A novel H₂O₂ responsive supramolecular hydrogel for controllable drug release

Abstract

Due to the important significance of hydrogen peroxide (H₂O₂) in physiology, aging and disease in living organisms, tremendous effort has been devoted to develop H₂O₂ responsive materials for the detection of its over production or for controlled drug release. However, it is still challenging to develop H₂O₂ responsive supramolecular hydrogels. In this study, we designed and synthesized a novel H₂O₂ responsive peptide hydrogelator bearing the thiazolidinone group. A supramolecular hydrogel based on peptide self-assembly was prepared through a heating–cooling process and its gel–sol phase transition could be triggered by the removal of thiazolidinone groups upon H₂O₂ oxidization. The excellent H₂O₂ responsive property of the supramolecular hydrogel was investigated by LC-MS, rheology and TEM. A drug release study in vitro demonstrated that the gel–sol phase transition could be applied for releasing gemcitabine sustainedly and controllably. Our study could provide a new way for the design of H₂O₂ responsive materials and hold great potential in the application of anticancer drug delivery.

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Peptide-based supramolecular hydrogels have attracted extensive research interest in the past few decades due to their inherent advantages, such as ease of design and synthesis, good biocompatibility and degradability.¹ Since supramolecular hydrogels are formed by non-covalent interactions (hydrogen bond, π–π, hydrophobic, and charge interactions), they are therefore extremely sensitive to external stimuli, including pH,² light,³ enzymes,⁴ ions,⁵ and redox agents.⁶ Increasing numbers of intelligent responsive supramolecular hydrogels have been developed so far, and they have shown great promise in the applications of drug delivery,⁷ cancer cell inhibition,⁸ vaccine adjuvants⁹ and detection of important analytes.¹⁰

Hydrogen peroxide (H₂O₂), as a second messenger for intracellular signal transduction,¹¹ usually results from cellular metabolism of molecular oxygen. In most cases, it is maintained at well-balanced concentration levels and plays crucial roles in cell proliferation, cell differentiation and cell migration.¹² The over-production of H₂O₂ can lead to high oxidative stress and therefore impaired cellular structures.¹³ Many reports have demonstrated that a series of pathologies are associated with elevated levels of H₂O₂ including inflammation,¹⁴ cancer,¹⁵ cardiovascular disorders,¹⁶ and neurodegenerative diseases.¹⁷ Consequently, enormous efforts have been made to develop H₂O₂ responsive nano-systems for controlled drug release to treat these diseases or for the detection of its over-production. These nano-systems are mostly based on the conventional peroxalate ester that can react with H₂O₂. For example, Murthy and co-workers have reported on nanoparticles formulated from peroxalate and fluorescent dyes capable of imaging hydrogen peroxide in vivo with high specificity and sensitivity.¹⁸ There are also several reports regarding H₂O₂ responsive supramolecular hydrogels. For example, Hamachi and co-workers have recently reported on responsive peptide-based hydrogels containing a H₂O₂-reactive boronoarylmethoxycarbonyl group and capable of retaining the activity of encapsulated enzymes.¹⁹ They have demonstrated that the programmable hybridization of the hydrogel and oxidases enables the resulting materials responding to not only H₂O₂ molecules but also a variety of disease-related biomarkers. These pioneering works highlight the importance of H₂O₂ responsive materials including the supramolecular hydrogels.

Recently, a novel H₂O₂ responsive group, thiazolidinone modified carboxylic acid, has been reported, and it can release free carboxylic acid upon the activation of H₂O₂.²⁰ Inspired by this work, we opted to develop a novel H₂O₂ responsive supramolecular hydrogelator bearing the thiazolidinone group. We therefore designed a short peptide derivative Nap-GFFYGD(Thi) (compound 1) bearing a H₂O₂ responsive thiazolidinone at the C-terminal of the peptide. As shown in Scheme 1, compound 1 was expected to form nanofibers and a hydrogel by supramolecular self-assembly at a given concentration. The removal of thiazolidinone through the oxidation/elimination reaction by H₂O₂ was expected to produce a more hydrophilic peptide Nap-GFFYGD (2), resulting the dis-assembly of nanofibres and a gel–sol transition. The synthesis route and purification process of Fmoc-aspartic acid decorated with thiazolidinone (D(Thi)) were described in Scheme S1.† We then prepared compound 1 by standard solid phase peptide synthesis (SPPS) and obtained the pure compound by reverse-phase high performance liquid chromatography (HPLC).

Scheme 1 (a) Chemical structures of Nap-GFFYGD(Thi) (compound 1) and Nap-GFFYGD (compound 2) and H₂O₂ triggered conversion. (b) Schematic representation of possible H₂O₂ triggered hydrogel degradation.

After obtaining the designed compound, the gelation property of compound 1 was firstly examined. Results in Fig. 1A indicated that it could form a hydrogel at a minimum gelation concentration (MGC) of 0.1 wt% within 5 minutes upon a heating–cooling process. We then tested the H₂O₂ responsive property of the hydrogel. A gel–sol transformation was observed after adding H₂O₂ (40 mM) to the gel at 37 °C for 4 hours (Fig. 1B). H₂O₂-response sensitivity of the hydrogel was evaluated through the gel–sol transition after the addition of 0–100 mM H₂O₂ to the gel. The results showed that at least 4 mM H₂O₂ was required to induce a complete collapse of the gel after 24 hours (Fig. S9†) and the time required for the gel–sol transition ranged from 1.5 to 24 hours depending on the amount of H₂O₂ (Fig. S10†). Liquid chromatography mass spectrometer (LC-MS) was employed to analyse the gel–sol phase transition. As shown in Fig. 1C, compound 1 gradually converted to compound 2 after the addition of H₂O₂. For instance, the conversion percentage was about 50% at one hour time point and reached an equilibrium after about four hours with a conversion rate of about 96% (Fig. S11†). The mass spectra of resulting solution in Fig. 1B (Fig. S7†) indicated the molecular weight peak of Nap-GFFYGD (2). These observations clearly demonstrated the success of our design, and the H₂O₂ responsive property of our hydrogel suggested its potential application in controlled drug release.

