Barium-triggered β-sheet formation and hydrogelation of a short peptide derivative

Abstract

We reported on a short peptide–taurine conjugate that could adopt β-sheet conformation and form hydrogels triggered by Ba²⁺, which might be applied for the removal of Ba⁺ from water with high Ba²⁺ content.

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Stimuli-responsive hydrogels of peptides lead to biomaterials with controllable property and utility for a wide range of biomedical applications.¹ Many peptides or peptide derivatives can respond rapidly to external stimuli including enzymatic triggers,² light irradiation,³ redox,⁴ addition of polymer and protein additives,⁵ addition of salts,⁶etc.⁷ Among these external stimuli, the addition of metal ion has been of particular interest. Inspired by the naturally occurring protein–metal and peptide–metal interactions, several peptides that can form hydrogels via the addition of metal ions have been rationally designed and reported. For example, Stupp and co-workers have reported a calcium-triggered peptide hydrogel system for the biomineralisation of calcium phosphate.⁸ Schneider and co-workers have developed a peptide that can form β-hairpin structure and hydrogels upon the addition of zinc ion.⁹ They have also reported a peptide hydrogel formed by the addition of heavy metal ions.¹⁰ Xu and co-workers have used calcium ions to trigger the formation of short peptide-based hydrogels and adjust the elasticity of the resulting hydrogels.¹¹ They have also reported hydrogel systems responsive to potassium ion and nickel ion.¹² To our knowledge, a barium-triggered peptide hydrogel system has not been reported.

Barium chloride, along with other water-soluble barium salts, is highly toxic.¹³ At low doses, barium ions act as a muscle stimulant, whereas at high doses they affect the nervous system, causing cardiac irregularities, tremors, weakness, anxiety, dyspnea and paralysis. This may be due to the ability of Ba²⁺ to block potassium ion channels which are critical to the proper function of the nervous system.¹⁴ Therefore, the long-term drinking the water with high Ba²⁺ content would bring about serious harm to the human health.

Stimulated by aforementioned pioneering works, we hypothesise that the peptide containing sulfonic acid group can interact with barium ion, resulting in the formation of hydrogel. To test our hypothesis, we designed and synthesised a dipeptide molecule bearing taurine, named as Nap-FF-Taurine (Fig. 1). The synthesis of Nap-FF-Taurine as shown in Fig. S1† was easy and straightforward. We firstly obtained Nap-FF by solid phase peptide synthesis. The active N-hydroxylsuccinimide (NHS) ester of Nap-FF was then used to couple with taurine to yield Nap-FF-Taurine in a moderate yield of 85%. The pure Nap-FF-Taurine (Fig. 1) was obtained by reverse phase high performance liquid chromatography (HPLC).

Fig. 1 Chemical structure of the Nap-FF-Taurine and an optical image of the hydrogel of Nap-FF-Taurine (0.5 wt%) with 1 equiv. of Ba²⁺.

We found that the Nap-FF could not well dissolve in PBS solution due to strong hydrophobic interaction. But the Nap-FF-Taurine could form homogeneous solutions of in PBS at pH 7.4 when its concentration was lower than 0.7 wt% (Fig. S3†). After the addition of one equiv. of Ba²⁺ to the solution of Nap-FF-Taurine. We observed a rapid hydrogel formation (Fig. 1) and the minimum gelation concentration of Nap-FF-Taurine with one equiv. of Ba²⁺ was about 0.2 wt%. For a PBS solution containing 0.2 wt% of Nap-FF-Taurine, the minimum equiv. of Ba²⁺ to trigger the formation of a gel was about 0.50. However, the addition of one equiv. of other metal ions to the solution of Nap-FF-Taurine including Fe²⁺, Ca²⁺, Pd²⁺, Mg²⁺, Sr²⁺, Cu²⁺, and Zn²⁺ could not induce the hydrogel formation (Fig. S3†). These observations clearly indicated that Ba²⁺ could selectively induce the formation of hydrogels of Nap-FF-Taurine and demonstrated the success of our design.

We first used a rheometer to study the mechanical properties of solution of Nap-FF-Taurine and gels formed by treating solution of Nap-FF-Taurine with different equiv. of Ba²⁺. As shown in Fig. 2A, before the addition of barium ions, the solution of Nap-FF-Taurine possessed the lowest elasticity (G′) value of about 40 Pa at the frequency of 0.1 rad s⁻¹. Upon the addition of 0.1, 0.5, and 0.7 equiv. of Ba²⁺ to the solution, the G′ value changed to bigger one of about 52, 650, 750 Pa, respectively which indicated that the addition of more Ba²⁺ would lead to bigger G′ value and there was a sharp increase in G′ value for the solution with 0.3–0.6 equiv. of Ba²⁺ (Fig. 2B). The G′ value of samples with 0.7 and 0.9 equiv. of Ba²⁺ were similar. These observations correlated with the fact that 0.5 equiv. of Ba²⁺ would lead to the hydrogelation of solution of Nap-FF-Taurine (0.2 wt%).

Fig. 2 (A) Rheology with the mode of dynamic frequency sweep of solutions of Nap-FF-Taurine with different equiv. of Ba²⁺ (■ no Ba²⁺ ● 0.1 equiv. ▲ 0.5 equiv and ▼ 0.7 equiv. of Ba²⁺); (B) the plot of elasticity (G′) vs. the different equiv. of Ba²⁺ ([Nap-FF-Taurine] = 0.2 wt%).

To investigate the effect of Ba²⁺ on the self-assembled microstructure of the hydrogels, we used transmission electron microscopy (TEM) to examine the morphology of the solution and gels. The solution of Nap-FF-Taurine (0.5 wt%) presented a morphology of irregular aggregates (Fig. 3A). upon the addition of 0.5 equiv. of Ba²⁺ to the solution of Nap-FF-Taurine, the irregular aggregates changed to small and short fibrils with diameter of about 35 nm and length shorter than 1.2 μm (Fig. 3B). For the gel formed by adding 0.7 equiv. of Ba²⁺, it exhibited long and uniform fibers with the diameter of about 45 nm and length longer than 7.9 μm (Fig. 3C). These fibrils were entangled with each other forming a network for hydrogelation. These results agreed with the observations of rheological measurements and indicated that addition of barium ions could lead to fibril formation, enhancement of G′ value, and the formation of hydrogels.

Fig. 3 TEM images of (A) solution of Nap-FF-Taurine (0.5 wt%), (B) gel of Nap-FF-Taurine (0.5 wt%) with 0.5 equiv. of Ba²⁺, and (C) gel of Nap-FF-Taurine (0.5 wt%) with 0.7 equiv. of Ba²⁺.

Circular dichroism (CD) spectroscopy is a powerful tool which provides information about the secondary structure of the self-assembled peptide in the gel phase.¹⁵ So we explored CD spectroscopy to monitor the triggered folding and self-assembly of Nap-FF-Taurine in the presence of different equiv. of Ba²⁺ (Fig. 4A) and different other metal ions (Fig. 4B and C). As shown in Fig. 4A, Nap-FF-Taurine adopted a random coil conformation when there was no barium ion. When 0.1 equiv. of BaCl₂ was added to the peptide solution, the β-sheet structure was observed, indicated by the positive peak at 196 nm and negative one at 213 nm.¹⁵ Moreover, more amounts of Ba²⁺ would lead to more pronounced peaks, suggesting that the bind between Ba²⁺ and Nap-FF-Taurine initiated the transition of conformation and subsequently peptide assembly into a β-sheet-rich network. For other metal ions including Zn²⁺, Sr²⁺, Pb²⁺, Mg²⁺, Ca²⁺, Cu²⁺ and Fe²⁺, they could not induce β-sheet formation of Nap-FF-Taurine (Fig. 4B and C), indicating that there were the weak affinity interaction between these ions and Nap-FF-Taurine molecules. These observations correlated well with the results of hydrogel formation test and indicated the specific interaction between Ba²⁺ and Nap-FF-Taurine.

Fig. 4 Circular dichroism (CD) spectra of solutions of Nap-FF-Taurine (0.05 wt%) with (A) different equiv. of Ba²⁺ (B and C) one equiv. of different metal ions.

To further investigate the molecular arrangement in the hydrogels, we obtained fluorescence emission spectra of Nap-FF-Taurine in both solution and gel phases. As shown in Fig. S8,† the emission peaks at 330 nm appeared in both solution and gel phase. Compared with solution phase, the center of the emission peaks didn't show red shift in the gel phases, suggesting that there was no clearly π–π stacking between naphthalene rings in its gel phase. It was reported that under proper condition, the Nap-FF molecular have excellent self-assembly ability to form hydrogels due to π–π interaction.¹⁶ However, the conjugation of taurine with Nap-FF would decrease (even completely destroy) the self-assembly ability of Nap-FF due to the increase of flexibility of Nap-FF molecule. Therefore, the resulting conjugate of Nap-FF-Taurine could form homogeneous solutions in phosphate buffer saline (pH = 7.4) solution. Interestingly, the Nap-FF-Taurine can form hydrogels in the presence of Ba²⁺ but not in the presence of Zn²⁺, Sr²⁺, Pb²⁺, Mg²⁺, Ca²⁺, Cu²⁺, and Fe²⁺. The reason for this phenomenon might be that the Ba²⁺ could specific bind with sulfonic group of Nap-FF-Taurine, leading to form Nap-FF-Taurine–Ba complex which had extremely low solubility similar to the barium sulfate (BaSO₄).

