Backbone Engineered Î³-Peptide Amphitropic Gels for Immobilization

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Backbone Engineered #-Peptide Amphitropic Gels for Immobilization of Semiconductor Quantum dots and 2D Cell Culture Rajkumar Misra, Aman Sharma, Anjali Shiras, and Hosahudya N Gopi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01283 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Backbone

Engineered

γ-Peptide

Amphitropic

Gels

for

Immobilization of Semiconductor Quantum dots and 2D Cell Culture Rajkumar Misra,† Aman Sharma,‡ Anjali Shiras, ‡ Hosahudya N. Gopi*† †

R. Misra, Dr. H. N. Gopi, Department of Chemistry, Indian Institution of Science Education

and Research, Homi Bhabha Road, Pune-411008, India. ‡

Dr. A. Sharma, Dr. Anjali Shiras, National Center for Cell Science, Pune University

Campus, Pune-411 007, India. Corresponding Author *E-mail address: [email protected]

Keywords: Supramolecular-assembly, Injectable hydrogel, γ-peptides, biocompatibility, quantum dots.

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ACS Paragon Plus Environment

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ABSTRACT: We are reporting a spontaneous supramolecular assembly of backbone engineered γ-peptide scaffold and its utility in the immobilization of semiconductor quantum dots and in cell culture. The stimulating feature of this γ-peptide scaffold is that it efficiently gelate both aqueous phosphate buffers as well as aromatic organic solvents. A comparative and systematic investigation reveals that the greater spontaneous self-aggregation property of γ-peptide over the α-, and β-peptide analogues is mainly due to the backbone flexibility, increased hydrophobicity and π-π stacking of γ-phenylalanine residues. The hydrogels and organogels obtained from the γ-peptide scaffold have been characterized through field emission scanning electron microscopic (FE-SEM), transmission electron microscopic (TEM), FT-IR, circular dichroism (CD), wide-angle X-ray diffraction and rheometric study. Additionally, the peptide hydrogel has displayed a stimuli responsive and thixotropic signature, which leads to the injectable hydrogels. 2D cell culture studies using normal and cancer cell lines reveal the biocompatibility of γ-peptide hydrogels. Further, the immobilization of semiconductor core-shell quantum dots in the transparent γ-peptide organogels showed ordered arrangement of quantum dots along the peptide fibrillar network with retaining photophysical property. Overall, γ-peptide scaffolds may serve as potential templates for the design of new functional biomaterials.

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INTRODUCTION Hierarchical self-assembling ability of peptides has been widely explored to design functional biomaterials. Peptides have been shown to adopt various diverse nanoarchitectures such as vesicles, micelles, fibers, ribbons, tapes and tubes.1-10 The supramolecular assembly of peptides is mainly governed by intermolecular non-covalent interactions such as hydrophobic, van der Waals, electrostatic, hydrogen bonds, and π-π interactions. Among various non-covalent interactions, aromatic-aromatic interactions play a crucial role in selfassembly of short peptides.11-21 The discovery of peptide nanotubes from a simple Phe-Phe dipeptide motif by Gazit et. al. gave a new dimension to the peptide based biomaterials.12 The peptide nanotubes have been explored as biosensors, delivery agents, templates for casting metal nanowires, energy storage materials, light harvesting, and nanopiezoelectrics.22-24 In addition to the peptide nanotubes, low molecular weight peptide gels specially hydrogels are becoming increasingly popular in tissue engineering, wound healing, drug delivery and antimicrobial candidates.25-31 In this context, the groups of Gazit,32 Uljin,33 Xu34 and others3539

exploited aromatic-aromatic interactions to derive peptide hydrogels from Phe-Phe

dipeptide motif by coupling with different aromatic rigid moieties. In continuation, Banerjee et. al. showed the supramolecular hydrogels of Phe-Phe by coupling with long fatty acid tail.40 Further, the self-assembling properties of modified dipeptides ∆Phe-∆Phe40 and sidechain homologated Phe-Phe42 dipeptides have also been investigated. Besides the α-peptides, supramolecular aggregation of β-peptides43-48 and oxazaolidinones49 have also been examined. We have been interested in understanding the conformational properties and the supramolecular assemblies of hybrid peptide foldamers composed of γ-amino acids. Recently, we showed the spontaneous self-assembly of γ- and hybrid γ-peptides into 3


