Supramolecular hydrogel of non-steroidal anti-inflammatory drugs: preparation, characterization and ocular biocompatibility

Abstract

A supramolecular hydrogel based on a peptide (GFFY) and non-steroidal anti-inflammatory drugs (naproxen and ibuprofen) was synthesized for use as a topical gel. Using a heating–cooling strategy, the drug–peptide derivatives could self-assemble into nanofibers in aqueous solution to produce a supramolecular hydrogel. The different drug molecules used as the capping group had a profound effect on the self-assembly behaviour measured by circular dichroism. The supramolecular hydrogels were confirmed by rheological measurements to be thixotropic and had no apparent toxicity in vitro against HCEC and L-929 cells after 24 h incubation. More importantly, an in vivo ocular biocompatibility test indicated that the supramolecular hydrogels were well tolerated in rabbits and had satisfactory ocular biocompatibility, suggesting that they are promising candidates for the delivery of ocular drugs.

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1. Introduction

Ocular inflammation, both anterior and posterior, is a serious, potentially sight-threating disorder. Clinically, two classes of drugs – corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) – have been approved for the treatment of various types of ocular inflammation.^1–3 Although corticosteroids can control ocular inflammation, there is a high risk of complications, such as cataracts and glaucoma, with long-term treatment.^4,5 A number of studies have shown that the topical administration of NSAIDs (e.g. diclofenac) has a comparable efficiency to corticosteroids in the treatment of ocular inflammation.^6–8 However, the topical administration of NSAIDs in ophthalmology is limited as a result of their poor water solubility and the inherent ocular irritation.^9,10 Several strategies, including nanoparticles, hydrogels and polymer complexation, have been adopted to improve the ocular safety, tolerability and efficacy of NSAIDs.^2,11–13

Hydrogel biomaterials, especially peptide-based supramolecular hydrogels, have received much attention in recent years for various applications in drug delivery and tissue engineering as a result of their inherent favourable properties, such as biocompatibility, biodegradability and bioactivity.^14–19 Peptide hydrogelators are both easy and cost-effective to prepare on a large scale.^20–24 Recent research in the field of peptide hydrogels has oriented to the development and evaluation of the practical applications of functional hydrogels with different active end-caps (e.g. carbamazepine or naproxen) at the N-terminal end.^17,25,26 Xu and coworkers^25,26 reported the conjugation of NSAIDs with small peptides to generate multifunctional supramolecular nanofibers/hydrogels. Inspired by their work, we designed and constructed a supramolecular hydrogel based on the capping of the GFFY peptide by NSAID molecules for potential use in the delivery of topical ocular drugs.^25,27,28 The supramolecular hydrogel was characterized by transmission electron microscopy (TEM), rheological measurements and circular dichroism (CD) spectrometry and its ocular biocompatibility was evaluated in a rabbit model.

2. Experimental

2.1 Materials

Ibuprofen (IPF) and naproxen (NPX) were purchased from J&K Scientific Ltd (Beijing, China). N-Fmoc-protected amino acids were provided by GL Biochem Ltd (Shanghai, China). Distilled water was used to prepare all aqueous solutions.

2.2 Synthesis and characterization of NPX-GFFY and IPF-GFFY

The NPX-GFFY and IPF-GFFY hydrogelators were prepared using a classic solid-phase peptide synthesis from 2-chlorotrityl chloride resin and N-Fmoc-protected amino acids.²⁵ The 2-chlorotrityl chloride resin was allowed to swell in dry dichloromethane (DCM) for 20 min and then the first amino acid was loaded onto the resin in a DMF solution of Fmoc-protected amino acid (1.5 equiv.) and N,N-diisopropylethylamine (DIEA; 2 equiv.) for 2 h. After washing five times with DCM, the unreactive sites of the resin were blocked by a solution of DCM/MeOH/DIEA (17 : 2 : 1) for 10 min. Thereafter the Fmoc protective group was removed by the addition of 20% piperidine (in DMF) followed by coupling the Fmoc-protected amino acids (2 equiv.) to the free amino group on the resin using DIEA (2 equiv.) and HBTU (1 equiv.) as the coupling agent in DMF for 2 h. These two steps were repeated to elongate the peptide chain. Finally, the NPX or IPF was coupled onto the peptide using DIEA (2 equiv.) and HBTU (1 equiv.) as the coupling agent in DMF for 2 h. The resultant NPX-GFFY and IPF-GFFY hydrogelators were cleaved from the resin with washing solution (TFA/TIS/H₂O 95 : 2.5 : 2.5). The crude product obtained was purified by reversed-phase HPLC and lyophilized for further applications.

2.3 Formation of supramolecular hydrogels

The NPX-GFFY and IPF-GFFY hydrogelators were suspended in a phosphate-buffered solution (pH 7.4) with the aid of ultrasound, followed by the addition of Na₂CO₃ solution (1 mM, 1 equiv.) to obtain a clear solution on heating to 80 °C. The supramolecular hydrogels were formed after cooling to room temperature.

2.4 Characterization

2.4.1 Transmission electron microscopy. TEM observations of the supramolecular hydrogel were performed using a negative staining technique. The hydrogels were pipetted onto the TEM grid, rinsed three times with distilled water and stained with 0.5 wt% phosphotungstic acid.

2.4.2 Circular dichroism spectrometry. The secondary structure of the hydrogelators was detected using a spectropolarimeter in the wavelength range 180–260 nm. The aqueous solution of hydrogelator was transferred into a quartz cell and the spectra were recorded.

2.4.3 Rheological properties. Rheological tests were conducted on a TA AR2000 rheometer using a 40 mm cone-plate. A 0.3 mL volume of aqueous hydrogelator solution was loaded onto the cone-plate and immediately monitored at 25 °C with a frequency of 1 Hz and a strain of 0.5%. A frequency sweep from 0.1 to 100 rad s⁻¹ at a strain rate of 0.5% and a steady shear test from 0.01 to 10 s⁻¹ were performed.

2.5 In vitro cytotoxicity test

The HCEC and L-929 cells were seeded into 96-well plates (1 × 10⁴ cells per well) and incubated overnight in a CO₂ incubator at 37 °C. The medium was then replaced with another 100 μL of DMEM containing different concentrations of a number of compounds (0–200 μM). After 24 h of incubation, 20 μL of MTT solution was added to each well for another 2 h of incubation, followed by the addition of DMSO to dissolve the formazan. The untreated cells were used as a reference. The absorbance of the whole solution at 570 nm was detected by a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The cell viability was calculated by the equation: cell viability (%) = absorbance test/absorbance reference × 100.

2.6 Ocular biocompatibility test

The ocular biocompatibility test was performed in rabbits. The animal experiments complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources and were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Twelve rabbits were randomly divided into two groups (n = 6). The rabbits were treated with 50 μL of 0.3% IPF-GFFY supramolecular hydrogel daily for three days and the normal saline (NS) solution was used as a reference. The congestion, swelling, discharge, and redness of the conjunctiva were scored and recorded by an experienced doctor and the corneal epithelial integrity was observed by fluorescein staining. The intraocular pressure (IOP) was measured using a hand-held applanation tonometer (Tono-Pen Avia, USA) and the corneal thickness and morphology were measured three times by the same operator with a iVue-OCT instrument (Optovue Inc., Fremont, CA, USA). The corneal endothelial cell counts were performed using a non-contact specular microscope (Topcon SP-3000P, Topcon Corporation, Tokyo, Japan) in the automatic mode.

2.7 Statistical analysis

The data were subjected to a t-test analysis of variance using the Origin 7.5 software package. Statistical significance was taken to be p < 0.05.

3. Results and discussion

3.1 Formation of supramolecular hydrogels

Scheme 1 shows the molecular design of the hydrogelator in which the NSAID drugs (NPX and IPF) were directly coupled to the GFFY peptide. The successful synthesis of the hydrogelator was confirmed by ¹H-NMR and mass spectrometry (Fig. S1 and S2†).

Scheme 1 Molecular design of supramolecular hydrogel based on the coupling of NSAIDs and the GFFY peptide.

After the successful synthesis of the designed hydrogelator, the gelation test indicated that both NPX-GFFY and IPF-GFFY could form stable supramolecular hydrogels at a concentration of 0.5 wt% via a heating–cooling strategy, but the supramolecular hydrogels exhibited different appearances. The 0.5 wt% IPF-GFFY supramolecular hydrogel was transparent, whereas the 0.5 wt% NPX-GFFY supramolecular hydrogel was opaque. The morphologies of the supramolecular hydrogels were characterized by TEM. Fig. 1A and B showed that the IPF-GFFY supramolecular hydrogel formed long, thin, flexible nanofibers several micrometres long with widths in the range 25–50 nm, whereas the NPX-GFFY supramolecular hydrogel formed short, thick, rigid nanofibers several micrometres long with widths in the range 50–75 nm. The morphological difference between the IPF-GFFY and NPX-GFFY supramolecular hydrogels suggested that the aromatic capping groups at the N-terminal end had a significant influence on its self-assembly behavior in aqueous solution.

Fig. 1 (A) TEM image of 0.5% IPF-GFFY hydrogel; (B) TEM image of 0.5% NPX-GFFY hydrogel; (C) CD spectrum of IPF-GFFY hydrogel; and (D) CD spectrum of NPX-GFFY hydrogel.

3.2 Circular dichroism spectrometry

Circular dichroism spectrometry was used to understand the self-assembly of the supramolecular hydrogels. As shown in Fig. 1C and D, the IPF-GFFY hydrogelator solution had a negative band at 206 nm, indicating the presence of an α-helix nanostructure (P2 conformation).^14,25 By contrast, the NPX-GFFY hydrogelator had a positive band at 198 nm and a negative band at 217 nm, suggesting the existence of a β-sheet nanostructure.^17,25 Therefore it is reasonable to believe that the aromatic capping groups at the N-terminal end greatly influenced the secondary structure of the peptide, resulting in the different nanostructures. We concluded from the results of the spectroscopic characterization that non-covalent interactions such as π–π stacking, hydrogen bonds and van der Waals forces were the major driving force for the formation of the NPX-GFFY and IPF-GFFY supramolecular hydrogels.^16,26,29

3.3 Rheological properties

The mechanical properties of the supramolecular hydrogels were characterized by rheology. Fig. 2A and B clearly show that the value of G′ for all the samples was larger than that of the corresponding G′′ values over the whole measurement period, suggesting the formation of a supramolecular hydrogel.¹⁷ With an increase in the concentration of NPX-GFFY or IPF-GFFY, the G′ value of the hydrogel increased accordingly. The G′ values of the 0.5% NPX-GFFY hydrogel and the 1% IPF-GFFY hydrogel were one order of magnitude larger than those of the 0.1% NPX-GFFY hydrogel and the 0.25% IPF-GFFY hydrogel, respectively. This result indicated that the mechanical properties of the hydrogels could be tailored by changing the concentrations of the components. Both supramolecular hydrogels showed a weak dependence on frequency in the range 0.1–100 rad s⁻¹, suggesting that all the samples were highly elastic materials (Fig. 2C and D). A dynamic viscosity sweep test showed that both supramolecular hydrogels were thixotropic (Fig. 2E and F). The viscosity of both supramolecular hydrogels rapidly decreased to nearly zero at a shear rate of 1 s⁻¹. The initial viscosity was dependent on the concentrations of the components, i.e. the initial viscosity increased with an increase in the component concentration. This thixotropic property may mean that the developed supramolecular hydrogel is a promising candidate for the delivery of ocular drugs.

Fig. 2 (A and B) Dynamic time sweep of NPX-GFFY and IPF-GFFY supramolecular hydrogel; (C and D) dynamic frequency sweep of NPX-GFFY and IPF-GFFY supramolecular hydrogel; and (E and F) dynamic viscosity sweep of NPX-GFFY and IPF-GFFY supramolecular hydrogel.

3.4 In vitro cytotoxicity

To investigate the potential application of the IPF-GFFY and NPX-GFFY supramolecular hydrogels in the field of drug delivery, the biological safety of these novel materials was assessed. Fig. 3 shows that there was no apparent cytotoxic effect against HCEC and L-929 cells at any concentration for either the IPF-GFFY or the NPX-GFFY supramolecular hydrogel compared with the native drugs after 24 h of incubation. Based on this result, we concluded that neither of the supramolecular hydrogels was toxic in vitro and that they might be a promising system for the delivery of ocular drugs.