We attempted to use Nap-FF-Taurine to remove Ba²⁺ from water. 3 mg of Nap-FF-Taurine could decrease the concentration of Ba²⁺ in 5 mL of water from 850 ppm to 200 ppm, which indicated that Nap-FF-Taurine could act as potential treatment agent of water with high Ba²⁺ content.

In summary, we demonstrated that taurine is a useful functional group for the construction of a peptide conjugate that could respond to Ba²⁺. The specific binding of taurine with barium ion could lead to β-sheet formation of the peptide conjugate and subsequent hydrogel formation. This is another example of a hydrogel system that uses metal–ligand coordination bonding. In addition, this rapid and specific hydrogelation system might be applied for the removal of toxic Ba²⁺ from water with high Ba²⁺ content. However, a low-cost method for the peptide's preparation needs to be developed to ensure economic feasibility, and the structure and components of the peptide also need to be further optimized so that it can bind Ba²⁺ as much as possible.

Experiments

Synthesis of Nap-FF-Taurine

Nap-FF was first synthesised by solid-phase peptide synthesis (SPPS) methods as described in the ESI.† For synthesis of Nap-FF-Taurine, the DMF solution containing Nap-FF and NHS was mixed with aqueous solution of equal amount of Taurine to form a homogeneous solution in a flask. After being cooled to 0 °C in the ice bath, DIEPA was added to adjust the pH to about 8. EDC was then added in the flask. The resulting reaction mixture was stirred overnight and directly separated by HPLC with MeOH–water (0.1%) as the eluents and the target product was collected. As shown in Fig. S1(B),† there is only one narrow single peak on the LC-MS spectrum indicating the target product was very pure. The MS data (Fig. S2†) of target product (MS: (M + 1)⁺ = 588.69, HR-MS: (M + 1)⁺ = 588.2165) was very consistent with that of Nap-FF-Taurine (calc: M⁺ = 587.69), proving the pure Nap-FF-Taurine was obtained.

Characterization of Nap-FF-Taurine

Structure of Nap-FF-Taurine was characterized by ¹H NMR (DMSO-d6) and ¹³C NMR (DMSO-d6). The characteristic peaks of ¹H NMR were assigned as follows: ¹H NMR (400 MHz, DMSO-d6): δ 8.25 (m, 2H), δ 7.93 (m, 1H), δ 7.86 (d, J = 8.0 Hz, 1H), δ 7.79 (d, J = 8.0 Hz, 1H), δ 7.76 (d, J = 8.0 Hz, 1H), δ 7.61 (s, 1H), δ 7.18 (m, 12H), δ 4.53 (m, 1H), δ 4.38 (m, 1H), δ 3.56 (m, 3H), δ 3.31 (m, 3H), δ 3.01 (m, 2H), δ 2.78 (m, 2H). ¹³C NMR (DMSO-d6): δ 171.1, 170.3, 169.9, 138.0, 137.8, 134.1, 133.0, 131.8, 129.4 (2 signals), 129.3 (2 signals), 128.2 (2 signals), 128.0 (2 signals), 127.7, 127.6 (2 signals), 127.5, 127.4, 126.4, 126.2, 126, 125.6, 54.4, 53.9, 50.4, 42.3, 37.7, 37.5, 35.7 ppm.

Hydrogel formation

In order to investigate the hydrogel formation, The Nap-FF-Taurine was first dissolved in a small amount of PBS, then sodium carbonate solution were added to adjust the pH to about 7.4, afterward, a certain amount of PBS was supplemented to obtain Nap-FF-Taurine solution with desired concentration of Nap-FF-Taurine. As shown in Fig. S4,† the gelation occurred after the addition of 0.5 or 0.7 equiv. of Ba²⁺. However, the gelation didn't occur after the addition of Ca²⁺, Cu²⁺, Fe²⁺, Pb²⁺, Sr²⁺, Mg²⁺ and Zn²⁺ (Fig. S5†).

Removal of Ba²⁺ from water

To investigate the removal of Ba²⁺ from water, 5 mL of the solution containing 850 ppm of Ba²⁺ was prepared. 3 mg of Nap-FF-Taurine was then added with final concentration of 0.06%. Several minutes later, the floccule rather than hydrogel was formed due to lower concentration than that of gelation. After centrifugalization, the concentration of Ba²⁺ in supernatant was determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES).

Acknowledgements

This work is supported by NSFC (31070856 and 51003049) and Tianjin MSTC (12JCYBJC11300).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization of the peptide, rheology, ¹H NMR, mass spectrum (MS) and fluorescence spectra, etc. See DOI: 10.1039/c3ra45023f

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