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Nontoxic Block Copolymer Nanospheres: Design and Characterization Corinne Nardin, Durgadas Bolikal, and Joachim Kohn* Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, and The New Jersey Center for Biomaterials, Piscataway, New Jersey 08854 Received April 17, 2004. In Final Form: August 22, 2004 Biodegradable polymers capable of self-assembly into hollow nanospheres of less than 100 nm have significant potential for biotechnology applications such as drug delivery and gene therapy. Here we describe the synthesis of a novel ABA-type triblock copolymer made from a hydrophobic tyrosine-derived core and two hydrophilic poly(ethylene glycol) end groups (poly(ethylene glycol)-block-oligo(desaminotyrosyltyrosine octyl ester suberate)-block-poly(ethylene glycol)). We describe the self-assembly of this triblock copolymer and characterize its particles as 100 nm size vesicular nanospheres. The vesicular nature of these particles was determined by light scattering and electron microscopy. The nanospheres did not exhibit any short-term cytotoxicity toward UMR-106 cells at a concentration up to 2 mg/mL.
Introduction There have been many studies to develop efficient systems for the delivery of therapeutics using carrier systems. These systems can help by providing a concentration of drugs in an aqueous milieu above the solubility limit of the free drug, by increasing its stability, and by targeting delivery to the required cells, thus lowering the required dosage.1,2 Self-assembling block copolymers are being actively explored as drug carriers.3-5 Similarly to low molecular weight lipid or surfactant molecules, amphiphilic block copolymers consist of at least one hydrophilic and one hydrophobic domain. Driven by their hydrophobicity, amphiphilic block copolymers self-assemble into particles in aqueous solution. At high concentrations, these particles build lamellar liquid crystalline phases, whereas in more dilute aqueous solutions they form superstructures of various shapes that include micelles and vesicles.1,6-13 Compared to the self-assembled superstructures formed by lower molecular weight amphiphilic molecules (such * To whom correspondence should be addressed at Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854. Phone: (732) 445-3888. E-mail: joachim@ rutchem.rutgers.edu. (1) Kakizawa, Y.; Kataoka, K. Adv. Drug Deliv. Rev. 2002, 54, 203222. (2) Emerich, D. F.; Thanos, C. G. Expert Opin. Biol. Ther. 2003, 3, 655-663. (3) Kabanov, A. V.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A. Polym. Mater. Sci. Eng. 2000, 82, 303-304. (4) Foerster, S.; Antonietti, M. Adv. Mater. (Weinheim, Ger.) 1998, 10, 195-217. (5) Nardin, C.; Meier, W. Chimia 2001, 55, 142-146. (6) Discher, B. M.; Bermudez, H.; Hammer, D. A.; Discher, D. E.; Won, Y.-Y.; Bates, F. S. J. Phys. Chem. B 2002, 106, 2848-2854. (7) Huang, H.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1999, 121, 3805-3806. (8) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. J. Am. Chem. Soc. 1998, 120, 9941-9942. (9) Luo, L.; Eisenberg, A. Angew. Chem., Int. Ed. 2002, 41, 10011004. (10) Rheingans, O.; Hugenberg, N.; Harris, J. R.; Fischer, K.; Maskos, M. Macromolecules 2000, 33, 4780-4790. (11) Shen, H.; Eisenberg, A. Angew. Chem., Int. Ed. 2000, 39, 33103312. (12) Stewart, S.; Liu, G. Chem. Mater. 1999, 11, 1048-1054. (13) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035-1041.
as lipid or surfactant), amphipathic block copolymers self-assemble into significantly more stable superstructures.14-16 It is this increased stability, along with their nontoxic, biodegradable, vesicular, and nanometersized structure that makes self-assembled amphipathic block copolymers so attractive for applications as drug delivery vectors.17-20 Encapsulation provides protection of the active molecules, while self-assembly ensures ease of preparation. With these characteristics in mind, we have designed a new ABA triblock copolymer, poly(ethylene glycol)-blockoligo(desaminotyrosyltyrosine octyl ester suberate)-blockpoly(ethylene glycol) (abbreviated as PEG-oligo(DTO suberate)-PEG). The PEG end blocks are well characterized and known to be noncytotoxic and biodegradable,21 and the protein-repellant character of this block makes it attractive for in vivo applications.22,23 The choice of the oligo(DTO suberate) for the middle block was based on its glass transition temperature Tg (294 K), which should ensure that each triblock copolymer will be sufficiently flexible to self-assemble into a dynamic and soluble structure under physiological conditions. In addition, this middle block is degradable under physiological conditions.24,25 The self-assembly of amphiphilic molecules depends on several correlated properties of the underlying material, i.e., its chemical structure, architecture, molecular weight, (14) Nardin, C.; Winterhalter, M.; Meier, W. Langmuir 2000, 16, 7708-7712. (15) Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143-1146. (16) Aranda-Espinoza, H.; Bermudez, H.; Bates, F. S.; Discher, D. E. Phys. Rev. Lett. 2001, 87, 208301/1-208301/4. (17) Delie, F.; Berton, M.; Allemann, E.; Gurny, R. Int. J. Pharm. 2001, 214, 25-30. (18) Oupicky, D.; Carlisle, R. C.; Seymour, L. W. Gene Ther. 2001, 8, 713-724. (19) Raghavachari, N.; Fahl, W. E. J. Pharm. Sci. 2002, 91, 615622. (20) Ding, J.; Liu, G. J. Phys. Chem. B 1998, 102, 6107-6113. (21) Greenwald, R. B.; Choe, Y. H.; McGuire, J.; Conover, C. D. Adv. Drug Delivery Rev. 2003, 55, 217-250. (22) Soppimath, K. S.; Aminabhavi, T. M.; Kakarni, A. R.; Rudzinski, W. E. J. Controlled Release 2001, 70, 1-20. (23) Vila, A.; Sanchez, A.; Perez, C.; Alonso, M. J. Polym. Adv. Technol. 2002, 13, 851-858.
10.1021/la0490285 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/16/2004
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and its hydrophobic-to-hydrophilic balance.11,26-28 In particular, low molecular weight amphiphilic ABA triblock copolymers (ca. 10 000 g mol-1) with a hydrophobic-tohydrophilic ratio of about 1.5 have been shown to selfassemble into 200 nm diameter vesicular structures.29 These values were used to design this novel PEG-oligo(DTO suberate)-PEG triblock copolymer. In this study, we describe the synthesis of PEG-oligo(DTO-suberate)-PEG and demonstrate its self-assembly into hollow, nontoxic nanospheres with diameters of approximately 100 nm. Materials and Methods Chemicals. Desaminotyrosyltyrosine octyl ester (DTO) was prepared as previously described.30,31 Methylene chloride (HPLC grade), 2-propanol, and methanol were obtained from Fisher Scientific (Pittsburgh, PA), suberic acid, 4-(dimethylamino)pyridine, 4-toluenesulfonic acid, and poly(ethylene glycol) monomethyl ether (Mw ) 2000 g mol-1) were from Aldrich Chemical Co. (Milwaukee, WI), and diisopropylcarbodiimide (DIPC) was from Tanabe Chemicals (San Diego, CA); chemicals were used without further purification. Synthesis. PEG-oligo(DTO suberate)-PEG was synthesized as follows: In a 100 mL round-bottomed flask were combined 2.21 g (0.005 mol) of DTO, 0.96 g (0.0055 mol) of suberic acid, 0.59 g (0.002 mol) of 4-dimethylaminopyridinium-p-toluene sulfate, and 25 mL of methylene chloride at 293 K. With continuous stirring at room temperature, 1.8 g (0.014 mol) of DIPC was added to the suspension, and at regular intervals, aliquots were withdrawn for gel permeation chromatography (GPC). When the Mn and Mw of the reaction mixture reached approximately 7000 and 15 000, respectively (relative to polystyrene standards), 1.1 g of poly(ethylene glycol) monomethyl ether (Mw ) 2000 g mol-1) and 0.4 g of DIPC were added. Following 2 h of additional stirring, the reaction mixture was filtered through a sintered glass funnel; the filtrate was evaporated to 10 mL and then precipitated with 2-propanol. This precipitate was dried, dissolved in 10 mL of methylene chloride, and reprecipitated with 50 mL of methanol. The product was isolated by centrifugation, washed with 20 mL of methanol, and dried under vacuum at room temperature. The resulting PEG-oligo(DTO suberate)-PEG was characterized by elemental analysis, GPC (Mw and Mn), and 1H NMR (CDCl3, 400 MHz): 6.98-7.20 (Ar-H), 5.98 (d, NH), 4.86 (d, CH of tyrosine), 4.08 (m, OCH2 of DTO), 3.65 (CH2CH2 of PEG), 3.38 (s, OCH3 of PEG). Preparation of Vesicles. Vesicle self-assembly was performed as previously described.5,6,9,13,14,25 Briefly, 10 mg of PEGoligo(DTO suberate)-PEG, dissolved in 0.2 g of THF, was added dropwise to 4.79 g of water (18 MΩ cm-1) under mild agitation. To achieve uniform particle size, the resulting turbid dispersion was sequentially filtered through 0.45, 0.22, and 0.1 µm size syringe filters (Millipore). All subsequent characterizations were performed using this final filtered preparation. Trace organic solvent contamination was removed by either gentle nitrogen blow-drying or size exclusion chromatography with Sephacryl S-400 HR (Bio-Rad, Piscataway, NJ). Static Light Scattering. Static light scattering (SLS) was performed at 303 K using a multiangle light scattering instrument (Wyatt Technology Corp., Santa Barbara, CA) equipped with a GaAs laser (wavelength λ ) 690 nm). Prior to injection, samples were cleared by filtration through a 0.1 µm Millipore syringe (24) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 1076110768. (25) Bourke, S. L.; Kohn, J. Adv. Drug Delivery Rev. 2003, 55, 447466. (26) Supramolecular Polymers; Ciferri, A., Ed; Marcel Dekker, Inc.: New York, 2000. (27) Luo, L.; Eisenberg, A. J. Am. Chem. Soc. 2001, 123, 1012-1013. (28) Svensson, M.; Alexandridis, P.; Linse, P. Macromolecules 1999, 32, 637-645. (29) Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Chem. Commun. (Cambridge, U.K.) 2000, 1433-1434. (30) Hooper, K. A.; Kohn, J. J. Bioact. Compat. Polym. 1995, 10, 327-340. (31) Kohn, J. MRS Bull. 1996, 21, 18-19.
Nardin et al. filter. The refractive index increment was obtained at the corresponding wavelength and temperature by using a commercial Waters 410 differential refractometer to measure the refractive index of the vesicle’s dispersion at different concentrations. Given the unexpectedly high value of the refractive index increment of the nanosphere solution, 1.23 mL g-1, this measurement was confirmed using an ALV-DR1 refractometer (ALV, Langen, Germany). SLS measurements at several angles and concentrations were extrapolated in a Zimm plot, and self-assembly properties, including average molecular weight, radius of gyration, and second virial coefficient, were calculated.32 The critical aggregation concentration (cac) was calculated using the close association model restrictions.33 Dynamic Light Scattering. A submicron particle analyzer (PSS Nicomp, Particle Sizing Systems, Santa Barbara, CA) was used to calculate the photon intensity autocorrelation function.34 The samples were clarified by filtering the solutions through 0.1 µm Millipore syringe filters into 6 × 50 mm borosilicate round cells (Kimble). Dynamic light scattering (DLS) was performed at 303 K. DLS results were subjected to Cumulant analysis,34 and (assuming small spherical, noninteracting particles) the hydrodynamic radius Rh was calculated using the Stokes-Einstein equation.34 Electron Microscopy. A drop of the vesicle preparation was frozen rapidly in liquid propane that had been chilled previously to 123 K with liquid nitrogen. For freeze-fracture electron microscopy, frozen samples were freeze-fractured to shape platinum/carbon replicas (High Vacuum Freeze-Etch Unit, Balzers Union Limited, FL-9496, Principality of Lichtenstein). The resulting replicas were collected on 200-mesh copper grids and analyzed by transmission electron microscopy using a JEM100CXII electron microscope (JEOL Ltd., Tokyo, Japan) operating at 80 kV. For cryotransmission electron microscopy, frozen samples were deposited on carbon-coated copper grids and examined using a Philips CM 20-FEG microscope operating at 200 kV. Cytotoxicity Assay. UMR-106 cells (ATCC, Manassas, VA) were seeded into 24-well plates at a density of 50 000 cells per well and propagated in DMEM (containing 100 units mL-1 penicillin, 100 µg mL-1 streptomycin, and 10% FBS) at 37 °C under 95% air/5% CO2. Following an initial 24 h incubation period, the culture medium was discarded and cells were exposed to 400 µL of serum-free DMEM containing polymeric vesicles prepared in phosphate-buffered saline ranging in concentration from 0.1 to 2 mg mL-1. After incubation for an additional 4 h at 37 °C cytotoxic effects were quantitated as a measure of reduced metabolic activity using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as described by the manufacturer. Absorbance at 490 nm was determined using a PowerWave X absorbance plate reader (Bio-Tek Instruments, Inc., Highland Park, VT).
Results and Discussion The synthesis of PEG-oligo(DTO suberate)-PEG is presented in Scheme 1. The hydrophobic middle block, oligo(DTO suberate), was prepared by reacting DTO with a slight excess of suberic acid, driving the reaction to self-terminate at a low molecular weight, resulting in an oligomer terminated with carboxylic acid end groups. In the following reaction step, the carboxylic acid end groups of oligo(DTO suberate) were reacted with methoxy-terminated PEG (molecular weight 2000) to produce the desired PEG-oligo(DTO suberate)-PEG triblock copolymer, which after purification was obtained as a white powder. Surprisingly, both the condensation polymerization and chain-coupling reactions can be performed in one vessel at room temperature. The (32) Ross-Murphy, S. B.; Shatweel, K. P. Biorheology 1993, 30, 217227. (33) Schaedler, V.; Nardin, C.; Wiesner, U.; Mendes, E. J. Phys. Chem. B 2000, 104, 5049-5052. (34) Schmitz, K. S. An Introduction to Dynamic Scattering by Macromolecules; Academic Press: New York, 1990.
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Scheme 1. Synthesis of Poly(ethylene glycol)-block-oligo(DTO suberate)-block-poly(ethylene glycol), Abbreviated as PEG-oligo(DTO suberate)-PEG
Table 1. Summary of Light Scattering Parameters and Results Obtained with Poly(ethylene glycol)-block-oligo(DTO suberate)-block-poly(ethylene glycol), Self-Assembled into Spherical Structures in Dilute Aqueous Solution investigated parametersa
value
dn/dc (mL g-1) cac (10-7 g mL-1) M (109 g mol-1) A2 (10-7 mol mL g-2) Rg (nm) Rh (nm) Do (108 cm2 s-1) P (105) F d (103 kg m-3)
1.23 2.6 9.0 -5.0 50 49 5.6 6.38 1.04 1.2
a dn/dc ) refractive index increment of nanosphere solution; cac ) critical aggregation concentration; M ) weight-average molecular weight of the self-assembled vesicle; A2 ) second virial coefficient; Rg ) radius of gyration; Rh ) hydrodynamic radius; P ) aggregation number; F ) Rg/Rh; d ) density of hydrophobic core.
number-average molecular weight of the triblock copolymer was determined by GPC to be 10 300, in complete agreement with the theoretically expected value of 10 200 (6200 for the hydrophobic core and two PEG chains of 2000 each). From this, the hydrophobic-to-hydrophilic ratio of the triblock copolymer was calculated to be 1.5. The weight-average molecular weight, Mw, was 14 100. Using a conventional injection technique, the triblock copolymer spontaneously self-assembled, forming particles. Characterization of these particles by light scattering and electron microscopic techniques revealed them to be hollow nanospheres. To increase nanosphere size uniformity, extrusion through membrane filters of a pore size close to the equilibrium diameter of the nanospheres was performed. Both angular and concentration dependent DLS and SLS studies were performed. The results are summarized in Table 1 and in Figure 1. The SLS concentration profile of the inversed scattered intensity increased drastically at high dilutions (Figure 1a). This feature is an indication for the existence of a critical aggregation concentration (cac). Since the nanosphere formation is driven by noncovalent interactions, the vesicles disintegrate upon dilution below the cac. A cac of 0.26 µg mL-1 is noteworthy since it is significantly lower than cac values previously reported for selfassembling block copolymer systems;29,33,35,36 this suggests that these PEG-oligo(DTO suberate)-PEG nanospheres are highly resistant to dilution. The high value of 1.23 mL g-1 for the refractive index increment of the nanosphere solution is caused by the vesicles themselves and is not (35) Pillay Narrainen, A.; Pascual, S.; Haddelton, D. M. J. Polym. Sci., Polym. Chem. 2002, 40, 439-450. (36) Nam, Y. S.; Kang, H. S.; Park, J. Y.; Park, T. G.; Han, S.-H.; Chang, I.-S. Biomaterials 2003, 24, 2053-2059.
Figure 1. Light scattering data. (a) Zimm plot obtained for self-assembled structures of PEG-oligo(DTO suberate)-PEG triblock copolymer in dilute aqueous solution. For clarity, only the extrapolated values to zero scattering angle of the inverse scattered intensity are represented as a function of the concentration. Experimental points, ]; result of the close association model, s. (b) Concentration profile of the diffusion coefficient of self-assembled structures of PEG-oligo(DTO suberate)-PEG triblock copolymer in dilute aqueous solution.
Figure 2. Freeze-fracture transmission electron microscopic (TEM) images of vesicles made from PEG-oligo(DTO suberate)PEG polymer in dilute aqueous solution (the largest spheres are 100 nm in size, magnification 54000×).
a property of the individual polymer molecules. The refractive index increment has been confirmed by independent measurement on two different refractometers and does not appear to be an artifact. We speculate that the high value of the refractive index increment may be due to the polarization properties of oligo (DTO suberate) which
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Figure 4. Metabolic activity of UMR-106 cells exposed to PEGoligo(DTO suberate)-PEG triblock copolymer vesicles prepared in phosphate-buffered saline (PBS). The assay was performed with increasing vesicle concentration in the presence of serumfree medium. Metabolic activity is expressed as number of viable cells remaining following a 4 h exposure to vesicles. Vesicle concentration: (1) 2 mg mL-1; (2) 1 mg mL-1; (3) 0.5 mg mL-1; (4) 0.4 mg mL-1; (5) 0.2 mg mL-1; (6) 0.1 mg mL-1; (7) PBS.
Figure 3. Transmission electron microscopic (TEM) images of vesicles made from PEG-oligo(DTO suberate)-PEG polymer in dilute aqueous solution. (a) Unstained transmission electron images (scale bar 50 nm). (b) Cryotransmission electron image (scale bar 100 nm).
may be further enhanced when the block copolymers selfassemble into vesicles. A conventional Zimm plot suggested a radius of gyration, Rg, of 50 nm, consistent with the largest spherical structures observed by transmission electron microscopy (Figure 2). The hollow nature of the self-assembled nanospheres was demonstrated by a combination of DLS and SLS measurements. The linearity of the angular profile of the diffusion coefficient (data not shown) indicated that a single diffusive process occurs in this system. The size polydispersity index was determined to be 0.3 (nonuniformity of 1.3), indicating a relatively narrow size distribution of the vesicles. The hydrodynamic radius Rh ) 49 nm was calculated using the Stokes-Einstein equation
with the diffusion coefficient obtained following extrapolation to a concentration of zero (Figure 1b). Within the accuracy limit of the light scattering techniques, this value is identical to the radius of gyration. This observation supports the conclusion that PEG-oligo(DTO suberate)PEG self-assembles into nanosized hollow nanospheres in dilute aqueous solution.37 Electron microscopy was used to characterize directly the morphology of the nanoparticle preparation. Freezefracture electron micrographs of the vesicles revealed rounded structures with a diameter of about 100 nm (Figure 2), which is in agreement with the results of the light scattering analysis presented above. The self-assembled particles could be imaged using conventional nonstained transmission and cryotransmission electron micrography. This is probably due to the high electron density of the aromatic rings present in the hydrophobic core (DTO suberate) of the triblock copolymer, causing the nanosphere membrane to exhibit a higher contrast than both the background and the vesicle inner pool (Figure 3). In these images, the hollow nanospheres appear to be smaller in size than suggested by light scattering analysis. This discrepancy may be due to the need for vacuum drying or freezing during the preparation of specimens for electron microscopy. These procedures may have altered the size of the nanospheres. Cytotoxicity was examined by incubating UMR-106 cells in serum-free media with the addition of PEG-oligo(DTO suberate)-PEG nanospheres to the media at concentrations ranging from 0.1 to 2 mg mL-1. Preliminary results have thus far revealed no significant decrease in cell metabolic activity in the presence of the nanospheres, suggesting that PEG-oligo(DTO suberate)-PEG nanospheres do not produce short-term cytotoxic effects. Conclusion A new ABA triblock copolymer, PEG-oligo(DTOsuberate)-PEG, has been synthesized from noncytotoxic, biodegradable blocks. In dilute aqueous solution, the triblock copolymer self-assembles into vesicular structures, which were shown to be vesicular nanospheres approximately 100 nm in diameter. These nanospheres exhibited an extremely low cac, and preliminary results revealed no short-term cytotoxic effects toward UMR-106 cells in vitro. (37) Burchart, W. In Physical Techniques for the Study of Food Biopolymers; Ross-Murphy, S. B., Ed.; Blackie Academics and Professional: New York, 1994; p 151.
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Acknowledgment. This work was supported by a fellowship from the Swiss National Fund to C.N., by NIH Grant EB00286, and by the New Jersey Center for Biomaterials. The authors thank Olivier Vebert for his assistance with light scattering measurements, M. Libera (Stevens Institute of Technology) for assistance with TEM, and Valentine Starovoytov for his assistance with freeze-
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fracture techniques. The authors also acknowledge the helpful discussions provided by R. Prud’homme (Princeton University) and the assistance in the final edits of the manuscript provided by R. Dubin (Rutgers University). LA0490285
