Tailored Polymeric Materials for Controlled Delivery Systems

Chapter 13

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 16, 2015 | http://pubs.acs.org Publication Date: September 24, 1998 | doi: 10.1021/bk-1998-0709.ch013

Shell Cross-Linked Knedels: Amphiphilic Core-Shell Nanospheres with Unique Potential for Controlled Release Applications 1

2

K. Bruce Thurmond II and Karen L. Wooley

Department of Chemistry, Washington University, One Brookings Drive, Campus Box 1134, St. Louis, MO 63130-4899 Shell cross-linked knedels (SCK's) are stable, covalently bound macromolecular assemblies that possess spherical shape, a core-shell morphology, and nanometer dimensions. The SCK's are prepared by a three-step procedure beginning with covalent construction of an amphiphilic diblock copolymer, followed by self-assembly into a three-dimensional structure and lastly, stabilization via covalent cross-links within selective regions. This is reminiscent of the method by which proteins are created. The chemistry is performed in water and the SCK's contain a hydrogel-like cross-linked surface layer that surrounds a hydrophobic core. Investigation of the SCK's for encapsulation and binding applications is discussed.

There is currently a great interest in the design, formation, and development of novel polymeric systems, including dendrimers, polymer micelles, etc. The motivation for the creation of novel three-dimensional macromolecular architectures comesfromthe ability of structures to affect properties, which then leads to different functions. It is not the goal of this chapter to debate which of these materials is the best or holds the most promise, but rather to provide information on the preparation, characterization, and potential applications of cross-linked polymer micelles, specifically shell cross-linked knedels (SCK's) (/). Polymer micelles (2-5) consist of block copolymer chains that form a micellar structure in a selective solvent for one block or in solvent mixtures that have differing solubilities for the different blocks. Micellar systems exist in a constant dynamic equilibrium between mono- and multi-molecular micelles and in each case the micelle maintains a core-shell morphology, where the soluble block shields the insoluble portionfromthe solvent system. The ability to control or change the

'Current address: Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, M O 63167. Corresponding author.

©1998 American Chemical Society

In Tailored Polymeric Materials for Controlled Delivery Systems; McCulloch, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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morphology allows for production of an assortment of polymeric micelle structures (6 7). Changes in the solvent system, the block copolymer content, or the ratios of the polymeric blocks leads to changes in the core, the shell, the surface, and the interface between the core and the shell thus providing some degree of control over these systems. The properties of the core and the shell enable encapsulation of molecules and subsequent release, which allows for polymer micelles to be studied as agents for pharmaceutical delivery. A variety of potential applications may be envisioned for these systems including areas such as drug delivery (#), gene therapy (9), and encapsulation technologies (10-12). Polymer micelles are stable systems at concentrations above their critical micelle concentration (cmc), but destruction of the system once going below this concentration may be of great concern when trying to apply these systems. In addition to concentration effects upon polymer micelle structures, deformation can result under the action of shear forces. Nevertheless, the use of polymer micelles for in vivo drug delivery has been reported (8). For example, an anti-tumor drug, adriamycin, was covalently-attached to the poly(aspartic acid) block, PASP, of a block copolymer composed of poly(ethylene oxide), PEO, and PASP. Formation of the micelle with PASP-adriamycin nucleating to form the core of the polymer micelle was then carried out. Release of the adriamycin was accomplished by cleavage from the PASP chains making up the core. PEO is commonly used in the shell of polymeric micelles (10,11) and on the surface of cationic liposomes (13) for biological (in vivo) applications (14), because of its flexibility, high hydration, resistance to proteins through the steric exclusion mechanism, and its strong hydrogen bonding character, which may be important at the interface between the polymer micelle and the target site (8). The properties of the polymer micelle core are also very important. Using polymer micelle systems with glassy cores (high Tg) can stabilize the multi-molecular micelles and prevent destruction of the micelles that can occur upon infinite dilution under in vivo conditions (8). However, such a high T and an impenetrable core may not be conducive to the loading or the release of guest molecules. The emphasis here is on static SCK structures, which possess mechanical stability and also are not affected by changes in concentration. The design of these materials has gained much from the in-depth studies of polymer micelle synthesis, characterization, structure, and function. The SCK's are polymer micelles containing covalent cross-links that serve to reinforce the micellar architecture. The preparation of such structures requires only a three step approach: synthesis of a functionalized diblock copolymer, self-assembly into a micellar structure, and cross-linking the polymer chains to form a stable, covalently bound particle system as seen in Figure 1. Examples of cross-linked polymer micelle systems in which the core is cross-linked have been reported with particle sizes typically between 0.02 and 1 nm (15-18). These materials contain hydrophobic polystyrene shells, which limits them to non-biological applications. Although core cross-linked micro- and nanoparticles are useful systems, the focus of this chapter is on polymer micelles with a hydrophobic polystyrene (PS) core and a cross-linked, hydrophilic, quaternized poly(4-vinylpyridine) (PVP) shell. These materials maintain water solubility and


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should provide additional volume in the non-cross-linked core for uptake or encapsulation of small molecules, thus allowing the SCK's to be envisioned for a variety of biological applications.