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nanofibers, vesicles, nanotubes and polyhedrons.50-52 Despite the remarkable folding properties and ordered supramolecular aggregations, the use of backbone engineered γpeptides as low molecular weight gelator are still unknown. In addition, influence of the backbone flexibility on the supramolecular assembly has not been systematically investigated. Nevertheless, Bianco and co-workers recently reported self-assembly of β- and γ-Phe-Phe dipeptide motifs in combination with functionalized carbon nanotubes.53 We envisioned that increasing the backbone flexibility from α→β→γ may lead to greater hierarchical architectures. The potential applications of easily accessible and biocompatible Phe-Phe dipeptide motif motivated us to inspect the supramolecular assembly of α-, β- and γPhe-Phe dipeptide motifs under identical conditions. Herein, we are reporting the stimuli responsive, injectable, bio-compatible and bio-stable transparent hydrogels and organogels from γ-Phe-Phe dipeptide motif. In comparison, no hydrogels are observed from the αpeptide and opaque gels are observed from the β-peptide analogue under identical conditions. These results demonstrate the advantage of γ-peptides in the hydrogelation process compared to that of α- and β-peptides. EXPERIMENTAL SECTION Materials

and

Methods.

All

amino

acids,

triphenylphosphine,

Quantum

dots,

Trifluoroacetic acid (TFA), Ethyl bromoacetate, N,N′-Dicyclohexylcarbodiimide (DCC) , 1Hydroxybenzotriazole ( HOBt) and Pd/C (10%), were purchased from commercial sources. Dulbecco Modified Eagle Medium (DMEM), 3-(4, 5- dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT), kanamycin sulfate, trypsin−EDTA solution, and fetal bovine

serum

were

purchased

from

Sigma

aldrich.

Dichloromethane

Dimethylformamide (DMF), ethyl acetate and pet-ether (60-80

(DCM),

o

C) have used after

distillation. Tetrahydrofuran (THF) was dried over sodium and distilled immediately prior to 4


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use. Column chromatography was performed on silica gel (120-200 mesh). 1H spectra were recorded on 400 MHz (or

13

C on 120 MHz) using residual solvents as internal standards

(Dimethyl sulfoxide-d6 δH 2.5 ppm, δC 39.51 ppm). Chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in Hz. Matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF/TOF) was used to obtain mass of the peptides. Rheometer was used for rheological studies. Infrared spectroscopy (IR) spectra were recorded on FT-IR spectrometer. Data for X-ray structure determination were obtained using Mo-Kα (λ= 0.71073 Å) graphite monochromated radiation. The Boc group was used for the N-terminal protection and the C-terminus was protected either with ethyl or methyl esters. The Boc-(S)-β-Phe was synthesized by Arndt–Eistert homologation of BocPhe.54 The Boc-(S)-γ-Phe was synthesized by Wittig reaction starting from Boc-Phe followed by catalytic hydrogenation and saponification of the ethyl ester of Boc-γ-phenylalanine as reported

earlier.55

Couplings

were

carried

out

using

N-Ethyl-N′-(3-

dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1- hydroxybenzotriazole (HOBt). The final compound was fully characterized by mass spectrometry, 1H NMR spectroscopy and 13C NMR spectroscopy. General procedure for the Peptide Synthesis: Synthesis of peptide P1, P2, P3, P4 and P5 were carried out by conventional solution phase chemistry. Briefly, Boc-amino acid (1 mmol) was dissolved in 10 mL of DCM and cooled to 0 °C. To this cold solution, ethyl or methyl ester of amino acid (1 mmol) was added, followed by EDC.HCl (1 mmol) and HOBt (1 mmol). The reaction mixture was stirred for about 4 hr. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with 100 mL ethyl acetate and washed with 10% Na2CO3 (3 × 30 mL), 5% HCl ( 3 × 30 mL), water ( 3 × 30 mL), brine solution (2 ×30 mL) and dried over 5


