Synthesis of β-cyclodextrin-lysozyme conjugates and their physicochemical and biochemical properties

J Incl Phenom Macrocycl Chem (2017) 87:341–348 DOI 10.1007/s10847-017-0706-8 ORIGINAL ARTICLE Synthesis of β-cyclodextrin-lysozyme conjugates and their physicochemical and biochemical properties Tomasz Marek Goszczyński1 · Maciej Gawłowski1 · Beata Girek2 · Konrad Kowalski1 · Janusz Boratyński1,2 · Tomasz Girek2 Received: 19 October 2016 / Accepted: 25 February 2017 / Published online: 8 March 2017 © The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Recently a great interest in the field of protein engineering and the design of innovative drug delivery systems employing specific ligands such as cyclodextrins is observed. The paper reports the solid state, thermal method for protein coupling with β-cyclodextrin and the physicochemical and biological properties of the obtained conjugates. The structure of the obtained conjugates was investigated via liquid chromatography-mass spectrometry, dynamic light scattering and circular dichroism analysis. The presented conjugates were biologically active and covalently bound β-cyclodextrin preserved the ability to form inclusion complexes with the model compound. This report demonstrates the great potential of cyclodextrin as a modifying unit that can be used to modulate the properties of therapeutic proteins, additionally giving such conjugates the possibility to transport many therapeutic substances in the form of inclusion complexes. In addition, the paper presents the potential of protein-cyclodextrin conjugates to construct innovative bioactive molecules for biological and medical applications. Electronic supplementary material The online version of this article (doi:10.1007/s10847-017-0706-8) contains supplementary material, which is available to authorized users. * Tomasz Marek Goszczyński * Tomasz Girek 1 Laboratory of Biomedical Chemistry, Department of Experimental Oncology, Hirszfeld Institute of Immunology and Experimental Therapy, PAS, 12 Rudolf Weigl St., 53‑114 Wrocław, Poland 2 Institute of Chemistry, Environmental Protection and Biotechnology, Jan Dlugosz University, Armii Krajowej Ave., 13/15, 42 201 Częstochowa, Poland Keywords Conjugates · Cyclodextrin · Inclusion complex · Protein · Thermal reaction in solid state Introduction Cyclodextrins (CDs) are cyclic oligosaccharides with significant application in the pharmaceutical, food and cosmetic industries [1, 2]. Naturally occurring CDs are composed of six, seven or eight d-glucopyranoside units in a toroidal structure, possessing a hydrophobic interior and hydrophilic exterior. Accordingly, CDs have an ability to encapsulate and solubilize hydrophobic guest species in water through host–guest complexation [3, 4]. The characteristic properties of the substance, such as solubility, chemical reactivity, or spectral property, are changed after guest compound is encapsulated. Due to these major features, CDs can be utilized in different areas of medicinal chemistry. Recently, these host–guest interactions have been adopted to assemble polymer nanoparticles for drug and gene delivery. Given their biocompatibility, CDs have been used as host units for the synthesis of host–guest delivery carriers. One example of such carriers is diamide linked γ-cyclodextrin (γ-CD) dimers that have been described as molecular-scale delivery capsules for the anticancer agent curcumin [5]. In vivo therapeutic efficacy has been reported for nanoparticles assembled from camptothecin conjugated β-CD polymers [6]. Additionally, cationic β-CD polymer derived nanoparticles have been found to be efficient non-viral delivery vectors for siRNA in humans [7]. Also cyclodextrin-based nanosponges can form complexes with many lipophilic and hydrophilic drugs. They can be used for protecting easily degradable molecules and also could be used to improve the solubility of poorly soluble drugs. For this reason CD-based nanosponges could be 13 Vol.:(0123456789) 342 used as stable delivery systems and innovative drug carries for therapeutic purposes [8–10]. CDs based rotaxans and polyrotaxanes have been employed as delivery carriers [11, 12]. Furthermore, by affecting the intermolecular interactions of proteins CDs can interfere with their oligomerization and aggregation processes causing a stabilizing effect [13]. Cyclodextrins, due to their possibility to form host–guest complexes with many drugs, can be used to modify the structure of proteins. Supramolecular and covalent CDprotein conjugates has been of great interest in the field of protein engineering. These conjugates are promising for delivery of anticancer drugs [14, 15]. The solid state, thermal reaction between proteins and reducing sugar was first described by Lea [16]. Then, this approach was further developed by Boratyński and Roy [17] as a method for protein glycation and the synthesis of biologically active neoglycoconjugates [18]. Also, experiments on albumin, fibrinogen-methotrexate and lysozyme glycation show that careful choice of temperature and reaction time ensures the retention of the biological activity of the proteins [19, 20]. We have previously reported the usefulness of mono-6-O-formyl-β-CD in the modification of proteins in thermal reactions in solid state. In that paper we presented the possibility of creating CD-protein conjugates, and the best conditions for carrying out this reaction have been determined. The obtained CD-BPTI and CD-lysozyme conjugates were characterized by mass spectrometry. MALDI-TOF spectra clearly indicate that in both obtained conjugates, one or two molecules of β-CD to the protein molecule were attached. We proved that thermal reaction in solid state between lysozyme and mono-6O-formyl-β-CD is temperature and solvent dependent. The use of higher temperature increases the reaction yield but also results in more than one β-CD molecules attached to protein [21]. Herein, we report the effect of β-CD conjugation on protein physicochemical and biological properties. As a model protein, we have chosen lysozyme from egg white, molecule with a well-defined structure and biological functions. In our present paper we used DMSO with addition of Scheme 1  Synthesis of β-CD/lysozyme conjugate 13 J Incl Phenom Macrocycl Chem (2017) 87:341–348 phosphate buffer (1%) as reaction medium (for high reaction yield) and after freeze drying, temperature 100 °C for 10 min (to obtain predominantly one β-CD molecule per lysozyme). The proposed procedure give a new perspectives for the use this method in the synthesis of biologically active conjugates. The obtained conjugates could be promising in drug delivery systems especially for hydrophobic drugs with low solubility (Scheme 1). Experimental Reagents and solvents Crystalline β-CD, lysozyme from chicken egg white, imidazole, p-toluenesulfonyl chloride, collidine (2,4,6-trimethylpyridine), dimethylsulfoxide (DMSO) were obtained from Sigma–Aldrich, St. Louis, USA. Inorganic salts and other solvents were kindly p (...truncated)