COMMUNICATION DOI: 10.1002/asia.201402271
The Synthesis of Dendritic EDOT–Peptide Conjugates and their Multistimuli-Responsive Self-Assembly into Supramolecular Nanorods and Fibers in Water Patrick Ahlers,[a] Hendrik Frisch,[a] Daniel Spitzer,[a] Zuzana Vobecka,[b] Filipe Vilela,*[b, c] and Pol Besenius*[a]
Abstract: We report the synthesis of amphiphilic dendritic EDOT–peptide conjugates and discuss their stimuli-responsive self-assembly into polyanionic nanorods in water. In order to expand the general concept of frustrated growth, whereby attractive supramolecular interactions within the enlarged p-system of the hydrophobic core of a dendritic peptide are hampered by repulsive interactions in the hydrophilic periphery, we show that changes in the pH and ionic strength are both able to independently trigger the self-assembly of the dendritic monomers into supramolecular nanorods and nanofibers. These transitions are analyzed using circular dichroism and fluorescence spectroscopic methods, and the resulting supramolecular polymers are characterized by transmission electron microscopy.
not only alkyl chains but also aromatic surfaces can be introduced to direct the self-assembly into one-dimensional (1D) morphologies based on hydrophobic dipeptide building blocks.[10] In the design of supramolecular synthons, we have recently focused on establishing routes to manipulate 1D supramolecular polymerizations[11] in order to form well-defined nanorods in aqueous buffers by balancing out positive with repulsive contributions.[3b] After functionalizing branched nonaphenylalanines with peripheral carboxylic acid Newkome dendrons,[12] the supramolecular reactivity of the weakly acidic monomer could be tuned via electrostatic repulsive forces. We were thereby able to investigate their pH-dependent self-assembly, similarly to the examples reported by the groups of van Esch,[13] Hartgerink,[14] Goldberger,[15] and others.[16] By studying the ionic strength dependency of the supramolecular polymerization processes,[17] we were furthermore able to establish state diagrams that describe the independent pH- and ionic strength-triggered self-assembly of polyanionic nanorods.[3b] The dendritic amphiphilic peptide monomers we have investigated so far have been based only on benzene-1,3-5-carboxamide-type cores because of the commercial availability of this popular branching unit. The resulting C3-symmetrical designs are indeed known to direct the supramolecular polymerization into strictly one-dimensional morphologies with negligible secondary aggregation.[3b, c, 13, 17a, b, 18] In order to expand the general concept of frustrated growth, whereby attractive supramolecular interactions within the enlarged p-system of the hydrophobic core of a dendritic peptide are balanced out with repulsive interactions in the hydrophilic periphery, we have designed 3,4-ethylenedioxythiophene (EDOT)[19]extended 1,3,5-substituted benzene branching units. This should increase the thermodynamic driving force for the formation of ordered and stable aggregates. At the same time the spectroscopic window will be expanded, which would allow for a successful characterization of the polymerization process in more complex biological environments, to regions outside of the far and middle UV region that is typical for standard peptide-based materials. Stimuli-responsive and well-defined nanomaterials[19b, 20] that are designed to undergo morphological transitions induced by specific changes in a physiological environment hold much promise for biomed-
Control of the self-assembly of macromolecular architectures in water has opened new avenues in the design of supramolecular hydrogel materials,[1] compartmentalized systems,[2] and responsive or switchable nanorods.[3] Interactions of these dynamic materials with natural systems,[4] like proteins, membranes, or living cells have accelerated exciting developments in tissue engineering,[5] imaging,[6] and delivery vectors.[7] In order to produce ordered supramolecular architectures in water, peptide-based building blocks have been shown to be extremely versatile,[8] particularly the bsheet-encoded peptide amphiphiles as developed by the group of Stupp.[5c, 9] Ulijn and co-workers have shown that [a] P. Ahlers, H. Frisch, D. Spitzer, Dr. P. Besenius Organic Chemistry Institute and CeNTech Westflische Wilhelms-Universitt Mnster Corrensstrasse 40, 48149 Mnster (Germany) E-mail: [email protected] [b] Dr. Z. Vobecka, Dr. F. Vilela Department of Colloid Chemistry Max-Planck-Institute of Colloids and Interfaces Am Mhlenberg 1, 14476 Potsdam (Germany) [c] Dr. F. Vilela School of Engineering&Physical Sciences, Chemical Sciences, Heriot-Watt University Edinburgh EH14 4AS (United Kingdom) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402271.
