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Notes Synthesis and Characterization of Peptide-Functionalized Polymeric Nanoparticles Shelby Taylor,† Liangwei Qu,† Alex Kitaygorodskiy,† Jesse Teske,† Robert A. Latour,‡ and Ya-Ping Sun*,† Department of Chemistry, Howard L. Hunter Chemistry Laboratory, and Department of Bioengineering, Rhodes Hall, Clemson University, Clemson, South Carolina 29634 Received August 31, 2003 Revised Manuscript Received October 29, 2003
Introduction There have been many studies of polymeric nanoparticles as carriers for biomolecules such as proteins and peptides. Among widely discussed applications are targeted drug delivery, controlled releases, bioseparations, and immunoassays and immunodiagnostics.1-12 For drug delivery, as an example, the nanoparticles have been shown to be able to protect drugs from degradation and thus to increase their bioavailability, to modify the biodistribution, and to carry drugs across the blood-brain barrier.1-4,7 A variety of polymeric nanoparticles have been prepared for such purposes. The design principle and architecture of the nanoparticles used in drug delivery are often shared by other biofunctionalized polymeric nanoparticle systems for different applications. In some of these systems, the conjugation of biomolecules such as proteins and peptides with the synthetic polymeric nanoparticles represents an important issue.10,12 Typically, the proteins and peptides are loaded onto the polymeric nanoparticles via either encapsulation or physical adsorption. For some applications, however, it is more advantageous for the biomolecules to be attached covalently to the polymeric nanoparticles. For example, Bastos-Gonzalez et al. used carboxylated latexes for covalent coupling with antibodies in immunoassays.10 The covalent attachments prevent the desorption of the biomolecules and allow the biomolecules to be directed toward the solution (thus more accessible by other species in the solution).12 Here, we report on the synthesis of polystyrene copolymerbased nanoparticles with carboxylic acid-terminated oligomeric poly(ethylene glycol) (PEG) chains as surface functionalities and the covalent coupling of the synthetic tripeptide, enkephalin 1-3 (Tyr-Gly-Gly), onto the nanoparticles. The tripeptide was selected as a relatively simple model compound to allow a study of the biofunctionalization by using conventional instrumental techniques. The polymeric nanoparticles before and after the peptide functionalization were * Corresponding author. † Department of Chemistry. ‡ Department of Bioengineering.
characterized by dynamic light scattering, transmission electron microscopy (TEM), NMR, and the modified Lowry assay. Experimental Section Materials. Enkephalin 1-3 (Tyr-Gly-Gly) was purchased from Sigma, N-hydroxysuccinimide (93+%) and MES hydrate (C6H13NO4SCH2O, 99%) from Acros, azobisisobutyronitrile (AIBN, 98%) from Aldrich, 1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride (EDAC) from Alfa Aesar, and ethanol from Fisher. Deuterated water (99.9%) and chloroform (99.8%) for NMR experiments were supplied by Cambridge Isotope Laboratories. Biuret reagent and Folin and Ciocalteu’s phenol reagent for the modified Lowry assay were obtained from Sigma. Measurements. Dynamic light scattering measurements were carried out on a Coulter N4 Plus particle sizer. NMR measurements were performed on a JEOL Eclipse +500 NMR spectrometer. TEM analyses were conducted on a Hitachi H7000 TEM system. UV/vis absorption spectra were recorded on a Shimadzu 3100 spectrophotometer. Nanoparticles. The polystyrene-PEG nanoparticles were prepared via the dispersion polymerization of the macromonomer 1 or 2 with styrene.13 Details on the synthesis of 1 and 2 are provided in the Supporting Information. In a typical reaction, styrene (1.042 g, 10 mmol) and 1 (1.1 g, 0.5 mmol) were added to an ethanol/water mixture (4/1, 25 mL). The mixture was cooled to 0 °C, and the polymerization initiator AIBN (16 mg, 0.1 mmol) was added to the mixture. Upon degassing, the mixture was stirred at 60 °C for 24 h, yielding a milky suspension. The suspension was transferred into a membrane tubing (cutoff molecular weight 12 000) for dialysis against fresh deionized water for 3 days to yield a purified aqueous suspension of the nanoparticles I. The same procedures were used for the copolymerization of 2 with styrene and for the purification to yield a purified aqueous suspension of the nanoparticles II. Functionalization with Peptide. MES hydrate was added to an aqueous suspension of the nanoparticles (10 mL, 5.2 wt % nanoparticles, 3 mol % -COOH on the nanoparticles according to NMR signal integration) to result in the buffer condition (0.1 M) of pH equal to 5.6, followed by the addition of N-hydroxysuccinimide (15 mg) and EDAC (30 mg). The suspension was stirred at room temperature for 3 h. The mixture was centrifuged to remove the MES buffer (along with the soluble reagents such as excess EDAC), washed with a phosphate buffer twice, and suspended in the phosphate buffer (10 mL). To the suspension was added enkephalin 1-3 (60 mg, 0.2 mmol). The resulting suspension was stirred at room temperature for 24 h, followed by dialysis
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Notes
Table 1. Parameters of the Nanoparticles average diametera before functionalization
after functionalization
nanoparticle
light scattering (nm)
TEM (nm)
light scattering (nm)
I II
175(25) ∼190
160(18) 180(20)
174(21) 190(60)
a
The number in parentheses is the size distribution standard deviation.
