pubs.acs.org/Langmuir © 2010 American Chemical Society
Protein PEGylation Attenuates Adsorption and Aggregation on a Negatively Charged and Moderately Hydrophobic Polymer Surface Sheetal S. Pai,† Todd M. Przybycien,*,†,‡ and Robert D. Tilton*,†,‡ †
Center for Complex Fluids Engineering, Department of Chemical Engineering and Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
‡
Received July 6, 2010. Revised Manuscript Received October 4, 2010 Covalent grafting of poly(ethylene glycol) chains to proteins (“PEGylation”) is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/ aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surfaceinduced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface.
Introduction Protein conjugates with grafted linear or branched polymers are an emerging class of biopharmaceuticals. The most prevalent of these are poly(ethylene glycol)-grafted, or “PEGylated,” proteins. Compared to the unmodified protein, these conjugates experience longer in vivo circulating half-lives due to decreased renal clearance rates, proteolysis, immune response, and/or inhibitor binding.1-3 Depending on the size and number of PEG chains grafted per protein, the conjugates can retain most or all of their biological activity.1,4 While early work on the pharmaceutical properties of these conjugates was based on grafting several 5000 molecular weight PEG chains, the industry trend is toward grafting a smaller number of larger chains, with 20 000 molecular weight linear PEG or 40 000 molecular weight branched PEG grafts providing improved performance.1,5 In addition to PEGylated proteins, grafted conjugates of immunoglobulin proteins and polysaccharides have been investigated for enhanced wound healing applications6 and the attachment of albumin to peptides or proteins has been explored to enhance stability and half-life.7 The interaction of protein-polymer conjugates with surfaces is an important determinant of the performance of these conjugates *To whom correspondence should be addressed. (R.D.T.) E-mail: tilton@ andrew.cmu.edu. Telephone: 412-268-1159. (T.M.P.) E-mail: todd@andrew. cmu.edu. Telephone: 412-268-3857. (1) Greenwald, R. B.; Choe, Y. H.; McGuire, J.; Conover, C. D. Adv. Drug Delivery Rev. 2003, 55(2), 217–250. (2) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40(7), 539–551. (3) Michaelis, M.; Cinatl, J.; Cinatl, J.; Pouckova, P.; Langer, K.; Kreuter, J.; Matousek, J. Anti-Cancer Drugs 2002, 13(2), 149–154. (4) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Biotechnol. Bioeng. 2005, 90(7), 856–868. (5) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discovery 2003, 2(3), 214–221. (6) Sun, L. T.; Bencherif, S. A.; Gilbert, T. W.; Farkas, A. M.; Lotze, M. T.; Washburn, N. R. Wound Repair Regener. 2010, 18(3), 302–310. (7) Kratz, F. J. Controlled Release 2008, 132(3), 171–183.
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in multiple applications. Purification of PEGylated proteins will typically be conducted on a commercial scale by chromatography,8,9 and the relevant process design requires an understanding of how the conjugates adsorb to solid chromatographic media. These surfaces may display a variety of surface energies and ionization states. Furthermore, protein PEGylation has been proposed as a means to improve both the extent of release and the retention of bioactivity of protein therapeutics administered by sustained release delivery depots, including poly(lactide-coglycolide) (PLG) microspheres.4,10-12 Protein delivery from such depots suffers from detrimental effects on the conformation, aggregation, and solubility of the protein caused by adsorption to PLG/aqueous interfaces.13-16 PEGylation is proposed to alleviate these effects by controlling the adsorptive contact between proteins and the surface, as well as lateral aggregation among tightly packed adsorbed proteins. There is also interest in the use of PEGylation to control order in adsorbed protein arrays that are designed to serve as templates for electronic materials fabrication. Kumashiro and co-workers17 showed that PEGylated ferritin, but not unmodified ferritin, produced ordered adsorbed arrays on cationically modified silica. In order to interpret adsorption mechanisms for proteinpolymer conjugates, it is informative to consider these conjugates (8) M€uller, E.; Josic, D.; Schr€oder, T.; Moosmann, A. J. Chromatogr., A 2010, 1217, 4696–4703. (9) Cisneros-Ruiz, M.; Mayalo-Deloisa, K.; Przybycien, T. M.; Rito-Palomares, M. Sep. Purif. Technol. 2009, 65, 105–109. (10) Diwan, M.; Park, T. G. J. Controlled Release 2001, 73(2-3), 233–244. (11) Diwan, M.; Park, T. G. Int. J. Pharm. 2003, 252(1-2), 111–122. (12) Castellanos, I.; Al-Azzam, W.; Griebenow, K. J. Pharm. Sci. 2005, 94(2), 327–340. (13) Pai, S. S.; Tilton, R. D.; Przybycien, T. M. AAPS J. 2009, 11(1), 88–98. (14) Crotts, G.; Sah, H.; Park, T. G. J. Controlled Release 1997, 47(1), 101–111. (15) Jiang, G.; Woo, B. H.; Kang, F.; Singh, J.; DeLuca, P. P. J. Controlled Release 2002, 79(1-3), 137–145. (16) Jiang, W.; Schwendeman, S. P. Pharm. Res. 2001, 18(6), 878–885. (17) Kumashiro, Y.; Ikezoe, Y.; Tamada, K.; Hara, M. J. Phys. Chem. B 2008, 112, 8291–8297.
