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Carbon Dioxide Emulsion Assisted Loading of Polymer Microspheres toward Sustained Release Materials Gary A. Baker,† Mary L. Campbell,‡,§ Matthew Z. Yates,‡,| and T. Mark McCleskey*,‡ Bioscience or Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received November 20, 2004. In Final Form: February 24, 2005 An organic solvent-free method for encapsulating progesterone at high loadings within micron-sized inert latex polymer beads is reported. This approach makes use of a polymeric surfactant to emulsify carbon dioxide into an aqueous latex suspension. In this way, preformed ∼4 µm polystyrene (PS) microparticles surface-grafted with poly(N-vinylpyrrolidone) (PVP) were plasticized and swollen followed by rapid partitioning of progesterone into the polymer matrix. The as-prepared polystyrene beads incorporated over 10% progesterone by weight while maintaining their initial size and morphological uniformity. Dissolution experiments were also carried out to obtain the release profile of progesterone entrapped within the PVP/PS particles.
Introduction Apart from the conspicuous, if nontrivial, goal of delivering a targeted (temporally and spatially) chemical payload in the correct dosage, modern drug delivery also seeks to address unmet needs such as patient compliance, affordable formulations, and reduced systemic toxicity with minimal adverse side effects. While the primary goal addresses improving the release profile, therapeutic efficacy, and bioavailability/absorption of drugs, next generation drug delivery systems need to consider intangibles such as patient comfort whatever the route of administration. In either case, the solution depends in part or wholly on the creation of new materials or the improved manipulation and control of existing ones.1,2 Supercritical fluid science, particularly that involving compressed carbon dioxide, is an established technology for polymer synthesis and processing. Fundamental “green” strategies for producing micronized particles with minimal residual solvent at moderate temperatures either involve rapid expansion of a supercritical solution (RESS) or supercritical antisolvent precipitation. Apart from being an environmentally benign medium for materials fabrication, the intrinsic properties of CO2 can also induce substantial and often beneficial changes in preexisting polymers leading to processing applications such as plasticization, swelling, foaming, crystallization, viscosity reduction, glass transition depression, and phase segregation or blending.3,4 Importantly, copolymers exposed to CO2 will experience various extents of swelling and enhanced chain mobility, significantly facilitating solute mass transport to sites within existing polymer matrixes * To whom correspondence should be addressed. Phone: 505667-5636. E-mail:
[email protected]. † Bioscience Division. ‡ Chemistry Division. § Current address: Chemistry Department, Stanford University, Stanford, CA 94305. | Current address: Department of Chemical Engineering, Laboratory for Laser Energetics, University of Rochester, Rochester, NY 14627-0166. (1) Henry, C. M. Chem. Eng. News 2002, 80 (34), 39-47. (2) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487-492. (3) Tomasko, D. L.; Li, H.; Liu, D.; Han, X.; Wingert, M. J.; Lee, L. J.; Koelling, K. W. Ind. Eng. Chem. Res. 2003, 42, 6431-6456. (4) Kazarian, S. G.; Chan, K. L. A. Macromolecules 2004, 37, 579584.