(membrane tubing cutoff molecular weight 12 000) against fresh deionized water for 3 days. Results and Discussion The dispersion copolymerizations of 1 and 2 with styrene yielded polymeric nanoparticles I and II, respectively, in aqueous suspensions. The suspensions appeared milky but remained stable (without precipitation) for as long as they were kept (at least 6 months). Each nanoparticle is expected to have a polystyrene core and a shell of PEG chains that are terminated with carboxylic acids. Such a structural arrangement is likely responsible for the stability of the aqueous suspensions.14,15 The dynamic light scattering results of the suspensions are shown in Table 1. These nanoparticles are apparently of relatively narrow size distributions. The formation of nanoparticles and their average sizes and size distributions are confirmed by the results from TEM characterizations. The TEM specimen was prepared by depositing a small drop of the nanoparticle suspension onto a carbon-coated copper grid, followed by the evaporation of the solvent. Shown in Figure 1 is a typical TEM image of the nanoparticle sample II. The halo surrounding each particle may be attributed to the PEG shell. The average sizes determined from the TEM images are slightly smaller than those obtained from the dynamic light scattering experiments (Table 1). This seems understandable because the dynamic light scattering measures hydrodynamic volumes of the nanoparticles, corresponding
Figure 1. A TEM image of nanoparticles II before peptide attachment.
to a more expanded surface structure of the nanoparticles in a suspension. Between the two nanoparticle samples, the difference in their average sizes has to do with the PEG chain length in the starting macromonomers. The macromonomer 1 with a longer PEG chain corresponds to smaller particles, while the macromonomer 2 of a shorter PEG chain corresponds to larger particles.13 The functionalization of the polymeric nanoparticles with the peptide Tyr-Gly-Gly resulted in no meaningful changes in the size characterizations. The dynamic light scattering and TEM results are essentially unchanged from those of the nanoparticles before the peptide functionalization. This also seems reasonable considering the small size of the peptide. The nanoparticle samples can be dissolved in organic solvents such as chloroform, resulting in the destruction of the nanoparticles because of the solubility of the polystyrene copolymers. The resulting homogeneous solutions in deuterated chloroform were used in NMR measurements. However, the solution-phase 1H NMR spectra thus obtained are hardly informative because they are overwhelmed by the extremely broad signals in the aromatic region arising from
Notes
Figure 2. 1H NMR spectra in the aromatic region for enkephalin 1-3 in D2O solution (top) and for enkephalin-functionalized nanoparticles I in a concentrated aqueous suspension (bottom).
