Improved insulin loading in poly(lactic-co-glycolic) acid (PLGA) nanoparticles upon self-assembly with lipids María García-Díaz *, Camilla Foged, Hanne Mørck Nielsen Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark A R T I C L E I N F O Article history: Received 13 September 2014 Received in revised form 18 November 2014 Accepted 20 November 2014 Available online xxx Keywords: PLGA nanoparticles Loading capacity Oral delivery Protein–lipid complexes Self-assembly Insulin A B S T R A C T Polymeric nanoparticles are widely investigated as drug delivery systems for oral administration. However, the hydrophobic nature of many polymers hampers effective loading of the particles with hydrophilic macromolecules such as insulin. Thus, the aim of this work was to improve the loading of insulin into poly(lactic-co-glycolic) acid (PLGA) nanoparticles by pre-assembly with amphiphilic lipids. Insulin was complexed with soybean phosphatidylcholine or sodium caprate by self-assembly and subsequently loaded into PLGA nanoparticles by using the double emulsion-solvent evaporation technique. The nanoparticles were characterized in terms of size, zeta potential, insulin encapsulation efficiency and loading capacity. Upon pre-assembly with lipids, there was an increased distribution of insulin into the organic phase of the emulsion, eventually resulting in significantly enhanced encapsulation efficiencies (90% as compared to 24% in the absence of lipids). Importantly, the insulin loading capacity was increased up to 20% by using the lipid–insulin complexes. The results further showed that a main fraction of the lipid was incorporated into the nanoparticles and remained associated to the polymer during release studies in buffers, whereas insulin was released in a non-complexed form as a burst of approximately 80% of the loaded insulin. In conclusion, the protein load in PLGA nanoparticles can be significantly increased by employing self-assembled protein-lipid complexes. ã 2014 Elsevier B.V. All rights reserved. 1. Introduction Oral delivery of biopharmaceuticals currently represents one of the main challenges for the pharmaceutical industry. Upon administration via the oral route, biopharmaceuticals such as therapeutic proteins must withstand the wide pH variation and the presence of proteolytic enzymes in the gastrointestinal (GI) tract. In addition, they have to penetrate the mucus layer lining the epithelial surface and subsequently permeate the gut epithelial cell layer without loss of biological activity. This harsh environment, combined with an inherent poor physical and chemical stability and low membrane permeability, eventually results in a very low bioavailability of protein therapeutics after oral administration. To overcome these delivery limitations, there has been continu- ous efforts towards developing formulations that can protect and release the molecules at the epithelial surface of the GI tract, as well as enhancing the intestinal permeability (Li et al., 2012; Singh et al., 2008). Among the different strategies, polymeric drug delivery systems have been widely tested and used as carriers for therapeutic proteins and other macromolecules (Ensign et al., 2012; Magnusson et al., 2011). Poly-(lactic-co-glycolic) acid (PLGA) is a well-known biodegradable and biocompatible polymer. It is approved by the US Food and Drug Administration (FDA) and the European Medicine Agency (EMA), and has been used in a variety of biomedical devices and tissue engineering scaffolds (Gentile et al., 2014; Jain, 2000). Different therapeutics have been encapsulated in PLGA nano- and microparticles for several purposes such as vaccination or cardiovascular and cancer treatments (Danhier et al., 2012; Lü et al., 2009; Mundargi et al., 2008). However, the hydrophobic nature of the PLGA polymer limits the efficient entrapment of hydrophilic proteins such as insulin. Two main strategies have been adopted to overcome this challenge of drug-carrier incompatibility to improve the loading efficiency and thus loading capacity of the drug delivery systems: (i) The first includes reduction of the hydrophobicity of the matrix- forming polymer by using hydrophilic polyesters such as poly (lactic-co-hydroxymethyl glycolic acid) (PLHMGA), which has been used to encapsulate different hydrophilic macromolecules into microspheres (Chaisri et al., 2011; Ghassemi et al., 2009, 2012). However, the loading capacity in those particles is usually only * Corresponding author. Tel.: +45 3532 0008. E-mail address: garcia.diaz@sund.ku.dk (M. García-Díaz). http://dx.doi.org/10.1016/j.ijpharm.2014.11.047 0378-5173/ ã 2014 Elsevier B.V. All rights reserved. International Journal of Pharmaceutics xxx (2014) xxx–xxx G Model IJP 14486 No. of Pages 8 Please cite this article in press as: M. García-Díaz, et al., Improved insulin loading in poly(lactic-co-glycolic) acid (PLGA) nanoparticles upon self-assembly with lipids, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.047 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsev ier.com/locate /ijpharm