Letter pubs.acs.org/macroletters
Tandem Coordination, Ring-Opening, Hyperbranched Polymerization for the Synthesis of Water-Soluble Core−Shell Unimolecular Transporters Chris S. Popeney,†,‡,§ Maike C. Lukowiak,‡,§ Christoph Böttcher,‡ Boris Schade,‡ Pia Welker,∥ Dorothea Mangoldt,∥ Gesine Gunkel,‡ Zhibin Guan,*,† and Rainer Haag*,‡ †
Department of Chemistry, University of California, 1102 Natural Sciences 2, Irvine, California 92697, United States Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany ∥ mivenion GmbH, Robert-Koch-Platz 4, 10115 Berlin, Germany ‡
S Supporting Information *
ABSTRACT: A water-soluble molecular transporter with a dendritic core−shell nanostructure has been prepared by a tandem coordination, ring-opening, hyperbranched polymerization process. Consisting of hydrophilic hyperbranched polyglycerol shell grafted from hydrophobic dendritic polyethylene core, the transporter has a molecular weight of 951 kg/mol and a hydrodynamic diameter of 17.5 ± 0.9 nm, as determined by static and dynamic light scattering, respectively. Based on evidence from fluorescence spectroscopy, light scattering, and electron microscopy, the core−shell copolymer transports the hydrophobic guests pyrene and Nile red by a unimolecular transport mechanism. Furthermore, it was shown that the core−shell copolymer effectively transports the hydrophobic dye Nile red into living cells under extremely high and biologically relevant dilution conditions, which is in sharp contrast to a small molecule amphiphile. These results suggest potential applicability of such core−shell molecular transporters in the administration of poorly water-soluble drugs.
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Specifically, in this study we combined two unique and efficient hyperbranched polymerization methods in tandem: a late transition metal-catalyzed chain walking polymerization (CWP) for generating a highly nonpolar dendritic polyethylene (PE) core, followed by anionic ring-opening polymerization of glycidol to graft a hydrophilic hyperbranched polyglycerol (PG) shell. Previously, our group has prepared core−shell nanocarriers containing preformed polyethylene glycol (PEG) chains introduced either during or after polymerization.15,16 The current PE-PG nanocarrier offers a number of unique advantages over these and other previous systems. First, both polymerization methods involved in the synthesis are highly versatile. As we have shown previously, both the size and the branching topology of the polyethylene core can be easily tuned by CWP.17 Meanwhile, the density and thickness of the PG shell can also be controlled. Second, the polarity gradient between the hydrophobic polyolefin core and the hydrophilic polyglycerol shell is high, which should enhance the drug loading efficacy and favor unimolecular transport. Furthermore, PG has been shown previously to be biocompatible and protein resistant,18 which are important for in vivo administration. Lastly, the surface hydroxyl groups of the PG shell offers convenient sites for further functionalization of various
he poor solubility of hydrophobic compounds into aqueous phases is a major limitation for their efficient dispersal and biomedical applications.1,2 The delivery of hydrophobic drugs into the human body is one particularly important example.3 Amphiphiles, composed mainly of smallmolecule or block copolymer based surfactants, find widespread use for this purpose, owing to their supramolecular selfassembly into micelles or vesicles.4,5 However, due to their supramolecular nature, these assemblies will dissociate and their carrier capabilities will be lost upon high dilution. As a result, they often lack the long-term stability necessary in many situations. Amphiphilic unimolecular transporters avoid this problem as they resemble micelles that are covalently held together3,6,7 and are attractive for use in biomedicine because of their stability in the high dilution environments of living systems.8 Despite the recent progress in this field,9,10 the synthesis of dendritic unimolecular nanocarriers is generally tedious and many of the systems studied are not soluble in water.1,11,12 Lastly, there remain questions on the mechanism of guest transport, as some systems exhibit encapsulation of the guest within large supramolecular aggregates of the transporter rather than by the unimolecular route.13,14 Herein, we report the first tandem coordination, ring-opening, hyperbranched polymerization to access biocompatible core−shell nanoparticles having a high polarity gradient and behaving as unimolecular transporters for hydrophobic compounds in water. © 2012 American Chemical Society
Received: February 20, 2012 Accepted: April 4, 2012 Published: April 13, 2012 564
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bioconjugates, such as for the attachment of specific cell targeting ligands.19 The synthesis (Scheme 1) consists of an initial copolymerization of ethylene and a siloxy-functionalized comonomer Scheme 1. Preparation of Core−Shell Copolymer 2
Figure 1. (a) Particle diameter distributions by volume from DLS of pure copolymer 2 (blue curve) and copolymer 2 with encapsulated pyrene in water (red curve). (b) Schematic of pyrene transport within the core of copolymer 2. (c) Cryo-TEM images of vitrified aqueous solutions of pure copolymer 2. (d) Cryo-TEM images of vitrified aqueous solutions of copolymer 2 with encapsulated pyrene. The full field images can be found as Figures S7 and S8.
under low ethylene pressure conditions20 using Brookhart’s Pd(II) α-diimine CWP catalyst.21−23 Deprotection of the siloxy group afforded hydroxyl-functionalized hyperbranched copolymer 1, which was subsequently used as a macroinitiator for the anionic ring-opening polymerization of glycidol.24 This process resulted in core−shell copolymer 2, which consisted of hydrophilic hyperbranched PG grafted onto a core of hydrophobic dendritic PE. Copolymer 2 is water-soluble and transports the hydrophobic dyes pyrene and Nile red by a unimolecular mechanism within the hydrophobic core. Copolymer 1 is typical of CWP under these conditions (0.1 atm of ethylene pressure), with a number-averaged molecular weight Mn of 191 kg/mol and polydispersity (Mw/Mn) of 1.7, as determined by gel permeation chromatography and multiangle laser light scattering (GPC-MALLS) in THF. Based on 1H NMR spectroscopy, it consists of 13.1 mol % of OH-bearing comonomer, corresponding to almost 640 OH groups per PE molecule. Due to its low polarity, copolymer 1 is soluble only in nonpolar solvents. Following the grafting of PG, a bimodal molecular weight distribution was obtained, which we attribute to self-initiated PG. At the cost of a reduction in copolymer 2 recovery, dialysis in methanol through large pores (MWCO = 300 kDa) was employed to effectively remove this PG homopolymer. The resulting molecular weight distribution by GPC-MALLS was monomodal (Figure S1). In contrast to copolymer 1, copolymer 2 is soluble only in water and methanol. Analysis of the molecular weight of copolymer 2 by GPC-MALLS shows it to have gained considerable mass in the grafting reaction (Mn = 951 kg/mol, Mw/Mn = 1.5). Its composition was determined by 1H NMR spectroscopy to consist of a 1.51:1 molar ratio of glycerol to olefin (either comonomer) (Figure S2), corresponding to an average of 11.6 polymerized glycerol monomer units per OH group. Further characterization by dynamic light scattering (DLS) in water revealed particles of 17.5 ± 0.9 nm in diameter (Figure 1a). Images of cryogenically vitrified solutions of copolymer 2, as
observed by transmission electron microscopy (cryo-TEM), also confirmed the presence of particles in this size range (Figure 1c). With the amphiphilic core−shell copolymer 2 in hand, we investigated its capability as molecular transporters for hydrophobic compounds. Two commonly used hydrophobic fluorescent dyes, pyrene and Nile red, were used in the current study. In uptake studies, copolymer 2 was found to be effective in the solubilization of pyrene. Transport was performed by overnight agitation of solid pyrene in a solution of the copolymer in water followed by filtration. Pyrene absorbance (Figure 2a,b) increased linearly with concentration of copolymer 2. From the absorbance data, a loading of pyrene in the copolymer of 4.0 mg/g, corresponding to 19 pyrene molecules per copolymer, was achieved. Analysis of the resulting solutions by DLS indicated particles measuring 17.7 ± 0.3 nm in diameter (Figure 1a). These sizes are comparable to the sizes obtained for the pure copolymer. Furthermore, as shown by cryo-TEM, no appreciable change in appearance was noted between the copolymer particles with encapsulated pyrene and the pure copolymer (Figure 1c and d). Both the DLS and the cryo-TEM data support that encapsulation occurs unimolecularly within the PE core of the copolymer (Figure 1b). Based on the well-known sensitivity of pyrene fluorescence on solvent polarity,25 detailed information regarding the local environment of encapsulated pyrene molecules was gathered by analysis of the fluorescence of pyrene/2 solutions (Figure 2c). The intensity ratio (I3/I1) of the third emission band to the first band (at 385 and 373 nm, respectively) is a frequently used quantity, with higher values indicating increasingly nonpolar environments. A value of 1.08 was reached at copolymer concentrations of 1.0 mg/mL (Figure 2d), which is indicative of an environment of relatively low polarity. Among common solvents compositionally similar to copolymer 2, this value most approximates that of isopropanol (1.10), 1-pentanol 565
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Figure 2. (a) Absorbance spectra of pyrene encapsulated in various concentrations (in mg/mL) of copolymer 2 and (b) plot of pyrene absorbance at 337.5 nm as a function of copolymer 2 concentration. (c) Fluorescence of pyrene with intensity normalized at the first emission line (constant I1) at different concentrations of copolymer 2 (in mg/mL) and (d) plot of I3/I1 ratios as a function of copolymer 2 concentration.
Figure 3. (a) Structure of amphiphile 3 and NR. (b) Image of A549 cells at 650× magnification showing the fluorescence of NR (red) and DAPI nuclear stain (blue) for cells incubated with copolymer 2 at 1.0 × 10−7 M and NR. (c) Corresponding image of same cell line incubated with amphiphile 3 at 2.1 × 10−5 M and NR. Images showing NR fluorescence alone can be seen in Figure S11.
(1.07), and diethyl ether (1.02).25 Although the ratio is below that typical of pure aliphatic hydrocarbons (1.65−1.8), it is significantly above that of ethylene glycol (0.63) and methanol (0.75), which closely resemble the PG shell compositionally. A more relevant comparison can be made to other nanocarrier systems. Within micelles of sodium dodecyl sulfate, an I3/I1 ratio of 0.96 has been reported.25 Brooks and coworkers have reported systems composed of PG with a hydrophobicized interior that exhibited ratios of only 0.81, indicating significantly higher overall polarity compared to our PE−PG system.26 Mecking and co-workers reported a ratio identical to ours, of 1.08, for amorphous PE nanoemulsions,27 which is reasonable considering the similar composition of their system and the core of copolymer 2. Based on comparison of our data with the results from other transporters, we conclude that the encapsulated pyrene molecules reside in the relatively nonpolar environment of the PE core of copolymer 2. The transport of the dye Nile red (NR) was also achieved using copolymer 2 following similar agitation treatments. Analysis of particle sizes by DLS revealed sizes of 16.4 ± 0.8 nm (Figure S3), which is comparable to the copolymer without guests and with encapsulated pyrene. From absorbance data, a loading of 0.16 mg NR per gram 2, or 0.49 mols of NR per mole of 2, was determined (Figure S4). The fluorescence of NR increased linearly with copolymer concentration, which gives further evidence in support of unimolecular transport (Figures S5 and S6). Lastly, no appreciable difference in the appearance of copolymer 2 particles was discernible by cryo-TEM, whether or not NR or pyrene was transported (Figures S7−9). From all of these observations, we can rule out a supramolecular or aggregate-based mechanism for pyrene and Nile red transport for copolymer 2. We have previously demonstrated the uptake of PG derivatives into cells.28 To demonstrate the applicability of unimolecular transport in dilute biological environments, we compared the uptake of NR into living cells using copolymer 2 as transporter with that of a compositionally similar low molecular weight amphiphile 3 (Figure 3a).29 Following
encapsulation with NR at high concentration, the copolymer 2/NR mixture was diluted with cell growth media so that the concentration of 2 was 10−7 M, while the amphiphile 3/NR mixture was similarly diluted to 2.1 × 10−5 M of 3 (well below its critical micelle concentration of 6.4 × 10−4 M). We expected the concentration of NR in both of these mixtures, following dilution, to be equivalent based on the calculated loadings of NR in both transport systems (2.28 mmol of NR per mole of 3, for example, see Figure S10), which allows a meaningful comparison in cell fluorescence studies. A549 lung tumor cells were incubated in both mixtures for 1 h and the cells were then visualized by fluorescence microscopy. The results clearly indicated that successful uptake of NR into the cells was aided by copolymer 2 (Figure 3b). Furthermore, the NR dye appeared to be well distributed within the interior of the cells and not just within the cell membrane. Fluorescence appeared to be absent in the vicinity of the cell nuclei, which was not expected if the dye was confined to the external membrane only. These observations suggest an endocytosis process. In contrast, only weak NR fluorescence was seen after the cells were incubated with the diluted amphiphile 3 mixture (Figure 3c), in which the micelles solubilizing and transporting the NR had disassembled. The dye uptake into the cells was also quantitatively measured by fluorescence-activated cell sorting (FACS). From cytometry measurements the cellular fluorescence was more than twice as intense after incubation with copolymer 2/ NR than with a similar treatment with the amphiphile 3/NR mixture. Measured cell fluorescence values from the FACS analysis can be seen in Table S1. From both of these experiments, it is clear that core−shell copolymer 2 remains an effective transporter of NR into cells at high dilution because of its covalently linked unimolecular structure. Amphiphile 3, however, does not form micelles at such low concentrations which limits the transport of NR to the cells because of poor dye solubility and cell accessibility. The results of these uptake experiments with NR provide definitive support for the use of 566
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(7) Hawker, C. J.; Wooley, K. L.; Frechet, J. M. J. J. Chem. Soc., Perkin Trans. 1 1993, 1287−1297. (8) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99, 3181−3198. (9) Lee, C. C.; MacKay, J. A.; Fréchet, J. M. J.; Szoka, F. C., Jr. Nat. Biotechnol. 2005, 23, 1517−1526. (10) Gupta, U.; Agashe, H. B.; Asthana, A.; Jain, N. K. Biomacromolecules 2006, 7, 649−658. (11) Stevelmans, S.; van Hest, J. C. M.; Jansen, J. F. G. A.; van Boxtel, D. A. F. J.; van den Berg, E. M. M. D.; Meijer, E. W. J. Am. Chem. Soc. 1996, 118, 7398−7399. (12) Krämer, M.; Stumbé, J.-F.; Türk, H.; Krause, S.; Komp, A.; Delineau, L.; Prokhorova, S.; Kautz, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 4252−4256. (13) Radowski, M. R.; Shukla, A.; von Berlepsch, H.; Böttcher, C.; Pickaert, G.; Rehage, H.; Haag, R. Angew. Chem., Int. Ed. 2007, 46, 1265−1269. (14) Kurniasih, I. N.; Liang, H.; Rabe, J. P.; Haag, R. Macromol. Rapid Commun. 2010, 31, 1516−1520. (15) Chen, G.; Guan, Z. J. Am. Chem. Soc. 2004, 126, 2662−2663. (16) Chen, G.; Huynh, D.; Felgner, P. L.; Guan, Z. J. Am. Chem. Soc. 2006, 128, 4298−4302. (17) Guan, Z.; Cotts, P. M.; McCord, E. F.; McLain, S. J. Science 1999, 283, 2059−2062. (18) Weinhart, M.; Becherer, T.; Haag, R. Chem. Commun. 2011, 47, 1553−1555. (19) Calderon, M.; Quadir, M. A.; Sharma, S. K.; Haag, R. Adv. Mater. 2010, 22, 190−218. (20) Chen, G.; Ma, X. S.; Guan, Z. J. Am. Chem. Soc. 2003, 125, 6697−6704. (21) Johnson, L. K.; Killian, C. M.; Brookhart, M. J. Am. Chem. Soc. 1995, 117, 6414−15. (22) Johnson, L. K.; Mecking, S.; Brookhart, M. J. Am. Chem. Soc. 1996, 118, 267−8. (23) Mecking, S.; Johnson, L. K.; Wang, L.; Brookhart, M. J. Am. Chem. Soc. 1998, 120, 888−899. (24) Sunder, A.; Hanselmann, R.; Frey, H.; Mü lhaupt, R. Macromolecules 1999, 32, 4240−4246. (25) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039−2044. (26) Kainthan, R. K.; Mugabe, C.; Burt, H. M.; Brooks, D. E. Biomacromolecules 2008, 9, 886−895. (27) Tong, Q.; Krumova, M.; Gottker-Schnetmann, I.; Mecking, S. Langmuir 2008, 24, 2341−2347. (28) Reichert, S.; Welker, P.; Calderon, M.; Khandare, J.; Mangoldt, D.; Licha, K.; Kainthan, R. K.; Brooks, D. E.; Haag, R. Small 2011, 7, 820−829. (29) Trappmann, B.; Ludwig, K.; Radowski, M. R.; Shukla, A.; Mohr, A.; Rehage, H.; Böttcher, C.; Haag, R. J. Am. Chem. Soc. 2010, 132, 11119−11124.
unimolecular transport systems for the uptake of hydrophobic targets into cells. In conclusion, we report the first tandem coordination, ringopening, hyperbranched polymerization for the efficient synthesis of a unimolecular core−shell nanocarrier derived from a dendritic polyethylene core and a hyperbranched polyglycerol shell. The dendritic core−shell nanostructure was prepared from a straightforward and versatile two-step polymerization process that avoids tedious chemical transformations. The core−shell nanoparticle has a high internal polarity gradient and is capable of transporting poorly watersoluble hydrophobic guests into aqueous solution. In addition, we have unambiguously demonstrated the unimolecular transporting behavior of copolymer 2 using a combination of absorbance and fluorescence spectroscopy, light scattering, and electron microscopy. Lastly, we have shown the advantages of unimolecular transport in the delivery of hydrophobic guests, a fluorescent dye in this case, to living cells under highly dilute conditions. Thus, these core−shell unimolecular nanocarriers are promising candidates for formulation and delivery of poorly water-soluble active agents across cellular membranes.
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ASSOCIATED CONTENT
S Supporting Information *
Detailed experimental procedures, including polymer synthesis and characterization by SEC-MALLS, NMR spectra, DLS, and cryo-TEM, fluorescence and loading determinations, and FACS data and fluorescence microscopy. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (R.H.);
[email protected] (Z.G.). Tel.: +49 30 838 53357 (R.H.); +1 949 824 2210 (Z.G.). Author Contributions §
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the U.S. National Science Foundation (CHE0723497) and the Deutsche Forschunsgemeinschaft (DFG HA 2549/10-1) under joint grant for funding. Z.G. acknowledges an Alexander von Humboldt fellowship. M.L. thanks Dr. Guobin Sun and Florian Paulus for assistance and the Frauenförderung der FU Berlin for financial assistance. C.P. thanks Michal Radowski for helpful discussions and the Allergan Foundation, the UCI Department of Chemistry, and Joan Rowland for financial assistance.
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REFERENCES
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