Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles
Introduction
The need for biocompatible, high water content materials that facilitate the transport of metabolites was emphasized by Wichterle and Lim in 1960 [1] who highlighted likely structural and biological incompatibility between conventional plastics and living tissue. Preparation of the first synthetic hydrogel by these scientists marked the beginning of a new era in modern hydrogel research. Since then, hydrogels – highly hydrated, porous materials produced through chemical and/or physical crosslinking of molecules – have received remarkable scientific interest in the field of biomedical research [2], [3], [4], [5]. The ability to conveniently tune the physical and chemical properties of hydrogels through changes in the constituent molecule chemical functionality, architecture as well as microenvironment (solution conditions etc.) have led to their application in fields such as tissue engineering, drug delivery and medical implants [3], [5], [6].
For delivery applications, hydrogels have been used to incorporate drug molecules into the gel matrix to create reservoirs that deliver bioactive agents [7]. Some of the key issues that drug delivery studies try to address include increasing bioavailability of drugs, reducing side effects, controlling kinetic release profiles, increasing clinical ease of use and reducing pain from administration. Localized drug delivery is especially desirable to increase the efficacy of highly metabolized drugs when administered conventionally, to circumvent natural barriers such as the blood–brain barrier and to reduce serious side effects of systemically administered toxic drugs. The necessity to deliver drugs for the efficacious treatment of diseases has resulted in significant advances in the development of injectable drug delivery vehicles [8], [9].
Currently, hydrogels are primarily designed to exist as free flowing polymer solutions ex vivo to enable injection as a low viscosity liquid and subsequently gel in vivo through crosslinking induced by stimuli such as radiation, enzymes, salt or temperature [10], [11], [12]. Although there are a number of advantages associated with injectable liquids that can be crosslinked into hydrogels after injection, the most important being that they facilitate local delivery of drug molecules, there are also significant limitations. Some of these limitations are toxicity of un-reacted monomers, a high degree of crystallinity in pre-crosslinked synthetic polymers, shrinkage or brittleness of the polymer gels post-crosslinking, and a large amount of drug discharge during the initial burst release in drug delivery vehicles [8]. In addition, ultraviolet radiation used during many in situ network formation strategies, or high local temperatures caused by covalent crosslinking chemical reactions, can be detrimental to cells or drug payloads mixed with the network-forming molecules. Finally, even with successful in vivo hydrogel formation, the final material and in vivo area are affected unavoidably by dilution from bodily fluids, before and during crosslinking, and flow into neighboring tissues prior to significant crosslinking, respectively. In this context, nano-engineered delivery systems can provide innovative solutions to overcome the limitations of conventional injectable liquid, polymer-based systems [13].
Self-assembling systems in which water soluble peptides undergo sol–gel transition in response to changes in ionic strength [14], temperature [15], [16], or pH [17], [18], [19], [20], [21], [22], [23] of the medium represent an area of growing interdisciplinary research. Mixing induced, two-component physical hydrogels and enzyme catalyzed self-assembly have also been reported [24], [25], [26], [27]. Gelation in such systems is achieved through non-covalent, physical crosslinking by secondary forces such as hydrophobic and van der Waals interactions, as well as ionic and hydrogen bonding [28]. Biocompatibility, hydrophilicity and physiologically benign processing conditions are some of the beneficial properties that make physically crosslinked peptide hydrogels attractive candidates as injectable delivery vehicles for therapeutic agents [28]. Importantly, the ability to modulate the bulk physical behavior, such as network stiffness and porosity, of the hydrogel by changing its local nanostructure or network architecture is a potent feature of peptide-based hydrogels. Such changes may be introduced by changing the peptide primary sequence (constituent amino acids) that dictates the assembled nanostructure, network architecture or the assembly kinetics of the hydrogel material [29], [30], [31].
We have developed a self-assembling peptide hydrogel system that can form a solid physical hydrogel by in vitro assembly at physiological conditions such as provided by cell culture medium. The physical gelation at physiological conditions allows three-dimensional, homogeneous encapsulation of desired molecules and/or cells [30], [32]. Moreover, these peptide hydrogels display shear-thinning and immediate recovery properties that make them excellent candidates for injectable therapies. More specifically, a gel formed ex vivo; with desired stiffness, porosity, nanostructure, encapsulated payload, etc.; has the ability to flow with low viscosity while under shear and immediately recover back to a solid hydrogel on cessation of shear [14], [30], [33]. This shear-thinning and immediate recovery enables injection, without syringe-clogging, to a specific site and provides site specificity due to immediate solid-like properties of gel after injection. Also, this design yields hydrogels with the same material properties (stiffness, porosity, nanostructure, payload distribution), both before and after injection [30], [33], as opposed to injected liquids where changes in materials properties and volume from observed in vitro behavior can occur due to dilution and flow. In addition, the peptide hydrogels do not swell after formation, either before or after injection, when exposed to additional solution or bodily fluids. Rather, new solutes and aqueous fluids can begin to diffuse into and within the stable hydrogels as defined by the porosity of the self-assembled peptide network. A schematic showing the self-assembly pathway of the peptide MAX8 used in this study is displayed in Fig. 1(a). MAX8 [30], a 20 amino acid peptide, is composed of two arms of alternating lysine and valine residues surrounding a four residue sequence (-VDPPT-) which is known to adopt a type II′ turn under appropriate solution conditions. A single lysine residue is replaced with a glutamic acid residue at the fifteenth position for the overall composition of VKVKVKVKVDPPTKVEVKVKV-NH2. Due to the charged nature of lysine groups, the peptide adopts an unfolded, random coil structure when dissolved in deionized (DI) water. Self-assembly of peptides can be triggered by changing the ionic strength [14], pH [19] and temperature [15] of the aqueous environment. An increase in ionic strength is obtained by adding cell culture media to the peptide dissolved in water. The salt ions in cell culture media screen the electrostatic repulsions between lysine residues allowing the peptide to fold into a β-hairpin [14], [30]. When folded, MAX8 peptide is stabilized by intrastrand hydrogen bonding and hydrophobic contacts and displays hydrophobic valine and hydrophilic lysine residues on opposite faces of the β-hairpin [34]. Hydrophobic interactions between valine faces of the hairpins cause peptide bilayer formation through hydrophobic collapse. Fibril formation is achieved by intermolecular hydrophobic side chain contacts and lateral hydrogen bonding, along the long axis of the fibril. Fig. 1(b) displays a transmission electron microscope (TEM) image of negatively stained MAX8 fibrils. Fibril branching [35] and fibril entanglements account for the mechanical rigidity of the hydrogel.
The polyphenolic compound curcumin (diferuloylmethane) is isolated from the rhizomes of turmeric (Curcuma longa), which is a member of the ginger family [36]. The potent therapeutic properties of curcumin for a variety of conditions such as respiratory diseases, liver disorders and diabetic wounds have been documented in ancient Indian literature [37]. Over the last half century, known pharmacological effects of curcumin have been expanded to encompass antioxidant [38] and anti-inflammatory properties [39] as well as inhibition of tumorigenesis [40], [41], [42] and metastasis [43]. Despite its great potential for the treatment of diseases, curcumin’s poor aqueous solubility, degradation and low bioavailability constitute major obstacles toward its medical deployment [44]. Heat treatment of curcumin [43] and the discovery of natural water soluble curcumin analogs [44], [45] have been suggested to overcome the stability and solubility issues in aqueous environments. Another approach to improve clinical applicability of curcumin has been to develop delivery vehicles that successfully transport the hydrophobic drug to in vivo target sites. Several formulations have been suggested for curcumin delivery. These include vehicles such as PLGA nanoparticles [46], micellar polymer aggregates [47], polyvinyl alcohol hydrogels [48], and pluronic block copolymer micelles [36] as well as curcumin-casein micelles [49].
In this paper, we combine curcumin with self-assembling peptide where curcumin encapsulation and peptide gelation is achieved concurrently in aqueous, physiological conditions. The curcumin-loaded peptide hydrogel combines the features of minimally invasive delivery with the advantages of stabilizing curcumin and controlling its release.
Section snippets
Peptide synthesis
MAX8 (95% purity) was produced and purified by New England Peptide (Gardner, MA, USA) according to previously published protocols [19], [50].
Preparation of MAX8 hydrogels
For the preparation of 0.5 wt% MAX8 hydrogel (0.5 mg MAX8 in 100 μl of hydrogel), 0.5 mg of MAX8 peptide is first dissolved in DI water. Self-assembly of the peptide is initiated with the addition of an equal volume of salt solution buffered to pH 7.4 or cell growth medium (pH 7.4). Buffer solutions used to trigger the self-assembly varied according to the
Curcumin interaction with MAX8 peptide hydrogel
The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. The solubility of curcumin in aqueous solutions is limited but is enhanced in organic solvents such as dimethyl sulfoxide (DMSO), ethanol, methanol or acetone. We used DMSO, a non-toxic aprotic polar solvent, to help solubilize curcumin in aqueous solutions [51].
Curcumin, due to its polyphenolic structure, is a naturally fluorescent molecule
Conclusion
The results demonstrate a minimally invasive curcumin delivery strategy that can encapsulate and deliver sustained concentrations of curcumin locally to a delivery site. Oscillatory rheology measurements have revealed that β-hairpin peptide hydrogels with encapsulated curcumin concentrations as high as 4 mm immediately display solid-like properties after shear-thinning and reheal quickly over time to stiffnesses close to pre-shear values. Also, hydrogels loaded with 4 mm curcumin were found to
Acknowledgments
AA and SJL have contributed equally to this work. This work was supported by the NIH COBRE P20 RR017716, American Cancer Society Research Grant RSG-09-021-01-CNE and funds from the Nemours Foundation to Sigrid A. Rajasekaran. We thank C. Ni, F. Kriss and the College of Engineering at the University of Delaware, for partial funding of W.M. Keck Electron Microscopy Facility.
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