Research Paper On prilled Nanotubes-in-Microgel Oral Systems for protein delivery Jan Kendall de Kruif a,b , Gisela Ledergerber a , Carla Garofalo b,e , Elizaveta Fasler-Kan c,d,e , Martin Kuentz b,⇑ a Department of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland b Institute of Pharma Technology, University of Applied Sciences and Arts Northwestern Switzerland, 4132 Muttenz, Switzerland c Institute of Chemistry and Bioanalytics, University of Applied Sciences and Arts Northwestern Switzerland, 4132 Muttenz, Switzerland d Department of Biomedicine, University of Basel, 4031 Basel, Switzerland e Department of Pediatric Surgery and Department of Clinical Research, Inselspital, Bern University Hospital, Bern, Switzerland article info Article history: Received 15 June 2015 Revised 19 November 2015 Accepted in revised form 20 January 2016 Available online 4 February 2016 Keywords: Halloysite Hydrogels Macromolecular drug delivery Multi-compartment system Nanocomposite Nanotubes NiMOS Oral protein delivery Prilling abstract Newly discovered active macromolecules are highly promising for therapy, but poor bioavailability hin- ders their oral use. Microencapsulation approaches, such as protein prilling into microspheres, may enable protection from gastrointestinal (GI) enzymatic degradation. This would increase bioavailability mainly for local delivery to GI lumen or mucosa. This work’s purpose was to design a novel architecture, namely a Nanotubes-in-Microgel Oral System, by prilling for protein delivery. Halloysite nanotubes (HNT) were selected as orally acceptable clay particles and their lumen was enlarged by alkaline etching. This chemical modification increased the luminal volume to a mean of 216.3 lLg 1 (+40.8%). After load- ing albumin as model drug, the HNT were entrapped in microgels by prilling. The formation of Nanoparticles-in-Microsphere Oral System (NiMOS) yielded entrapment efficiencies up to 63.2%. NiMOS shape was spherical to toroidal, with a diameter smaller than 320 lm. Release profiles depended largely on the employed system and HNT type. Protein stability was determined throughout prilling and after in vitro enzymatic degradation. Prilling did not harm protein structure, and NiMOS demonstrated higher enzymatic protection than pure nanotubes or microgels, since up to 82% of BSA remained unscathed after in vitro digestion. Therefore, prilled NiMOS was shown to be a promising and flexible multi-compartment system for oral (local) macromolecular delivery. Ó 2016 Elsevier B.V. All rights reserved. 1. Introduction New proteins as active pharmaceutical ingredients (API) have drawn much attention to scientists in modern pharmaceutics [1–3]. The oral delivery of these compounds is challenging in terms of bioavailability, which is substantially reduced by the conditions in the gastrointestinal (GI) tract [4,5]. The GI barriers to overcome consist mainly of enzymatic drug digestion, mucus penetration of the API or of the delivery system, and absorption of the API [6]. If primarily luminal activity is required for the therapeutic action of the macromolecule, only enzymatic protection must be achieved, which is a still challenging but realistic pharmaceutical objective. Herein, microencapsulation has shown potential to over- come this major hurdle by protecting proteins from the GI environ- ment [7–10]. Among several other techniques, prilling can be a way to formulate proteins as microparticles [11]. The mild condi- tions of the process avoid thermally induced protein degradation. Prilling is also known as vibrating nozzle technique. This approach embeds the macromolecular API in a polymeric microgel by drop- ping a solution of both components in a hardening bath. Herein, the API-containing polymeric solution is extruded through a noz- zle. The liquid stream is then broken into droplets by applying vibration. The droplets pass through a ring electrode that charges them electrostatically to avoid mid-air coalescence [12]. Finally, the droplets are collected in a hardening bath where crosslinking occurs and the API is efficiently entrapped. Both polymer and http://dx.doi.org/10.1016/j.ejpb.2016.01.014 0939-6411/Ó 2016 Elsevier B.V. All rights reserved. Abbreviations: ANOVA, analysis of variance; API, active pharmaceutical ingre- dient; APS, ammonium persulfate; BET, Brunauer–Emmett–Teller theory; BSA, bovine serum albumin; DTT, dithiothreitol; GI, gastrointestinal; HNT, halloysite nanotube (comprising modified and non-treated halloysite nanotube); bHNT, base- modified halloysite nanotube; nHNT, non-treated halloysite nanotube; MCC, mono- N-carboxymethyl chitosan; NiMOS, Nanoparticles-in-Microsphere Oral System (comprising microgels loaded with bHNT or nHNT); bNiMOS, NiMOS loaded with base-modified HNT; nNiMOS, NiMOS loaded with non-treated HNT; PBS, phosphate buffer saline; SDS-PAGE, sodium dodecyl sulfate gel electrophoresis; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TEMED, tetramethylethylenediamine. ⇑ Corresponding author at: University of Applied Sciences and Arts Northwestern Switzerland, Institute of Pharma Technology, Gründenstrasse 40, 4132 Muttenz, Switzerland. Tel.: +41 61 467 46 88; fax: +41 61 467 47 01. E-mail address: martin.kuentz@fhnw.ch (M. Kuentz). European Journal of Pharmaceutics and Biopharmaceutics 101 (2016) 90–102 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb