Langmuir 2008, 24, 83-90
Composite Block Copolymer Stabilized Nanoparticles: Simultaneous Encapsulation of Organic Actives and Inorganic Nanostructures Marian E. Gindy, Athanassios Z. Panagiotopoulos, and Robert K. Prud’homme* Department of Chemical Engineering, Princeton UniVersity, Princeton, New Jersey 08544 ReceiVed September 18, 2007 We describe the preparation and characterization of hybrid block copolymer nanoparticles (NPs) for use as multimodal carriers for drugs and imaging agents. Stable, water-soluble, biocompatible poly(ethylene glycol)-block-poly(caprolactone) NPs simultaneously co-encapsulating hydrophobic organic actives (β-carotene) and inorganic imaging nanostructures (Au) are prepared using the flash nanoprecipitation process in a multi-inlet vortex mixer. These composite nanoparticles (CNPs) are produced with tunable sizes between 75 nm and 275 nm, narrow particle size distributions, high encapsulation efficiencies, specified component compositions, and long-term stability. The process is tunable and flexible because it relies on the control of mixing and aggregation timescales. It is anticipated that the technique can be applied to a variety of hydrophobic active compounds, fluorescent dyes, and inorganic nanostructures, yielding CNPs for combined therapy and multimodal imaging applications.
Introduction Nanoparticles (NPs) have become increasingly important in the development of new materials for enhanced drug delivery and imaging applications, particularly in cancer research.1,2 Colloidal drug carriers such as liposomal,3 polymer vesicle,4 and micellar dispersions5,6 consisting of particles 50-400 nm in diameter have shown great promise in the formulation of highly insoluble anticancer therapeutics, permitting selective tumor targeting and more potent drug delivery. More recently, inorganic nanoparticles, including quantum dots,7 gold nanospheres,8 nanoshells,9 and superparamagnetic metals,10 have been explored for nanoparticle-based biomedical functions, such as tagging, medical imaging, sensing, and separation. Despite extensive innovation over the past decade, there remains a need for integrated, easily adaptable drug delivery and imaging modalities, especially those for the delivery and monitoring of highly toxic compounds in vivo. Polymer NPs in particular are a versatile medium for this purpose, due to their enhanced drug loading capacity, biological stability, and extended in vivo circulation.11 Initial research efforts have focused on the (1) Adams, M. L.; Lavasanifar, A.; Kwon, G. S. Amphiphilic block copolymers for drug delivery. J. Pharm. Sci. 2003, 92 (7), 1343-1355. (2) Portney, N. G.; Ozkan, M. Nano-oncology: drug delivery, imaging, and sensing. Anal. Bioanal. Chem. 2006, 384 (3), 620-630. (3) Kim, S. Liposomes As Carriers Of Cancer-Chemotherapy - Current Status And Future-Prospects. Drugs 1993, 46 (4), 618-638. (4) Discher, D. E.; Eisenberg, A. Polymer vesicles. Science 2002, 297 (5583), 967-973. (5) Allen, C.; Maysinger, D.; Eisenberg, A. Nano-engineering block copolymer aggregates for drug delivery. Colloids Surf., B-Biointerfaces 1999, 16 (1-4), 3-27. (6) Kwon, G. S. Diblock copolymer nanoparticles for drug delivery. Crit. ReV. Ther. Drug Carrier Syst. 1998, 15 (5), 481-512. (7) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005, 307 (5709), 538-544. (8) West, J. L.; Halas, N. J. Engineered nanomaterials for biophotonics applications: Improving sensing, imaging, and therapeutics. Annu. ReV. Biomed. Eng. 2003, 5, 285-292. (9) Loo, C.; Lin, A.; Hirsch, L.; Lee, M. H.; Barton, J.; Halas, N.; West, J.; Drezek, R. Nanoshell-enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treat. 2004, 3 (1), 33-40. (10) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. Magnetic nanoparticle design for medical diagnosis and therapy. J. Mater. Chem. 2004, 14 (14), 21612175. (11) Kwon, G. S.; Kataoka, K. Block-Copolymer Micelles As Long-Circulating Drug Vehicles. AdV. Drug DeliVery ReV. 1995, 16 (2-3), 295-309.
modification of polymer drug carriers with organic fluorescent dyes for particle visualization. Fluorescent NPs have been prepared through the reaction of water-soluble fluorophores to preformed nanoparticle surfaces12 or, more commonly, through the self-assembly of modified amphiphilic block copolymers, in which a fluorescent dye has been chemically tethered to the hydrophobic block terminus.13 Nonetheless, the selection of suitable organic dyes often limits the use of these fluorescent nanoparticles to in vitro applications, particularly for nanoparticle cellular uptake and localization studies.14 Alternatively, inorganic contrast agents consisting of a metallic core that defines the magnetic, optical, or fluorescence properties of the particle have recently proven more suitable for in vivo biomedical imaging applications such as magnetic resonance imaging and computed tomography X-ray.15,16 A variety of polymer coating strategies have been employed to impart biocompatibility and ensure stability of the generally hydrophobic nanostructure surfaces in aqueous environments.17-21 Researchers22-24 have additionally demonstrated the ability to (12) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L. Facile syntheses of surface-functionalized micelles and shell cross-linked nanoparticles. J. Polym. Sci., Part A: Polym. Chem. 2006, 44 (17), 5203-5217. (13) Luo, L. B.; Tam, J.; Maysinger, D.; Eisenberg, A. Cellular internalization of poly(ethylene oxide)-b-poly(-caprolactone) diblock copolymer micelles. Bioconjugate Chem. 2002, 13 (6), 1259-1265. (14) Savic, R.; Luo, L. B.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003, 300 (5619), 615-618. (15) Bulte, J. W. M.; Kraitchman, D. L. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 2004, 17 (7), 484-499. (16) Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M.; Smilowitz, H. M. Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol. 2006, 79 (939), 248253. (17) Azzam, T.; Eisenberg, A. Monolayer-protected gold nanoparticles by the self-assembly of micellar poly(ethylene oxide)-b-poly(-caprolactone) block copolymer. Langmuir 2007, 23 (4), 2126-2132. (18) Kim, B. S.; Taton, T. A. Multicomponent nanoparticles via self-assembly with cross-linked block copolymer surfactants. Langmuir 2007, 23 (4), 21982202. (19) Butterworth, M. D.; Illum, L.; Davis, S. S. Preparation, of ultrafine silicaand PEG-coated magnetite particles. Colloids Surf., A 2001, 179 (1), 93-102. (20) Gupta, A. K.; Gupta, M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26 (18), 39954021. (21) Soo, P. L.; Sidorov, S. N.; Mui, J.; Bronstein, L. M.; Vali, H.; Eisenberg, A.; Maysinger, D. Gold-labeled block copolymer micelles reveal gold aggregates at multiple subcellular sites. Langmuir 2007, 23 (9), 4830-4836. (22) Paciotti, G. F.; Kingston, D. G. I.; Tamarkin, L. Colloidal gold nanoparticles: A novel nanoparticle platform for developing multifunctional tumortargeted drug delivery vectors. Drug DeV. Res. 2006, 67 (1), 47-54.
10.1021/la702902b CCC: $40.75 © 2008 American Chemical Society Published on Web 11/29/2007
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Figure 1. Preparation of multicomponent NPs through block copolymer self-assembly via flash nanoprecipitation in a multi-inlet vortex mixer (MIVM). The co-encapsulation of organic soluble molecules and inorganic colloidal nanostructures is illustrated.
functionalize inorganic nanoparticle surfaces with receptorspecific peptides or protein ligands, allowing for targeted localization of the imaging particles. By extension, combined drug delivery and imaging has been realized through the covalent attachment of drug molecules to the surfaces of coated inorganic nanoparticles,25-27 although this strategy is generally restricted to water-soluble compounds. A more difficult problem has been the development of multifunctional polymer NPs that allow for the encapsulation of hydrophobic organic compounds and inorganic nanostructures within a water-soluble vehicle for combined drug delivery and detection.28,29 Encapsulation of drug compounds within NP cores has been shown to result in increased drug loading capacity, reduced toxicity, and enhanced protection of the drug molecule from the surrounding environment.6,30 Encapsulation may additionally allow for tailored drug release kinetics.6,30 Despite these advantages, only limited literature on the successful preparation of hybrid organic-inorganic NP formulations exists. For example, Gao and co-workers have described the preparation of composite poly(ethylene glycol)-block-poly(D,L-lactide) micelles encapsulating the chemotherapeutic doxorubicin and superparamagnetic iron oxide nanoparticles.31 More recently, the successful preparation of antibody conjugated poly(D,Llactide-co-glycolide) nanoparticles incorporating doxorubicin and magnetic iron nanocrystals was reported by Haam et al.32 In both works, enhanced cancer cell affinity and increased (23) Zhang, Y.; Kohler, N.; Zhang, M. Q. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 2002, 23 (7), 1553-1561. (24) Zhou, J. K.; Leuschner, C.; Kumar, C.; Hormes, J. F.; Soboyejo, W. O. Sub-cellular accumulation of magnetic nanoparticles in breast tumors and metastases. Biomaterials 2006, 27 (9), 2001-2008. (25) Paciotti, G. F.; Myer, L.; Weinreich, D.; Goia, D.; Pavel, N.; McLaughlin, R. E.; Tamarkin, L. Colloidal gold: A novel nanoparticle vector for tumor directed drug delivery. Drug DeliVery 2004, 11 (3), 169-183. (26) Yu, S.; Chow, G. M. Carboxyl group (-CO2H) functionalized ferrimagnetic iron oxide nanoparticles for potential bio-applications. J. Mater. Chem. 2004, 14 (18), 2781-2786. (27) Gupta, A. K.; Curtis, A. S. G. Lactoferrin and ceruloplasmin derivatized superparamagnetic iron oxide nanoparticles for targeting cell surface receptors. Biomaterials 2004, 25 (15), 3029-3040. (28) Liu, Y. Y.; Miyoshi, H.; Nakamura, M. Nanomedicine for drug delivery and imaging: A promising avenue for cancer therapy and diagnosis using targeted functional nanoparticles. Int. J. Cancer 2007, 120 (12), 2527-2537. (29) Fahmy, T. M.; Peter, F. M.; Park, J.; Constable, T.; Saltzman, W. M. Nanosystems for Simultaneous Imaging and Drug Delivery to T Cells. AAPS J. 2007, 9 (2), E171-E180. (30) Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E. Biodegradable polymeric nanoparticles as drug delivery devices. J. Controlled Release 2001, 70 (1-2), 1-20. (31) Nasongkla, N.; Bey, E.; Ren, J. M.; Ai, H.; Khemtong, C.; Guthi, J. S.; Chin, S. F.; Sherry, A. D.; Boothman, D. A.; Gao, J. M. Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems. Nano Lett. 2006, 6 (11), 2427-2430.
sensitivity to MRI were reported in vitro. Unfortunately, the preparative techniques employed, namely, solvent evaporation and emulsification processes, suffer from several disadvantages. First, they often require the use of stabilizing surfactants and numerous purification stages for the preparation of uniformly sized NPs.31,32 Additionally, the loading capacity of the hydrophobic components is generally limited by compound solubility within the NP cores.33 Last, these preparative processes do not allow for independent specification component loadings and furthermore do not ensure uniform distribution of actives within NP interiors. Flash nanoprecipitation is a process for production of NPs using rapid micromixing to effect high supersaturations and kinetically controlled aggregation of hydrophobic compounds using block copolymer self-assembly.34,35 The process of preparing multicomponent composite nanoparticles (CNPs) using a multi-inlet vortex mixer (MIVM) is illustrated in Figure 1. The molecularly dissolved organic solutes, inorganic nanostructures, and amphiphilic block copolymers are solubilized in a watermiscible organic solvent such as tetrahydrofuran, dimethyl sulfoxide, or ethanol. Intense mixing of the organic solvent stream with water in the MIVM induces supersaturations as high as 1000 in times on the order of milliseconds to initiate rapid precipitation of all hydrophobic components, including the hydrophobic block of the block copolymer.36 The process depends on tuning three time scales: (1) time to attain homogeneous mixing (τmix), (2) time for nucleation and growth of the hydrophobic actives (τng), and (3) time of block copolymer self-assembly (τsa). The detailed design and mixing characterization of the MIVM are reported elsewhere.36 A characteristic mixing time in the range of milliseconds is obtained for a Reynolds number (related to stream velocities) greater than 1600.36 The mixing time is shorter than the time scale for nucleation and growth of dissolved organic solutes (τng). By balancing the nucleation and growth times with the block copolymer assembly time, it is possible to block further particle (32) Yang, J.; Lee, C.-H.; Park, J.; Haam, S. Antibody conjugated magnetic PLGA nanoparticles for diagnosis and treatment of breast cancer. J. Mater. Chem. 2007 (Advance Article). (33) Shuai, X. T.; Ai, H.; Nasongkla, N.; Kim, S.; Gao, J. M. Micellar carriers based on block copolymers of poly(-caprolactone) and poly(ethylene glycol) for doxorubicin delivery. J. Controlled Release 2004, 98 (3), 415-426. (34) Johnson, B. K.; Prud’homme, R. K. Flash NanoPrecipitation of organic actives and block copolymers using a confined impinging jets mixer. Aust. J. Chem. 2003, 56 (10), 1021-1024. (35) Johnson, B. K.; Prud’homme, R. K. Mechanism for rapid self-assembly of block copolymer nanoparticles. Phys. ReV. Lett. 2003, 91 (11). (36) Liu, Y.; Cheng, C.; Lui, Y.; Prud’homme, R. K.; Fox, R. O. Mixing in a Multi-Inlet Vortex Mixer (MIVM) for Flash Nanoprecipitation. Chem. Eng. Res. Submitted, 2007.
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growth and control NP size. Polymer self-assembly that is too rapid consumes the stabilizer and results in uncontrolled growth, while nucleation and growth that are too rapid result in largerthan-desired particle sizes. The average NP size is thus controlled by the supersaturation levels and kinetics of aggregation of both the block copolymer and hydrophobic compounds. In the present work, we expand the utility of the flash nanoprecipitation process by preparing not only NPs of hydrophobic organic compounds but also of inorganic solid NPs. These multicomponent nanoparticles enable the development of new diagnostic nanostructures that include, for example, drug compounds, imaging agents, and a protective biocompatible polymer coating. Particles based on the self-assembly of poly(ethylene glycol)-block-poly(-caprolactone) (PEG-b-PCL) are prepared using a four-stream MIVM. In particular, we describe the synthesis and characterization of polymeric CNPs encapsulating colloidal gold (Au) as an illustrative imaging contrast agent and β-carotene as a model therapeutic. While colloidal Au NPs are employed in this work primarily as a proof of principle, their use in biomedical applications is well-established. Gold colloids are presently employed in immunodiagnostic37 and biosensing applications.8,38 They have additionally been utilized in studies of nanoparticle cellular uptake and localization,21,39,40 targeted drug delivery,22,25,41-43 and photothermal destruction of cancer cells.9,44,45 Most recently, colloidal Au NPs have been investigated as an alternative to iodine-based contrast agents for in vivo X-ray computed tomography imaging.16,46 The goal of the present work is to generate stable NPs with controllable particle size, narrow polydispersity, high loading capacity, and precise manipulation of encapsulated component composition for potential use in integrated drug delivery and imaging applications. The data presented are intended to demonstrate the ability to generate nanoparticles under a wide range of composition. Optimization of a particular nanoparticle formulation will depend on the specific requirements of the system under consideration. Experimental Section Materials. All materials were purchased from Aldrich and, unless otherwise noted, used as received. Water, purified by reverse osmosis, (37) Thanh, N. T. K.; Rosenzweig, Z. Development of an aggregation-based immunoassay for anti-protein A using gold nanoparticles. Anal. Chem. 2002, 74 (7), 1624-1628. (38) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature (London) 1996, 382 (6592), 607-609. (39) Huff, T. B.; Hansen, M. N.; Zhao, Y.; Cheng, J. X.; Wei, A. Controlling the cellular uptake of gold nanorods. Langmuir 2007, 23 (4), 1596-1599. (40) Shukla, R.; Bansal, V.; Chaudhary, M.; Basu, A.; Bhonde, R. R.; Sastry, M. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: A microscopic overview. Langmuir 2005, 21 (23), 1064410654. (41) Yang, Y. C.; Wang, C. H.; Hwu, Y. K.; Je, J. H. Synchrotron X-ray synthesis of colloidal gold particles for drug delivery. Mater. Chem. Phys. 2006, 100 (1), 72-76. (42) Bergen, J. M.; Von Recum, H. A.; Goodman, T. T.; Massey, A. P.; Pun, S. H. Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery. Macromol. Biosci. 2006, 6 (7), 506-516. (43) Yang, P. H.; Sun, X. S.; Chiu, J. F.; Sun, H. Z.; He, Q. Y. Transferrinmediated gold nanoparticle cellular uptake. Bioconjugate Chem. 2005, 16 (3), 494-496. (44) Hirsch, L. R.; Gobin, A. M.; Lowery, A. R.; Tam, F.; Drezek, R. A.; Halas, N. J.; West, J. L. Metal nanoshells. Ann. Biomed. Eng. 2006, 34 (1), 15-22. (45) Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Lett. 2005, 5 (4), 709-711. (46) Kattumuri, V.; Katti, K.; Bhaskaran, S.; Boote, E. J.; Casteel, S. W.; Fent, G. M.; Robertson, D. J.; Chandrasekhar, M.; Kannan, R.; Katti, K. V. Gum arabic as a phytochemical construct for the stabilization of gold nanoparticles: In vivo pharmacokinetics and X-ray-contrast-imaging studies. Small 2007, 3 (2), 333341.
Langmuir, Vol. 24, No. 1, 2008 85 ion exchange, and filtration (Milli-Q water) was used for NP preparation and dialysis. Synthesis of Poly(ethylene glycol-block-caprolactone) Block Copolymer. PEG-b-PCL block copolymers were synthesized by acid-catalyzed ring-opening polymerization of -caprolactone (PCL) using monomethoxy poly(ethylene glycol) (mPEG) as an initiator according to published procedure.47 Dichloromethane and PCL were distilled from calcium hydride under reduced pressure shortly before use. Hydrochloric acid in diethyl ether was used as received. mPEG (5000 g/mol) was dissolved in tetrahydrofuran (THF), precipitated into cold hexane, and dried under vacuum. The polymer was further dried by azeotropic distillation of toluene under reduced pressure. To a solution of mPEG in dichloromethane was added PCL. Polymerization was catalyzed by addition of hydrochloric acid solution, and the reaction was carried out at room temperature for 24 h. The copolymer was precipitated into cold hexane, filtered, and dried at room temperature under reduced pressure. Synthesis of Hydrophobic Gold NPs. Dodecanethiol modified gold NPs (C12-Au) were prepared by a two-phase reduction of hydrogen tetrachloroaurate (AuCl4-) in the presence of dodecanethiol according to the method of Brust.48 In brief, an aqueous solution of AuCl4- was mixed with a solution of tetraoctylammonium bromide in toluene. The mixture was vigorously stirred and the organic layer separated. Dodecanethiol was added to the organic phase and followed by the addition of aqueous sodium borohydride. The organic phase was separated and evaporated under vacuum. Gold NPs were precipitated into cold ethanol, filtered, and dried at room temperature under reduced pressure. Preparation of PEG-b-PCL Protected Gold NPs. A representative synthesis of block copolymer NPs incorporating preformed nanostructures prepared via flash nanoprecipitation is as follows. To a solution of PEG-b-PCL (5000-b-6000 g/mol) (55 mg) in THF (HPCL grade) (5 mL) was added dry C12-Au NPs (8.6 mg). The organic solution was fed (12 mL/min, stream 1), along with water (40 mL/min, streams 2-4), into a four-stream MIVM (Figure 1) using two digitally controlled syringe pumps (Harvard Apparatus, PHD 2000 programmable, Holliston, Massachusetts) to yield a final solvent composition of 1:10 v/v % THF/water. The concentrations of C12-Au and PEG-b-PCL in final NP solution were 0.016 wt % and 0.1 wt %, respectively. NPs were dialyzed against Milli-Q water using a Spectra/Por dialysis bag with MWCO of 6000-8000 (g/ mol) (Spectrum Laboratories Inc., California) and stored at room temperature. Characterization. Polymer molecular weights and polydispersity indices were measured by gel permeation chromatography (GPC) using a GPC unit (Waters Inc., Milford, MA) equipped with a series of Phenogel columns and a differential refractive index detector, calibrated with polystyrene standards (Polysciences Inc., Warrington, PA). High-resolution 1H NMR spectra were obtained using a Varian Inova 400 MHz spectrometer. Nanoparticle size and size distributions were characterized via dynamic light scattering (DLS) (Brookhaven Instruments, BI-200SM, Holtsville, NY), consisting of double-pumped continuous NdYAG laser (Coherent Inc., wavelength 532 nm, 100 mW, Santa Clara, CA), and a photomultiplier with detection angle of 90°. The signal of the photomultiplier was analyzed by autocorrelation (ALV-Laser Vertriebsgesellschaft mbH, ALV-5000/E, Langen, Germany), yielding the time-averaged scattered average particle size and polydispersity index (PDI). The particle size distribution was calculated using the ALV-5000/E software, from the decay-time distribution function with the assumption that the scattering particles behave as hard spheres.49 (47) Shibasaki, Y.; Sanada, H.; Yokoi, M.; Sanda, F.; Endo, T. Activated monomer cationic polymerization of lactones and the application to well-defined block copolymer synthesis with seven-membered cyclic carbonate. Macromolecules 2000, 33 (12), 4316-4320. (48) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. Synthesis Of Thiol-Derivatized Gold Nanoparticles In A 2-Phase Liquid-Liquid System. J. Chem. Soc.: Chem. Commun. 1994 (7), 801-802. (49) Bohren, G. F.; Huffman, D. R. Absorption and Scattering of Light by Small Particles; John Wiley and Sons, Inc.: New York, 1983.
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Figure 2. Hydrodynamic diameter, determined by DLS, for PEGb-PCL (5000-b-6000 g/mol) protected Au NPs as a function of Au loading (Au weight divided by Au and block copolymer weight) at fixed block copolymer concentration (0.1 wt % in final solution). Symbols are from experiments with line as guide to eye. The standard deviations (σ) of repeated experiments are represented by error bars. Figure inset demonstrates the cubic root dependence of PEG-b-PCL protected Au NP size (R) on Au volume fraction (φAu). NP size has been normalized by the size of unloaded PEG-b-PCL NPs (R0). UV-visible absorbance spectra of NPs were collected at room temperature using an Evolution 300 spectrometer (Thermo Electron Inc., Madison, Wisconsin) in the wavelength range 200-800 nm, with a resolution of 1 nm. Transmission electron microscopy (TEM) images were obtained on a JEOL 2010 TEM microscope (Tokyo, Japan) working under an acceleration voltage of 200 kV. For the analysis, a drop of NPs dispersed in water was deposited onto a carbon film supported by a copper grid and dried under reduced pressure. Observations were performed directly following grid preparation.
Results and Discussion Encapsulation of Colloidal Imaging Agents. Mean particle diameters of PEG-b-PCL protected Au particles prepared using the MIVM as a function of Au NP loading are presented in Figure 2. The error bars represent the standard deviation in measured diameters of several experimental runs generated at each condition. NPs were prepared at fixed block copolymer composition (0.1 wt % in the final solution) and Au loading is reported as solids weight percent (Au weight divided by Au and block copolymer weight). The mean size of unfilled polymer nanoparticles as prepared in the MIVM is 50 ( 2 nm. The term “unfilled” refers to nanoparticles prepared using only the block copolymer stabilizer and which do not encapsulate any Au colloids. The average NP diameter is shown to increase with increasing Au concentration, reaching a value of 103 ( 6 nm at a loading of 23 wt % Au. The inset of Figure 2 shows the NP radius, R, normalized by the unfilled micelle radius, R0, which scales with the gold colloid volume fraction (φAu) as R/R0 ∝ (1 - φAu)-1/3. As developed in a later section of this paper, the experimentally observed trend is predicted by a reaction model of colloid coagulation in the diffusion-limited regime.50 The corresponding particle size distributions, shown in Figure 3, remain narrow, with PDI values less than 0.25 ( 0.02 obtained in all cases. For reference, particle size distributions of polystyrene calibration standards of similar sizes (80 and 170 nm) are also shown in Figure 3, with measured PDI values of 0.11 ( 0.03 and 0.13 ( 0.03, respectively. Since no postsynthesis purification of NP solutions was performed, no material losses are associated with the particle (50) Fennell Evans, D.; Wennerstrom, H. The Colloidal Domain: Where Physics, Chemistry, Biology, and Technology meet. In AdVances in Interfacial Engineering Series, 2nd ed.; Wiley-VCH: Weinheim, 1999; pp 417-424.
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Figure 3. Representative particle size distributions, determined by DLS, for PEG-b-PCL protected Au NPs at the conditions of Figure 2. NP Au loading, calculated as Au weight percent in final solution, is indicated in legend. Particle size distributions of calibration grade polystyrene (PS) latex spheres (Ted Pella, Inc.) of sizes 81 ( 3 nm (solid line) and 170 ( 5 nm (dashed line) are shown for reference.
preparation process and high volumetric productivity is achieved. Typical precipitation processes operate at concentrations below 0.05 mg/mL18 of the block copolymer stabilizer and often require postprocessing purification for the removal of macroscopic aggregates, resulting in significant material losses and reduced colloid loadings, on average, less than 10 wt % with respect to the block copolymer.31 Using the MIVM, NPs with Au loadings of greater than 20 wt % have been prepared at block copolymer concentrations in the range of 1.0-4.0 mg/mL, demonstrating both enhanced NP loading capacity and improved volumetric productivity. A representative TEM micrograph of PEG-b-PCL protected Au NPs is shown in Figure 4. Contrast in the TEM image is provided only by the Au colloid, as the block copolymer is unstained. Individual Au monomers, approximately 5 nm in diameter, are clearly visible. For a representative Au loading of 23.3 wt %, nearly spherical particles with a mean diameter of 103 ( 6 nm, as determined by DLS, are produced. Particle size and size distributions, as determined by DLS, are in good agreement with TEM and SEM (see Supporting Information) observations. Co-Encapsulation of Organic Drug and Colloidal Imaging Agent. The flash nanoprecipitation technology is used to simultaneously load hydrophobic organic actives and inorganic colloids for integrated drug delivery and imaging applications. The vitamin A precursor β-carotene is selected as a model hydrophobic compound and encapsulated, in conjunction with Au colloid, within the cores of PEG-b-PCL NPs using the MIVM as described. Using ratios of β-carotene/Au/block copolymer of 30.5:5.0:64.5 wt % (fractional weight of β-carotene, Au, and block copolymer with respect to total solids mass), CNPs of approximately 80 nm in diameter were prepared. To confirm capture of components within the NP interiors, an as-prepared NP solution was filtered through a 10K Omega nanoseparation centrifuge filter membrane (Pall Corporation, East Hills, NY), which allows for the retention of CNPs on the membrane surface and permits passage of free β-carotene and unprotected Au colloidal particles. The encapsulation of Au monomers and β-carotene within CNPs is first examined visually as shown in Figure 5A. The CNP solution prior to filtration is deep red (vial 1), whereas the filtrate is clear (vial 2). Total recovery of unprotected Au colloid through the nanoseparation filter (vial 3) is confirmed independently via UV-visible absorbance measurements at 520 nm, where Au colloids of this size exhibit a maximum in the
Composite Block Copolymer Stabilized Nanoparticles
Figure 4. Representative TEM micrograph of PEG-b-PCL (5000b-6000 g/mol) protected Au NPs prepared at a loading of 23.3 wt % Au. The random, close-packing nature of Au monomers within the particle core is clearly evident in the TEM image.
Figure 5. Composite PEG-b-PCL (5000-b-6000 g/mol) NPs encapsulating Au (9.4 wt %) and β-carotene (30.2 wt %) prepared via flash nanoprecipitation. (A) CNP solution (1) before and (2) after filtration via 10 000 MW ultrafiltration membrane. Complete capture of Au and β-carotene is indicated by transparency of supernatant (2) when compared to a solution of nonprotected Au colloid suspended in THF filtered via the same membrane (3). (B) UV-visible absorbance spectra of nanoparticle solution before and following ultrafiltration. Complete capture of organic solute and inorganic colloid is again confirmed by absence of β-carotene and Au UV absorbance peaks in filtrate.
absorbance spectrum (see Supporting Information). Transfer of β-carotene through the filter is expected on the basis of the molecular weight of the molecule (536.9 g/mol). Corresponding UV-visible absorbance spectra of the CNP solution and filtrate are shown in Figure 5B. A distinct absorbance peak at 520 nm resulting from the presence of Au colloids is seen in the spectrum of the CNP solution, whereas this peak is essentially undetected in the solution following filtration. Quantification of Au concentration in the CNP and filtrate solutions is made on the basis of calibrated measurements of absorbance values at 520 nm. The UV-visible spectrum of β-carotene does not interfere with that of Au in the wavelength range 400-800 nm, and thus the absorbance value at 520 nm can be utilized to calculate Au concentration in the CNP
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Figure 6. Particle size distributions, determined by DLS, of PEGb-PCL (5000-b-6000 g/mol) NPs encapsulating Au (9.4 wt %) and β-carotene (30.2 wt %) in 155 mM NaCl initially (solid line) and following 28 day storage (dashed line) at room temperature. NP solutions are not purified prior to analysis. NP size increases from 83 ( 3 nm initially to 97 ( 4 nm after 28 days. Increase in NP size is attributed to Ostwald ripening of β-carotene over time.52
formulation. On the basis of this calibration, an Au encapsulation efficiency in excess of 99.5 wt % is estimated. See the Supporting Information for Au NP UV calibrations. The quantification of β-carotene CNP loading is complicated by the overlap in absorption spectra of the two components in the UV region, where solutions of β-carotene exhibit an absorbance maximum at 290 nm. As such, we have alternatively prepared PEG-b-PCL NPs in which β-carotene is independently encapsulated. NPs are isolated as previously described, and the concentration of free β-carotene in the filtrate measured. On the basis of UV calibration at 290 nm, a β-carotene concentration of approximately 0.05 mg/mL is estimated. This concentration corresponds to the solubility limit of β-carotene in the final solvent composition of 1:10 v/v % THF/H2O (see Supporting Information for NP preparation conditions, β-carotene UV calibration, and solubility data). Thus, all β-carotene in excess of the solubility limit is incorporated within the NPs. Because NP loading relies on compound solubility, the encapsulation efficiency of organic molecules will remain unaffected in multiple-component formulations. The ability of this technology to provide quantitative homogeneous incorporation of actives arises from the very high level of supersaturation of all components, leading to spinodal decomposition and diffusion-limited aggregation.51 The significant advantage of the process is that component loadings can be accurately specified a priori, in contrast to slow, quasi-equilibrium formation processes which lead to unequal incorporation of individual components depending on their solubilities. In our process, drug and imaging agent loadings can be optimized independently and subsequently formulated into a single multifunctional delivery vehicle. The extended stability of these particles in the presence of physiological salt concentrations was also investigated. Figure 6 shows particle size distributions of PEG-b-PCL protected β-carotene/Au CNPs immediately after preparation and dialysis and after 1 month of storage in 155 mM saline at room temperature. The mean particle diameter and size distribution of the unfiltered solutions increases to a minor extent from approximately 85 to 100 nm over this time, indicating particle stability in aqueous environments for extended time periods. (51) Brick, M. C.; Palmer, H. J.; Whitesides, T. H. Formation of colloidal dispersions of organic materials in aqueous media by solvent shifting. Langmuir 2003, 19 (16), 6367-6380.
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Figure 7. Hydrodynamic diameter of PEG-b-PCL protected Au NPs, determined by DLS, as a function of PCL (3200 g/mol) volume fraction. NP size is shown to be a nearly linear function of homopolymer volume fraction (φPCL) for φPCL values above approximately 33%. The nearly constant particle size for φPCL less than 33% is attributed to filling of the interstitial voids of the NP cores, created through the random packing of Au colloid (estimated to be approximately 37%54), by homopolymer PCL. PCL addition beyond this point contributes to increasing particle diameters. Particle size and agent content can thus be selectively and precisely controlled a priori.
The slight increase in particle diameter arises from Ostwald ripening inherent in all nanometer scale particles.52 Particle Size Control. NPs in the size range 100-300 nm are specifically of interest, as they have been exploited for passive delivery of anti-cancer agents to solid tumor vasculature, where defective vascular architecture and impaired lymphatic drainage allow for improved particle uptake and localization through the enhanced permeation and retention (EPR) effect.53 We demonstrate the ability to control the size of CNPs within the above specified range in a predictable fashion in Figure 7. PEG-b-PCL protected Au CNPs in which the particle size was “tuned” through the addition of an inert component, homopolymer PCL (3200 g/mol) were prepared. For fixed colloid concentration (0.016 wt % in solution), CNP size in the range 75-275 nm is shown to be a nearly linear function of homopolymer volume fraction (φPCL) for PCL loadings above 33 vol %. The relatively constant NP size with PCL addition for volume fractions below this value is speculated to result from initial filling of the interstitial voids created by the random packing of Au monomers in the NP core, estimated at approximately 37 vol %.54 The dense, random nature of monomer packing is supported by TEM imaging as shown in Figure 4. PCL addition beyond this point quantitatively contributes to increasing particle diameters. NP size and active loading can thus be specified independently of one another, yielding a highly flexible NP formation platform. Prediction of Encapsulated Colloid Number and NP Size. In Figure 2, the size of polymer-protected Au NPs was shown to be a function of colloid concentration. In this section, we illustrate that this behavior is predicted well using a simple representation of colloid self-assembly in the diffusion-limited regime, as outlined by Evans and Wennerstrom.50 In this model, a system of spherical particles each of uniform radius R undergoing Brownian motion is considered. The spheres are assumed to interact according to a square well potential of infinite energy with an interaction distance of 2R. At steady state and in the diffusion-limited regime, the rate constant for colloid (52) Liu, Y.; Kathan, K.; Saad, W.; Prud’homme, R. K. Ostwald ripening of beta-carotene nanoparticles. Phys. ReV. Lett. 2007, 98 (3). (53) Duncan, R.; Sat, Y. N. Tumour targeting by enhanced permeability and retention (EPR) effect. Ann. Oncol. 1998, 9, 39-39. (54) Torquato, S.; Truskett, T. M.; Debenedetti, P. G. Is random close packing of spheres well defined? Phys. ReV. Lett. 2000, 84 (10), 2064-2067.
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Figure 8. Plot of aggregate distribution N*[PN]/[P0] as a function of the number of monomers N in aggregate [PN], normalized by the initial number of monomers in solution [P0]. Distributions are calculated for varying Au concentrations (Au weight percent in solution) as indicated in legend and an estimated block copolymer self-assembly time of 40 ms. Au concentrations in this figure are equal to Au concentrations as indicated in Figure 2.
association is shown to be independent of aggregate size and can be used universally to ascertain the kinetics of aggregation, yielding the following general solution to the aggregation process:
[PN] ) [P]tot 0
(τt ) (1 + τt ) N-1
where [PN] is the concentration of particles each composed of N monomers, [P] tot 0 is the monomer concentration at t ) 0, and τ ) 2/(k[P] tot ), where k is the universal rate constant given by 0
8 kBT 3 µ
for which, kB is the Boltzmann constant, T is the solution temperature, and µ is the solvent viscosity. In the MIVM, rapid micromixing in the range of milliseconds is attained, yielding a homogeneous system in which colloid aggregation and block copolymer precipitation occur in the diffusion-limited regime. In this manner, colloid aggregation persists until block copolymer deposition on the assembly surface limits further coagulation. Thus, the time allowed for colloid assembly will be dictated by the sum of the characteristic mixing time in the MIVM and the block copolymer induction time. In the case of PEG-b-PCL, the copolymer self-assembly time is estimated on the basis of a value for comparable molecular weight poly(ethylene glycol)-b-polystyrene (1000-b-3000 g/mol) block copolymers as reported in the literature, where the induction time is approximated to be 37 ms.34 Accounting for an estimated mixing time of 3 ms36 in the MIVM, snapshots of the particle size distributions at a time of 40 ms are calculated using eq 1 for varying colloid concentrations (wt % in solution). The results of these calculations are shown in Figure 8. For a given initial monomer concentration [P0], the final distribution of aggregates, each composed of N monomers, at an assembly time of 40 ms is calculated. The normalized fraction of monomers participating in an aggregate, N * [PN]/[P0], is shown to reach a maximum for each colloid concentration studied, with a shift toward larger maximum values as the colloid concentration is increased. The model additionally predicts a corresponding increase in cluster distribution dispersity as the colloid loading is increased. This trend is supported experimentally, as evidenced by the slightly increasing PDI values of PEG-b-PCL protected Au NPs with increasing Au content shown in Figure 3. Model predictions of cluster sizes calculated at t ) 40 ms are compared to particle diameter values, as determined by DLS, for
Composite Block Copolymer Stabilized Nanoparticles
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Figure 9. UV-visible absorbance spectra of dodecane capped Au (C12-Au) NPs dispersed in THF (solid line) and PEG-b-PCL protected C12-Au NPs dispersed in 1:10 v/v THF/water (dashed line). The maximum in absorbance spectra (at ∼520 nm), indicative of position of Au SPR, remains unaltered in the block copolymer protected sample, indicating no overlap in Au electronic structure in the assembled NPs.
PEG-b-PCL protected Au NPs prepared in the MIVM. For particles in the Rayleigh scattering range, the intensity of scattered light is proportional to the sixth power of the size for each particle.49 This leads to the following expression for particle radius as obtained by DLS measurements: max ,∞
R h ≡ R6-5 )
where nN is the number of particles of a given radius RN. For a given colloid concentration, particle size can be calculated analytically using eq 3 in conjunction with the particle size distributions, [PN], calculated from eq 1. Calculation of NP core volume is made assuming each C12-Au monomer occupies a radius of 4 nm (2.5 nm for Au core and 1.5 nm for C12 extended chain length55). Additionally, packing of the monomers within the NP core is assumed to be close-packed and random in nature, occupying a volume fraction of 0.63.54 To account for the PEGb-PCL stabilizing copolymer, the diameter of unloaded PEGb-PCL NPs prepared using the MIVM (50 nm) is added to the cluster diameters computed through the model. Calculated cluster diameters as a function of colloid concentration are reported in Table 1 (column 2). The standard deviation in particle size calculated from simulations at each Au concentration is reported as the uncertainties. When compared to experimentally determined particle diameters, as obtained by DLS (column 3), the results show that particle size is predicted well using this simple model of colloid aggregation. The model also allows for the prediction of colloid number density within the NP core. The intensity-averaged particle aggregation number, N6-5, is similarly calculated according to eq 3 and the results shown in Table 1 (column 4). The average aggregation number increases with increasing colloid loading, reaching 126 (σ ) 5) for the highest Au concentration investigated. The estimated colloid loading density for this Au composition is well-supported by TEM imaging for a similarly prepared sample as shown in (55) Terrill, R. H.; Postlethwaite, T. A.; Chen, C. H.; Poon, C. D.; Terzis, A.; Chen, A. D.; Hutchison, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.; Superfine, R.; Falvo, M.; Johnson, C. S.; Samulski, E. T.; Murray, R. W. Monolayers in three dimensions: NMR, SAXS, thermal, and electron hopping studies of alkanethiol stabilized gold clusters. J. Am. Chem. Soc. 1995, 117 (50), 12537-12548.
Figure 4. This characterization thus allows for accurate a priori determination of particle size and colloid loading based solely on process inputs, permitting incorporation of multiple inorganic colloidal components at independently specified concentrations. Properties of Self-Assembled NPs. Physical properties of colloidal particles are expected to be preserved upon incorporation within the NP cores. The case of gold colloid encapsulation shown here is particularly interesting owing to the electronic behavior of nanometer-sized gold crystals. Gold NPs exhibit localized collective oscillation of surface conduction electrons, leading to distinctive surface plasmon resonance (SPR) peaks in the UV-visible region.56 The SPR frequency of a particular sample of gold colloid is shown to depend strongly on the size, shape, dielectric properties, and aggregation state of the NPs.56 Encapsulation of Au particles within a block copolymer shell using the MIVM is shown to preserve the metallic properties of isolated Au NPs. When Au particles are in close proximity, they are able to interact electromagnetically, primarily through a dipole-dipole coupling mechanism. This mechanism broadens and red-shifts the plasmon resonance bands.56 Figure 9 shows the recorded absorbance spectra for dispersions of C12-Au in THF and PEG-b-PCL protected C12-Au NPs in a THF/water mixture (1:10 v/v %). The peak in the extinction spectra, lying at approximately 520 nm, remains unaltered in the NP assembly, suggesting that no overlap in the electronic structure of neighboring Au particles has occurred. The dodecane capping layer not only dictates the properties surrounding the gold NP (medium dielectric constant and refractive index), but its thickness, estimated between 1 and 2 nm,55 controls the separation distance between neighboring Au monomers, maintaining the particles in an electronically independent state. The interparticle separation distance, and thus electronic properties of the aggregate, can be precisely controlled through selection of an appropriate capping ligand. We have utilized this capacity for control over interparticle distance to generate composite fluorophore-gold assemblies, in which enhanced fluorescence from the organic dye in the NP assembly is observed. This system is expected to provide a photostable imaging platform with the capacity for particle size control, multimodal imaging, and reduced toxicity effects. The details of this work will be the subject of a subsequent paper. Finally, we want to place the process of flash nanoprecipitation we have used here in context with other block copolymer-based nanoparticle formation processes described previously. There are fundamental thermodynamic constraints which limit the ability of processes used by previous researchers31,32 to produce multifunctional NPs at high loadings and with controlled particle size. Those limitations can be summarized in Figure 10, which shows the precipitation concentrations, or solubility boundaries, for two components as a function of antisolvent addition. Previous researchers have slowly added antisolvent to initially soluble solutions of block copolymers and imaging agents or drugs.57,58 Figure 10 displays the solubility of the organic active β-carotene and the solubility (critical micelle concentration) of a block copolymer stabilizer (poly(ethylene glycol)-block-polystyrene) as a function of THF content at 35 °C.59 While the solubility data shown in Figure 10 are specific to PEG-b-PS stabilized β-carotene nanoparticles, as detailed in previous work,34 the operating line (56) Link, S.; El-Sayed, M. A. Shape and size dependence of radiative, nonradiative and photothermal properties of gold nanocrystals. Int. ReV. Phys. Chem. 2000, 19 (3), 409-453. (57) Allen, C.; Han, J. N.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. Polycaprolactone-b-poly(ethylene oxide) copolymer micelles as a delivery vehicle for dihydrotestosterone. J. Controlled Release 2000, 63 (3), 275-286. (58) Kim, S. Y.; Lee, Y. M. Taxol-loaded block copolymer nanospheres composed of methoxy poly(ethylene glycol) and poly(-caprolactone) as novel anticancer drug carriers. Biomaterials 2001, 22 (13), 1697-1704.
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Table 1. Calculated vs Experimental Size of Block Copolymer Protected Au NPs Prepared via Flash Nanoprecipitationa
calculated average diameter (D6-5) of PEG-b-PCL protected Au NPs with σ in value reported as uncertainty
experimental diameter of PEG-b-PCL protected Au NPs as determined by DLS
calculated average number of Au monomers (N6-5) in PEG-b-PCL protected Au NPs with σ in value reported as uncertainty
0.004 wt % 0.008 wt % 0.016 wt % 0.031 wt %
81 ( 3 nm 89 ( 3 nm 98 ( 3 nm 105 ( 2 nm
69 ( 5 nm 73 ( 6 nm 92 ( 5 nm 103 ( 6 nm
38 ( 2 70 ( 3 109 ( 5 126 ( 5
a Au concentration in first column is reported as weight fraction of Au in solution. The standard deviations (σ) in calculated and experimentally determined values, rounded to the nearest integer, are reported as error.
Conclusions and Summary This work demonstrates the utility of flash nanoprecipitation using a multi-inlet vortex mixer (MIVM) for the preparation of multicomponent polymer NPs for integrated drug delivery and imaging applications. NPs composed of PEG-b-PCL copolymers encapsulating spherical gold colloids are prepared for possible application as X-ray contrast agents. The encapsulation of colloidal imaging agents within biocompatible polymer NPs is expected to permit prolonged in vivo circulation, potentially allowing for increased tumor accumulation, enhanced imaging capacity, longer imaging times, and increased solid tumor localization through the EPR effect. Figure 10. Solubility of β-carotene and a block copolymer stabilizer as a function of THF concentration at 35 °C.59 The straight line represents mixing operating line for dilution of a solution initially with 2.6 wt % β-carotene and 2.6 wt % block copolymer (designated A) with the addition of water. The rate of change in the average solvent content is based on the mixing time for the system. Under a slow mixing condition, β-carotene precipitates (designated B), prior to the block copolymer (designated C), leading to unprotected β-carotene particle growth. In the case of fast mixing, the final solvent content is rapidly established (designated D) and the competitive rate at which β-carotene nucleates and grows versus the rate at which the amphiphilic colloidal stabilizer precipitates and stabilizes the growing particles dictates the final particle size distribution.
shown can be generally applied to describe the flash nanoprecipitation process. The method of slow antisolvent addition involves traversing the operating line from the initially pure solvent condition (designated A) in which all components are soluble, to the intersection with the solubility curve for β-carotene (designated B) at 2.5 wt % water in THF. At this point, β-carotene will begin precipitating. The stabilizing polymer does not start aggregating on the particle surface until the water concentration reaches 23 wt % (designated C). However, at this point over 70% of the β-carotene has precipitated as unprotected crystals. In the case of fast mixing, as achieved by the flash nanoprecipitation process,60 such high levels of supersaturation are produced in milliseconds that at the final solvent composition (designated D) all species aggregate by a diffusion-limited, nonspecific process. The composition of the resulting nanoparticles reflects the stoichiometry of the feeds, and no unincorporated material is produced. In this manner, block copolymer NPs at high drug loadings (2.6 wt % PEG-b-PS to 2.6 wt % β-carotene for the case shown) are easily prepared.34
We have demonstrated the ability to incorporate hydrophobic organic compounds and inorganic colloids, quantitatively, into compound nanoparticles. The NP sizes can be precisely tuned from 75 to 275 nm by varying the ratio of colloid and organic additive. As discussed by previous researchers,29,31,32 hybrid nanoparticles such as the composite PEG-b-PCL stabilized β-carotene/gold NPs described in this work may provide new capabilities for applications in medical treatment. A single hybrid delivery vehicle can potentially allow for independent optimization of each encapsulated component and ultimately provide a platform for rational drug dosing strategies based on quantification of local drug concentrations in vivo. Furthermore, established surface hydrophobization techniques should permit a variety of inorganic-based imaging agents, including iron oxide, quantum dots, gold nanorods, and nanoshells to be incorporated within NP or CNP cores, yielding NPs for integrated therapy and multimodal imaging applications. CNP formulations could additionally address the treatment of cancers through photodynamic therapy in which the photosensitizer drug and an inorganic light source are co-administered as a single nanoparticle formulation.61 Acknowledgment. Financial support for this work was provided by the National Science Foundation through a Graduate Research Fellowship to M.G. and by the NSF NIRT center on Nanoparticle Formation. M.G. additionally acknowledges financial support from Merck and Company through a Doctoral Research Fellowship. Supporting Information Available: Additional experimental information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. LA702902B
(59) Johnson, B. K. Flash nanoprecipitation of organic actives via confined micromixing and block copolymer stabilization. Ph.D. Thesis, Princeton University, Princeton, 2003. (60) Johnson, B. K.; Prud’homme, R. K. Chemical processing and micromixing in confined impinging jets. AIChE J. 2003, 49 (9), 2264-2282.
(61) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. Photodynamic therapy. J. Natl. Cancer Inst. 1998, 90 (12), 889-905.