(impregnation).5,6 To date, the most common application of impregnation in CO2 is in the area of organic dyeing of polymeric materials, with some excellent examples from the Eckert group.7-9 Surprisingly, even for dyes with very low solubility in the supercritical medium, favorable partitioning into the polymer phase is often observed.7-10 In fact, distribution coefficients are often 102-103 times higher than that with conventional liquid solvents. As a recent example, using confocal fluorescence microscopy Wang et al.11 showed that a uniform distribution of organic solute in polypropylene could be achieved despite low solubility in the fluid phase and unfavorable modifierpolymer interactions. Still, in many applications it is advantageous to achieve a high interfacial area by employing fluorinated or hydrocarbon surfactants to generate stable water and CO2 emulsions.12-15 For example, we have recently reported a new microencapsulation approach for infusing additives into sterically stabilized aqueous polymer colloids by using CO2 in water emulsions.16-18 Keys to this work are CO2 acting simultaneously as the swelling agent for the (5) Clarke, M. J.; Cooper, A. I.; Howdle, S. M.; Poliakoff, M. J. Am. Chem. Soc. 2000, 122, 2523-2531. (6) Gross, S. M.; Givens, R. D.; Jikei, M.; Royer, J. R.; Khan, S.; DeSimone, J. M.; Odell, P. G.; Hamer, G. R. Macromolecules 1998, 31, 9090-9092. (7) Kazarian, S. G.; Brantley, N. H.; Eckert, C. A. CHEMTECH 1999, 29, 36-41. (8) Kazarian, S. G.; Brantley, N. H.; West, B. L.; Vincent, M. F.; Eckert, C. A. Appl. Spectrosc. 1997, 51, 491-494. (9) West, B. L.; Kazarian, S. G.; Vincent, M. F.; Brantley, N. H.; Eckert, C. A. J. Appl. Polym. Sci. 1998, 69, 911-919. (10) Perman, C. A.; Bartkus, J. M.; Choi, H. O.; Reichert, M. E.; Witcher, K. J.; Kao, R. C.; Stefely, J. S.; Gozum, J. E. U.S. Patent 5,508,060, 1994. (11) Wang, Y.; Yang, C.; Tomasko, D. Ind. Eng. Chem. Res. 2002, 41, 1780-1786. (12) Hoefling, T. A.; Beitle, R. R.; Enick, R. M.; Beckman, E. J. Fluid Phase Equilib. 1993, 83, 203-212. (13) Johnston, K. P.; Harrison, K. L.; Clarke, M. J.; Howdle, S. M.; Heitz, M. P.; Bright, F. V.; Carlier, C.; Randolph, T. W. Science 1996, 271, 624-626. (14) Sarbu, T.; Styranec, T.; Beckman, E. J. Nature 2000, 405, 165168. (15) Kane, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Langmuir 2000, 16, 4901-4905. (16) Yates, M. Z.; Birnbaum, E. R.; McCleskey, T. M. Langmuir 2000, 16, 4757-4760. (17) Liu, H.; Yates, M. Z. Langmuir 2002, 18, 6066-6070. (18) Liu, H.; Yates, M. Z. Langmuir 2003, 19, 1106-1113.
10.1021/la047146m CCC: $30.25 © 2005 American Chemical Society Published on Web 03/29/2005
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Figure 1. The carbon dioxide assisted microparticle impregnation process. The first step involves emulsification of the CO2 into the continuous water phase by the action of an appropriate fluorinated or hydrocarbon surfactant. The CO2 acts as both a carrier for the drug and a plasticizer of the polymer matrix. After the release of CO2 pressure, the particles revert to their glassy state and regain their former size, entrapping the agent within.