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anhydrous sodium sulphate. The organic layer was evaporated under reduced pressure to get crude peptides. The crude peptides were further purified through column chromatography using ethyl acetate/hexane gradient solvent system. The pure peptides were further subjected ester hydrolysis in methanol using 1N NaOH. The peptide acids were directly used for the gelation studies. RESULTS AND DISCUSSION The sequences of peptides under investigation (P1-P5) are shown in Figure 1. Peptides were synthesized through conventional solution phase method using EDC and HOBt as coupling agents and purified. The dipeptide acids were subjected to the gelation studies.

Figure 1. Chemical structures of peptides P1 to P5

Gelation study. In order to understand the ordered aggregation in aqueous buffers, all purified peptides, P1-P5, were subjected to gelation in phosphate buffer at pH 7.4. The gelation behavior peptides P1-P5 is shown in Figure 2. The α-peptide P1 was found to be soluble in phosphate buffer after gentle heating, while peptide P2 was found to be sparingly soluble in phosphate buffer and showed opaque hydrogels. In a marked contrast to P1 and 6


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P2, the γ-peptide P3 showed the highly stable transparent hydrogel in phosphate buffer. Interestingly, the gels were found to be stimuli responsive in nature. The gel to sol transition was observed either by increasing the temperature or by external mechanical forces. Instructively, γ-peptides P4 with Leucine side-chains and P5 with conformationally restricted backbone double bonds failed to produce hydrogels. P4 was found to be highly soluble, whereas P5 was found to be sparingly soluble in phosphate buffer.

The spontaneous

hydrogelation property of P3 could be attributed to its flexible backbone, which enhances the rotational freedom of the peptide and hydrophobicity as well as π-π staking between the phenyl groups. The notable gelation properties of P3 motivated us to investigate its molecular level aggregation in single crystals. Several attempts have been made to crystalize peptide P3, however due to its very high tendency for self-aggregation led to the fibers. Nevertheless, we were able to get single crystals for Boc–γPhe-γPhe-OEt and NH2-γPhe-γPhe-COOH (Figure 2). Both peptides adopted β-sheet type conformation in single crystals, stabilized by intermolecular hydrogen bonds as well as π-π staking between the phenyl groups of adjacent peptides.

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Figure 2. Examination of the gelation properties of peptides P1 to P5 in phosphate buffer (6 mg/mL) pH at 7.4 (A) Crystal structure of BocNH-γPhe-γPhe-COOEt (B) Crystal structure of H2N-γPhe-γPhe-COOH.

Morphology study. To gain insight into the nature of supramolecular assembly in aqueous buffer of these peptides, field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) were carried out under identical conditions in phosphate buffer. The FE-SEM and TEM images are shown in Figure 3. Peptide P1 showed the microtubular assembly. The dilute samples of opaque P2 and transparent P3 hydrogels in phosphate buffer showed nanofibrillar assembly, however with different entangled network. The average diameter of the nanofibrils of P3 was found to be around 30 nm as suggested by the TEM analysis. Peptide P4 with γ-leucine residues showed tubular or rod shape morphology, while the γ-peptide P5 showed spherical morphology in SEM analysis (Figure 3). The formation of ordered supramolecular assembly observed in the peptides P1-P5, is the combined effect of various intermolecular noncovalent interactions like hydrogen bonding, π-π stacking, electrostatic, hydrophobic and van der wall’s interactions. The sidechains and backbone conformations of the peptides are mainly responsible for all these noncovalent interactions. Peptides P1, P2 and P3 have similar side chain functionality however with different backbones. As envisioned the increasing backbone flexibility which simultaneously increases the hydrophobicity, molecular entropy and H-bonding favours the gelation of peptides in aqueous buffer. The SEM images of peptide P1 revealed the thermodynamically favourable crystalline peptide self-assembly. In spite of having similar γpeptide backbone as P3, peptide P4 failed to gelate aqueous buffers due to the lack of aromatic-aromatic interactions. The self-assembled morphology observed for P4 is purely due to the hydrophobic interactions between the Leu side-chains and H-bonding. In peptide 8


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P5, the backbone rotational freedom is restricted through the α, β-unsaturated double bond and therefore failed to gelate, suggesting the importance of both π-π staking of the sidechains and rotational freedom of the peptide backbone in gelation. The driving forces of the peptide P3 gelation are found to be the optimum balance of π–π stacking, hydrogen bonding, and hydrophobic interactions. Due to the rotational freedom arising through additional C-C bond in the γ-peptide backbone, the external triggers like sonication assisted the self-assembly process kinetically and that leads to the gelation.