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Amphiphilic Block Copolymer

Figure 1. Schematic representation of the basic procedure for the formation of shell cross-linked polymer micellesfromamphiphilic block copolymers. SCK Synthesis The preparation of the SCK's begins by synthesizing the diblock copolymer, polystyrene-6-poly(4-vinylpyridine) (PS-6-PVP), under anionic conditions. Three different block copolymers were formed with PS:PVP ratios of 1:2, 1:1, and 2:1, and with molecular weights of 20700, 14600, and 14400, respectively. A percentage, 15% to 50%, of the pyridyl groups were quaternized with /?-chloromethylstyrene to provide hydrophilicity to the VP portion of the block copolymer and to introduce the cross-linkable sites along the hydrophilic segment. The quaternized block copolymer was dissolved in a mixture of 30% tetrahydrofuran/H20 at concentrations that allowed for formation of the polymer micelle. Under these conditions, the hydrophobic PS nucleated to form the core and the quaternized hydrophilic PVP comprised the surrounding shell. After formation of the polymer micelle, cross-linking through the /7-chloromethylstyrene groups was carried out using a water-soluble radical initiator, 4,4'-azotos(4-cyanovaleric acid), and irradiating for 24


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hours. Polymerization of the styrene side groups along the VP chains resulted in cross-links within the shell and formation of the SCK's, Figure 2. Following cross-linking, no cmc was detectable by pyrene excitation spectroscopy and the SCK's maintained spherical shape upon adsorption onto a mica substrate as observed by atomic force microscopy (AFM) (19). In contrast, polymer micelles exhibited cmc's at ~10" M and experienced severe deformation (flattening) upon mica. The SCK's were determined by AFM to be of narrow size dispersities with average diameters between 5 and 30 nm, depending upon the makeup of the SCK (20). For example, increasing relative hydrophobicity gave SCK's with increasing diameters, where the 1:2, PS:PVP block copolymers resulted in SCK's with average diameter of 9 ± 3 nm, the 1:1, PS:PVP gave 15 ± 2 nm, and the 2:1, PS:PVP system was 27 ± 5 nm.


7

Shell Cross-linked Knedel (SCK)

Amphiphilic Diblock Copolymer

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Figure 2. Chemistry involved in the synthesis of a shell cross-linked knedel (SCK), in which polystyrene comprises the core and the cross-linking is accomplished by radical polymerization of styrenyl side groups along the backbone of poly(4vinylpyridine) located in the polymer micelle outer-shell. The SCK's are a diverse material with a high degree of tailorability of the macromolecular structure and composition (Figure 3). It is possible to alter the shell surface, the shell layer, the core, and thus the interface between the two, either by selecting different starting block copolymers, using different cross-linking reactions or modifying regions of the SCK after construction. This ability to manipulate large facets of the structure of the SCK makes for a variety of potential applications.


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Specifically, encapsulation within the hydrophobic core and binding throughout the hydrophilic shell, where charge interactions are key, have been studied.

Figure 3. Schematic representation of an SCK displaying the diversity of the macromolecular structure and composition, selectively controlled within either the core domain, the shell layer, the surface, or any combination thereof. Core Encapsulation Excitation spectroscopy experiments showed that large hydrophobic molecules, such as pyrene, could be encapsulated within the core (27). Additionally, the solution-state H NMR spectra obtained in D2O indicated that tetrahydrofuran (THF) was able to penetrate the several nanometer thick cross-linked shell, migrate to the core, and solvate the mobile PS (21). A third molecule was investigated, a,y-bisdiphenylene-P-phenylallyl radical or BDPA radical (1), a large hydrophobic molecule. ]

1 BDPA radical has no visually detectable solubility in a 10% THF/H 0 solution, and gives no absorbance by UV-Vis spectroscopy. However, addition of BDPA radical (10% by weight) to a solution of 10% THF/H 0 containing SCK (1:1, PS:PVP) results in complete dissolution. Encapsulation of 1 in the SCK's was also evident based upon the UV-Vis spectrum which gave a peak at 485 nm (Figure 4) and a change in color of the solutionfrompale yellow to a bright yellow/orange. 2

2



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Figure 4. UV-Vis Spectra of (a) SCK and (b) SCK + BDPA radical, taken in 10% THF/H 0. 2


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Shell Binding


The complexation of small molecules within the positively-charged shell layer of the SCK structure was also studied through binding interactions of SCK's with negatively charged small molecules. Specifically, the dye molecules Coomassie Blue 2 and Copper (II) phthalocyanine tetrasulfonic acid tetrasodium salt 3 were used.

