Full length Article Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase Elita Montanari a , Chiara Di Meo a , Simona Sennato b,c , Antonio Francioso d , Anna Laura Marinelli a , Francesca Ranzo a,b,c , Serena Schippa e , Tommasina Coviello a , Federico Bordi b,c , Pietro Matricardi a, * a Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy b Institute for Complex Systems, National Research Council, Via dei Taurini 19, 00185 Rome, Italy c Physics Department, Sapienza University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy d Department of Biochemical Sciences, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy e Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy A R T I C L E I N F O Article history: Available online xxx Keywords: Nanohydrogel Hyaluronan Self-assembly Protein delivery Alginate lyase A B S T R A C T Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan- riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA- CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121  C for 20 min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Young’s modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm- associated infections. ã 2016 Elsevier B.V. All rights reserved. Introduction In recent years, self-assembled NHs, based on various polymer systems and prepared using different protocols, have been widely studied as carriers for drugs, therapeutic proteins and genetic material, showing a broad spectrum of effectiveness and draw- backs. NHs are nanosized, three-dimensional networks able to absorb a high amount of water, showing swelling and de-swelling properties. NHs are usually soft, hydrophilic and biocompatible and represent a highly versatile nanosystem able to deliver a variety of bioactive molecules [1–3]. The porosity of the NH network provides a reservoir for loading molecular and macromo- lecular therapeutics, showing, in many cases, a protective action against degradation due to environmental conditions. In particular, self-assembled NHs have been recognised as promising carriers for macromolecular therapeutics [4], since their hydrophobic domains, surrounded by a hydrophilic outer shell, can serve as a reservoir for various hydrophobic and/or hydrophilic macromo- lecular drugs. Furthermore, Akiyoshi and collaborators have studied extensively the molecular chaperone-like activity of such self-assembled NHs based on pullulan [5]. Abbreviations: AL, alginate lyase; ALG, alginate; HA, hyaluronan; HA-CH, hyaluronan-cholesterol; HA-Rfv, hyaluronan-riboflavin; NHs, nanohydrogels; PDI, polydispersity index. * Corresponding author. E-mail addresses: pietro.matricardi@uniroma1.it, p.matricardi@libero.it (P. Matricardi). http://dx.doi.org/10.1016/j.nbt.2016.08.004 1871-6784/ã 2016 Elsevier B.V. All rights reserved. New Biotechnology xxx (2016) xxx–xxx G Model NBT 913 No. of Pages 10 Please cite this article in press as: E. Montanari, et al., Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase, New Biotechnol. (2016), http://dx.doi.org/10.1016/j.nbt.2016.08.004 Contents lists available at ScienceDirect New Biotechnology journa l homepage: www.e lsevier.com/loca te/nbt