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ical applications.[21] We are currently developing strategies whereby the acidity in the microenvironment of a tumor interior is able to induce the pH-triggered self-assembly of small molecules to large nanorods for selective tumor retention and accumulation in cancer therapy and diagnosis.[15, 22] Using an orthogonal protecting group strategy, diphenylalanine was functionalized with a Newkome-type dendron to obtain building block 8 based on a slightly adapted procedure from the literature.[3b] Molecule 8 was then coupled to benzene-1,3,5-tri(3,4-ethylenedioxythiophen-5-yl-2-carboxylic acid) (10) to obtain, after deprotection, the dendritic EDOT–diphenylalanine conjugate 1 in a convergent synthesis (Figure 1 A). As reported previously, all of the peptidebased intermediate compounds could be isolated and purified by precipitation or by conventional flash column chromatography on silica gel.[3b]
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The dendritic EDOT–peptide conjugate 1 turned out to be highly soluble in aqueous buffers at neutral pH. First, we studied the self-assembly behavior in phosphate buffer using fluorescence and circular dichroism (CD) spectroscopy (Figure 2). At pH 7.5, the weak CD bands suggest the presence of molecularly dissolved species.[3b] The weak positive CD band at 223 nm and the weak negative band at 360 nm are similar to the bands observed in buffer/CH3CN (1:2) mixtures (Figure S1, Supporting Information). The addition of CH3CN is known to disrupt the self-assembly in water because it diminishes the hydrophobic shielding of the hydrogen-bonding amide sequences.[3b, 17a, b] Coulomb repulsive interactions from the negatively charged carboxylate groups in the hydrophilic rim of the peptides most probably prevent the polymerization into large macromolecular structures. As we reported previously,[3b] the strength of the repulsive electrostatic interactions can be modulated by adjusting the
Figure 1. A) Synthetic scheme for the final two steps of the preparation of the amphiphilic dendritic EDOT–hexaphenylalanine conjugate 1: a) PyBOP, N,N-diisopropylethylamine (DIPEA), DMF (31 %); b) TFA/CH2Cl2 (1:1) (quantitative). B) Schematic representation of the self-assembly of 1 into 1D nanorods.
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creases and shifts slightly to 440 nm, and a small shoulder at 414 nm appears. This quenching is typically observed in the aggregation of organic fluorophores and is thereby consistent with the observations from CD spectroscopy. When comparing the normalized CD bands with the normalized quenching efficiency (Figure 2 C), it becomes apparent that both spectroscopic techniques can be used as a sensitive tool to monitor the monomer-to-polymer transition: At neutral pH, the ionic strength-induced polymerization occurs over a wide range of NaCl concentrations and reaches a plateau at about 0.8 m NaCl.[23] These results are in good agreement with previously established state diagrams for a dendritic nonaphenylalanine with a smaller benzene-tricarboxamide core.[3b] This confirms that the enlarged hydrophobic core of the EDOT–hexaphenylalanine conjugate 1 is able to compensate the loss of non-covalent interactions due to the omission of one phenylalanine per side arm in the dendritic supramolecular monomer. Using ionic strength to trigger the self-assembly of charged monomers into supramolecular polymers is an intriguing approach to study the mechanistic details of the selfassembly and the cooperativity involved in the supramolecular polymerization,[17a, d] which in the past allowed us to correlate the degree of cooperativity with the observed morphology[24] and to extract quantitative information about the effective surface charge densities of nanorods in solution.[17d] However, for the purpose of developing stimuli-responsive supramolecular materials[19b, 20] for biomedical applications, pH-triggered morphological transitions are much more desirable than ionic strength-triggered events. Therefore, we studied the effects of pH between pH 7.5 and pH 5.1 using fluorescence, circular dichroism (CD), and UV/Vis spectroscopy (Figure 3 and Figure S3, Supporting Information). Very similar to the ionic strength titrations, the monomer-specific weak positive CD bands at 223 nm and 319 nm, and the weak negative band at 359 nm disappear to form weak negative bands at 230 nm and 303 nm, and a weak positive band at 359 nm, thereby indicating the formation of supramolecular polymers. In addition, fluorescence titrations confirm the pH-triggered formation of aggregates in solution: the emission band at 430 nm decreases significantly and shifts slightly to 440 nm, while at low pH a small shoulder at 414 nm appears, exactly like in the ionic strength-dependent luminescence spectra. The transition observed in the normalized quenching efficiency correlates very well with the one determined using the normalized CD bands: the pH-induced polymerization is very sharp and occurs in a pH window between pH 5.8 and pH 5.5. Finally, pH-dependent UV/Vis spectra were recorded (Figure S3 A, Supporting Information): the normalized data show a sharp change between pH 6.0 and pH 5.5 (Figure S3 B, Supporting Information), which further supports the monomer-to-polymer transition indicated in the CD and fluorescence spectroscopic investigations. Interestingly, (partial) screening of the repulsive interactions by protonation of the dendritic carboxylic acids occurs at a pH value of 5.5, which is at least one pH unit higher than expected for a small molecule-based organ-
Figure 2. Ionic strength-dependent CD (A) and fluorescence (B, lex = 333 nm) spectra for amphiphilic dendritic EDOT–peptide conjugate 1 (3 10 5 m) at pH 7.5 in 10 mm phosphate buffer at 293 K; (C) normalized spectroscopic data comparing the CD bands at 230 nm and 364 nm to the fluorescence emission at 434 nm.
