Insulin particles as building blocks for controlled insulin release multilayer nano-films Xiangde Lin, Daheui Choi, Jinkee Hong ⁎ School of Chemical Engineering & Material Science, Chung-Ang University, 47 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea abstract article info Article history: Received 13 February 2015 Received in revised form 6 April 2015 Accepted 15 May 2015 Available online 18 May 2015 Keywords: Diabetic therapy Insulin delivery Insulin nanoparticles Layer-by-layer assembly Long-acting release Insulin nanoparticles (NPs) were prepared by pH-shift precipitation and a newly developed disassembly method at room temperature. Then, an electrostatic interaction-based, layer-by-layer (LbL) multilayer film incorporating insulin NPs was fabricated with poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH), which is described herein as Si/(PAH/PAA) 5 (PAH/PAA-insulin NPs) n . The positively charged insulin NPs were introduced into the LbL film in the form of biocompatible PAA-insulin NP aggregates at a pH of 4.5 and were released in phosphate-buffered saline (pH 7.4), triggered by changes in the charges of the insulin molecules. In addition, the insulin-incorporated multilayer was swollen because of the different ionic environment, leading also to insulin release. Eighty percent of the insulin was released from the LBL film in the first stage of 3 h, and sustained release could be maintained in the second stage for up to 7 days in vitro, which is very critical for specific diabetic patients. These striking findings could offer novel directions to researchers in establishing insulin delivery systems for diabetic therapy and fabricating other protein nanoparticles applied to various biomedical platforms. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Diabetes therapy incorporating various nanomaterials and ap- proaches to effectively control glycemic stability has received increased attention over the past decades [1–6]. In implementing new methods, different forms of insulin such as supramolecular insulin assemblies (SIA) [7], insulin analogs [8], insulin nanoparticles (NPs) [9], nano- networks [10], and nanocomplexes [11] have been prepared and widely employed for insulin delivery, in particular via traditional subcutaneous injection. For instance, nano-sized insulin particles could serve as appropriate drug depots at the injection site for long-term release, according to related research by Gupta et al. [7]. A great deal of effort has also been made to establish various insulin delivery systems such as oral administration [12], intranasal therapy [13], gastrointestinal route [14], pulmonary delivery [15], and tablet implantation [16]. However, a number of challenging issues involved in the process of diabetes treatment, including high cost, low compatibility in vivo, common infections, patient compliance, and sudden hypoglycemia, have not yet been overcome in a flawless manner. The layer-by-layer (LbL) technique for multilayer assembly provides a superb route to build up desired films using a range of functional materials, including polymer polyelectrolytes, DNA, proteins, graphene, nanocomplexes, nanoparticles, nanowires, and nanotubes [17–23]. Particular advantages of the LbL method, such as the precise control of film thickness, specific functionality, optional compositions, and versatile morphology, have been investigated and demonstrated in the field of multilayered structures ranging from the nano- to micro- scale [24–26]. Thus, inorganic NPs, also applicable for nearly any type of charged components, were introduced into desired functional multi- layers by virtue of electrostatic interactions [22]. Similarly, LbL multilay- ered films with building blocks of different drugs can be obtained by taking advantage of the molecular interactions among materials, in particular electrostatic interactions or hydrogen bonding [27,28]. Specifically, researchers have been attracted to the design of insulin delivery systems based on LbL thin films. For example, Chen et al. successfully fabricated a glucose-sensitive multilayer film based on a 21-armstar polymer, showing an on–off switch of insulin release in response to in vivo glucose levels [29]. Their group then further devel- oped LbL films constructed from supramolecular insulin assemblies, which were useful for super long-term glycemic control for up to 295 days [30]. Unlike the glucose-sensitive system, insulin release triggered by variations in pH was also observed by Yoshida et al. after exposing a template containing insulin to weakly acidic or neutral solutions [31]. After transcutaneous protein drug delivery was created, we proposed a method for creating insulin-encapsulated nanofilms by LbL, which could be regarded as a nano-container for controlled insulin release [32]. Herein, we aimed to rapidly prepare insulin NPs through pH-shift precipitation and crystal disassembly, which is a novel technique, compared with conventional growth means that can be time- consuming, limited in high temperature or carried out under denaturation condition. Following preparation, insulin NPs in the form of PAA-insulin NP aggregation were assembled into pH-sensitive Materials Science and Engineering C 54 (2015) 239–244 ⁎ Corresponding author. E-mail address: jkhong@cau.ac.kr (J. Hong). http://dx.doi.org/10.1016/j.msec.2015.05.046 0928-4931/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec