Article pubs.acs.org/Macromolecules
Self-Assembly of Chiral Supramolecular Ureido-Pyrimidinone-Based Poly(ethylene glycol) Polymers via Multiple Pathways Mellany Ramaekers,†,§ Isja de Feijter,†,§ Paul H. H. Bomans,†,∥ Nico A. J. M. Sommerdijk,†,∥ Patricia Y. W. Dankers,*,†,§ and E. W. Meijer*,†,‡,§ †
Institute for Complex Molecular Systems, ‡ Laboratory of Macromolecular and Organic Chemistry, §Laboratory of Chemical Biology, and ∥Laboratory for Macromolecular and Organic Chemistry, and Soft Matter Cryo-TEM Research Unit. Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands S Supporting Information *
ABSTRACT: The recently developed supramolecular hydrogelator system based on poly(ethylene glycols) end-functionalized with ureido-pyrimidinone (UPy) units has been shown to be eminently suitable as a drug delivery vehicle in soft tissues such as the heart and kidney. To understand the assembly behavior of this system in more detail, we here report on the introduction of a stereogenic center. This allowed for the investigation of the self-assembly mechanism of this system by circular dichroism, which showed the presence of helical fibers. Additionally, fluorescence spectroscopy and scattering techniques in combination with cryoTEM showed elongated rod-like structures as the major species, next to spherical micelles. Interestingly, different self-assembly pathways occurred when using two aggregate preparation methods based on different cooling rates. Both positive and negative bisignate Cotton effects were observed only by changing the method of preparation, indicating that the UPy-polymer constructs self-assemble via multiple pathways. A similar phenomenon is observed in biology, which illustrates the versatility of the system. This versatility is key to the optimization of material properties for biomedical applications.
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INTRODUCTION
diimide/peptide amphiphilic conjugates, forming different nanofibers.31 Furthermore, supramolecular systems in water are proposed to be eminently suitable for their use as biomaterials in the field of regenerative medicine. Recently, studies of peptide amphiphiles showed progress in spinal cord injury treatment, hearth tissue regeneration and replacement, and induction of angiogenesis.32 In addition, it has been demonstrated that scaffolds of molecular peptide gels, built up of alternating positive and negative L-amino acids, can be used for the regrowth of nerve cells.33 All these examples show how progress in understanding the self-assembled structures helps to develop applications in regenerative medicine. Our goal is to investigate a supramolecular system applicable in regenerative medicine and study its self-assembly behavior to obtain structure−property relationships which are important for the design of biomaterials. We recently developed a ureido−pyrimidinone- (UPy-) based poly(ethylene glycol) (PEG) system that forms a transient network in water.34 In this system, the UPy-units dimerize by 4-fold hydrogen bonding and stack laterally by
Studies of self-assembling systems in an aqueous environment are increasingly popular because nature is based on such complex self-assembled structures.1,2 However, far fewer examples exist of synthetic supramolecular structures that self-assemble in water3−10 as opposed to organic solvents.11−24 An example of a water-soluble structure is the amphiphilic perylene derivative, composed of a hydrophobic aromatic core and four hydrophilic oligo(ethylene glycol) arms, which selfassembles into monomolecular wires.6 Control of the physical and biological properties of peptide amphiphiles based on a hydrophobic alkyl tail and a hydrophilic peptide head, when arranged in nanofibers, were investigated for the use as drug delivery agents.25,26 In another study, the self-assembly mechanism of a 20-residue peptide (MAX1) was elucidated, which composed of intramolecular folding into β-hairpins and cluster formation into nanofibrils.27 By using chirality as a probe to study self-assembly and the relation between microscopic organization and macroscopic structure, new insights into growth mechanisms were gained.28−30 For example, a supramolecular sandwich structure of L-glutamic acid derivatives and bipyridines led to twist formation into chiral ribbons.28 Control over self-assembly pathways into kinetically trapped aggregates was obtained with perylene © XXXX American Chemical Society
Received: March 25, 2014 Revised: May 25, 2014
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Scheme 1. Synthetic Route to Telechelic PEGs (Mn: 10 000 g/mol) Functionalized with Alkyl Spacers and Chiral Dimethylheptyl−UPy Groups
Figure 1. CryoTEM images of a 0.09 mM solution of compound 4a in ultra pure water (gold particles added for tomography). (a) Scale bar represents 200 nm. (b) Magnification of Figure 4a. A striped dark and light gray pattern can be observed indicating a helical pitch. Some micelles are indicated with arrows. Scale bar represents 100 nm.
tyl−UPy−PEG polymers were synthesized, and their behavior was investigated using cryoTEM, DLS, small-angle X-ray scattering (SAXS), CD, UV−vis, and fluorescence spectroscopy. Furthermore, different methods of preparation of the supramolecular fibers were investigated to elucidate the dynamics of the system on the final structure and ordering.
additional urea motifs in a hydrophobic core formed by an alkyl spacer.35 These UPy-based polymers form long, well-defined fibers and small spherical micelles as shown by cryogenic transmission electron microscopy (CryoTEM) and dynamic light scattering (DLS). However, knowledge on the molecular ordering inside the self-assembled structures was lacking. Low weight percentage (5 wt %) networks in water were obtained by mixing of monofunctional and bifunctional UPy−PEG polymers, and the resulting self-assembled architectures were tuned in a dual-fiber network by changing the ratio of the composition.36 Our group investigated several applications of this UPy−PEG based system in regenerative medicine, as drug delivery vehicle for growth factors injection into infarcted pig hearts,37 and into ischemic rat kidneys.34 Here we report on the introduction of a stereogenic center to study the self-assembly behavior of UPy−PEG supramolecular polymers in more detail by means of circular dichroism (CD), in order to obtain structural information on the molecular ordering in the previously mentioned fibers. Both (R)- and (S)-dimethylhep-
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RESULTS Synthesis, Characterization, and Assembly Conditions. Two chiral UPy−PEG compounds, 4a and 4b, were synthesized (Scheme 1), with an (S)- and (R)-dimethylheptyl group at the 6-position of the UPy, respectively. The synthesis of the chiral isocytosines was adapted from a previously established route34 and further expanded into a CDI-activated UPy synthon. Between the UPy moiety and the PEG backbone, an alkyl spacer was coupled, enabling the formation of a hydrophobic core in water.34 In the first step, the hydroxyl-terminated PEG (Mn: 10 000 g/mol) was reacted with 4.9 equiv of 1,1-carbondiimidazole, B
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Figure 2. (a) Autocorrelation curve of 0.44 mM 4a in water at 20 °C, at an angle of 90°, and in the inset a weighted distribution. The insert in the upper right corner shows the q-dependence of the decay rate at multiple angles. (b) Scattering curves of 0.44 mM 4a in water at 20 and 40 °C and a fit of the data obtained at 20 °C.
resolution of the reconstruction was not sufficient to visualize the handedness of the helix, (Figure S7) and no distinct differences were observed when comparing the 2D projection images from both chiral compounds 4a and 4b (Figure S5). In addition, a second preparation method was investigated. Supramolecular aggregates were prepared by dissolving the sample in ultrapure water, heating at 70 °C for 2 h and quenching the sample to 0 °C. This resulted in fibers with the same average diameter as the fibers prepared via the first preparation method (Figure S6). Scattering Techniques. In contrast to CryoTEM measurements, scattering methods provide ensemble measurements that represent the total sample. Therefore, multiangle dynamic light scattering (DLS) measurements were performed on 4a at room temperature (Figure 2a). Samples were dissolved in water, heated at 70 °C for 2 h and slowly cooled to room temperature. The obtained second-order correlation functions were fitted using a CONTIN routine,38,39 and showed a bimodal distribution of decay rates. A fit of the decay rates versus q2 afforded apparent diffusion coefficients of 3.1 and 16.3 nm2s−1, indicative of the presence of small and larger species, respectively. More insight into the morphology was gained by small-angle X-ray scattering experiments at 20 and 40 °C (Figure 2b). At these two temperatures the scattering patterns overlap, indicating that there are no major structural differences between the assemblies at these temperatures. The scattering curve obtained at 20 °C shows a q−1 slope in the low q region of the curve, which is characteristic for long, relatively rigid rods with a length that is beyond the resolution of the measurement. The data was fitted to a model for long cylindrical core−shell structures to give a minimum length of 36 nm and a diameter of approximately 10 nm. The smaller species are not observed in SAXS because the contrast is heavily dominated by the larger structures. Fluorescence Spectroscopy. To obtain more insight into the morphology and the hydrophobic core of the supramolecular aggregates, fluorescence spectroscopy was combined with a Nile red assay. Nile red is a phenoxazone dye that fluoresces intensely with varying color in organic solvents and hydrophobic lipids. In water hardly any signal is visible because Nile red has a poor solubility.40,41 A heating experiment was executed from 20 to 70 °C with compound 4a and Nile red in
resulting in activated compound 2. Subsequently, an excess of 1,12-dodecyldiamine was used for the coupling to the activated PEG. Purification by precipitation and filtration over Celite was applied to obtain compound 3 in a 76% yield. The last coupling reaction was performed with either the (S)- or (R)dimethylheptyl−UPy synthon. An amine functionalized resin was added to the reaction mixture to remove the excess of the UPy-synthon. The compounds were purified by filtration over Celite and precipitation in diethyl ether, resulting in compounds 4a and 4b with overall yields of 40% and 54%, respectively. 1H NMR spectroscopy confirmed the successful modification of the polymer end groups by monitoring the disappearance of the protons next to the amine at 2.7 ppm (Figure S1 and S2, Supporting Information). Furthermore, 13C NMR, IR and MALDI-TOF-MS corroborates successful formation of the products. The optical purity of the chiral starting materials (S)-citronellol and (R)-citronellol were determined to be 98.4 and >99%. The 1H NMR spectra are similar, but MALDI shows a mass difference of 602 g/mol for its most abundant species between 4a and 4b (Supporting Information). Since the polymers are polydisperse and several precipitation steps are involved in the total synthesis, this difference can be neglected. CryoTEM. From previous work on the methyl-UPypolymers it has been shown that supramolecular assemblies can be visualized by CryoTEM.34 Supramolecular aggregates were prepared by dissolving compounds 4a or 4b in ultrapure water, heating at 70 °C for 2 h and slowly cooling to room temperature. Direct visualization of the morphology of the expected aggregates with cryoTEM showed spherical micelles and micrometer long fibers in accordance with the methyl-UPypolymers34 (Figure 1). Additionally, at larger magnification a clear pattern of alternating dark and light gray was seen, which is indicative of a helical pitch (Figure 1b). The average diameter of the fibers is 6 ± 1 nm as predicted by estimation of the core with ChemBio3D (6.2 nm) and the helical pitch is 28 ± 3 nm (Figure S3 and S4). Some micelles are indicated with an arrow in the magnified picture and the average diameter is similar to the fiber diameter (Figure 1b). Only the hydrophobic core is visualized by cryoTEM, since PEG gives no contrast relative to the aqueous environment. Cryo-tomograms were obtained by recording a tilt series at a pixel size of 0.37 nm, however, the C
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water (Figure 3). The sample was prepared by dissolving compound 4a in water and heating at 70 °C for 2 h, after which
the sampled was slowly cooled to room temperature. Then, finally Nile red was added from a 2.5 mM stock solution in methanol. In all measurements a clear signal was observed, indicative of a hydrophobic core. A slight red-shift was observed from 20 to 40 °C, consistent with the heating of Nile red in a hydrophobic environment.41 At 70 °C the maximum shifted 6 nm and slightly decreased in intensity. This observation can be assigned to a small change in the hydrophobic environment. In a cooling experiment, the maximum did not shift back to 615 nm although the intensity did increase to its starting value (Figure S8). CD Spectroscopy. To confirm the helicity of the supramolecular fibers visualized with cryoTEM, and to study the self-assembly behavior in more detail, temperature-dependent CD spectroscopy measurements were performed on compounds 4a and 4b. Two preparation methods were applied to obtain supramolecular aggregates. In the first method the samples were dissolved in water, heated to 70 °C for 2 h and slowly cooled down to room temperature (Figure 4a−c). In the second method the samples were dissolved in water, heated to 70 °C and quenched to 0 °C (Figure 4d). Compound 4a, prepared with the first method, showed a positive bisignate Cotton effect with a maximum of 22 mdeg at 20 °C, which decreased to 0 mdeg at 70 °C (Figure 4a). The corresponding
Figure 3. Fluorescence spectra of 0.44 mM 4a in water, with 5 μM Nile red, during heating from 20 to 70 °C.
