Programming pH-Triggered Self-Assembly Transitions via

4 Dec 2014 - Programming pH-Triggered Self-Assembly Transitions via. Isomerization of Peptide Sequence. Arijit Ghosh,. †. Eric T. Dobson,. †. Chri...
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Programming pH-Triggered Self-Assembly Transitions via Isomerization of Peptide Sequence Arijit Ghosh,† Eric T. Dobson,† Christian J. Buettner, Michael J. Nicholl, and Joshua E. Goldberger* Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: While the ordering of amino acids in proteins and peptidebased materials is known to affect their folding patterns and supramolecular architectures, tailoring self-assembly behavior in stimuli responsive peptides by isomerizing a peptide sequence has not been extensively explored. Here, we show that changing the position of a single hydrophobic amino acid in short amphiphilic peptides can dramatically alter their pH-triggered self-assembly transitions. Using palmitoylIAAAEEEE-NH2 and palmitoyl-IAAAEEEEK(DO3A:Gd)-NH2 as controls, moving the Isoleucine away from the palmitoyl tail preferentially induces nanofiber formation over spherical micelles. Shifting the Isoleucine one residue away makes the transition pH more basic by 2 units. When in the third or fourth position, nanofibers are formed exclusively above 10 μM. We propose that moving the Isoleucine away from the tail enhances its ability to promote β-sheet formation instead of folding back into the palmitoyl core. These findings reveal a novel strategy for programming pH-triggered self-assembly by isomerizing a peptide sequence.

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applications.16 While the hydrophobic β-sheet forming region promotes nanofiber formation due to its strong propensity to form hydrogen bonds along the PA backbone, the charged amino acids destabilize the β-sheets via electrostatic repulsion and induce formation of spherical micelles instead. Hence, an optimal balance between hydrophobic and repulsive interactions is required to enable the micelle-to-nanofiber transition in the desired pH range. We have shown this pH-trigger of selfassembly for these molecules to be tunable via alterations in the identity of the amino acid residues in the β-sheet region. By synthesizing and characterizing the pH-triggered self-assembly behavior of palmitoyl-IAAAEEEE-NH 2 and palmitoylIAAAEEEEK(DO3A:Gd)-NH2 at various concentrations, we have demonstrated that a ratio of one strongly hydrophobic amino acid (I) to three Alanines (A) is necessary to enable the transition in the desired pH window at 10−100 μM and that addition of a (DO3A:Gd) imaging moiety induces a spherical micellar morphology.16 Furthermore, substituting Isoleucine for less strongly hydrophobic amino acids such as Valine, Phenylalanine, and Tyrosine, can lower the nanofiber-to-micelle transition pH by 0.6 pH units.16 Despite the recent progress in PA-based therapeutics and diagnostics, there is a general lack of knowledge regarding the effect of peptide sequence variation on PA self-assembly behavior. The studies that exist involve only static self-

INTRODUCTION The development of biomaterials that can undergo selfassembly transitions in vivo in response to specific physiological stimuli has led to an emerging class of next-generation sensing, diagnostic, and therapeutic agents.1−4 Numerous studies have demonstrated that self-assembling peptide amphiphiles (PAs) can serve as artificial supramolecular polymers in applications such as regenerative medicine.5−9 However, most studies to date have focused on tuning the self-assembly of PAs into static morphologies.10−12 Recent research has indicated that the size and shape of intravenously-injected nanomaterials significantly affect their in vivo biodistribution.13,14 Therefore, designing PAbased nanomaterials that spontaneously change shape and size in response to specific physiological stimuli can allow for the exploitation of different diffusion kinetics of nanosized carriers, enhancing their accumulation selectively at targeted diseased sites. Previously, we have developed a general PA-based vehicle design scheme that allows for fine-tuning a pH-triggered selfassembly morphology transition from spherical micelles to bulky, slow-diffusing nanofibers between pH values of 7.4 (normal physiological pH) and 6.6 (extracellular pH of most tumors),15 as a novel mechanism to achieve high concentrations of these agents at the tumor site relative to normal tissue.16 This design involves four main segments: a hydrophobic alkyl (palmitic acid) tail, followed by a β-sheet forming peptide region (-XAAA-, where X is a hydrophobic amino acid), a charged amino acid sequence (-EEEE-), and a macrocyclic Gd3+chelate bound to a lysine for MRI-imaging © XXXX American Chemical Society

Received: September 20, 2014 Revised: November 14, 2014

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trations at which aggregates (either micelles or nanofibers) began to form at pH values between 5 and 10. Circular dichroism (CD) spectroscopy was used to determine the pHdependent secondary structure of PAs at various concentrations. It has previously been shown that the random coil pattern in CD spectrum corresponds to either single molecules or micelles, and the β-sheet pattern corresponds to the nanofiber morphology.16 In PAs that undergo pH-triggered transitions between the single molecule/micelle and nanofiber morphologies, the CD transition point was defined as the pH value at which the ellipticity at 205 nm rose from a negative value (random coil) to zero along with the appearance of a negative band near 218 nm. In addition to CAC and CD measurements, conventional transmission electron microscopy (TEM) was used to confirm the PA morphology at different pH values.

assembling systems rather than systems that undergo morphological changes in response to environmental triggers (pH in our case).17,18 For instance, Zhao et al. have shown that by rearranging the amino acid sequence in a short peptide pentamer, a variety of morphologies including nanofibrils, nanotubules, and a lack of regular structure can be induced depending on the programmed sequence.19 By creation of Nmethylated glycine derivatives and alanine mutants, Paramonov et al. have shown that for an amphiphilic system (palmitoylGGGGGGGERGDS-COOH), the H-bonding in the four amino acids near the hydrophobic core is most essential for self-assembly of nanofibers.20 Recently, Cui et al. have demonstrated via creation of a series of tetrapeptides (VVEE, EEVV, VEVE, EVEV) that the relative positions of Valine and Glutamic acid in the peptide sequence lead to distinctly different self-assembly morphologies.21 In this Letter, we establish a general relationship between the position of the β-sheet forming Isoleucine (I) residue in the primary PA sequence and the pH of self-assembly morphology transition. To accomplish this, palmitoyl-AIAAEEEE-NH2, palmitoyl-AAIAEEEE-NH2, palmitoyl-AAAIEEEE-NH2 and palmitoyl-AIAAEEEEK(DO3A:Gd)-NH2 were synthesized and their pH-triggered self-assembly behavior compared to well characterized control systems, palmitoyl-IAAAEEEE-NH2 and palmitoyl-IAAAEEEEK(DO3A:Gd)-NH2 (Table 1, Chart 1). We show that moving the Isoleucine farther away from the palmitoyl tail preferentially induces nanofiber formation over spherical micelles.

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RESULTS AND DISCUSSION The concentration-pH phase diagrams for the control molecules PA 1 and PA 5 were previously established in simulated serum salt solutions containing 150 mM NaCl and 2.2 mM CaCl2.16 In order to understand the effects of sequence isomerization on pH-triggered self-assembly, concentration-pH phase diagrams were mapped out for PAs in which the Isoleucine was shifted away from the palmitoyl tail by one residue (PA 2 and PA 6). The single molecule to micelle/ nanofiber transitions at various pH values were determined using CAC measurements, and the pH-dependent transitions from either spherical micelles or isolated molecules into nanofibers at various PA concentrations were determined via CD spectroscopy, in the same salt solution. The phase diagram of PA 2 is overlaid with PA 1 (Figure 1c). The CAC values for PA 2 are pH-dependent, ranging from 1.1 μM to 7.5 μM at pH 5 and 10, respectively (Figure SI-3a). Both PA 1 and PA 2 show an increase in CAC values in more basic conditions due to the greater electrostatic repulsion among deprotonated glutamic acids. However, the CACs for PA 2 are 3−5 times lower than those for PA 1 at comparable pH values. This indicates an increase in the attractive forces in these surfactant-like molecules when the Isoleucine is moved by one residue away from the tail. CD spectra of PA 2 confirmed that the nanofibers were formed at lower concentrations relative to PA 1 (Figure SI-4). At 8.4 μM and above, PA 2 formed nanofibers across all pH values. TEM images collected for 100 μM PA 2 at pH 5.0 and 8.3 (Figure 1a,b) confirmed the nanofiber morphology. The PA 2 nanofiber diameter 9.5 ± 0.8 nm is close to the observed diameters for PA 1 nanofibers (9.1 ± 1.5 nm) from previous TEM measurements.16 Therefore, the self-assembled morphology does not significantly change upon changing the position of the Isoleucine in the hydrophobic region. The lowering of the CAC values upon moving the Isoleucine away from the palmitoyl tail was also observed in PAs containing a sterically bulky Gd:DO3A moiety. The phase diagram of PA 6 was measured and overlaid with the previously characterized PA 5 (Figure 2c). CAC values for PA 6 are also pH-dependent, ranging from 1.3 to 4.8 μM at pH 5 and 10, respectively (Figure SI-3b). Furthermore, the CAC values for PA 6 are consistently less than those for PA 5. For example, PA 6 exhibits over a 6-fold lower CAC value than PA 5 at pH ∼7. This indicates that PA 6 has greater attractive supramolecular interactions relative to PA 5. The sterically bulky Gd:DO3A moiety induces a spherical micelle morphology at high pH values and high concentrations. Moving the Isoleucine away