ionic strength of the aqueous buffer, thereby leading to a pronounced change in the CD spectra (Figure 2 A and Figure S2 A, Supporting Information). The weak positive bands at 223 nm and 319 nm, and the weak negative band at 359 nm disappear upon increasing the salt concentration to 1 m NaCl to form weak negative bands at 230 nm and 342 nm, as well as a weak positive band at 375 nm. These changes in the CD spectra are indicative for the formation of ordered supramolecular polymers. To corroborate these results, we have performed similar ionic strength titrations and monitored the fluorescence emission of the benzene1,3,5-tri(EDOT-carboxyamide) core upon excitation at 333 nm (Figure 2 B). Upon increasing the ionic strength from 0 m NaCl to 1 m NaCl, the emission band at 430 nm de-
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experiments under two sets of conditions in aqueous buffer, one at high ionic strength (1 m NaCl, Figure 4) and the other in acidified buffer at pH 5.1 (Figure 5). The first conditions lead to micrographs that show rods that are few microme-
Figure 4. TEM image of the amphiphilic dendritic EDOT–peptide conjugate 1 deposited on Formvar grids from a 1 mg mL 1 solution in 10 mm Tris buffer (pH 7.5) containing 1 m NaCl (negative staining was performed with 1 % w/v uranyl actetate). Scale bar, 50 nm; the black arrows highlight rods with a thickness of 4 nm.
Figure 3. pH-dependent CD (A) and fluorescence (B, lex = 333 nm) spectra for amphiphilic dendritic EDOT–peptide conjugate 1 (3 10 5 m) in 10 mm phosphate buffer at 293 K; (C) normalized spectroscopic data comparing the CD bands at 230 nm and 359 nm to the fluorescence emission at 434 nm.
Figure 5. TEM images of amphiphilic dendritic EDOT–peptide conjugate 1 deposited on Formvar grids from a 1 mg mL 1 solution in 10 mm Tris buffer, pH 5.2 (negative staining was performed with 1 % w/v uranyl actetate). Scale bar, 50 nm; the black arrows highlight rods with a thickness of 4 nm while the white arrows indicate fibers with a thickness of 8 nm.
ic carboxylic acid in water (the pKa for a Newkome-type dendritic carboxylic acid is about 4[25]). We have previously observed a similar shift in the apparent pKa value for an amphiphilic dendritic carboxylic acid;[3b] this is a well-known phenomenon in the case of polymeric weak acids and selfassembled fatty acids.[26] We have thereby been able to show that frustrated growth into ordered supramolecular polymers can be independently triggered by adjusting both the ionic strength and the pH. In order to confirm the formation of 1D nanorod-like supramolecular polymers, we investigated solutions of amphiphilic dendritic EDOT–peptide conjugate 1 (1 mg mL 1) by transmission electron microscopy (TEM). We performed
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ters long and 4.0 0.4 nm in thickness (indicated by the black arrows in Figure 4). The latter correlates well with the diameter of the hydrophobic core of the dendritic molecular building block (Figure 1) of 4.2 nm. Even though the surface density of the self-assembled materials on the carbon-coated TEM grid is high, the observed morphology can be referred to as a nanorod since the 1D molecular assemblies do not form higher ordered or bundled architectures often observed for peptide materials, for example, fibrils or fibers.[1a, h, 8c–e, 21] Depositing the materials on the grids from an acidic solution reveals morphologies that are slightly different. The
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presence of nanorods with a thickness of 4 nm can still be observed, however, with a lower stiffness compared to those obtained from solutions with higher ionic strength. The protonated rods with a diameter of 4 nm (marked with black arrows in Figure 5) also seem to have a higher tendency to intertwine and to form bundled fibers with a thickness of 8 nm (marked with white arrows). The findings we present here are significant: electrostatic screening of interactions between dendritic polycarboxylate-based monomers enhances elongation into very long and stiff nanorods in solution. On the other hand, protonation of the carboxylate side chains in the monomers also induces formation of 1D supramolecular polymers, but these are less stiff compared to screened polyanionic nanorods and have a higher tendency to form bundles. In summary, we report the synthesis of a new dendritic amphiphilic EDOT–peptide conjugate. This stimuli-responsive supramolecular synthon self-assembles into 1D nanorod-like morphologies based on two independent triggers, an increase in the ionic strength through the addition of NaCl or a decrease in the pH from neutral to pH 5.5. With these dendritic amphiphiles we have further expanded our methodology of frustrated growth that allows us to manipulate supramolecular polymerizations by compensating attractive non-covalent interactions with repulsive electrostatic forces. These materials have great potential as carrier materials for therapeutic applications and as contrast agents for molecular imaging. We are currently developing strategies based on responsive supramolecular nanorods, whereby the acidity in the microenvironment of a tumor interior is able to induce the pH-triggered self-assembly of small molecules to large nanorods. This would further enable their selective retention and accumulation in the tumor tissue for applications in cancer therapy and diagnosis.
Keywords: nanostructures · pH switch · self-assembly · stimuli-responsive · supramolecular polymer
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Experimental Section Synthetic procedures, material characterizations, and full details about the instrumentation can be found in the Supporting Information. TEM images were recorded at the University of Twente on a Philips CM300ST-FEG transmission electron microscope operated at 300 kV, equipped with a GATAN Tridiem energy filter and a GATAN Ultrascan1000 (2kx2k CCD camera). CD spectra were recorded on a J-815 (JASCO) spectrometer, and fluorescence spectra on a FP-6500 (JASCO) spectrometer.
Acknowledgements We thank Rico Keim (MESA + , University of Twente) for the TEM images, Dr. Martin Peterlechner (Institute of Material Physics, WWU Mnster) for preliminary TEM experiments, the Fonds der Chemischen Industrie (FCI) for a Liebig [P.B.] and a doctoral [H.F.] Fellowship, and Marie Curie Actions FP7 for a Career Integration Grant (SupraBioMat, PCIG10-GA-2011–303872). We acknowledge COST Action CM1005 (Supramolecular Chemistry in Water). This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Mnster, Germany.
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Leenders, L. Albertazzi, T. Mes, M. Koenigs, A. R. A. Palmans, E. W. Meijer, Chem. Commun. 2013, 49, 1963 – 1965. a) H.-A. Klok, A. Rosler, G. Gotz, E. Mena-Osteritz, P. Bauerle, Org. Biomol. Chem. 2004, 2, 3541 – 3544; b) S. R. Diegelmann, J. M. Gorham, J. D. Tovar, J. Am. Chem. Soc. 2008, 130, 13840 – 13841; c) D. A. Stone, A. S. Tayi, J. E. Goldberger, L. C. Palmer, S. I. Stupp, Chem. Commun. 2011, 47, 5702 – 5704; d) I. D. Tevis, L. C. Palmer, D. J. Herman, I. P. Murray, D. A. Stone, S. I. Stupp, J. Am. Chem. Soc. 2011, 133, 16486 – 16494; e) L. Tian, R. Szilluweit, R. Marty, L. Bertschi, M. Zerson, E.-C. Spitzner, R. Magerle, H. Frauenrath, Chem. Sci. 2012, 3, 1512 – 1521; f) J. A. Lehrman, H. Cui, W.-W. Tsai, T. J. Moyer, S. I. Stupp, Chem. Commun. 2012, 48, 9711 – 9713; g) S. Schmid, A. Mishra, M. Wunderlin, P. Buerle, Org. Biomol. Chem. 2013, 11, 5656 – 5665.. a) A. Kishimura, T. Yamashita, K. Yamaguchi, T. Aida, Nat. Mater. 2005, 4, 546 – 549; b) J. Baram, E. Shirman, N. Ben-Shitrit, A. Ustinov, H. Weissman, I. Pinkas, S. G. Wolf, B. Rybtchinski, J. Am. Chem. Soc. 2008, 130, 14966 – 14967; c) F. Tian, D. Jiao, F. Biedermann, O. A. Scherman, Nat. Commun. 2012, 3, 1207; d) Z. Huang, S.-K. Kang, M. Banno, T. Yamaguchi, D. Lee, C. Seok, E. Yashima, M. Lee, Science 2012, 337, 1521 – 1526; e) J. del Barrio, P. N. Horton, D. Lairez, G. O. Lloyd, C. Toprakcioglu, O. A. Scherman, J. Am. Chem. Soc. 2013, 135, 11760 – 11763; f) S. Biswas, K. Kinbara, T. Niwa, H. Taguchi, N. Ishii, S. Watanabe, K. Miyata, K. Kataoka, T. Aida, Nat. Chem. 2013, 5, 613 – 620; g) M. Nakahata, Y. Takashima, A. Hashidzume, A. Harada, Angew. Chem. Int. Ed. 2013, 52, 5731 – 5735; Angew. Chem. 2013, 125, 5843 – 5847. R. J. Mart, R. D. Osborne, M. M. Stevens, R. V. Ulijn, Soft Matter 2006, 2, 822 – 835. C. C. Lee, J. A. MacKay, J. M. J. Fr chet, F. C. Szoka, Nat. Biotechnol. 2005, 23, 1517 – 1526. While increasing the salt concentration further from 1 m NaCl to 2 m NaCl, we observed more pronounced changes in the CD spectroscopic data (Figure S2 A, Supporting Information) and a sharp transition between 1.2–1.4 m NaCl (Figure S2 B, Supporting Information): the appearance of very strong negative bands at 223 nm and 292 nm, and a strong positive band at 320 nm. Since depletion forces and salting out effects cannot be excluded at these high ionic strengths, we have restricted the discussion to titrations performed below 1 m NaCl. I. de Feijter, P. Besenius, L. Albertazzi, E. W. Meijer, A. R. A. Palmans, I. K. Voets, Soft Matter 2013, 9, 10025 – 10030. T. J. Cho, R. A. Zangmeister, R. I. MacCuspie, A. K. Patri, V. A. Hackley, Chem. Mater. 2011, 23, 2665 – 2676. a) A. V. Dobrynin, M. Rubinstein, Prog. Polym. Sci. 2005, 30, 1049 – 1118; b) A. V. Dobrynin, Curr. Opin. Colloid Interface Sci. 2008, 13, 376 – 388; c) J. R. Kanicky, D. O. Shah, Langmuir 2003, 19, 2034 – 2038. Received: March 21, 2014 Revised: April 9, 2014 Published online: && &&, 0000