Fig 3. (A,B) SEM images of peptide P1, P2, at minimum gelation condition(6mg/mL). (C, D) SEM and TEM images of peptide hydrogels peptide P3. (E,F), SEM, images of peptide P4, P5, at minimum gelation condition (6mg/mL).

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CD and UV Spectroscopy. We further invoke circular dichroism (CD) and UV Vis absorption spectroscopy to gain insight into the peptide secondary structure and to understand molecular level aggregation of P3 in concentration dependent manner. The dilute solution (1.06 mM) of P3 in phosphate buffer displayed CD minima at 220 nm, supporting the extended conformation in solution.56-57 Instructively, with increasing concentration of P3 from 0.5 mg to 5 mg/mL leads to the gradual decrease in CD minima (Figure 4B) and complete loss of CD signal at 5 mg/mL. In addition, the observed red shift of the CD minima along with signal broadening support the aggregation of peptide through the intermolecular hydrogen bonding. In addition, the concentration dependent UV-Vis absorption spectroscopy studies of P3 suggested the red shift and broadening of amide (220 nm) and aromatic (260 nm) groups (Figure 4A). This was further supported by the thin and thick fibrillar networks of P3 in SEM analysis at 3 mg/mL and 5 mg/mL, respectively (Figure 4). These results demonstrated the key role of both hydrogen bonding and π- π staking of phenyl groups in hierarchical aggregation.58

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Fig 4. (A) UV spectra of peptide P3 at different concentration in phosphate buffer. (B) CD spectra of peptide P3 at different concentration in phosphate buffer. (C, D) SEM images of peptide fibril at different concentration of peptide P3

FT-IR spectroscopy and PXRD study. To understand the conformations of the peptide in solid as well as in xerogel, FT-IR and powder XRD (PXRD) studies were carried out. The characteristic C=O stretching (amide I) and N-H bending (amide II) peaks observed at 1646 cm-1 and 1528 cm-1 in both solid sample as well as in xerogel support the β-sheet signature of the peptide (Figure 5A).40 The β-sheet conformation of the peptide in xerogel was further confirmed by the wide angle PXRD. The peaks around 18o and 23o observed in the PXRD correspond to the d-spacing value 5 Å and 3.85 Å, respectively indicating a β-sheet arrangement of P3 in gel state. The d-spacing of 3.5 Å (2θ = 26o) and 2.85Å (2θ=32o) could be attributed to the π-stacking distance between the two aromatic groups across the β-strands and layer arrangement of hydrogelator in the gel phase (Figure 5B), respectively.59-60

Fig 5. (A) IR spectra of solid sample (red) and dried gel(blue) of P3 (B) PXRD pattern of dried gel P3

Viscoelastic and Stimuli responsive property. In order to understand the viscoelasticity and self-healing properties of γ-peptide hydrogels, rehological measurements of P3 hydrogel 11


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were carried out. The larger storage modulus (G′) over the loss modulus (G″) in a strainsweep experiment at constant oscillating frequency suggests the strong elastic behavior of the hydrogels (Figure 6A). Further, the frequency sweep experiment where the storage modulus (G′) and loss modulus (G″) were measured as a function of angular frequency at 0.1% constant strain suggest that G′ and G″ values are feebly dependent on frequency, inferring the formation of stable gels (Figure 6B ).

Figure 6. (A) Step-strain time dependent rheological analysis of the hydrogel P3 (6mg/mL) at a fixed angular frequency of 1 rad/s. (B) Frequency sweep rheological analysis of the hydrogel P3 (6mg/mL) at a constant strain of 0.1%.