polystyrene, as well as broad peaks resulting from the polymer corona. There are additional problems associated with the difference in solubility between the polystyrene core and the PEG-peptide shell in organic solvents.16,17 The relatively low peptide content overall in the nanoparticle sample also makes the solution-phase NMR characterization virtually impossible. Thus, the gel-phase NMR approach18-20 was employed to characterize the peptide-functionalized polymeric nanoparticles in an aqueous suspension. In principle, the gel-phase NMR technique allows selective detection of the more mobile PEG-peptide moieties on the relatively stationary polymeric nanoparticle core. The gel-phase NMR measurements were carried out with the same NMR probe as that for solutions, except that highly concentrated nanoparticle suspensions were used.21 As shown in Figure 2, the proton NMR spectrum of the peptide-attached nanoparticles I in an aqueous (D2O) suspension consists of broad signals in the aromatic region in addition to the strong peaks associated with the protons in the PEG corona of the nanoparticles, but there are no meaningful contributions from the polystyrene core. The aromatic signals are attributed to the phenyl protons in the tyrosine unit of the peptide, in comparison with the sharp signals observed in the same region for the peptide in solution (Figure 2). In the gel-phase 1 H NMR spectrum of nanoparticles I without peptide attachment, there are the same strong peaks associated with the protons in the PEG corona of the nanoparticles but no aromatic signals. The comparison of 1H NMR results in Figure 2 provides strong evidence that the peptide species are indeed attached to the polymeric nanoparticles. More quantitatively, the peptide species attached to the polymeric nanoparticles were analyzed by using the modified Lowry assay.22,23 The assay is commonly used in the determination of peptide and protein contents through targeting the tyrosine or tryptophan moiety. The testing procedure involves the mixing of the specimen with the Biuret reagent and then Folin and Ciocalteu’s phenol reagent.
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The tyrosine or tryptophan moiety is detected when the colorless solution turns blueish green, the extent of the color change (absorbance around 725 nm in the visible absorption spectrum) corresponding to the tyrosine or tryptophan concentration. The presence of a tyrosine unit in enkephalin 1-3 makes the peptide-functionalized polymeric nanoparticles responsive to the assay. A standard curve was obtained by using solutions of free peptide. To match the effects of light scattering on the absorption measurements, these solutions for the standard curve were also added with the same amount of unfunctionalized nanoparticles as the estimated nanoparticle content in the sample solution for determination. The result thus obtained for the peptide-functionalized nanoparticles I is 0.01 mg of peptide per mg of nanoparticles. By using the average diameter obtained from the TEM analysis (Table 1) for the spherical polystyrene core, with the same density of 1.047 g/cm3 as for bulk polystyrene, we estimated the average number of PEG tethers per nanoparticle and the average total mass per nanoparticle as 395 000 tethers/particle and 3.6 × 10-12 mg/particle, respectively. Thus, the average number of peptide species per nanoparticle is 73 000, which corresponds to ∼20% of the PEG tethers (-COOH heads) on the nanoparticle. The polymeric nanoparticles with tethers terminated with carboxylic acid groups represent an excellent platform for the covalent attachment of bioactive species, such as the peptide molecules reported here. The same carbodiimideactivated coupling reaction has been used for introducing other peptide moieties onto the polymer particle surface.24,25 For example, Melamed and Margel reported on the covalent binding of trypsin to poly(N-vinyl R-amino acid) microspheres.24 An obvious challenge has been the detailed characterization of the particle-bound biofunctional groups. As demonstrated here, the gel-phase NMR technique is a valuable tool in this regard, at least for the polymeric nanoparticles with tethered biofunctional groups. The traditional bioanalytical methods such as the modified Lowry assay are still applicable under the conditions of suspended polymeric particles,24 making it possible to quantify the population of the nanoparticle-bound bioactive species. In summary, we have shown the use of derivatized styrene macromonomers in the dispersion copolymerization with styrene to form nanoparticles of a polystyrene core and oligomeric PEG surface tethers with carboxylic acid headgroups. The dynamic light scattering and TEM results confirm the nanoparticle formation. The tethered carboxylic acid groups can be activated by carbodiimide to react with enkephalin peptide molecules. The gel-phase NMR and the modified Lowry assay results support the conclusion that the peptide molecules are covalently attached to the polymeric nanoparticles. Acknowledgment. We thank Weijie Huang, Mohammed Meziani, Bing Zhou, Yi Lin, and Huaping Li for experimental assistance. Financial support from USDA and NSF is gratefully acknowledged. J.T. is a participant of the Summer Undergraduate Research Program sponsored jointly by NSF (Grant REU/DMR-0243734) and Clemson University.