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COMMUNICATION Stimulus does work: The synthesis of an amphiphilic dendritic EDOT–peptide conjugate is reported and its stimuli-responsive self-assembly into polyanionic nanorods and fibers in water is discussed. Using the concept of frustrated growth, it is shown that changes in the pH and ionic strength are both able to independently trigger the selfassembly of the dendritic monomers into supramolecular nanostructures.
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Self-Assembly Patrick Ahlers, Hendrik Frisch, Daniel Spitzer, Zuzana Vobecka, Filipe Vilela,* Pol Besenius* &&&&—&&&& The Synthesis of Dendritic EDOT– Peptide Conjugates and their Multistimuli-Responsive Self-Assembly into Supramolecular Nanorods and Fibers in Water
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The Synthesis of Dendritic EDOT–Peptide Conjugates and their Multistimuli-Responsive Self-Assembly into Supramolecular Nanorods and Fibers in Water Patrick Ahlers,[a] Hendrik Frisch,[a] Daniel Spitzer,[a] Zuzana Vobecka,[b] Filipe Vilela,*[b, c] and Pol Besenius*[a]
Abstract: We report the synthesis of amphiphilic dendritic EDOT–peptide conjugates and discuss their stimuli-responsive self-assembly into polyanionic nanorods in water. In order to expand the general concept of frustrated growth, whereby attractive supramolecular interactions within the enlarged p-system of the hydrophobic core of a dendritic peptide are hampered by repulsive interactions in the hydrophilic periphery, we show that changes in the pH and ionic strength are both able to independently trigger the self-assembly of the dendritic monomers into supramolecular nanorods and nanofibers. These transitions are analyzed using circular dichroism and fluorescence spectroscopic methods, and the resulting supramolecular polymers are characterized by transmission electron microscopy.
not only alkyl chains but also aromatic surfaces can be introduced to direct the self-assembly into one-dimensional (1D) morphologies based on hydrophobic dipeptide building blocks.[10] In the design of supramolecular synthons, we have recently focused on establishing routes to manipulate 1D supramolecular polymerizations[11] in order to form well-defined nanorods in aqueous buffers by balancing out positive with repulsive contributions.[3b] After functionalizing branched nonaphenylalanines with peripheral carboxylic acid Newkome dendrons,[12] the supramolecular reactivity of the weakly acidic monomer could be tuned via electrostatic repulsive forces. We were thereby able to investigate their pH-dependent self-assembly, similarly to the examples reported by the groups of van Esch,[13] Hartgerink,[14] Goldberger,[15] and others.[16] By studying the ionic strength dependency of the supramolecular polymerization processes,[17] we were furthermore able to establish state diagrams that describe the independent pH- and ionic strength-triggered self-assembly of polyanionic nanorods.[3b] The dendritic amphiphilic peptide monomers we have investigated so far have been based only on benzene-1,3-5-carboxamide-type cores because of the commercial availability of this popular branching unit. The resulting C3-symmetrical designs are indeed known to direct the supramolecular polymerization into strictly one-dimensional morphologies with negligible secondary aggregation.[3b, c, 13, 17a, b, 18] In order to expand the general concept of frustrated growth, whereby attractive supramolecular interactions within the enlarged p-system of the hydrophobic core of a dendritic peptide are balanced out with repulsive interactions in the hydrophilic periphery, we have designed 3,4-ethylenedioxythiophene (EDOT)[19]extended 1,3,5-substituted benzene branching units. This should increase the thermodynamic driving force for the formation of ordered and stable aggregates. At the same time the spectroscopic window will be expanded, which would allow for a successful characterization of the polymerization process in more complex biological environments, to regions outside of the far and middle UV region that is typical for standard peptide-based materials. Stimuli-responsive and well-defined nanomaterials[19b, 20] that are designed to undergo morphological transitions induced by specific changes in a physiological environment hold much promise for biomed-
