European Journal of Pharmaceutics and Biopharmaceutics
Research paperComplex coacervates of hyaluronic acid and lysozyme: Effect on protein structure and physical stability
Graphical abstract
Introduction
Complex coacervation is the binding of two oppositely charged molecules driven mainly by electrostatic interactions that results in a liquid–liquid phase separation between the bulk solution and the soluble complexes [6]. In the majority of the cases these molecules are macromolecules, e.g. polyelectrolytes such as, proteins, nucleic acids, dendrimers or linear polymers. The coacervates are in a soluble state, in contrast to complex flocculates, which are formed upon precipitation of the interacting macromolecules in the case of highly efficient interactions, thereby expelling the water from the complex structure. In many cases, this soluble nature of complex coacervates leaves the biofunctionality of the complexed macromolecules intact, thereby making them interesting for formulation of pharmaceutical proteins [14]. A wide variety of complex coacervates systems have been reported and investigated in the literature, e.g. protein–protein [5], [32], protein–polymer [13], [31], nucleic acid–polymer [16], [26], [24] and nucleic acid–dendrimer [11], [19]. A more complete overview can be found in recent review papers [14], [30].
The physicochemical properties of complex coacervates make them interesting as potential drug delivery systems, mainly due to their earlier mentioned ability to retain the biofunctionality of a pharmaceutically relevant macromolecule. Different drug delivery strategies have been explored by exploiting the physicochemical nature of complex coacervates such as using them as stabilizing agents to preserve biofunctionality [35], reduce enzymatic cleavage as well as physical stability of the biological by inhibition of aggregation [7] or denaturation [23], or for entrapment of the molecule in complexes used for controlled release delivery. However, the effect is also dependent on the complexed macromolecule e.g. the physical stability of lysozyme (LZ) was reduced upon complexation with heparin [31].
Hyaluronic acid (HA) is a linear anionic polysaccharide consisting of N-acetyl-d-glucosamine and d-glucuronic acid disaccharide units bound by alternating β-(1 −> 3) β-(1 −> 4) glucosidic bonds first discovered by Meyer and Palmer [21]. It is mainly used for basic clinical applications like viscoaugmentation and viscoprotection [4], [34] due to its high viscosity caused by the arrangement of the molecules in structured networks based upon hydrophobic patches and intermolecular hydrogen bonds [28]. HA is biocompatible and biodegradable, and less likely to cause undesired immune responses due to its presence in almost all biological fluids and tissues of vertebrates, making it a promising polymer for drug delivery applications.
The complex coacervation behavior of HA has been investigated with different polyelectrolyte polymers such as chitosan [17] and silk fibroin [20]. Other formulations of HA for drug delivery applications include complex formation in the presence of cationic polyelectrolytes for delivery of nucleic acids [25], [1], and covalently bound or cross-linked HA molecules forming hydrogel networks and depots for controlled release [8], [12].
This work investigated the use of HA as a complex coacervate forming polyelectrolyte by binding positively charged proteins and thereby potentially increase their physical stability. Lysozyme was chosen as model protein, as it has been shown earlier that its stability is decreased upon complex coacervation with heparin [31]. BSA was also investigated, as it is mainly negatively charged at physiological pH and did not form macroscopic complex coacervates upon mixing with HA under the studied conditions. However, coacervation has been reported in literature by Du et al. [10] under different conditions. The binding stoichiometry of the HA–LZ complexes at low ionic strength conditions was investigated and the influence of the complexation behavior on the stability of the protein was determined. The thermal unfolding and structure of LZ and BSA was studied with differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), differential scanning fluorimetry (DSF) and the stoichiometry determined by isothermal titration calorimetry (ITC) and solution depletion.
Section snippets
Materials
Hyaluronic acid with an average molecular weight of 870 kDa was supplied by Novozymes A/S (Bagsvaerd, Denmark). Hen egg white lysozyme and bovine serum albumin (>98%, fraction V) were obtained from Sigma (St. Louis, US). HEPES buffer salt was purchased from Applichem (Darmstadt, Germany), sodium chloride (NaCl) (Sigma, St. Louis, US) and Milli-Q water (Millipore, Billerica, US) was used in all buffers and sample preparations.
Buffer and sample preparation
HEPES buffer (25 mM) pH 7.2 was prepared with an ionic strength of 6 mM.
Complex characterization
In order to characterize the complexation behavior of HA and LZ solution depletion experiments were performed. To obtain saturation of the HA binding sites at low concentrations of HA a high concentration of LZ (2.4 mg/mL, 164.4 μM) was added. Under these conditions transparent complex coacervates were observed upon mixing of the two compounds. Fig. 1 shows the ratio between free LZ in the supernatant and the initial concentration of free LZ vs. the HA:LZ ratio, at high (100 mM) and low (6 mM)
Discussion and conclusion
The solution depletion and ITC experiments were able to characterize the complexation behavior of LZ with HA. The data of both methods were in agreement about the derived binding stoichiometry, providing a value of approximately 0.1 LZ molecules bound per HA monomer. This binding stoichiometry is in agreement with the findings presented by Morfin et al. [22] of 0.125 per monomer, as obtained using Small Angle Neutron Scattering (SANS) in low ionic strength conditions, even though different
Acknowledgments
The authors acknowledge Novo Nordisk for partially funding the MicroCal VP-DSC, the Drug Research Academy for funding the NanoDrop 2000C, the Advanced Technology Foundation for funding the TA Instruments Nano-ITC system and Apotekerfonden af 1991 for funding the Bomem MB-100 FTIR spectrometer. Novozymes A/S is acknowledged for providing the hyaluronic acid used for this study and for financing a scholar grant.
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