Figure 4. Circular dichroism spectroscopy. (a) CD spectra of 0.44 mM 4a in water during heating from 20 to 70 °C, (b) UV−vis spectra of 0.44 mM 4a in water during heating from 20 to 70 °C, (c) CD spectra of 4a and 4b at 0.44 mM in water at 20 °C, and (d) CD spectra of assembly condition 2, in which the sample of 4a was quenched from 70 to 0 °C. D
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core is still present, which indicates that the molecules remain aggregated, either as a spherical or rod-like micellar structure. From recent research we propose that nanofibers and micelles exist in the gel and dilute state, which are stable up to 50 °C and higher.34 This was shown for our chiral UPy−PEGs in both SAXS and fluorescence measurements with the Nile red assay. Comparison of results obtained with respect to compounds 4a and 4b suggest that the (R)- and (S)-dimethylheptyl−UPy− PEG form helices of opposite handedness. Furthermore, heating and cooling experiments showed that at 70 °C no CD signal was visible, suggesting no ordered aggregates at higher temperatures. UV−vis measurements showed a tautomeric shift to the non dimerizing UPy-tautomer upon heating to 70 °C. Since our species were not molecularly dissolved at 70 °C, it was not possible to determine a growth mechanism, which can be of interest to determine the relation between the microscopic assembly and the material properties.27,30 Next to that, the lower critical solution temperature of the UPy−PEG compounds, around 90 °C, hinders temperature increase to reach a possible molecularly dissolved state. From CD spectroscopy measurements, comparing the two assembly conditions, opposite bisignate Cotton effects were observed with maxima at 208 and 218 nm while the UV−vis traces and cryoTEM pictures show no differences. The opposite Cotton effects might indicate the presence of two helices with different handedness, obtained from one stereogenic center by only changing aggregation protocol. This phenomenon was observed in the work on (S)-chiral oligo(pphenylenevinylene) aggregates, showing the presence of two competing self-assembling pathways that led to assemblies with opposite helicity.44 These pathways were investigated in kinetic experiments and distinguished to be a thermodynamically and a kinetically favored route. Hence, here we obtained new insights into the self-assembly behavior and fiber morphology of UPy− PEG compounds in water by introducing a chiral probe. Multiple aggregation pathways exist, and the desired helicity can be obtained by tuning of the assembly conditions.
UV−vis spectrum showed a maximum at 213 nm and a minimum at 284 nm (Figure 4b). These signals correspond to the 4[1H]-, and the 6[1H]-pyrimidinone tautomers, respectively.42,43 A decrease in intensity was observed for the first maximum upon increasing temperature, which correlates to the isosbestic point in the CD spectra (Figure 4a). The peak at 284 nm, showed an increase upon heating, and can be assigned to a tautomeric shift from the 4[1H]- to the 6[1H]-keto tautomer, which cannot dimerize. Upon cooling from 70 to 20 °C, the Cotton effect returned to its starting intensity (Figure S9a,b). Comparison of 4b with 4a showed an opposite Cotton effect with a maximum of 15 mdeg at 208 nm for 4b and 22 mdeg at 217 nm for 4a (Figure 4c), the deviation in signal intensity was attributed to a slight difference in concentration which is visible in the UV−vis spectrum (Figure S9c). Furthermore, the level of noise increases at lower wavelengths. To compare both assembly conditions, the CD spectrum of 4a was measured using assembly condition 2 (Figure 4d). A negative bisignate Cotton effect was observed with a maximum of 22 mdeg at 207 nm, which remained negative in time (measured after 1 day). The signal could be reversed by heating the sample to 40 °C. The corresponding UV−vis spectra for this heating experiment show no changes at all, indicating no conformational changes in the aggregate (Figure S9d). Furthermore, for all measurements, no linear dichroism was observed.
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DISCUSSION Two chiral UPy−PEG polymers were synthesized through modification of the 6-position of the UPy group with a dimethylheptyl chain. Both the self-assembly behavior of these chiral UPy−PEG compounds and the morphology obtained in dilute aqueous solutions were studied with different techniques. Strong evidence for the helicity of the fibers was found with cryoTEM. Striped fibers were visualized which suggest a helical pitch. Both fibers and micelles are visible for compounds 4a and 4b and no significant differences in fiber diameter or helical pitch were observed. Furthermore, two assembly conditions were investigated, in which the samples were either slowly cooled or quenched, which showed no distinct effect in terms of fiber properties. There was however a difference visible in CD spectroscopy. DLS showed two populations in dilute solution at 20 °C, where one of the populations could be assigned to the elongated rods consistent with SAXS. The SAXS data was fitted to give a diameter of approximately 10 nm, which is larger than the 6 nm determined by cryoTEM. This is in agreement with the core−shell model used, where both the hydrophobic core and the PEG shell are taken into account, whereas for cryoTEM only the core could be visualized. The second population, being smaller in size, is proposed to be spherical micelles. Temperature-dependent SAXS measurements showed no difference in morphology at 20 and 40 °C. To further investigate the chiral UPy−PEGs, a Nile red assay was performed to probe the hydrophobic core of the supramolecular aggregates during temperature-dependent experiments. Even at 70 °C, a hydrophobic core was observed, with only a slight decrease in intensity, indicating stable supramolecular aggregates. CD spectroscopy confirmed experimental evidence of fiber helicity. A bisignate Cotton effect was observed at 20 °C up to 50 °C indicating the existence of internally ordered structures at these temperatures. At higher temperatures a hydrophobic
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CONCLUSION In this study the formation of helical fibers with a diameter of 6 ± 1 nm and a helical pitch of 28 ± 3 nm, induced by the (S)and (R)-dimethylheptyl group at the 6-position of the UPy, were confirmed by CD spectroscopy and CryoTEM measurements. Additionally, scattering data confirmed the same distribution of species as CryoTEM, which showed both fibers and spherical micelles, and no significant changes upon heating to 40 °C were visible. Interestingly, the absence of a helix at higher temperatures was shown, while at 70 °C a hydrophobic core still exists. Furthermore, we showed that two self-assembly pathways exist, leading to opposite bisignate Cotton effects, which can be controlled by changing the assembly conditions. These findings illustrate that a chiral probe can be used to obtain structural data and in this case visualize control over the final state of the assembled structure. This control can be translated to optimize the material properties for future use in biomaterials.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details and materials and methods, 1H NMR spectra, cryoTEM micrographs, cryo electron tomography, fluorescence spectra, and CD and UV−vis spectra. This E
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected], tel 0031 40 247 3101 (E.W.M.). *E-mail:
[email protected], tel 0031 40 247 5451 (P.Y.W.D.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Ivo Filot for the table of content picture design, and Jolanda Spiering and Wilco Appel for synthesis of the UPy-precursors. The research leading to this manuscript has received funding from the Ministry of Education, Culture and Science (Gravity program 024.001.035), The Netherlands Organization for Scientific Research (NWO) and the European Research Council (FP7/2007-2013) ERC Grant Agreement 308045. This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, cofunded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged. N.A.J.M.S. and P.H.H.B. are supported by a VICI grant from The Netherlands Organization for Scientific Research− Chemical Sciences (NWO−CW).
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