Control of the self-assembly of macromolecular architectures in water has opened new avenues in the design of supramolecular hydrogel materials,[1] compartmentalized systems,[2] and responsive or switchable nanorods.[3] Interactions of these dynamic materials with natural systems,[4] like proteins, membranes, or living cells have accelerated exciting developments in tissue engineering,[5] imaging,[6] and delivery vectors.[7] In order to produce ordered supramolecular architectures in water, peptide-based building blocks have been shown to be extremely versatile,[8] particularly the bsheet-encoded peptide amphiphiles as developed by the group of Stupp.[5c, 9] Ulijn and co-workers have shown that [a] P. Ahlers, H. Frisch, D. Spitzer, Dr. P. Besenius Organic Chemistry Institute and CeNTech Westflische Wilhelms-Universitt Mnster Corrensstrasse 40, 48149 Mnster (Germany) E-mail: [email protected] [b] Dr. Z. Vobecka, Dr. F. Vilela Department of Colloid Chemistry Max-Planck-Institute of Colloids and Interfaces Am Mhlenberg 1, 14476 Potsdam (Germany) [c] Dr. F. Vilela School of Engineering&Physical Sciences, Chemical Sciences, Heriot-Watt University Edinburgh EH14 4AS (United Kingdom) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402271.
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ical applications.[21] We are currently developing strategies whereby the acidity in the microenvironment of a tumor interior is able to induce the pH-triggered self-assembly of small molecules to large nanorods for selective tumor retention and accumulation in cancer therapy and diagnosis.[15, 22] Using an orthogonal protecting group strategy, diphenylalanine was functionalized with a Newkome-type dendron to obtain building block 8 based on a slightly adapted procedure from the literature.[3b] Molecule 8 was then coupled to benzene-1,3,5-tri(3,4-ethylenedioxythiophen-5-yl-2-carboxylic acid) (10) to obtain, after deprotection, the dendritic EDOT–diphenylalanine conjugate 1 in a convergent synthesis (Figure 1 A). As reported previously, all of the peptidebased intermediate compounds could be isolated and purified by precipitation or by conventional flash column chromatography on silica gel.[3b]
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The dendritic EDOT–peptide conjugate 1 turned out to be highly soluble in aqueous buffers at neutral pH. First, we studied the self-assembly behavior in phosphate buffer using fluorescence and circular dichroism (CD) spectroscopy (Figure 2). At pH 7.5, the weak CD bands suggest the presence of molecularly dissolved species.[3b] The weak positive CD band at 223 nm and the weak negative band at 360 nm are similar to the bands observed in buffer/CH3CN (1:2) mixtures (Figure S1, Supporting Information). The addition of CH3CN is known to disrupt the self-assembly in water because it diminishes the hydrophobic shielding of the hydrogen-bonding amide sequences.[3b, 17a, b] Coulomb repulsive interactions from the negatively charged carboxylate groups in the hydrophilic rim of the peptides most probably prevent the polymerization into large macromolecular structures. As we reported previously,[3b] the strength of the repulsive electrostatic interactions can be modulated by adjusting the
Figure 1. A) Synthetic scheme for the final two steps of the preparation of the amphiphilic dendritic EDOT–hexaphenylalanine conjugate 1: a) PyBOP, N,N-diisopropylethylamine (DIPEA), DMF (31 %); b) TFA/CH2Cl2 (1:1) (quantitative). B) Schematic representation of the self-assembly of 1 into 1D nanorods.
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creases and shifts slightly to 440 nm, and a small shoulder at 414 nm appears. This quenching is typically observed in the aggregation of organic fluorophores and is thereby consistent with the observations from CD spectroscopy. When comparing the normalized CD bands with the normalized quenching efficiency (Figure 2 C), it becomes apparent that both spectroscopic techniques can be used as a sensitive tool to monitor the monomer-to-polymer transition: At neutral pH, the ionic strength-induced polymerization occurs over a wide range of NaCl concentrations and reaches a plateau at about 0.8 m NaCl.[23] These results are in good agreement with previously established state diagrams for a dendritic nonaphenylalanine with a smaller benzene-tricarboxamide core.[3b] This confirms that the enlarged hydrophobic core of the EDOT–hexaphenylalanine conjugate 1 is able to compensate the loss of non-covalent interactions due to the omission of one phenylalanine per side arm in the dendritic supramolecular monomer. Using ionic strength to trigger the self-assembly of charged monomers into supramolecular polymers is an intriguing approach to study the mechanistic details of the selfassembly and the cooperativity involved in the supramolecular polymerization,[17a, d] which in the past allowed us to correlate the degree of cooperativity with the observed morphology[24] and to extract quantitative information about the effective surface charge densities of nanorods in solution.[17d] However, for the purpose of developing stimuli-responsive supramolecular materials[19b, 20] for biomedical applications, pH-triggered morphological transitions are much more desirable than ionic strength-triggered events. Therefore, we studied the effects of pH between pH 7.5 and pH 5.1 using fluorescence, circular dichroism (CD), and UV/Vis spectroscopy (Figure 3 and Figure S3, Supporting Information). Very similar to the ionic strength titrations, the monomer-specific weak positive CD bands at 223 nm and 319 nm, and the weak negative band at 359 nm disappear to form weak negative bands at 230 nm and 303 nm, and a weak positive band at 359 nm, thereby indicating the formation of supramolecular polymers. In addition, fluorescence titrations confirm the pH-triggered formation of aggregates in solution: the emission band at 430 nm decreases significantly and shifts slightly to 440 nm, while at low pH a small shoulder at 414 nm appears, exactly like in the ionic strength-dependent luminescence spectra. The transition observed in the normalized quenching efficiency correlates very well with the one determined using the normalized CD bands: the pH-induced polymerization is very sharp and occurs in a pH window between pH 5.8 and pH 5.5. Finally, pH-dependent UV/Vis spectra were recorded (Figure S3 A, Supporting Information): the normalized data show a sharp change between pH 6.0 and pH 5.5 (Figure S3 B, Supporting Information), which further supports the monomer-to-polymer transition indicated in the CD and fluorescence spectroscopic investigations. Interestingly, (partial) screening of the repulsive interactions by protonation of the dendritic carboxylic acids occurs at a pH value of 5.5, which is at least one pH unit higher than expected for a small molecule-based organ-
Figure 2. Ionic strength-dependent CD (A) and fluorescence (B, lex = 333 nm) spectra for amphiphilic dendritic EDOT–peptide conjugate 1 (3 10 5 m) at pH 7.5 in 10 mm phosphate buffer at 293 K; (C) normalized spectroscopic data comparing the CD bands at 230 nm and 364 nm to the fluorescence emission at 434 nm.
ionic strength of the aqueous buffer, thereby leading to a pronounced change in the CD spectra (Figure 2 A and Figure S2 A, Supporting Information). The weak positive bands at 223 nm and 319 nm, and the weak negative band at 359 nm disappear upon increasing the salt concentration to 1 m NaCl to form weak negative bands at 230 nm and 342 nm, as well as a weak positive band at 375 nm. These changes in the CD spectra are indicative for the formation of ordered supramolecular polymers. To corroborate these results, we have performed similar ionic strength titrations and monitored the fluorescence emission of the benzene1,3,5-tri(EDOT-carboxyamide) core upon excitation at 333 nm (Figure 2 B). Upon increasing the ionic strength from 0 m NaCl to 1 m NaCl, the emission band at 430 nm de-
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experiments under two sets of conditions in aqueous buffer, one at high ionic strength (1 m NaCl, Figure 4) and the other in acidified buffer at pH 5.1 (Figure 5). The first conditions lead to micrographs that show rods that are few microme-
Figure 4. TEM image of the amphiphilic dendritic EDOT–peptide conjugate 1 deposited on Formvar grids from a 1 mg mL 1 solution in 10 mm Tris buffer (pH 7.5) containing 1 m NaCl (negative staining was performed with 1 % w/v uranyl actetate). Scale bar, 50 nm; the black arrows highlight rods with a thickness of 4 nm.
Figure 3. pH-dependent CD (A) and fluorescence (B, lex = 333 nm) spectra for amphiphilic dendritic EDOT–peptide conjugate 1 (3 10 5 m) in 10 mm phosphate buffer at 293 K; (C) normalized spectroscopic data comparing the CD bands at 230 nm and 359 nm to the fluorescence emission at 434 nm.