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Supporting Information Available. Details on the synthesis of 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Allemann, E.; Leroux, J.-C.; Gurny, R. AdV. Drug DeliVery ReV. 1998, 34, 171. (2) Sakuma, S.; Mayashi, M.; Akashi, M. AdV. Drug DeliVery ReV. 2001, 47, 21. (3) Takeuchi, H.; Yamamoto, H.; Kawashima, Y. AdV. Drug DeliVery ReV. 2001, 47, 39. (4) (a) Schroeder, U.; Sabel, B. A. Brain Res. 1996, 710, 121. (b) Schroeder, U.; Sommerfeld, P.; Ulrich, S.; Sabel, B. A. J. Pharm. Sci. 1998, 87, 1305. (c) Schroeder, U.; Sommerfeld, P.; Sabel, B. A. Peptides 1998, 34, 777. (5) Peracchia, M. T.; Gref, R.; Minamitake, Y.; Domb, A.; Lotan, N. Langer, R. J. Controlled Release 1997, 46, 223. (6) Cremaschi, D.; Porta, C.; Ghirardelli, R. Biochim. Biophys. Acta 1999, 1416, 31. (7) Tobio, M.; Sanchez, A.; Vila, A.; Soriano, I.; Evora, C.; Vila-Jato, J. L.; Alonso, M. J. Colloids Surf., B 2000, 18, 315. (8) Li, Y.-P.; Pei, Y.-Y.; Zhang, X.-Y.; Gu, Z.-H.; Zhou, Z.-H.; Yuan, W.-F.; Zhou, J.-J.; Zhu, J.-H.; Gao, X.-J. J. Controlled Release 2002, 71, 203. (9) Gibanel, S.; Heroguez, V.; Gnanou, Y.; Aramedia, E.; Bucsi, A.; Forcada, J. Polym. AdV. Technol. 2001, 12, 494. (10) Bastos-Gonzales, D.; Ortega-Vinuesa, J. L.; De Las Nieves, F. J.; Hidalgo-Alvarez, R. J. Colloid Interface Sci. 1995, 176, 232. (11) Ortega-Vinuesa, J. L.; Bastos-Gonzales, D.; Hidalgo-Alvarez, R. J. Colloid Interface Sci. 1995, 176, 240. (12) Peula, J. M.; Hidalgo-Alvarez, R.; de las Nieves, F. J. J. Colloid Interface Sci. 1998, 201, 139.
Notes (13) (a) Chen, M.-Q.; Serizawa, T.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 2155. (b) Serizwa, T.; Takehara, S.; Akashi, M. Macromolecules 2000, 33, 1795. (14) (a) Akashi, M.; Kirikihira, I.; Miyauchi, N. Angew. Makromol. Chem. 1985, 132, 81. (b) Akashi, M.; Chao, D.; Yashima, E.; Miyauchi, N. J. Appl. Polym. Sci. 1990, 39, 2027. (c) Capek, I.; Riza, M.; Akashi, M. Makromol. Chem. 1992, 193, 2843. (15) (a) Kawaguchi, S.; Winnik, M. A.; Ito, K. Macromolecules 1995, 28, 1159. (b) Shay, J. S.; English, R. J.; Spontak, R. J.; Balik C. M.; Khan, S. A. Macromolecules 2000, 33, 6664. (16) Carignano, M. A.; Szleifer, I. J. Chem. Phys. 1994, 100, 3210. (17) Cosgrove, T.; Crowley, T. L.; Ryan, K.; Webster, J. R. P. Colloids Surf. 1990, 51, 255. (18) Hrkach, J. S.; Peracchia, M. T.; Domb, A.; Lotan, N.; Langer, R. Biomaterials 1997, 18, 27. (19) Yamamoto, Y.; Yasugi, K.; Harada, A.; Nagasaki, Y.; Kataoka, K. J. Controlled Release 2002, 82, 359. (20) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Langmuir 2002, 18, 3669. (21) The results obtained from gel-phase 1H NMR measurements of the same samples using a magic-angle-spin probe at the Bruker BioSpin service center are not different from those reported here in any meaningful way. (22) Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (23) Ohnishi, S. T.; Barr, J. K. Anal. Biochem. 1978, 86, 193. (24) Melamed, O.; Margel, S. Colloids Surf., A 2002, 208, 147. (25) Banerjee, P.; Irvine, D. J.; Mayes, A. M.; Griffith, L. G. J. Biomed. Mater. Res. 2000, 50, 331.
BM034326M