Published on Web 11/10/2010
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as a special class of block copolymer. PEGylated proteins consist of a single globular protein “block” with one or more random coil polymer blocks. As with any block copolymer, the adsorption isotherm and adsorbed layer structure for protein-polymer conjugates reflect a competition among the different blocks for the surface. Adsorption tendencies depend on the relative solvent affinities of the blocks, their relative sizes, and their relative surface interaction energies. Steric interactions among nonadsorbed blocks will play a critical role in determining how tightly the conjugates can pack on a surface. Like most polymers, proteins or protein-polymer conjugates tend to display partial adsorption irreversibility due to their ability to have multiple points of contact with a surface. In addition to sharing common features with conventional block copolymers, protein-polymer conjugates introduce fundamentally distinct new pathways to alter adsorption energetics that are not available with conventional block copolymers. Globular proteins have well organized secondary and tertiary conformations. Surface-induced conformational changes often contribute favorably to the entropy of adsorption for proteins, and may therefore be expected to influence protein-polymer conjugate adsorption energies. Nevertheless, these conformational changes are normally not so profound as to eliminate the globular character of the protein, and the orientation of the protein on the surface has a large influence on the saturation surface excess concentration. Adsorption energies also vary with protein orientation. These factors will influence protein-polymer conjugate adsorption energies. Another distinctive feature that must be considered in protein-polymer conjugate adsorption is that adsorbed proteins may form aggregates mediated by intermolecular β-sheet formation among tightly packed molecules on a surface. In the only prior study of its kind, protein PEGylation has been shown to inhibit surface-induced aggregation on silica surfaces.18 PEG has protein repellant properties. Protein PEGylation is well-known to inhibit protein-protein interactions in solution.19-21 It is also well-known that PEG attachment to solid surfaces nearly eliminates protein adsorption to those surfaces due to the strong hydration of PEG chains and an unfavorable entropy change associated with protein binding to PEG.22-24 Nevertheless, these protein-repelling tendencies do not guarantee that protein PEGylation should prevent adsorption to surfaces that have not been modified by PEG. It should be noted that PEG chains are surface active on hydrophobic as well as acidic hydrophilic surfaces.25,26 As such, the PEG portion of the protein-polymer conjugates would compete with the protein portion for adsorptive contact with the surface, perhaps preferentially populating certain types of surfaces. This could protect the protein from direct denaturing exposure to the surface. We previously observed evidence for preferential adsorption of PEG (18) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Colloids Surf., B 2007, 57(1), 81–88. (19) Ducreux, J.; Tyteca, D.; Ucakar, B.; Medts, T.; Crocker, P. R.; Courtoy, P. J.; Vanbever, R. Bioconjugate Chem. 2009, 20(2), 295–303. (20) Scott, M. D.; Murad, K. L.; Koumpouras, F.; Talbot, M.; Eaton, J. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94(14), 7566–7571. (21) Lee, H.; Jang, I. H.; Ryu, S. H.; Park, T. G. Pharm. Res. 2003, 20(5), 818–825. (22) Gref, R.; Domb, A.; Quellec, P.; Blunk, T.; Muller, R. H.; Verbavatz, J. M.; Langer, R. Adv. Drug Delivery Rev. 1995, 16(2-3), 215–233. (23) Szleifer, I.; Carignano, M. A. Macromol. Rapid Commun. 2000, 21(8), 423–448. (24) Wei, J.; Ravn, D. B.; Gram, L.; Kingshott, P. Colloids Surf., B 2003, 32(4), 275–291. (25) Van der Beek, G. P.; Cohen Stuart, M. A.; Fleer, G. J.; Hofman, J. E. Macromolecules 1991, 24(25), 6600–6611. (26) Huang, Y.; Santore, M. M. Langmuir 2002, 18(6), 2158–2165.
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grafts during PEGylated ribonuclease A adsorption to PLG films.4 Here, we report the impact of protein PEGylation on adsorption to solid PLG films. PLG is moderately hydrophobic. Upon immersion in water, it carries a negative surface charge due to acidic chain end groups. Prior research shows that both net positively charged and net negatively charged proteins adsorb to solid PLG surfaces,4,14,27,28 in keeping with its moderate hydrophobicity. PLG films serve as a direct model for protein-polymer conjugate adsorption to sustained release drug delivery depots and also for many chromatographic surfaces that can be prepared in forms with widely varying surface charge and hydrophobicity. The protein-polymer conjugate examined here is chicken egg lysozyme grafted with either one or two 20 000 molecular weight PEG chains. Lysozyme serves as a benchmark model protein for adsorption studies owing to the large literature base on the conformation, orientation, and clustering of lysozyme adsorbed to many surfaces.18,29-33 The PEG molecular weight is representative of that commonly employed with PEG-conjugated protein drugs in current use.1,5 We employ total internal reflection fluorescence (TIRF), a surface-sensitive fluorescence spectroscopy platform, to compare adsorption of lysozyme and its monoPEG and diPEG conjugates. We use standard TIRF methods to determine how the degree of PEGylation controls adsorption isotherms and adsorption reversibility. In addition, we probe the extent of surface-induced aggregation using a TIRF-based thioflavin T dye-binding assay. We previously compared adsorption of unmodified lysozyme and PEGylated lysozyme conjugates on silica surfaces. Clean, base-treated silica surfaces are perfectly wetted by water and are more strongly negatively charged than PLG at neutral pH. Overall, PEGylation decreased the adsorption affinity to silica, made the adsorption more reversible, and inhibited surfaceinduced aggregation. These effects were significantly more pronounced for diPEGylated conjugates than for monoPEGylated conjugates. A transition in the adsorption isotherm for unmodified lysozyme was shown to correspond to a crowding-induced reorientation from a more random orientation distribution at low surface concentrations to an orientation at high surface concentrations that juxtaposes the positively charged patch containing the N-terminal lysine residue against the negatively charged silica surface.29,34 In the case of monoPEG-lysozyme on silica, a transition was observed that instead corresponded to a change in conjugate orientation from one in which both PEG and lysozyme portions of the conjugate lay in contact with the surface to one in which only the lysozyme portion contacted the surface while the PEG grafts extended in the manner of a polymer mushroom layer.30 The proposed difference in orientation was supported by independent atomic force microscopy force measurements.30 With a primary locus of PEGylation on the lysozyme N-terminus, PEGylation prevented lysozyme from adopting its preferred orientation on silica and increased the adsorption reversibility accordingly. This prior work provides a frame of (27) Crotts, G.; Park, T. G. J. Controlled Release 1997, 44(2-3), 123–134. (28) Butler, S. M.; Tracy, M. A.; Tilton, R. D. J. Controlled Release 1999, 58(3), 335–347. (29) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Langmuir 2003, 19(9), 3848– 3857. (30) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Langmuir 2005, 21(4), 1328– 1337. (31) Blomberg, E.; Claesson, P. M.; Fr€oberg, J. C.; Tilton, R. D. Langmuir 1994, 10(7), 2325–2334. (32) Buijs, J.; Speidel, M.; Oscarsson, S. J. Colloid Interface Sci. 2000, 226(2), 237–245. (33) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1–15. (34) Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12(25), 6104–6113.
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reference for interpreting adsorption to the more hydrophobic, less densely charged PLG surface. We report here that PEGylation effects on these key adsorption phenomena are qualitatively reproduced on the PLG surface, suggesting that the two types of surfaces have similar relative affinities for both the PEG grafts and the lysozyme core of the conjugates.
Experimental Section Materials. Chicken egg lysozyme (Sigma-Aldrich Company, St. Louis, MO, cat. no. L-6876), poly(ethylene glycol) homopolymer, average molecular weight 20 000 Da (Sigma-Aldrich Company, cat. no. 95172), and fluorescein-5-isothiocyanate (Molecular Probes/Invitrogen, Carlsbad, CA, cat. no. F143) were used without further purification. Methoxy-poly(ethylene glycol)-propionaldehyde (mPEG-propionaldehyde), molecular weight 19 772 Da with 1.04 polydispersity, was generously donated by Dr. Reddy’s Laboratories Ltd. (Cambridge, U.K.) and also used without further purification. The nominally 50:50 poly(lactide-co-glycolide (PLG) sample (54:46 actual molar ratio of L/G; Mw=24 kDa) was generously donated by Alkermes, Inc. (Cambridge, MA) and used as received. According to the certificate of analysis, the donated PLG had a free acid end group. While the polarity and charge of the PLG end group has been shown to influence PLG degradation rates,35 prior work has shown that is has no significant effect on protein adsorption to PLG.28 All adsorption experiments were conducted in a 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES, Sigma-Aldrich Company, g99.5% pure) buffer, at pH 7.4, containing 100 mM NaCl. In this buffer, the average molecular weight of 50:50 PLG decreases by approximately 80% over 40 days, according to Alkermes. Conjugation reaction and conjugate purification buffers were prepared from sodium phosphate monobasic and dibasic anhydrous salts (Fisher Scientific, 99% pure) and sodium chloride (Sigma-Aldrich Company, 99% pure). Sodium cyanoborohydride (95% pure) and thioflavin T (cat. no. 228850; dye content ∼75%) were from Sigma-Aldrich Company and used as received. All water was purified by reverse osmosis followed by treatment to 18 MΩcm resistivity using a Barnstead NANOpure Diamond system.
Lysozyme PEGylation and Conjugate Characterization. We reacted 2 mg/mL lysozyme with 34.5 mg/mL 20 kDa mPEGpropionaldehyde (1:6 mol ratio) in a pH 5.1, 100 mM sodium phosphate buffer containing 20 mM sodium cyanoborohydride. The reaction mixture was stirred rapidly, but carefully to avoid foaming, by using a magnetic stir bar for 17 h at 4 °C. While these reaction conditions have been established to strongly favor N-terminal PEGylation of the protein due to the lower pKa of the N terminal amino group relative to the ε-amino groups of lysine,36 lysozyme has six lysine residues with a range of solvent exposures and titration behaviors in addition to the N-terminal lysine that could also be conjugated. We purified the conjugation reaction mixture into fractions containing mono- and diPEGylated conjugates and residual unmodified protein via size:: exclusion chromatography (Akta Explorer, GE Healthcare, Uppsala, Sweden) using a Sephacryl S-200 column (1.6 cm inner diameter 60 cm length, GE Healthcare) with a 2 mL injection volume in a pH 7.2, 10 mM sodium phosphate buffer with 150 mM NaCl.30 A typical PEGylation reaction yielded 63% monoPEG-lysozyme, 22% diPEG-lysozyme, and 15% unmodified lysozyme. We used MALDI mass spectrometry (PerSeptive Voyager STR MS, Applied Biosystems, Carlsbad, CA) to verify the number of PEG molecules attached to lysozyme in each collected fraction, as described previously.30 The locus of the PEG modification was (35) Tracy, M. A.; Ward, K. L.; Firouzabadian, L.; Wang, Y.; Dong, N.; Qian, R.; Zhang, Y. Biomaterials 1999, 20(11), 1057–1062. (36) Kinstler, O. B.; Gabriel, N. E.; Farrar, C. E.; DePrince, R. B. N-Terminally chemically modified protein compositions and methods. U.S. Patent 5,824,784, 1998.
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determined previously by subjecting whole proteins and tryptic digests of unmodified and PEG-modified lysozyme to reversephase high performance liquid chromatography and MALDI MS.30 It was concluded that monoPEG-lysozyme was primarily N-terminally modified, with slight modification at lysine residues 33 and 97; diPEG-lysozyme was likely modified at the N-terminus and at residue 33, with slight modification at residues 97 and 116.30 We characterized the secondary and tertiary structure of the lysozyme conjugates using circular dichrosim (J-810 spectropolarimeter, Jasco, Inc., Easton, MD) fluorescence spectroscopy (Spex Fluorolog-3, HORIBA Jobin Yvon, Edison, NJ) and determined that PEGylation did not result in significant protein structural perturbations (see Supporting Information S1). Using dynamic light scattering (Zetasizer 3000 HS, Malvern Instruments, Malvern, PA), we measured intensity-weighted average hydrodynamic diameters (DH) of the conjugates, unmodified lysozyme, and free PEG in order to suggest plausible configurations for the conjugates in solution or on surfaces (see Supporting Information Table S1). The hydrodynamic diameters of unmodified lysozyme and free PEG, 3.9 ( 1.8 and 7.0 ( 2.7 nm, respectively, correspond well with the reported ellipsoidal dimensions of lysozyme, 3 3 4.5 nm3,37 and the hydrodynamic diameter reported elsewhere for a nominally 20 000 molecular weight PEG sample, 9.7 nm;38 error values correspond to the half width of the light scattering peak. The hydrodynamic diameters of monoPEG-lysozyme and diPEG-lysozyme, 11.6 ( 5.2 and 14.0 ( 6.8 nm, respectively, were approximately the sum of the diameters of one lysozyme molecule plus one or two PEG chains, respectively. This suggests, but does not prove, that the conjugates adopt dumbbell configurations wherein the protein and PEG portions of the conjugate occupy separate domains.4,30 This is consistent with separate conclusions drawn from molecular modeling39 and small-angle X-ray scattering experiments40 on PEG-conjugated hemoglobin.
Adsorption Substrate Preparation and Characterization. PLG films were spin-cast onto quartz microscope slides, coverslips, or oxidized silicon wafers for use in adsorption studies. Slides, coverslips, and wafers were cleaned following a protocol described elsewhere,29 except that the Chromerge cleaning solution was replaced with a solution of sulfuric acid and NoChromix (Sigma-Aldrich Company, cat. no. 328693). Optical grade silicon wafers (Virginia Semiconducter, Fredericksburg, VA) were oxidized in a 1000 °C oven to produce a 30 nm surface oxide layer prior to spin-casting. These were used for ellipsometric measurements of PLG film thickness and, since PEG is nonfluorescent, for measurements of unconjugated PEG adsorption to PLG films. Quartz slides (TIRF Technologies, formerly BioElectroSpec, Morrisville, NC) were coated with PLG and used for all TIRF experiments, and acid-treated glass coverslips, 25 mm in diameter (Ted Pella, Inc., Redding, CA, cat. no. 260372), were coated with PLG and used for PLG film contact angle and ζ- potential measurements. After cleaning the appropriate substrate, a PLG film was spincast onto the substrate from 1 mL of a 0.004 volume fraction PLG solution in ethyl acetate (Fisher Scientific, Pittsburgh, PA) at 2000 rpm for 1 min.28 Immediately after spin-casting, the films were baked in a 60 °C oven for 2 h to ensure evaporation of residual ethyl acetate. Films were used for adsorption experiments immediately after preparation. This spin-casting procedure yields a PLG film with a thickness of approximately 40 nm, as measured by phase modulation ellipsometry (Picometer, Beaglehole Instruments, Wellington, New Zealand),41,42 and a typical rms roughness (37) Blake, C. C.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206(4986), 757–761. (38) Fee, C. J.; Alstine, J. M. V. Bioconjugate Chem. 2004, 15(6), 1304–1313. (39) Manjula, B. N.; Tsai, A.; Upadhya, R.; Perumalsamy, K.; Smith, P. K.; Malavalli, A.; Vandegriff, K.; Winslow, R. M.; Intaglietta, M.; Prabhakaran, M.; Friedman, J. M.; Acharya, A. S. Bioconjugate Chem. 2003, 14(2), 464–472. (40) Svergun, D. I.; Ekstr€om, F.; Vandegriff, K. D.; Malavalli, A.; Baker, D. A.; Nilsson, C.; Winslow, R. M. Biophys. J. 2008, 94(1), 173–181.
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Article of 2.4 A˚ over a 1 μm2 area as measured by tapping mode atomic force microscopy (Nanoscope IIIa, Digital Instruments, Plainview, NY). The surface charge density and hydrophobicity of the PLG films were characterized via ζ-potential (ZetaSpin, ZetaMetrix Inc., Pittsburgh, PA) and contact angle (NRL C.A. Goniometer, Rame-Hart, Inc., Netcong, NJ) measurements, respectively. The ζ-potentials of PLG films spin-casted on a round glass coverslip were measured using the rotating disk streaming potential technique developed by Sides and co-workers and described in detail elsewhere.43-45 The streaming potential was converted to ζpotential in the thin double layer limit. The PLG film ζ-potential was measured periodically over several hours of immersion in sterile-filtered 10 mM HEPES buffer to determine how the surface charge density would vary over the duration of an adsorption experiment. NaCl was omitted from the buffer for these measurements in order to provide a measurable streaming potential; since this is an indifferent electrolyte, the surface charge density is not expected to change significantly in its presence. Initially, the PLG film surface is negatively charged with a ζ-potential of -34.0 ( 0.8 mV in 10 mM HEPES buffer. During buffer immersion, the ζ-potential shifts only slightly to -35.7 ( 0.4 mV over 30 min and then remains constant for several hours. The confidence limits represent the standard deviation of triplicate determinations. The latter ζ-potential is equivalent to a surface charge density of 1800 A˚2/charge. The initial advancing and receding contact angles of a sessile water drop on the PLG film were 62.0° and 47.0°, respectively, indicating a moderately hydrophobic surface.
Total Internal Reflection Fluorescence (TIRF) for Adsorption Isotherms of Unmodified and PEGylated Lysozyme on PLG Films. TIRF is a platform for surface-selective fluorescence spectroscopy and is routinely used to measure adsorption of molecules that are either intrinsically fluorescent or that have been labeled with fluorescent tags. We used TIRF to measure the adsorption extent and reversibility for lysozyme and its PEGylated conjugates on PLG films and also to probe orientation changes in unconjugated lysozyme layers using an electrostatic potential-sensitive fluorescein tag. (The latter is discussed in the Supporting Information.) The basic principles of TIRF have been described elsewhere.29,34,46-48 For this study, we used a modular Spex Fluorolog-3 (HORIBA Jobin Yvon) fluorescence spectrometer. A TIRF flow cell manufactured by TIRF Technologies Inc. was mounted into the spectrometer as described previously.29 We monitored intrinsic tryptophan fluorescence during adsorption at excitation and emission wavelengths of λex =295 nm and λem =340 nm, respectively, with slit widths fixed at 3 nm for both excitation and emission. The tryptophan emission intensity is proportional to the surface concentration of lysozyme. PEG does not contribute to the fluorescence intensity. Adsorption isotherms were determined from multiple independent TIRF experiments, each conducted at a different bulk concentration. All experiments were conducted at 25 °C with solutions undergoing laminar slit flow at a wall shear rate of 23 s-1. Surface excess concentrations were determined after the TIRF signal had reached a plateau and remained stable for at least 10 min. TIRF intensities were converted to surface excess concentrations using the calibration method based on N-acetyl tryptophan described elsewhere.48 (41) deFeijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17(7), 1759– 1772. (42) Beaglehole, D. Phys. B (Amsterdam, Neth.) 1980, 100(2), 163–174. (43) Hoggard, J. D.; Sides, P. J. Langmuir 2004, 20(26), 11493–11498. (44) Hoggard, J. D.; Sides, P. J.; Prieve, D. C. Langmuir 2005, 21(16), 7433– 7438. (45) Sides, P. J.; Newman, J.; Hoggard, J. D.; Prieve, D. C. Langmuir 2006, 22(23), 9765–9769. (46) Lok, B. K.; Cheng, Y. L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91(1), 87–103. (47) Lok, B. K.; Cheng, Y. L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91(1), 104–116. (48) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11(9), 3500–3509.
