance reading was used as a background measurement and subtracted from the baseline of both the SCK and nanocage solutions containing BODIPY. The data from this experiment confirmed the loss of the hydrophobic core material from within the SCK upon conversion to the nanocage. This is indicated by the profound loss of uptake of the BODIPY dye, despite the increased containment volume of the nanocage compared to the SCK. The elimination of nonspecific interactions of the hydrophobic dye with the nanostructures, as demonstrated above, is exciting. In addition to increased internal free volume, one advantage of utilizing these nanocages is the availability of aldehyde and ketone functionalities, lining the nanocage structure (Scheme 1) upon core removal. These functionalities provide a handle for covalent attachment strategies that may lay the foundation from which smart materials can be obtained. For example, lining the core volume of the nanocage with molecules designed to interact specifically with the guest of interest should serve well to move beyond the range of simple nonspecific hydrophobic interactions. In summary, hydrophilic nanoscopic cage-like structures were prepared by the excavation of the hydrophobic, olefinic material within a SCK nanoparticle template. The reaction conditions employed for the production of these materials were optimized to ensure complete core degradation while preserving shell integrity. Nanocages resulting from the optimized reaction conditions behave as expected, showing enhanced responses to environmental stimuli as compared to the parent SCK particles. Additionally, loss of the amphiphilic character of the particles inhibits effective uptake of hydrophobic guest molecules. Moreover, the degradation of the core material leaves aldehyde and ketone functionalities lining the nanocage. Studies are underway to explore the utilization of these reactive carbonyls and their potential for regioselective attachment of guests within the nanocage that are capable of transforming their properties. Acknowledgment. The authors thank Dr. Edward E. Remsen for valuable discussions throughout this work. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-27103 and by a NIH Chemistry-Biology Interface Pathway Training Grant Fellowship for J.L.T. (5T32GM08785-03). References (1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (2) Webber, S. E. J. Phys. Chem. B 1998, 102, 2618-2626. (3) Ma, Y.; Kolotuchin, S. V.; Zimmerman, S. C. J. Am. Chem. Soc. 2002, 124, 13757-13769. (4) Huang, H.; Kowalewski, T.; Wooley, K. L. J. Polym. Sci. Part A: Polym. Chem. 2003, 41, 1659-1668. (5) Orfanou, K.; Topouza, D.; Sakellariou, G.; Pispas, S. J. Polym. Sci. Part A: Polym. Chem. 2003, 41, 2454-2461. (6) Ma, Q.; Remsen, E. E.; Kowalewski, T.; Schaefer, J.; Wooley, K. L. Nano Lett. 2001, 1, 651-655. (7) Bu¨tu¨n, V.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. J. Am. Chem. Soc. 1999, 121, 4288-4289. (8) Lodge, T. P. Macromol. Chem. Phys. 2003, 204, 265-273. (9) Murthy, K. S.; Ma, Q.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Mater. Chem. 2003, 13, 2785-2795. 687

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Nano Lett., Vol. 4, No. 4, 2004