As hydrogen bonding and π-π staking are major contributors for the formation of hydrogels, they also responsible for gel-sol transition with different stimuli. For example hydrogels displayed gel-sol transition with increasing temperature and transformed back to gel state when it reached to room temperature. In addition, hydrogel also possesses thixotropic property, upon mechanical shaking gel transformed to sol and quickly transform back to gel state as soon as the force was removed (Figure 7A), which further confirmed by the step-strain rheology experiment (Figure S7). This mechanoresponsive property leads to injectability of the hydrogelator, which is very crucial for biodelivery systems (Figure 7B). We further investigated whether these hydrogels can be used as drug delivery agents, as a 12


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proof of concept, we encapsulated small molecules, rhodamine dye and proflavine inside the gel matrix and their slow release to the clear top layer of phosphate buffer was monitored with time. The gradual enrichment of color intensity of the buffer layer and subsequent decrease in color intensity of the gel suggested that slow release of encapsulated molecules from the gel matrix (Figure 7C).40

Figure 7. (A) Stimuli responsive behaviour of peptide (P3) hydorogels. (B) Injectable nature of peptide hydrogels P3. (C) Slow Release of proflavin (yellow) rodhamine (pink) from hydrogel matrix to the buffer layer after 16 hours.

Amphitropic gelation of peptide P3. Further, the spontaneous hydrogelation properties of P3 and its inherent hydrophobicity and π-staking ability motivated us to examine whether or not this peptide scaffold can also gelate organic solvents. Remarkably, P3 showed the 13


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spontaneous thermo-reversible gelation property in various aromatic organic solvents such as benzene, chlorobenzene, toluene, mesitylene and xylene (Figure 8A). The visco-elastic behavior of transparent organogels is shown in Figure 8C. The TEM analysis of dilute gel samples suggests the nanofibrillar morphology (Figure 8B) of P3 in organic solvents similar to its hydrogel. Overall, these results suggest the amphitropic signature of γ-peptide P3.61-62

Figure 8. (A) Inverted sample vial experiment to confirm the gel formation of the peptide P3 in (I) Benzene; (II) Chlorobenzene; (III) Toluene; (IV) Xylene; (V) Mesitylene; (B) TEM image of the dilute organogel of peptide P3 in toluene. (C) Step-strain time dependent rheological analysis of the organogel P3 (6 mg/mL in toluene) at a fixed angular frequency of 1 rad/s

Immobilization of quantum dots. The transparent nature of γ-peptide organogel and the inherent photophysical properties of emissive core-shell CdSe/ZnS QD motivated us to 14


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examine whether fluorescent peptide organogels can be synthesized in combination with CdSe/ZnS quantam dots for the applications in electronic and optical devices.63 To prepare fluorescent organogel composed with CdSe/ZnS quantum dots, we added quantum dots dispersed in toluene to the preheated solution of peptide P3. After cooling, the formation of stable transparent gel was observed with green fluorescent emission (Figure 9A). The photophysical property of quantum dot retained in the gel state, however with quenching of fluorescence intensity compared to the QDs in disperse medium. This could be because of the interaction between peptide ligands and nanoparticles.64 To gain better insight into the morphological features of this hybrid system, transmission electron microscopy (TEM) was carried out. The TEM analysis revealed the ordered assembly of quantum dots with their regular shape and size along the peptide fibrilar network.

Figure 9. (A) Vial inversion test of CdSe/ZnS QD incorporated organogel P3, irradiated under UV light at 365 nm. (B) Photoluminescence spectra of CdSe/ZnS QD (0.5 mg/mL) before and after gelation (λexc = 465 nm). (C) TEM images of CdSe/ZnS QD incorporated organogel P3.

Biocompatibilty of hydrogel P3. To test the biocompatibility of novel γ-peptide hydrogel, in vitro cytotoxicity was carried out using 2D cell culture. The 1:1 ratio of hydrogel of P3 in 15


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PBS buffer and culture medium was coated on the culture plate. After 3 hours, HEK293T cells (normal cell line) and LN229 cells (cancer cell line) were seeded on the coated plate. Live dead assay was carried out after 18 hrs using calcein staining. The calcein staining live cell images (green) along with control (culture media) are shown in Figure 10. These results suggest the biocompatibility of γ-peptide hydrogels.

Fig 10 (A) Calcein AM staining after 18 hr in HEK293T, LN229 cells showing viable cells in green color. (B) Merged images of the same after 48 hr

Conclusion: In summary, we have demonstrated the remarkable hydrogelation property of backbone flexible γ-peptide P3. Under identical conditions, α-peptide P1 failed to gelate and β-peptide P2 form opaque gel. Similarly, γ-peptides with leucine side-chains P4 and conformationally restricted γ-peptide P5 failed to give hydrogels. These results suggested the importance of both conformational flexibility and aromatic interactions in the hydrogelation of short peptide sequences. More importantly, these γ-peptide hydrogels were found to be sensitive to the external stimuli. The biocompatibility and thixotropic behaviour with their 16


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inherent biostability of γ-peptide hydrogels can be further explored as injectable and drug delivery agents. In addition, γ-peptide P3 spontaneously self-assembled to organogels in various organic solvents, and this property has been utilized to immobilize semiconductor core-shell QDs. Overall, the strategy to design low molecular weight amphitrophic peptide gelator and its applications described here may seed the generation of new functional biomaterials.

ACKNOWLEDGEMENT R. M is thankful to CSIR for fellowship. HNG thanks DST-SERB for financial support. Supporting Information Detailed experimental procedures of peptide synthesis, characterization, X-ray structures, gelation studies. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1) Stupp, S. I.; Palmer, L. C. Supramolecular Chemistry and Self-Assembly in Organic Materials Design. Chem. Mater., 2014, 26 , 507-518. (2) Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol., 2003, 21, 1171-1178. (3) Lakshmanan,A.; Zhang, S.; Hauser, C. A. E. Short self-assembling peptides as building blocks for modern nanodevices. Trends Biotechnol., 2012, 30, 155-165.

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(4) Wang, J.; Liu, K.; Xinga, R.; Yan, X. Peptide self-assembly: thermodynamics and kinetics Chem. Soc. Rev., 2016, 45, 5589-5604. (5) Zelzer, M.; Ulijn, R. V. Next-generation peptide nonmaterials: molecular networks, interfaces and supramolecular functionality. Chem. Soc. Rev., 2010, 39, 3351-3357. (6) Gazit, E. Self-assembled peptide nanostructures: the design of molecular building blocks and their technological utilization. Chem. Soc. Rev., 2007, 36, 1263-1269. (7) Santis, E. De.; Ryadnov, M. G. Peptide self-assembly for nanomaterials: the old new kid on the block. Chem. Soc. Rev., 2015, 44, 8288-8300. (8) Ulijn, R. V.; Woolfson, D. N. Peptide and protein based materials in 2010: from design and structure to function and application. Chem. Soc. Rev., 2010, 39, 3349-3350. (9) Smith, A. M.; Ulijn, R. V. Designing peptide based nanomaterials. Chem. Soc. Rev., 2008, 37, 664. (10) Hamley, I. W. Peptide nanotubes. Angew. Chem. Int. Ed. 2014, 53, 6866-6881. (11) Gazit, E. A possible role for pi-stacking in the self-assembly of amyloid fibrils. FASEB J., 2002, 16 , 77-83. (12) Reches, M.; Gazit, E.; Casting metal nanowires within discrete self-assembled peptide nanotubes. Science, 2003, 300, 625-627. (13) Levin, A.; Mason,T. O.; Abramovich, L. A.; Buell, A. K.; Meisl, G.; Galvagnion, C. Bram,Y.; Stratford,S. A.; Dobson, C. M.; Knowles, T. P. J.; Gazit, E. Ostwald’s rule of stages governs structural transitions and morphology of dipeptide supramolecular polymers. Nat. Commun., 2014, 5, 5219.

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Backbone Engineered Î³-Peptide Amphitropic Gels for Immobilization

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