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Ellipsometry for Adsorption of PEG Homopolymer to PLG Films. Due to its lack of fluorescence, PEG surface concentrations on PLG films were measured by phase modulation ellipsometry (picometer, Beaglehole Instruments).41,42 The effective optical thickness and refractive index of the adsorbed PEG layer were converted to surface excess concentration via the de Feijter equation41 using the PEG refractive index increment, 0.13 cm3/g, that was separately measured by differential refractometry.49
TIRF for Surface-Induced Aggregation Measurements. In addition to its use for measuring adsorption and desorption, TIRF was also used to spectroscopically probe aggregation in adsorbed layers using a dye binding technique. We adapted a thioflavin T fluorescence-based assay for β-sheet-mediated aggregation, previously used to probe amyloid fibril formation in solution,50-52 to a TIRF spectroscopy platform in order to investigate two-dimensional clustering of lysozyme adsorbed on planar surfaces.18 Thioflavin T undergoes significant excitation and emission red shifts when exposed to aggregated, fibril states of proteins; the fibril state is characterized by extensive formation of intermolecular β-sheet structures. In solution, unbound thioflavin T has excitation and emission maxima at 342 and 430 nm, respectively. When bound to intermolecular β-sheets in amyloid fibrils, the excitation and emission peaks are red-shifted to 440 and 482 nm, respectively.52 To track the extent of intermolecular β-sheet formation relative to the amount of protein adsorbed, we monitored the ratio of red-shifted thioflavin T fluorescence intensity (I) to tryptophan fluorescence intensity, ΔIThT/Itrp, where ΔIThT =I(λex =440 nm, λem =482 nm) - I(λex =342 nm, λem = 430 nm) and Itrp = I(λex = 295 nm, λem = 340). For all surface-induced aggregation experiments, thioflavin T was added to the buffer at a concentration of 15 μM, in some cases mixed with the protein solution used for adsorption and in other cases added to the protein-free buffer after rinsing any reversibly adsorbed protein from the surface. Validation of the TIRF thioflavin T assay was previously done using attenuated total reflection Fourier transform infrared spectroscopy for an adsorbed layer of lysozyme on silica.18 To quantify the percentage of β-sheet formation on the surface, we used a calibration based on molecules aggregated and probed in bulk solution by conventional fluorescence spectroscopy. We defined 0% aggregation as the ΔIThT/Itrp value (0.16) when a solution of native, unmodified lysozyme is mixed with a 15-fold molar excess of thioflavin T. We defined 100% aggregation as the ΔIThT/Itrp value (2.31) after lysozyme in solution had been visually fibrilized by vortexing vigorously and shaking, then centrifuged, separated from its supernatant, and then measured in the fluorescence spectrometer also with a 15-fold molar excess of thioflavin T. Both the fibrils and supernatant were assayed, and 100% aggregation was verified by the absence of any protein detected in the supernatant. The ΔIThT value for either state was corrected by subtracting the small ΔIThT value measured for thioflavin T in a protein-free solution. A 50% aggregation standard was prepared by diluting the completely aggregated lysozyme with an equal amount of unaggregated lysozyme. The percentage of aggregated protein followed the linear calibration % aggregation= 46.2 (ΔIThT/Itrp) - 4.5 (R2 =0.99). A similar calibration was attempted with monoPEG-lysozyme, but no visual fibrilization was observed despite several cycles of heating, shaking, and vortexing. We therefore verified aggregation of monoPEG-lysozyme using dynamic light scattering to measure size distributions. Aggregated solutions produced bimodal size distributions. The percentage of aggregated monoPEG-lysozyme conjugates was directly calculated from the relative areas under the large (aggregated) and small (unaggregated) (49) Braem, A. D.; Prieve, D. C.; Tilton, R. D. Langmuir 2001, 17(3), 883–890. (50) deFerrari, G. V.; Mallender, W. D.; Inestrosa, N. C.; Rosenberry, T. L. J. Biol. Chem. 2001, 276(26), 23282–23287. (51) Friedhoff, P.; Schneider, A.; Mandelkow, E. M.; Mandelkow, E. Biochemistry 1998, 37(28), 10223–10230. (52) LeVine, H. Methods Enzymol. 1999, 39, 274–284.
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Figure 1. Adsorption isotherms in 10 mM, pH 7.4 HEPES buffer containing 100 mM NaCl under laminar shear flow at a wall shear rate of 23 s-1: unmodified lysozyme (filled squares); monoPEGlysozyme (open triangles); diPEG-lysozyme (filled circles); homopolymer PEG (stars). The lines are linear least-squares regressions of the corresponding data ranges. For both unmodified and monoPEG lysozyme, the regression lines have been extended, with 95% confidence, to the bulk concentrations that correspond to the approximate midpoints of the transitions. For example, the regression line for unmodified lysozyme has been extended to a bulk concentration of 0.85 μM; the surface concentration with 95% confidence at this bulk concentration is calculated as 0.066 ( 0.008 molecules/nm2 below the transition and 0.102 ( 0.013 molecules/ nm2 above the transition. For monoPEG-lysozyme, the regression line has been extended to a bulk concentration of 2 μM; the surface concentration with 95% confidence at this bulk concentration is calculated as 0.040 ( 0.008 molecules/nm2 below the transition and 0.057 ( 0.011 molecules/nm2 above the transition. fractions. Aggregated and unaggregated monoPEG lysozyme solutions were subjected to bulk solution thioflavin T fluorescence spectroscopy as described above. The relationship between percent aggregation and ΔIThT/Itrp had a slope of 47.6 between solutions that were 0% aggregated and 95.1% aggregated. The extrapolated ΔIThT/Itrp value for 100% monoPEG-lysozyme (2.30) is very similar to that for unmodified lysozyme. Thus, all reported percent aggregation values are based on the calibration with unmodified lysozyme, and it is assumed that binding of the small molecule thioflavin T is not affected by any steric repulsion from PEG. To support this assumption, we observed that the ΔIThT/Itrp values for thioflavin T mixed in solution with either unmodified, unaggregated lysozyme or with unaggregated monoPEG-lysozyme were similar, 0.16 and 0.18, respectively.
Results and Discussion Adsorption Isotherms. Adsorption isotherms for unmodified lysozyme, monoPEG-lysozyme, and diPEG-lysozyme measured by TIRF on freshly cast PLG films are shown in Figure 1, together with the adsorption isotherm for unconjugated PEG homopolymer on PLG, as measured by ellipsometry due to the nonfluorescent nature of PEG. PEGylated lysozyme adsorption is significantly diminished relative to the unmodified form at all bulk concentrations but is not eliminated. The isotherms for unmodified lysozyme and monoPEG-lysozyme display two approximately linear regimes on a semilog scale separated by a transition. Such transitions are typically attributed to lateral (53) Asanov, A. N.; DeLucas, L. J.; Oldham, P. B.; Wilson, W. W. J. Colloid Interface Sci. 1997, 191(1), 222–235. (54) Luo, Q.; Andrade, J. D. J. Colloid Interface Sci. 1998, 200(1), 104–113. (55) Marsh, R. J.; Jones, R. A. L.; Sferrazza, M. Colloids Surf., B 2002, 23(1), 31–42.