Fig. 1 Optical images of the hydrogel of compound 1 (0.1 wt%) in PBS solution (A) and the resulting solution upon adding 40 mM H₂O₂ to the gel at 37 °C for 4 hours (B) and (C) HPLC traces to demonstrate the transformation triggered by 40 mM H₂O₂ at 37 °C within 4 hours.

Rheology was also performed to characterize the mechanical properties of the hydrogels before and after the addition of H₂O₂. The dynamic frequency sweep was carried at a fixed strain value of 0.5% in the frequency range from 0.1 to 100 rad s⁻¹. As shown in Fig. 2A and S12,† before the addition of H₂O₂, the G′ value of the hydrogel was an order of magnitude bigger than its G′′ value in the tested frequency range, indicating a true hydrogel formation.²¹ After incubation with H₂O₂ for two hours and four hours, the G′′ value of the samples become bigger than their corresponding G′ value at frequency value of 50 and 2 rad s⁻¹, respectively. At the same time, the G′ value decreased dramatically from 40 Pa to 0.4 Pa after four hours' incubation with H₂O₂ (Fig. 2B). These observations suggested the formation of viscous solutions after the addition of H₂O₂. Transmission electron microscopy (TEM) was then used to characterize the morphology of nanostructures in the hydrogel and the obtained solution. As shown in Fig. 2C, uniform nanofibers were observed in the hydrogel with the diameter of about 25 nm and the length of up to several microns. They entangled with each other to form dense networks for the hydrogel formations. Upon the addition of H₂O₂ overnight, the long nanofibers disappeared and only few short nanofibers could be observed (Fig. 2D).

Fig. 2 (A) Dynamic frequency sweep at the strain of 0.5% after the addition of H₂O₂ at different time points (solid symbols, G′; hollow symbols, G′′): 0 h (squares), 2 h (triangles) and 4 h (diamond), (B) the G′ value at the mode of dynamic frequency sweep at the frequency of 10 rad s⁻¹ and strain of 0.5% after the addition of H₂O₂ at different time points. TEM images of the gel without H₂O₂ (C) and with 40 mM H₂O₂ overnight (D).

We therefore tested the possible application of our responsive hydrogel in controlled drug release. We choose gemcitabine as a drug molecule to investigate its controlled release property from our hydrogel at 37 °C. A 0.25 mL of PBS solution with different amounts of H₂O₂ was placed on top of the gel (0.2 mL). The upper solution was totally taken out at different time intervals following by adding another 0.25 mL fresh PBS solution containing corresponding H₂O₂. The accumulation percentage of released gemcitabine from the hydrogel were quantified by LC-MS based on a standard curve. Results in Fig. 3 demonstrated that the release speed of gemcitabine depended on the amount of H₂O₂ in PBS solution. That was, the more H₂O₂ in PBS solution, the faster gemcitabine released from the hydrogel. In the absence of H₂O₂, the accumulative release percentage of gemcitabine was only about 10% in 12 hours. While in the presence of 4 and 10 mM H₂O₂, the accumulative release percentage of gemcitabine from the gel was about 30% and 50%, respectively in 12 hours. Since cancer cells exhibit elevated levels of H₂O₂ compared with normal cells,²² our H₂O₂ responsive peptide hydrogel might be used as a potential delivery system for anticancer drug delivery to tumor sites.

Fig. 3 Release profile of gemcitabine from the hydrogel with different amounts of H₂O₂.

In summary, we have developed a novel supramolecular hydrogel based on peptide self-assembly with a gel–sol phase transition triggered by H₂O₂. The hydrogel showed an excellent H₂O₂ responsive property and could release gemcitabine sustainedly and controllably. Compared with conventional H₂O₂ responsive materials involving peroxalate ester or boronoaryl groups, the synthesis of thiazolidinone modified hydrogelator were relatively easier and more straightforward, and the gelator could avoid being cleaved by ubiquitous esterases in vivo. However, due to the limited structure change of thiazolidinone modified hydrogelator upon H₂O₂ oxidization, the H₂O₂-response sensitivity of the hydrogelator developed by us was not as high as that of peroxalate ester and boronoaryl groups and should be further improved in the future study. As the most important marker for reactive oxygen species (ROS), H₂O₂ is increasingly investigated in physiology, aging and disease in living organisms. Our supramolecular hydrogel system could provide a new way for the detection of the overproduced H₂O₂ and hold great potential in the application of anticancer drug delivery.

Acknowledgements

This work is supported by National Natural Science Foundation of China (No. 81471727), Outstanding Young Faculty Award of Peking Union Medical College (YR1579), PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (3332015100), Fundamental Research Funds for CAMS & PUMC (2016ZX310082) and CAMS Initiative for Innovative Medicine (2016-I2M-3-022).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterization, hydrogel formation, determination of conversion percentage, rheological data. See DOI: 10.1039/c6ra26536g

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