polymer host and the carrier solvent for the additive. The efficiency of this process results from the dramatic increase in CO2/water interfacial area upon emulsification. The CO2-based impregnation process can be conceptually summarized in three stages (see Figure 1). Initially, amorphous polymer latex particles are suspended in water (W). After emulsification with a suitable surfactant, a Gibbs surface excess of CO2 at the polystyrene (PS)-water interface occurs, resulting in swelling of the PS latex with enhanced chain and segmental mobility. At higher pressures, CO2 diffuses into and liquefies the glassy polymer. The so-formed CO2 microdroplets also act as carriers for the solute (denoted as b in the second panel). After partitioning of the solute into the polymer matrix, the agent-loaded, solvent-free latex product is recovered upon slow, controlled venting during which the particles recover their former size and shape. Although it holds immense promise, application of CO2 fluid technology for impregnating pharmaceuticals and biopharmaceuticals into materials toward controlled drug delivery vehicles has been explored only recently.19-21 In this letter, we report the first application of a CO2 in water emulsion toward the creation of stable polymer colloids encapsulating a medically relevant species. In these studies, the choice of progesterone as the model drug was made because of its clear importance in hormone therapy.22,23 Apart from its main progestational action, natural progesterone also shows antiandrogenic and antimineralcorticoid activities as well. However, peroral administration generally shows poor bioavailability due to its intense hepatic metabolism. This work is designed as a proof-of-concept to show the potential for a wide variety of potential applications. Experimental Section Materials and Equipment. Styrene (99%), poly(N-vinylpyrrolidone) (Mw ) 40 kD), 2,2′-azobisisobutyronitrile (AIBN), and progesterone were purchased from Sigma-Aldrich and used as received. Absolute ethanol was a product of Pharmco Products, Inc., and supercritical fluid grade CO2 (Matheson) was passed through Alltech oxygen, water, and hydrocarbon traps prior to (19) Guney, O.; Akgerman, A. AIChE J. 2002, 48, 856-866. (20) Kazarian, S. G.; Martirosyan, G. G. Int. J. Pharm. 2002, 232, 81-90. (21) Liu, H.; Finn, N.; Yates, M. Z. Langmuir 2005, 21, 379-385. (22) Valenta, C.; Walzer, A.; Clausen, A. E.; Bernkop-Schnu¨rch, A. Pharm. Res. 2001, 18, 211-216. (23) Taghizadeh, S. M.; Mashak, A.; Jamshidi, A.; Imani, M. J. Appl. Polym. Sci. 2004, 91, 3040-3044.
use. The poly(ethylene oxide)-block-poly(butylene oxide) (PEOb-PBO, tradename SAM 185) diblock surfactant was kindly donated by BASF. Nanopure water (Barnstead, 18.2 MΩ cm) was used throughout. UV/vis spectra were taken on an HP8453. Dispersion Polymerization. The polymerization procedure follows previously reported methods.16 Briefly, poly(N-vinylpyrrolidone) (PVP, 1.5 g) was dissolved in 75 mL of ethanol followed by helium sparging for an additional 30 min to remove dissolved oxygen. The ethanolic PVP solution was then heated to 70 °C in an oil bath while under a blanket of helium. In a separate flask, 0.25 g of AIBN was dissolved in 25 mL of styrene after removing the inhibitor by elution through an inhibitor removal column just prior. The AIBN in styrene solution was then added by syringe to the stirring PVP in ethanol. The mixture was slightly turbid, becoming milky in ca. 10 min. The styrene was polymerized for 24 h at 70 °C after which ethanol was removed by rotary evaporation. The final product was highly monodisperse with a mean particle diameter of 3.6 ( 0.2 µm as determined by scanning electron microscopy (LEO 982 FE-SEM, 5.0 kV). Progesterone Impregnation and Release. PS particles (0.337 g) in the form of a dry powder were dispersed into 10 mL of Nanopure water on a mechanical shaker for several minutes. The resulting aqueous latex was loaded into a stainless steel variable-volume viewing cell along with 0.150 g of progesterone and 0.092 g of SAM 185. In a typical experiment, 3.5 g of CO2 was added using a computer-controlled high-pressure syringe pump (ISCO, Inc., model 260D). The impregnation was carried out under continuous stirring at 25 °C and 310 bar for 24 h. It is important that CO2 pressure be released slowly and in a wellcontrolled manner in order to allow the gas to fully escape prior to polymer recovery from the swollen state. In our experience, a depressurization time of 2 h is sufficient to completely avoid foaming lending itself to product uniformity. The treated aqueous latex was transferred to a centrifuge tube and spun at 10 000g for 20 min. The solvent was decanted, and the particles were resuspended in ethanol. Again, the sample was centrifuged followed by decanting to leave the progesterone-infused PVPgrafted PS beads. The particles were air-dried overnight and then dried under vacuum for 4 h before study. Complete removal of ethanol was confirmed by 1H NMR analysis for the progesterone-containing polymer beads in CD2Cl2. The 1H NMR spectrum from the final material shows three singlet peaks from progesterone (2.122, 1.220, and 0.680 ppm) and a broad PS matrix singlet corresponding to the phenyl protons of styrene (7.1 ppm). Based on their relative integrated peak intensities, the matrix contains 10 wt % progesterone compared to styrene. To study the release of progesterone from the loaded beads, 0.05 g of progesterone-loaded latex beads were suspended in 10 mL of ethanol. At various time increments, the sample was centrifuged and a UV/vis measurement was taken of the supernate. Particles were then suspended by shaking. Progesterone concentration was determined by monitoring absorbance at 240 nm ( ) 2670). The amount of progesterone released after
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Figure 2 displays the progesterone time release behavior using ethanol as the release medium. After dispersing the particles in ethanol, progesterone is slowly released from the PVP/PS particles by diffusion through the polymer matrix and into the surrounding medium. Aliquots were removed at suitable time intervals and assayed using absorbance (235 nm) to determine the amount of progesterone released. Indeed, the PVP/PS-encapsulated progesterone shows sustained release of the hormone over the course of about 5 h.