Figure 5. TEM images of amphiphilic dendritic EDOT–peptide conjugate 1 deposited on Formvar grids from a 1 mg mL 1 solution in 10 mm Tris buffer, pH 5.2 (negative staining was performed with 1 % w/v uranyl actetate). Scale bar, 50 nm; the black arrows highlight rods with a thickness of 4 nm while the white arrows indicate fibers with a thickness of 8 nm.
ic carboxylic acid in water (the pKa for a Newkome-type dendritic carboxylic acid is about 4[25]). We have previously observed a similar shift in the apparent pKa value for an amphiphilic dendritic carboxylic acid;[3b] this is a well-known phenomenon in the case of polymeric weak acids and selfassembled fatty acids.[26] We have thereby been able to show that frustrated growth into ordered supramolecular polymers can be independently triggered by adjusting both the ionic strength and the pH. In order to confirm the formation of 1D nanorod-like supramolecular polymers, we investigated solutions of amphiphilic dendritic EDOT–peptide conjugate 1 (1 mg mL 1) by transmission electron microscopy (TEM). We performed
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ters long and 4.0 0.4 nm in thickness (indicated by the black arrows in Figure 4). The latter correlates well with the diameter of the hydrophobic core of the dendritic molecular building block (Figure 1) of 4.2 nm. Even though the surface density of the self-assembled materials on the carbon-coated TEM grid is high, the observed morphology can be referred to as a nanorod since the 1D molecular assemblies do not form higher ordered or bundled architectures often observed for peptide materials, for example, fibrils or fibers.[1a, h, 8c–e, 21] Depositing the materials on the grids from an acidic solution reveals morphologies that are slightly different. The
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presence of nanorods with a thickness of 4 nm can still be observed, however, with a lower stiffness compared to those obtained from solutions with higher ionic strength. The protonated rods with a diameter of 4 nm (marked with black arrows in Figure 5) also seem to have a higher tendency to intertwine and to form bundled fibers with a thickness of 8 nm (marked with white arrows). The findings we present here are significant: electrostatic screening of interactions between dendritic polycarboxylate-based monomers enhances elongation into very long and stiff nanorods in solution. On the other hand, protonation of the carboxylate side chains in the monomers also induces formation of 1D supramolecular polymers, but these are less stiff compared to screened polyanionic nanorods and have a higher tendency to form bundles. In summary, we report the synthesis of a new dendritic amphiphilic EDOT–peptide conjugate. This stimuli-responsive supramolecular synthon self-assembles into 1D nanorod-like morphologies based on two independent triggers, an increase in the ionic strength through the addition of NaCl or a decrease in the pH from neutral to pH 5.5. With these dendritic amphiphiles we have further expanded our methodology of frustrated growth that allows us to manipulate supramolecular polymerizations by compensating attractive non-covalent interactions with repulsive electrostatic forces. These materials have great potential as carrier materials for therapeutic applications and as contrast agents for molecular imaging. We are currently developing strategies based on responsive supramolecular nanorods, whereby the acidity in the microenvironment of a tumor interior is able to induce the pH-triggered self-assembly of small molecules to large nanorods. This would further enable their selective retention and accumulation in the tumor tissue for applications in cancer therapy and diagnosis.
Keywords: nanostructures · pH switch · self-assembly · stimuli-responsive · supramolecular polymer
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Experimental Section Synthetic procedures, material characterizations, and full details about the instrumentation can be found in the Supporting Information. TEM images were recorded at the University of Twente on a Philips CM300ST-FEG transmission electron microscope operated at 300 kV, equipped with a GATAN Tridiem energy filter and a GATAN Ultrascan1000 (2kx2k CCD camera). CD spectra were recorded on a J-815 (JASCO) spectrometer, and fluorescence spectra on a FP-6500 (JASCO) spectrometer.
Acknowledgements We thank Rico Keim (MESA + , University of Twente) for the TEM images, Dr. Martin Peterlechner (Institute of Material Physics, WWU Mnster) for preliminary TEM experiments, the Fonds der Chemischen Industrie (FCI) for a Liebig [P.B.] and a doctoral [H.F.] Fellowship, and Marie Curie Actions FP7 for a Career Integration Grant (SupraBioMat, PCIG10-GA-2011–303872). We acknowledge COST Action CM1005 (Supramolecular Chemistry in Water). This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Mnster, Germany.
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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COMMUNICATION Stimulus does work: The synthesis of an amphiphilic dendritic EDOT–peptide conjugate is reported and its stimuli-responsive self-assembly into polyanionic nanorods and fibers in water is discussed. Using the concept of frustrated growth, it is shown that changes in the pH and ionic strength are both able to independently trigger the selfassembly of the dendritic monomers into supramolecular nanostructures.
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Self-Assembly Patrick Ahlers, Hendrik Frisch, Daniel Spitzer, Zuzana Vobecka, Filipe Vilela,* Pol Besenius* &&&&—&&&& The Synthesis of Dendritic EDOT– Peptide Conjugates and their Multistimuli-Responsive Self-Assembly into Supramolecular Nanorods and Fibers in Water
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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