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interactions between adsorbed proteins that induce either conformational changes or changes in orientation.53-55 Using linear least-squares regression to fit the data points above and below the transition, Figure 1 also illustrates predicted pre- and posttransition surface concentrations at the 95% confidence level for unmodified and monoPEG lysozyme at bulk concentrations corresponding to the putative midpoints. The pre- and posttransition difference in unmodified lysozyme adsorbed concentrations is significant at greater than the 95% confidence level; that for the monoPEGylated lysozyme is significant at slightly less than the 95% confidence level. The adsorption isotherm of diPEG-lysozyme shows no such transition on PLG within the concentration range studied. This behavior is very similar to the adsorption of unmodified and PEGylated lysozyme on silica, mentioned previously.29,30,34 The transition in the unmodified lysozyme isotherm occurs near the hexagonal close packing limit for an all-side-on configuration, 0.087 molecules/nm2, and adsorption continues above 0.128 molecules/nm2, the limit for an all-end-on configuration.34 Thus, the highest coverages observed are inconsistent with monolayer adsorption and likely indicate partial bilayer formation at high concentrations, similar to what has been observed in the surface force apparatus for adsorbed lysozyme layers on mica.31 To probe the orientation of unmodified lysozyme, we monitored the fluorescence emission of lysozyme labeled with the electrostatic potential-sensitive fluorophore fluorescein isothiocyanate (see Supporting Information S2).29,34 These experiments indicated that lysozyme adopts an orientation that juxtaposes a large positively charged patch on the protein molecule that contains the N terminus against the negatively charged PLG surface, just as it does on hydrophilic silica surfaces.29,34 This preferred orientation of the unmodified lysozyme has implications for the PEG-lysozyme adsorption mechanism. The PEGylation reaction conditions employed favor N-terminal grafting. Given that the large positive patch on lysozyme is now occupied by the PEG chain, the PEGylated lysozyme molecules cannot adopt their preferred orientation on the negatively charged PLG surface and the molecule is forced to adsorb in an orientation that has a weaker adsorption energy for the protein portion of the conjugate. This influences the adsorption reversibility, as discussed below. The similarity in behavior on silica and on PLG leads us to infer that the transition in the monoPEGlysozyme isotherm has similar origins here as it did on silica. At low surface concentrations, both PEG and lysozyme portions of the conjugate lay in contact with the surface and exclude area, while at higher coverages the PEG chains no longer lay on the surface but extend into solution in a mushroom-like configuration.30 This reorientation allows monoPEG-lysozyme conjugates to continue filling the surface with a smaller excluded area per molecule. The adsorption isotherms for PEG homopolymer and diPEGlysozyme in Figure 1 were quite similar, suggesting that PEGsurface interactions dominate the adsorption behavior of diPEGlysozyme. Bearing in mind that each diPEG-lysozyme carries two PEG chains, for each point in the adsorption isotherm, where diPEG-lysozyme and PEG homopolymer gave nearly identical numbers of molecules per unit area, the number of PEG chains per unit area is twice as large for diPEG-lysozyme as for the adsorbed PEG homopolymer. By comparing the average area per PEG chain at the surface to the area excluded by a PEG random coil with radius of gyration RG, πRG2, it is possible to probe the degree of overlap of adjacent chains. If the ratio of the average area per adsorbed chain to πRG2 is less than 1, it indicates greater chain overlap and implies that DOI: 10.1021/la102709y
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Figure 2. Schematic illustration of proposed layer structures at low and high surface concentrations for lysozyme (A), PEG homopolymer (B), monoPEG-lysozyme (C), and diPEG-lysozyme (D). The N-terminal positive patch of lysozyme can be identified by the lightly shaded portion of the lysozyme molecule in pictures (A), (C), and (D). A partial bilayer with unknown orientation of the outer adsorbed molecules may exist for unmodified lysozyme at high concentrations.
adsorbed chains are forced to extend further from the surface to minimize thermodynamically unfavorable chain interpenetration. Using the hydrodynamic radius of the 20 000 molecular weight PEG chains, we calculate RG =5.25 nm, where RG = 3/2RH for a random coil polymer.56 The plateau in the PEG homopolymer adsorption isotherm, approximately 0.008 molecules/nm2, corresponds to a normalized area per chain of 1.5, indicating essentially no overlap and minimal extension of PEG chains from the surface, as expected. The normalized area per PEG chain in the diPEG-lysozyme conjugates ranges from 0.3 to 1.4, indicating that chain overlap would likely occur and favor extension away from the surface. A plausible orientation for diPEG-lysozyme places one PEG chain lying in a relatively flat, homopolymer-like, conformation and orients the second chain toward the solution. Unlike a monoPEG-lysozyme conjugate, such a molecule has little opportunity to decrease its projected area on the surface by reorienting, leaving a featureless adsorption isotherm. Figure 2 illustrates plausible layer structures for all species at low and high surface concentrations, indicating how the unmodified lysozyme and monoPEG-lysozyme undergo reorientations from low to high surface concentration, while the homopolymer PEG and diPEG-lysozyme experience no significant changes in configuration at low and high surface concentrations. Adsorption Reversibility. It is common for a large fraction of an adsorbed protein layer to have an immeasurably slow desorption rate after rinsing, to be effectively irreversibly adsorbed.57-60 The effect of PEG grafting on adsorption reversibility was determined by rinsing layers of unmodified or PEGylated lysozyme with protein-free buffer. We report the reversibility of adsorption as the fraction of protein that is removed from the surface after approximately 15 min of desorption into pure buffer while maintaining the wall shear rate at 23 s-1. The desorption rate was observed to become immeasurably small by this time for all cases. The adsorption reversibility of unmodified and PEGylated lysozyme is plotted as a function of the surface concentration in Figure 3. Unmodified lysozyme is at most ∼30% reversibly adsorbed on PLG at all surface concentrations. The reversibility is relatively constant, with no discernible change near the isotherm transition. PEGylated conjugates are significantly more reversibly adsorbed than the unmodified lysozyme at all surface concentra(56) Kok, C. M.; Rudin, A. Makromol. Chem., Rapid Commun. 1981, 2(11), 655–659. (57) Feder, J.; Giaever, I. J. Colloid Interface Sci. 1980, 78(1), 144–154. (58) Norde, W. Adv. Colloid Interface Sci. 1986, 25(4), 267–340. (59) Norde, W.; MacRitchie, F.; Nowicka, G.; Lyklema, J. J. Colloid Interface Sci. 1986, 112(2), 447–456. (60) Schaaf, P.; Dejardin, P.; Johner, A.; Schmitt, A. Langmuir 1992, 8(2), 514–517.