2 3 Binding experiments were carried out using a constant mass of SCK (PS:PVP, 1:1), 0.58 mg in 5 mL of water, and masses of dyes ranging from 0.05 - 1.1 mg. After allowing the solutions to stir for 24 h, UV-Vis spectra were obtained. The Xmax of 2 shifted from 585 nm in pure H2O to 602 nm when in the presence of the SCK's, Figures 5a and 5b. This shift may be due to a solvatochromic effect upon binding of 2 to the SCK's, however, determination of binding could not be made from the UVVis spectra alone. Therefore, the sample was centrifiiged to precipitate 2/SCK complexes and measure the amount of dye remaining in solution, Figure 5c. The amount of binding/uptake was based upon the difference in intensities, at 602 nm for 2 and at 611 nm for 3, of the spectra before and after centrifugation. As shown in Figure 6, the amount of dye 2 uptake for the constant mass of SCK was plotted against the mass of the dye in die solution. The theoretical values in Figure 6 are based upon achievement of a charge balanced SCK-dye complex, which would occur at 0.43 mg 2 per 0.58 mg SCK. Charge balance can be described as the amount of dye needed to contain the same number of negative charges as there are positive charges in the SCK. Since a constant mass of SCK was used, a plateau of uptake is expected, but as shown in Figure 6 this is not observed. The plot can be broken down into three areas, low, intermediate, and high dye concentration. There is a deviation between actual and theoretical uptake at low and high dye concentrations, but it is much more pronounced at the high masses of 2 added. At intermediate dye concentrations the actual and theoretical binding correspond rather well, Figures 5b, 5c, and 6. The hypothesis for the behavioral differences over the entire range of dye concentrations is as follows. At low dye concentrations there is not enough of 2 to form a neutral complex, thus complete precipitation does not occur and a portion of the SCK-dye complexes remain in solution, suspended by excess positive charges of the SCK's



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Figure 5. UV-Vis Spectra of (a) coomassie blue, 2; (b) SCK+ 2; and (c) SCK + 2 after centrifugation, each collected in H2O.



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(with chloride counterfoils). At intermediate dye concentrations, the hydrophilicity of the SCK is reduced by interaction with a significant number of the dye molecules. This results in formation of an insoluble complex, which leads to comparable actual and theoretical binding values. At high dye concentrations the maximum uptake of 2 by the SCK is exceeded and provides an excess of 2, which forms weak interactions with the SCK-dye complexes. This helps to maintain solubility of the SCK-dye complex to be detected by UV-Vis spectroscopy and reduce the amount of precipitate formed. Therefore, the deviation between the actual and theoretical data points is apparently observed due to the formation of soluble complexes, and the extent of SCK-dye binding is underestimated by this method. Further analysis of the complexation is in progress. Strikingly similar data was observed when using 3 under the same conditions as the previous experiment (Figure 7). The dye molecule 3 contains two more negative charges per molecule than 2, thus charge balance was achieved with a lesser mass. The results provide additional support for the SCK-dye complexes remaining in solution with an excess of dye or SCK, but when there are sufficient numbers of dye molecules interacting with each SCK to reduce the effective hydrophilicity, the complexes precipitate. Although these experiments do not provide quantitative measurements of binding, they do offer insight into the nature of binding and solubility properties for the complexes of SCK's with small negatively charged organic molecules. This is expected to be important for binding and release applications. For example, when an excess of negatively-charged guests bind to the SCK's, there must be differences in the binding strengths and a gradient of release kinetics may be observed. Further investigation of these behaviors is in progress. The uptake of hydrophobic molecules within a hydrophilic system and the binding of negatively charged molecules are just two of the many unique characteristics of the SCK's. The results from these initial studies, in combination with the diversity of the SCK's structure and composition provides encouragement toward further evaluation and development of the SCK's for controlled release applications. The shell, the core, the surface, or any combination of these may be manipulated to provide a new system tailored to the desired environment. The core can be changedfroma glassy polymer to a rubbery polymer and the charge within the shell can bear charges or be neutral (22). This allows for control of the shell surface as well as the interface between the core and the shell. The SCK's are prepared by a simple self-assembly process, but they possess increased stability over polymer micelles because of the covalent linkages between the polymer chains. This increased stability may allow for longer and highly controlled release times and provide protection for the guest molecule when used as a carrier vehicle. Degradable cores, shells, and cross-links are being investigated for post-delivery decomposition i and release. The numerous methods in which an SCK can be tailored have shown promise in preliminary studies of areas such as encapsulation, drug delivery, and DNA compaction and transfection.


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Mass of 2 (mg) Figure 6. Plot of the mass of coomassie blue, 2, bound by the SCK (constant 0.58 mg mass in 5 mL H2O) and precipitated as a charge balanced complex (•), measured as the decrease in UV-vis intensity for the absorbance of 2 following binding and centrifugation. The theoretical binding (•) is based upon complete uptake of 2 until a charge balance between the dye molecules and the SCK's is reached. Deviation from theoretical values indicates that the 2/SCK complex remains suspended in the water, with excess 2 or excess SCK.

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Mass of 3 (mg) Figure 7. Plot of the mass of copper (II) phthalocyaninetetrasulfonic acid, tetrasodium salt, 3, bound by the SCK (constant 0.58 mg mass in 5 mL H 0) and precipitated as a charge balanced complex (•), measured as the decrease in UV-vis intensity for the absorbance of 3 following binding and centrifugation. The theoretical binding (•) is based upon charge balance between the dye molecules and the SCK's. Deviation from theoretical values indicates that the 3/SCK complex remains suspended in the water. 2


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Acknowledgements Financial support for this work from the National Science Foundation National Young Investigator Award DMR-9458025 and Monsanto Company is gratefully acknowledged. Fellowship support (K.B.T.) was provided from the Department of Education, Graduate Assistance in Areas of National Need (P200A4014795). The authors thank Professor Tomasz Kowalewski for A F M studies and Mr. Christopher G. Clark, Jr. for structural drawings. References: 1. (note: knedel is a Polish word to describe a dumpling-like food of filling surrounded by a dough layer; pronounced k∙ned' '1) 2. Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339. 3. Gao, Z.; Eisenberg, A. Macromolecules 1993, 26, 7353. 4. Qin, A.; Tian, M.; Ramireddy, C.; Webber, S.; Munk, P; Tuzar, Z. Macromolecules 1994, 27, 120. 5. Forder, C.; Patrickios, C. S.; Armes, S. P. Billingham, N. C. Macromolecules 1996, 29, 8160. 6. Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. 7. Spatz, J. P.; Möβmer, S.; Möller, M. Angew. Chem. Int. Ed. Engl. 1996, 35, 1510. 8. Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119. 9. Friedmann, T.; Felgner, P. L.; Blaese, R. M.; Ho, D. Y.; Sapolsky, R. M.; Mirsky, S.; Rennie, J. Scientific American June 1997, 276, 95. 10. Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600. 11. Peracchia, M. T.; Gref, R.; Minamitake, Y.; Domb, A.; Lotan, N.; Langer, R. J. Controlled Release 1997, 46, 223. 12. Kriz, J.; Masar, B.; Doskocilova, D. Macromolecules 1997, 30, 4391. 13. Lasic, D. D. ACS Polym. Prepr. 1997, 38, 543. 14. Harris, J. M. ACS Polym. Prepr. 1997, 38, 520. 15. Procházka, K.; Baloch, M. K.; Tuzar, Z. Makromol. Chem. 1979, 180, 2521. 16. Wilson, D. J.; Riess, G. Eur. Polym. J. 1988, 24, 617. 17. Ishizu, K.; Saito, R. Polym.-Plast. Technol. Eng. 1992, 31, 607. 18. Guo, A.; Liu, G.; Tao, J. Macromolecules 1996, 29, 2487. 19. The SCK shapes were found to be spherical with equivalent height-to-diameter ratios, after deconvolution of AFM tip effects. All SCK diameters were determined as the particle heights from tapping mode AFM images of SCK's adsorbed onto mica, and were averaged over measurement of ca. 300 SCK particles. 20. Thurmond II, K. B.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 6656 21. Thurmond II, K. B.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1996, 118, 7239. 22. Huang, H.; Kowalewski, T.; Remsen, E. E.; Gertzmann, R.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 11653.


Tailored Polymeric Materials for Controlled Delivery Systems

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