Table 1. Synthesized PA Molecules Molecule PA PA PA PA PA PA

1 2 3 4 5 6

Sequence palmitoyl-IAAAEEEE-NH2 palmitoyl-AIAAEEEE-NH2 palmitoyl-AAIAEEEE-NH2 palmitoyl-AAAIEEEE-NH2 palmitoyl-IAAAEEEEK(DO3A:Gd)-NH2 palmitoyl-AIAAEEEEK(DO3A:Gd)-NH2

Chart 1. PA Structure and Design

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EXPERIMENTAL SECTION All peptides were synthesized using solid phase Fmoc chemistry, purified via reverse-phase high performance liquid chromatography (HPLC) (Figure SI-1) and assessed for purity with Analytical HPLC, electrospray ionization mass spectrometry (Figure SI-2) and peptide content analyses. Critical aggregation concentration (CAC) measurements using the pyrene 1:3 method22 were used to determine the concenB

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The transition pH shifted to more acidic values (from 7.7 to 6.5) with lower PA concentrations (Figure SI-5). Above 45 μM, the pH of transition does not change with PA concentration, similar to PA 5. At all concentrations, PA 6 exhibited a transition pH that was more basic by 1.5 to 2.0 pH units relative to PA 5. Again, these morphologies were verified with TEM measurements (Figures 2a, 2b). At pH 6.6 (150 μM), nanofibers of ∼11.3 ± 1.1 nm width and in excess of 1 μm length were identified, often in bundles. At pH 10.0 (1 mM) PA 6 showed self-assembled spheres that were ∼10.1 ± 0.9 nm diameter. The diameters of both morphologies were comparable to previous TEM observations for PA 5, indicating that isomerization has a negligible effect on the size of the selfassembled structures.16 This shift in transition pH to more basic values reflects the increased β-sheet propensity of the Isoleucine when moved one amino acid away from the palmitoyl tail. Moving the Isoleucine to either the third or fourth amino acid position from the palmitoyl tail induces nanofiber formation across pH 5−10 and all detectable concentration values. CAC values for PA 3 were below the detectable limit (∼200-300 nM) at all pH values tested except at pH 10 (1.3 μM). PA 4 was observed to be in the aggregated state across all pH and concentration values and CAC values could not be detected via the pyrene 1:3 method. CD spectra for PA 3 revealed exclusively β-sheet formation irrespective of concentration and pH (Figure 3c, Figure SI-6). The nanofiber

Figure 1. TEM images of 100 μM PA 2 at (a) pH 5.0 (b) pH 8.3. (c) Concentration-pH phase diagram of PA 2 (blue) overlaid on the same for PA 1 (pink), as determined via CD (squares) and CAC (triangles) measurements. For both PAs, the top area corresponds to nanofiber morphologies, and the bottom area corresponds to unassembled single molecules. The self-assembled morphology in the region of the phase diagram between these two regions is uncertain due to the lack of experimental techniques in this concentration range. All measurements were performed in 150 mM NaCl and 2.2 mM CaCl2.

