Fluorescence study of freeze-drying as a method for support the interactions between hyaluronan and hydrophobic species

RESEARCH ARTICLE Fluorescence study of freeze-drying as a method for support the interactions between hyaluronan and hydrophobic species Petra Michalicová, Filip Mravec, Miloslav Pekař* Brno University of Technology, Faculty of Chemistry, Institute of Physical and Applied Chemistry and Materials Research Centre, Brno, Czech Republic * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Michalicová P, Mravec F, Pekař M (2017) Fluorescence study of freeze-drying as a method for support the interactions between hyaluronan and hydrophobic species. PLoS ONE 12(9): e0184558. https://doi.org/10.1371/journal. pone.0184558 Editor: Bing Xu, Brandeis University, UNITED STATES Received: April 28, 2017 Accepted: August 25, 2017 Published: September 8, 2017 Copyright: © 2017 Michalicová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Materials Research Centre at FCH BUTSustainability and Development, LO1211, with financial support from National Programme for Sustainability I (Ministry of Education, Youth and Sports, Czech Republic) (http://www.msmt.cz/). The funder had no role in study design, data Abstract A freeze-drying method enabling solubilization of hydrophobic species in aqueous solutions of native hyaluronan is described. The method is based on opening the access to supposed hydrophobic patches on hyaluronan by disturbing its massive hydration shell. Hydrophobic and/or polarity-sensitive fluorescence probes were used as hydrophobic models or indicators of interactions with hydrophobic patches. Fluorescence parameters specific to individual probes confirmed the efficiency of the freeze-drying method. This work is the first step in developing biocompatible and biodegradable carriers for hydrophobic drugs with targeted distribution of the active compound from native, chemically non-modified hyaluronan. Introduction Hyaluronan is a biopolymer which can be characterized as essential for living organisms of various complexity, ranging from microorganisms to vertebrates. It is a linear biopolysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by a β-1,3 glycosidic bond (Fig 1). These disaccharide units are linked by β-1,4 bonds. It is a part of the extracellular matrix in most tissues and also a major component of a variety of other tissues. Besides its mechanical functions, this compound is important for many biological processes [1–3]. Its contribution to the proliferation of tumour cells is one typical example. This area of cancer research involving hyaluronan is the subject of many studies. Literature shows that cancerous tissues are rich in the receptors CD44 and RHAMM [4–7]. These receptors are specific to hyaluronan and cause the packaging of tumour tissue using the biopolymer. This is the main reason for the application of hyaluronan in the area of drug-delivery systems (DDS) [8–12]. The function of DDS can be compared to a Trojan horse—tumour cells interact with hyaluronan from the surrounding cellular environment through the abovementioned receptors. Systems containing hyaluronan and cytostatic drugs release the drug and subsequently induce cell death. The majority of already utilized or potential drugs have a hydrophobic character. In contrast, hyaluronan is a highly hydrophilic polymer. Two strategies are used to tackle this problem—to prepare a hyaluronan-drug conjugate or to hydrophobize hyaluronan [13]. In the latter, hydrophobically modified hyaluronan can form polymeric micelles in an aqueous PLOS ONE | https://doi.org/10.1371/journal.pone.0184558 September 8, 2017 1 / 13 Freeze-drying—A support of the interactions between hyaluronan and hydrophobes collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. medium capable of solubilizing hydrophobic substances [14–17]. Both approaches are based on chemical reactions of hyaluronan conducted usually in some organic solvent. Chemical modification, however, can affect the biological functions and biocompatibility of hyaluronan; further, the organic solvent should be carefully removed from a product intended for use in the human body [13]. An alternative is to use hyaluronan-surfactant complexes formed by physical (electrostatic and hydrophobic) interactions [18–20]. However, due to hyaluronan’s negative charge, cationic surfactants should be used which have general cytotoxic effects though somewhat moderated in the presence of hyaluronan [21]. Scott published an idea about existence of “hydrophobic patches” on the hyaluronan chain [22]. Because of the beta configuration, all bulky groups occupy sterically favorable equatorial positions. On the other hand, small hydrogen atoms are moved into less sterically favorable axial positions. This gives rise to the amphiphilic character of the hyaluronan chain, which contains both a hydrophilic (prevalent) part and a hydrophobic part. We therefore hypothesized that these hydrophobic patches could be used to bind non-polar substances directly onto the native hyaluronan chain. However, in aqueous solution, the chain generates a twisting ribbon structure in which the hydrophilic face is oriented into the solution and the hydrophobic face is hidden inside the domain. The non-polar areas are then probably protected also by hydration shell—hyaluronan is reported to have a thick hydration layer resulting in a large hydrodynamic volume [23–26]. Therefore, interactions between native hyaluronan and hydrophobic species must somehow be supported by opening the hydration shell in order to make the hydrophobic patches accessible for interactions. The combination of hydrophilic and hydrophobic character as an intrinsic property of the hyaluronan backbone was demonstrated by atomic force microscopy investigations of hyaluronan deposited on various surfaces [27, 28]. The behavior of graphite deposition was very different from that on mica. Hyaluronan deposited on hydrophobic graphite surface without rinsing with water before drying interacted more strongly with the surface than the well-rinsed depositions. The authors hypothesized that hyaluronan chains in (excess) water tended to mask the hydrophobic patches and their interactions with the hydrophobic substrate were weakened. In this work, a freeze-drying method in the presence of an organic co-solvent was studied as a potential opener of the hydration shell and supporter of hydrophobic interactions between the hyaluronan chain and non-polar species. Recently, Průšová et al. studied the effect of Fig 1. Structure of hyaluronan biopolym (...truncated)