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Figure 3. Fraction of protein removed from the surface after ∼15 min of desorption into pure buffer, at a wall shear rate of 23 s-1, reported as a function of surface concentration: unmodified lysozyme (filled squares); monoPEG-lysozyme (open triangles); diPEG-lysozyme (filled circles). The start of the isotherm transitions are noted by dashed lines for monoPEG and unmodified lysozyme.
tions except at the lowest surface concentrations measured for diPEG-lysozyme. Unlike the unmodified lysozyme, reversibility increases as the surface concentration increases for both monoPEG- and diPEG-lysozyme. For equal surface concentrations, the diPEG conjugate is significantly more reversibly adsorbed than the monoPEG conjugate. There are many potential factors at work. The increased reversibility can reflect a weakening of the direct conjugate interaction with the surface, a net destabilization of the adsorbed layer due to the addition of repulsive steric interactions among PEG grafts on different proteins within the layer, or a combination of both. It is also possible that the reversibility is due in part to the aggregation state of adsorbed proteins, in which case the effect of PEGylation on reversibility could be related to disruption of aggregation. Finally, the reversibility may reflect the heterogeneity of the surface if different sites were to provide significantly different adsorption energies. Surface-induced aggregation will be addressed directly below, but first it is informative to examine the observed trends of reversibility with respect to surface concentration. Aggregation models, such as the isodesmic aggregation model, show that the extent of aggregation should increase with increasing surface concentration.61 If aggregation largely dictated the degree of reversibility, the reversibility would decrease as surface concentration increased, but there was no significant surface (61) Frieden, C.; Nichol, L. W. Protein-protein interactions; Wiley-InterScience: New York, 1981; p 403.
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concentration dependence for unmodified lysozyme and the opposite trend was observed for the PEG conjugates. Given that PLG hydrolyzes slowly in water, it is possible that the PLG surface is heterogeneous and may therefore present sites of varying adsorption energy for lysozyme or its PEGylated conjugates. The increasing reversibility with increasing surface concentration of PEGylated conjugates would be consistent with the preferential filling of the highest adsorption energy sites first, leaving the less favorable, and hence more readily reversible, sites to be filled only after the preferred sites were occupied. Nevertheless, further consideration suggests that adsorption site heterogeneity is not responsible for the observed reversibility behavior. Calling the reversible fraction R, the product of the irreversible fraction and the surface concentration, (1 - R)Γ, is a surrogate for the putative number density of occupied high energy sites. This product is observed to increase continuously with increasing surface concentration, rather than saturating as high energy sites become fully occupied. Although it is conceivable that high energy sites could under some circumstances also exhibit higher local activation energies for adsorption than surrounding sites,53 it is unlikely that the reversibly adsorbed fraction represents the heterogeneity of adsorption sites on the PLG surface. We hypothesize that the dominant factors underlying the increased reversibility of PEGylated lysozyme adsorption are a weakening of the direct protein-surface attraction and a destabilization of the layer due to steric repulsions between PEG grafts. Concerning the first factor: recall that the N-terminus is the preferential site of PEG conjugation. Recall also that the preferred orientation of unconjugated lysozyme places the N-terminus in direct contact with the surface. Thus, the PEG conjugate prevents the PEGylated lysozyme molecule from adopting its preferred orientation on the PLG surface. Concerning the second factor: steric repulsions between PEG grafts on neighboring conjugates would add an unfavorable component to the overall adsorption energy, the importance of which increases as the layers become more crowded. This would destabilize the adsorbed layer and thereby decrease the energy barrier against desorption. Surface-Induced Aggregation. Protein aggregation is typically accompanied by the formation of intermolecular β-sheet structures.62-65 We therefore measured the extent of surfaceinduced aggregation via the intermolecular β-sheet probe thioflavin T, using a protocol described previously.18 In order to use thioflavin T as a probe of adsorbed protein aggregation, the dye should not suffer significant changes to its excitation or emission maxima upon exposure to the bare PLG surface. This control experiment was performed by exposing a PLG-coated slide to a flowing 15 μM thioflavin T solution in the TIRF apparatus. The ΔIThT value measured in this control experiment is approximately equal to only 13% of the value attained for a saturated lysozyme layer, indicating that thioflavin T exposure to a PLG surface does not mimic the effect of binding to intermolecular β-sheet. This contribution was accounted for in all ΔIThT values reported for all experiments. Figure 4 indicates how the extent of intermolecular β-sheet formation, represented by ΔIThT/Itrp, evolves over time for unmodified, monoPEG- and diPEG-lysozyme. In these experiments, thioflavin T was introduced to the irreversibly adsorbed (62) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40(2), 6036–6046. (63) Nielsen, L.; Frokjaer, S.; Carpenter, J. F.; Brange, J. J. Pharm. Sci. 2001, 90(1), 29–37. (64) Sethuraman, A.; Vedantham, G.; Imoto, T.; Przybycien, T. M.; Belfort, G. Proteins: Struct., Funct., Bioinf. 2004, 56(4), 669–678. (65) Yokoyama, Y.; Ishiguro, R.; Maeda, H.; Mukaiyama, M.; Kameyama, K.; Hiramatsu, K. J. Colloid Interface Sci. 2003, 268(1), 23–32.