Figure 3. TEM images of 100 μM (a) PA 3 at pH 8.0 and(b) PA 4 at pH 8.0. pH-dependent CD spetra of (c) 5 μM PA 3 and (d) 10 μM PA 4. All measurements were conducted in 150 mM NaCl and 2.2 mM CaCl2.

morphology was confirmed at pH 8.0 via TEM measurements (Figure 3a). The observed fibers were >1 μm in length and ∼9.6 ± 0.5 nm in diameter. CD spectra for PA 4 also exhibited a β-sheet secondary structure regardless of concentration and pH (Figure 3d, Figure SI-7). TEM images collected at pH 8.0 of PA 4 additionally show the formation of nanofibers with lengths >1 μm and ∼9.0 ± 0.8 nm in diameter (Figure 3b). Together, these results clearly demonstrate that simply moving the position of a single β-sheet forming residue in a PA can dramatically alter the pH- and concentration-dependent self-assembly behavior. More specifically, moving the Isoleucine farther away from the palmitoyl tail enhances its ability to promote β-sheet formation, inducing the nanofiber morphology

Figure 2. TEM images of PA 6 at (a) 150 μM, pH 6.6 and (b) 1 mM, pH 10.0. (c) Concentration-pH phase diagram of PA 6 (blue) overlaid on the same for PA 5 (pink), as determined via CD (squares) and CAC (triangles) measurements. All measurements were performed in 150 mM NaCl and 2.2 mM CaCl2.

from the palmitoyl tail shifts the transition pH to more basic values. For PA 6, the transition pH between spherical micelles and nanofibers was concentration dependent below 45 μM. C

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powerful strategy that will allow additional flexibility in designing new classes of sensors, diagnostic and therapeutic vehicles that function via changing morphologies in response to specific stimuli. However, in order to precisely control and predict their self-assembly transition behavior, a more rigorous understanding of how to balance the intramolecular and supramolecular interactions via molecular design is essential.

at lower concentrations and more basic pH values. The effect of isomerizing the peptide sequence is much more significant than that of changing the identity of the amino acid. In our previous studies, substituting the Isoleucine of PA 1 with amino acids having weaker β-sheet propensity such as Valine, and Tyrosine, shifted the transition only by 0.4-0.6 pH units.16 Here, moving the Isoleucine to the second amino acid position from the palmitoyl tail, shifts the transition pH by 1.5-2.0 units, and in the third and fourth position, nanofibers form regardless of pH at all observable concentrations. We hypothesize that when positioned next to the palmitoyl acid tail, hydrophobic residues like Isoleucine can readily associate with the hydrophobic alkyl tail, reducing their preference to form a β-sheet secondary structure. This association interrupts β-sheet formation among neighboring PA molecules. Creating distance between the Isoleucine and the hydrophobic core via isomerization makes their hydrophobic association less favorable. Instead, the Isoleucine facilitates hydrogen-bonding interactions among neighboring PAs, thus adding to the attractive interactions of the hydrophobic core. Another possibility is that when placed in close proximity to the Glutamic acid residues, the hydrophobic Isoleucine could reduce the electrostatic repulsive interactions of these charged anionic residues. It is well established that the pKa of an anionic amino acid shifts to more basic values when placed in a more hydrophobic environment. These effects combined result in the PA adopting nanofiber morphologies at lower concentrations and at more basic pH values. This preference in forming nanofibers becomes even more pronounced as the distance between the hydrophobic residue and the palmitoyl core increases in PA 3 and PA 4. Indeed, previous studies using coarse-grained molecular dynamics simulations suggest that the hydrophobic amino acid positioned next to the palmitoyl tail strongly associates with the alkyl tail core in another peptide amphiphile system, palmitoylVVVAAAEEE-NH2.23 These simulations also suggest that the third and fourth amino acid residue away from the palmitoyl tail have the highest probability of forming a hydrogen bond with a neighboring peptide, thus promoting the β-sheet secondary structure.23 These simulations are consistent with our observation that moving the hydrophobic residue to the third and fourth position maximizes its β-sheet propensity exclusively forming the nanofiber morphology, irrespective of pH and concentration.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental methods, materials, HPLC chromatograms, ESIMS spectra, CD spectra, and CAC determination. 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]. Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS TEM images presented in this report were generated using the instruments and services at the Campus Microscopy and Imaging Facility at The Ohio State University. J.E.G. acknowledges the Pelotonia Intramural Research Program for funding. E.T.D. acknowledges the Pelotonia Undergraduate Research Fellowship for funding.

