Self-assembly of ciprofloxacin and a tripeptide into an antimicrobial nanostructured hydrogel

Abstract

This work reports the self-assembly of a sparingly soluble antibiotic (ciprofloxacin) and a hydrophobic tripeptide (^DLeu–Phe–Phe) into supramolecular nanostructures that yield a macroscopic hydrogel at physiological pH. Drug incorporation results in modified morphology and rheological properties of the self-assembled hydrogel. These changes can be correlated with intermolecular interactions between the drug and the peptide, as confirmed by spectroscopic analysis (fluorescence, circular dichroism, IR). The drug appears bound within the hydrogel by non-covalent interactions, and retains its activity over a prolonged release timescale. Antimicrobial activity of the ciprofloxacin-peptide self-assembled hydrogel was evaluated against Staphylococcus aureus, Escherichia coli, and a clinical strain of Klebsiella pneumoniae. Interestingly, the peptide hydrogel alone exhibited a mild anti-bacterial activity against Gram-negative bacteria. While toxic to bacteria, no major cytotoxicity was seen in haemolysis assays of human red blood cells or in mouse fibroblast cell cultures. This new approach of drug incorporation into the nanostructure of a simple tripeptide hydrogel by self-assembly may have important applications for cost-effective wound dressings and novel antimicrobial formulations.

Introduction

Today there is an intense research effort directed towards the development of cost-effective, antimicrobial materials for topical applications such as wound dressings [1], [2]. Nanomaterials offer the advantage of high surface area to volume ratio and the possibility to design their physical properties (such as porosity, mechanical strength etc.) to match natural tissue, and to selectively load drug molecules for their controlled release at the wound site [3]. In particular, the use of nanotechnology to develop innovative hydrogels is attractive for wound healing applications [4] since it combines the advantages described above with hydrogel properties known to accelerate the healing process, e.g. the moist and occlusive environment they provide, as well as their ability to allow for cell attachment and infiltration [1], [2], [5].

Tissue regeneration at the site of injury is often hampered by infection, which can be prevented or eradicated by the sustained release of relevant antimicrobials. Different approaches exist to prevent bacteria colonisation, such as the use of metal nanoparticles [6], or the sustained release of antibiotics at a specific site of application [7]. In particular, the fluoroquinolone ciprofloxacin is one of the most effective antibiotics used clinically and has become the gold standard for a variety of topical applications, such as skin and eye infections. Drug formulations capable of sustained release are highly sought after, providing a means for drug concentration to be maintained for long periods of time above the minimum inhibitory concentration (MIC) for relevant pathogens. In order to address these requirements, formulations such as liposomes and gels are typically studied, as they offer good vehicles for the incorporation of this hydrophobic and sparingly soluble drug [7], [8], [9], [10].

For delivery applications, hydrogels offer convenient drug delivery matrices. They are typically composed of natural polymers (e.g. alginate), or crosslinked synthetic macromolecules (e.g. polyethylene glycol, polyvinyl alcohol), which offer biocompatibility and desirable physical properties. However, there are also significant limitations, such as undesired burst release of drugs, polymer shrinkage post-crosslinking, or even toxicity of unreacted synthetic polymer precursors [11].

Peptide self-assembled systems in which non-covalent interactions are responsible for the physical assembly of peptide molecules offer a viable alternative to generate hydrogels [12]. Gelation can be triggered in a number of ways, for instance by changes in pH [13], ionic strength [14], temperature [15], enzyme-catalysis [16], or a combination of different stimuli [17]. These systems are attractive biomaterials in that they biodegrade into benign catabolic products, their processing involves mild conditions that are compatible with physiological systems, and low concentrations (in the millimolar range) are required for gelation [12]. Moreover, some of these systems facilitate wound healing, allowing cell infiltration and retention of viability [18], [19]. Materials such as these can also be used to entrap bioactive molecules and hydrophobic drugs for controlled release [18], [20], [21]. However, the majority of peptides capable of self-assembly into hydrogels consist of rather long molecules (>10 amino acids) [22], for which solid-phase synthetic preparation is expensive and difficult to scale-up.

In contrast, ultrashort peptides (i.e. made of 2 or 3 amino acids) are attractive candidates for hydrogels, as they can be readily prepared in solution-phase, making scale-up convenient [23]. Only a few systems of this kind have been described in the literature of which the majority exploit the presence of synthetic end-capping groups (e.g. Fmoc, naphthalene, etc.) to increase molecular hydrophobicity and to assist self-assembly [24], [25], [26]. However, concerns exist for the potential in vivo toxicity of certain aromatic synthetic groups, since examples of reduced viability of cells cultured on these gels have been reported [27], [28]. In addition, small amounts of organic solvents are often required to aid peptide dissolution and to assist with secondary structure stability [27], [29], [30].

For biological applications, alternative ultrashort gelling agents devoid of synthetic end-capping groups, and that do not require any organic solvent, are needed. We recently reported, to our knowledge, the first hydrogel systems that satisfy these requirements, and that are based on uncapped tripeptides capable of hydrogel formation under physiological conditions (^DVal–Phe–Phe, ^DPhe–Phe–Val, and ^DLeu–Phe–Phe) [31], [32]. Moreover, these systems display a d-amino acid at their N-terminus that could be advantageous for prolonged stability in human tissue. These ultrashort gelling peptides are composed of hydrophobic molecules where aromatic interactions between the phenylalanine rings are pivotal to self-assembly. As a result, they could be a useful vehicle for the sustained delivery of hydrophobic drugs that contain aromatic groups, which may favourably interact with the peptide at the nanostructural level. Moreover, these hydrogels are formed from the combination of two precursor solutions at acidic and alkaline pH respectively. Therefore, drugs that are sparingly soluble at physiological pH, but that present ionisable groups, could be dissolved in either of the precursor solutions (prior to entrapment into the hydrogel) at levels that are higher than their solubility limit at neutral pH.

In order to test this hypothesis, we chose the self-assembling peptide ^DLeu–Phe–Phe and the antibiotic ciprofloxacin (CIP) as a model hydrophobic drug that is sparingly soluble at physiological pH. To the best of our knowledge, this is the first report of self-assembly of a drug and a tripeptide into a hydrogel with distinctive nanostructure that allows for controlled release of the drug. These materials were used to challenge cultures of Staphylococcus aureus, Escherichia coli, and a clinical isolate of Klebsiella pneumoniae [34]. Sustained drug release from the hydrogels was reflected by antibiotic concentrations of the surrounding solutions and by agar-diffusion assays determining efficacy at bacterial killing. The formulations were also validated for non-cytotoxicity to mammalian cells.