Fig. 3 Relative cell viability of HCEC cells and L-929 cells incubated with different concentrations of IPF, IPF-GFFY supramolecular hydrogel, NPX and NPX-GFFY supramolecular hydrogel (0–200 μM) for 24 h.

3.5 Ocular biocompatibility test

Biocompatibility is a major requirement for the development of potential biomaterials for the delivery of ocular drugs.^30,31 Although the developed IPF-GFFY supramolecular hydrogel did not elicit a cytotoxic effect in vitro, further investigation of its ocular biocompatibility was carried out. We investigated the ocular biocompatibility of the IPF-GFFY supramolecular hydrogel via instillation in a rabbit model. No abnormal ocular response (e.g. corneal opacity, conjunctival redness, inflammation) was observed within 14 days of the instillation of the 0.3% IPF-GFFY supramolecular hydrogel (Fig. S3†). Fluorescence staining indicated that there was no obvious damage to the corneal epithelium after the instillation of the 0.3% IPF-GFFY supramolecular hydrogel (Fig. S3†). Fig. 4 shows typical OCT images of the cornea of both control groups and the 0.3% IPF-GFFY supramolecular hydrogel group. The corneal epithelium, intrastromal morphology and endothelium were clearly differentiated. There was no significant change in the corneal thickness and the corneal morphology for the control group and the 0.3% IPF-GFFY supramolecular hydrogel group. IOP measurements suggested that the instillation of the 0.3% IPF-GFFY supramolecular hydrogel did not obviously influence its IOP compared with that of the control group during the study period (Fig. S4†). The corneal endothelium is sensitive to foreign substances and low proliferative activity was observed in vivo. Pilot studies have reported that cellular hexagonality is a sensitive indicator of corneal endothelial damage.^32–34 Fig. 5 shows that the corneal endothelial cells in the rabbits' eyes exhibited a typical hexagonal shape, suggesting that the instillation of the 0.3% IPF-GFFY supramolecular hydrogel did not alter the morphology of the corneal endothelium. The corneal endothelial cell count analysis indicated that there was no significant difference in the cell density between the control group and the 0.3% IPF-GFFY supramolecular hydrogel group. All these results suggest that the IPF-GFFY supramolecular hydrogel was well tolerated and has a satisfactory in vivo biocompatibility, suggesting that it is a promising candidate for the delivery of ocular drugs.

Fig. 4 OCT images and corneal thickness of corneal tissue after instillation of 50 μL of 0.3% IPF-GFFY supramolecular hydrogel daily for three days.

Fig. 5 Corneal endothelial cell counts in rabbits after instillation of 50 μL of 0.3% IPF-GFFY supramolecular hydrogel daily for three days.

4. Conclusions

We investigated a supramolecular hydrogel composed of peptides and NSAIDs formed via a heating–cooling strategy. The aromatic capping groups at the N-terminal end of the peptide significantly influenced its self-assembly behavior, as indicated by CD measurements and TEM observations. Rheology tests indicated that the mechanical strength of the supramolecular hydrogel increased with an increase in the concentration of the hydrogelator. The supramolecular hydrogel was thixotropic and non-toxic towards L-929 and HCEC cells after 24 h of incubation. In vivo biocompatibility tests showed that the developed supramolecular hydrogel was a non-irritant and had satisfactory ocular biocompatibility and therefore might be a promising candidate for the treatment of ocular inflammation.

Acknowledgements

This work was financially supported by grants from the National Natural Science Foundation of China (Grant No. 51303136) and the Key Program for International S&T Cooperation Projects of China (2015DFA50310), National Science and Technology Major Project (2014ZX09303301).

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Footnotes

† Electronic supplementary information (ESI) available: ¹H-NMR and MS of NPX-GFFY and IPF-GFFY; slit lamp observation and fluorescence staining of corneal tissue; intraocular pressure (IOP) changes of rabbit. See DOI: 10.1039/c6ra09615h

‡ G. J. Pu did equal work with X. Y. Li, was co-first author.

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