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OUTLOOK The rational design of stimuli-responsive PA vehicles that undergo morphological transitions under precise sets of solution conditions is crucial to guarantee their functionality in vivo. This work shows that in short amphiphilic peptides, the peptide sequence plays a crucial role in tailoring the pHtriggered self-assembly transition. In these PAs, changing the position of a single β-sheet forming Isoleucine residue induces a much larger change in their micelle-to-nanofiber transition pH (> 2 pH units), compared to changing the identity of this residue (∼0.6 pH units).16 Moving this hydrophobic amino acid away from the palmitoyl core strengthens its ability to promote β-sheet formation, preferentially inducing a nanofiber morphology. This likely occurs as a result of the reduced tendency of the Isoleucine to associate with the palmitoyl core, as well as the reduction of electrostatic repulsive interactions among the Glutamic acid residues due to their closer proximity to Isoleucine. This work shows that sequence isomerization is a D

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Peptide Amphiphile Supramolecular Structure and Interactions. Langmuir 2011, 27, 6163−6170. (12) Pashuck, E. T.; Cui, H.; Stupp, S. I. Tuning Supramolecular Rigidity of Peptide Fibers through Molecular Structure. J. Am. Chem. Soc. 2010, 132, 6041−6046. (13) Albanese, A.; Tang, P. S.; Chan, W. C. W. The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems. Annu. Rev. Biomed. Eng. 2012, 14, 1−16. (14) Decuzzi, P.; Pasqualini, R.; Arap, W.; Ferrari, M. Intravascular Delivery of Particulate Systems: Does Geometry Really Matter? Pharm. Res. 2009, 26, 235−243. (15) Hashim, A. I.; Zhang, X.; Wojtkowiak, J. W.; Martinez, G. V.; Gillies, R. J. Imaging pH and metastasis. NMR Biomed. 2011, 24, 582− 591. (16) Ghosh, A.; Haverick, M.; Stump, K.; Yang, X.; Tweedle, M. F.; Goldberger, J. E. Fine-Tuning the pH Trigger of Self-Assembly. J. Am. Chem. Soc. 2012, 134, 3647−3650. (17) Lee, N. R.; Bowerman, C. J.; Nilsson, B. L. Effects of Varied Sequence Pattern on the Self-Assembly of Amphipathic Peptides. Biomacromolecules 2013, 14, 3267−3277. (18) Lee, N. R.; Bowerman, C. J.; Nilsson, B. L. Sequence Length Determinants for Self-Assembly of Amphipathic β-Sheet Peptides. Biopolymers 2013, 100, 738−750. (19) Zhao, Y.; Wang, J.; Deng, L.; Zhou, P.; Wang, S.; Wang, Y.; Xu, H.; Lu, J. R. Tuning the Self-Assembly of Short Peptides via Sequence Variations. Langmuir 2013, 29, 13457−13464. (20) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. Self-Assembly of Peptide−Amphiphile Nanofibers: The Roles of Hydrogen Bonding and Amphiphilic Packing. J. Am. Chem. Soc. 2006, 128, 7291−7298. (21) Cui, H.; Cheetham, A. G.; Pashuck, E. T.; Stupp, S. I. Amino Acid Sequence in Constitutionally Isomeric Tetrapeptide Amphiphiles Dictates Architecture of One-Dimensional Nanostructures. J. Am. Chem. Soc. 2014, 136, 12461−12468. (22) Aguiar, J.; Carpena, P.; Molina-Bolivar, J. A.; Ruiz, C. C. On the Determination of the Critical Micelle Concentration by the Pyrene 1:3 Ratio Method. J. Colloid Interface Sci. 2003, 258, 116−122. (23) Fu, I. W.; Markegard, C. B.; Chu, B. K.; Nguyen, H. D. The Role of Electrostatics and Temperature on Morphological Transitions of Hydrogel Nanostructures Self-Assembled by Peptide Amphiphiles via Molecular Dynamics Simulations. Adv. Healthcare Mater. 2013, 2, 1388−1400.

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