Figure 2. Time release profile for progesterone from a sample of PVP/PS latex prepared by CO2-assisted impregnation; kobs ) 0.712 h-1. Ethanol was used as the extraction solvent at a level of 2 mL per mg of progesterone-loaded latex. Results for a negative control PS latex made without using SAM 185 are shown by the open squares. 8 h is statistically equivalent (within 1.2%) to the level after 24 h (the absorbance at 24 h is thus taken as A∞). A negative control was also done with progesterone in the absence of SAM 185.
Results and Discussion Based on scanning electron microscopic evidence, controlled release materials formed using this approach starting with preexisting amorphous polymer microbeads exhibit no agglomeration. Further, no particle growth, distortion, deformation, surface roughening, or foaming were observed compared with the unadulterated material. In addition, the CO2 is readily removed by simply vaporizing under reduced pressure. Despite the low solubility of progesterone in CO2, the latex beads were readily loaded with 10% progesterone. The CO2 in this process acts both as a swelling agent for the polymers in the latex bead and as a transfer agent for the progesterone. The poor solvation properties of CO2, that limit potential applications for chemical reactions, offer an advantage for a transport medium. The progesterone is transported effectively by the supercritical phase of CO2 with low mass transport barriers and readily deposits into the polymer matrix where it is more soluble.
Summary In this work, we have used CO2-assisted impregnation to formulate polymer microspheres incorporating high levels of a steroid hormone for controlled release. As is not generally the case with alternative routes in controlledrelease drug formulation, there is no exposure at any stage of the process to harmful organic solvents, mechanical stresses, or raised temperature. This approach has broad scope and can be applied to the encapsulation of a variety of bioactive or therapeutic factors including nutrients, antioxidants, pharmaceuticals, preservatives, peptides, hormones, plasmids, growth factors, photodynamic therapy agents, sunscreens, or antimicrobial agents. Only limited solubility of the drug in CO2 is needed since it is an equilibrium process that leads to encapsulation. It is more important that the drug be compatible with the polymer used for encapsulation. Any of the wide range of polymer latex suspensions could be used. As an example, biodegradable or bioerodible scaffolds based on poly(R-hydroxyacids) impregnated via this procedure have potential as sutures, bone implants, and drug delivery reservoirs. New avenues of application can open up after the production of such materials. Indeed, dispersion of superparamagnetic nanoparticles within suitable hosts may lead to magnetically responsive vehicles for localized and targeted drug delivery,24 a consideration of special importance when delivering highly toxic drugs. These prospects and others are currently under exploration in our laboratories. Acknowledgment. G.A.B. acknowledges generous support from a Frederick Reines Fellowship. LA047146M (24) Chattopadhyay, P.; Gupta, R. B. Ind. Eng. Chem. Res. 2002, 41, 6049-6058.