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Figure 4. Representative time-dependent measures of the extent of β-sheet formation, as ΔIThT/Itrp, for surface concentrations of ∼0.02 molecules/nm2 for lysozyme (filled squares), monoPEGlysozyme (open triangles), and diPEG-lysozyme (filled circles). Thioflavin T was introduced to a preadsorbed and rinsed layer of protein. The inset represents the average final values of ΔIThT/Itrp for triplicate experiments reported with 95% confidence limits for unmodified lysozyme (U), monoPEG-lysozyme (M), and diPEGlysozyme (D). (*) DiPEGylation significantly (p < 0.05) reduced the extent of β-sheet formation.
protein of a preadsorbed and rinsed layer. Results are summarized in Table 1. Solution concentrations were selected so that the final surface number concentration, Γ ∼ 0.02 molecules/nm2, would be the same for each species. This surface concentration corresponds to the “low” surface concentration for unmodified lysozyme and monoPEG-lysozyme listed in Table 1 and the “high” surface concentration for diPEG-lysozyme. The bulk concentrations required to achieve this surface concentration were 0.034, 0.63, and 18.32 μM for unmodified lysozyme, monoPEG-lysozyme, and diPEG-lysozyme, respectively. Intermolecular β-sheet formation is decreased, although not completely eliminated, upon PEGylation. The addition of two PEG chains significantly decreases intermolecular β-sheet formation as might be expected with the addition of this more extensive steric barrier to interaction. We also monitored ΔIThT/Itrp for preadsorbed and rinsed layers containing higher surface concentrations of 0.12 and 0.063 molecules/nm2 for unmodified and monoPEG-lysozyme, respectively. The final ΔIThT/Itrp ratios observed when thioflavin T was introduced alone to the irreversibly adsorbed remnant of a preadsorbed and rinsed layer were 1.62 ( 0.08 and 1.22 ( 0.02 for unmodified lysozyme and monoPEG-lysozyme at the high surface concentrations, respectively. The ΔIThT/Itrp values are all reported as the mean ( 95% confidence limits for triplicate experiments. Using the calibration described above, the percent aggregation values at low and high surface concentrations for each species are reported in Table 1. It is evident that higher surface concentrations favor aggregation and that PEGylation significantly decreases the degree of lysozyme aggregation on PLG surfaces at both low and high surface concentrations. The only species for which surface concentration did not affect aggregation was diPEG-lysozyme, where only 31% was aggregated at either higher or lower surface concentrations. We also monitored thioflavin T fluorescence when the dye was added at a concentration of 15 μM during the adsorption process, rather than adding it to a preadsorbed and rinsed layer. This was performed at the higher surface concentrations for unmodified (0.12 molecule/nm2) and monoPEG-lysozyme (0.063 molecule/nm2). These results indicate the degree of aggregation in an unrinsed layer that contains both the irreversibly and reversibly adsorbed DOI: 10.1021/la102709y
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Table 1. Extent of β-Sheet Formation, ΔIThT/Itrp, and Corresponding % Aggregation for All Species at a Similar Surface Concentration (Γ); % Aggregation at High Surface Concentrations Is Also Reporteda low surface concentration species
Γ molecules/nm2
ΔIThT/Itrp
high surface concentration % agg
Γ molecules/nm2
ΔIThT/Itrp
% agg
unmodified lysozyme 0.020 1.46 ( 0.09 63 0.12 1.62 ( 0.08 70 monoPEG-lysozyme 0.025 1.10 ( 0.07 46 0.063 1.22 ( 0.02 52 diPEG-lysozyme 0.012 0.80 ( 0.03 31 0.021 0.80 ( 0.02 31 a Thioflavin T was added to the irreversibly adsorbed remnant of a pre-adsorbed and rinsed layer in these experiments. The mean values are reported with 95% confidence intervals.
species. This experiment was not conducted for diPEG-lysozyme due to material constraints. Final ΔIThT/Itrp values of 1.22 ( 0.08 and 1.13 ( 0.06 were observed for unmodified lysozyme and monoPEG-lysozyme, respectively. These values correspond to 52% and 48% aggregation for unmodified lysozyme and monoPEG-lysozyme, respectively, as determined by the calibration equation described previously. The fraction of aggregated unmodified and monoPEGylated lysozyme is therefore slightly higher for rinsed, irreversibly adsorbed molecules compared to the unrinsed layer that contains both reversibly and irreversibly adsorbed molecules. This suggests that lateral aggregation does play some role in determining adsorption irreversibility. In these experiments, the thioflavin T fluorescence was also monitored before and during the rinse step. This is discussed in more detail in Supporting Information S3. In all cases, the aggregation percentage was higher after the buffer rinse, suggesting that the protein that remained on the surface was more aggregated on average than the protein that desorbed during the rinse. That is, unaggregated species were more likely to desorb, as would be expected. The effect was most pronounced for unmodified lysozyme which was most prone to surface-induced aggregation, while there was little difference between prerinse and postrinse aggregation for diPEG-lysozyme, which suffered the least surfaceinduced aggregation in general.
Conclusions Lysozyme PEGylation fundamentally alters the adsorption mechanism of this protein to a solid surface. The extent of lysozyme adsorption to PLG films is significantly decreased by PEGylation of the protein. PEGylated lysozyme suffers significantly less surface-induced aggregation and is more reversibly adsorbed than unmodified lysozyme. These effects are more pronounced for diPEGylated lysozyme than for monoPEGylated lysozyme, though neither degree of PEGylation is sufficient to completely eliminate adsorption, or to completely eliminate some irreversible adsorption or surface-induced aggregation. Each
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of the observed effects can be attributed in some part to steric repulsions among grafted PEG chains. Adsorption energies for PEGylated lysozyme conjugates are also weakened by the biased attachment of PEG chains to the N-terminus of lysozyme, which prevents this protein from assuming its preferred orientation on a negatively charged surface. All of the key behaviors observed on moderately hydrophobic, negatively charged PLG surface were similar to observations on hydrophilic, negatively charged silica. PEG is surface active on both of these surfaces, so the similarity in behaviors between these two disparate surfaces suggests that the relative affinities for PEG adsorption versus for lysozyme adsorption are similar on the two materials. The common feature is the presence of a negative surface charge, which evidently dictated the key observations made here. With respect to practical applications, particularly drug delivery, the increased adsorption reversibility and decreased aggregation at the solid PLG/aqueous interface support the notion that PEGylation has the potential to increase the amount of bioactive protein released from degradable PLG microspheres. Practical implications of this work are discussed more thoroughly in Supporting Information S4. Acknowledgment. This material is based on work supported by the National Science Foundation under Grant CBET 0755284. The authors thank TIRF Technologies Inc., Alkermes, Inc., and Dr. Reddy’s Laboratories Ltd. for their generous donations of a TIRF flow cell, PLG, and mPEG-propionaldehyde, respectively. Supporting Information Available: Light scattering and spectroscopic characterization of PEGylated lysozyme conjugates, TIRF probe of lysozyme reorientation on PLG, the relationship between irreversibility and aggregation, and discussion of broader implications for sustained release drug delivery. This material is available free of charge via the Internet at http://pubs.acs.org.
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