A novel strategy for the treatment of chronic wounds based on the topical administration of rhEGF-loaded lipid nanoparticles: In vitro bioactivity and in vivo effectiveness in healing-impaired db/db mice Garazi Gainza a,b , Marta Pastor a,b , José Javier Aguirre c , Silvia Villullas d , José Luis Pedraz a,b , Rosa Maria Hernandez a,b , Manoli Igartua a,b, ⁎ a NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country, Vitoria, Spain b Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria, Spain c Hospital Universitario de Álava (HUA) Txagorritxu, Vitoria 01009, Spain d Biopraxis Research AIE, Miñano 01510, Spain abstract article info Article history: Received 24 February 2014 Accepted 18 April 2014 Available online 29 April 2014 Keywords: EGF (epidermal growth factor) Lipid nanoparticles SLN (solid lipid nanoparticles) NLC (nanostructured lipid carriers) Chronic wounds Tissue engineering Lipid nanoparticles are currently receiving increasing interest because they permit the topical administration of proteins, such as recombinant human epidermal growth factor (rhEGF), in a sustained and effective manner. Because chronic wounds have become a major healthcare burden, the topical administration of rhEGF-loaded lipid nanoparticles, namely solid lipid nanoparticles (SLN) and nanostructured lipid carries (NLC), appears to be an interesting and suitable strategy for the treatment of chronic wounds. Both rhEGF-loaded lipid nanoparti- cles were prepared through the emulsification-ultrasonication method; however, the NLC-rhEGF preparation did not require the use of any organic solvents. The characterisation of the nanoparticles (NP) revealed that the encapsulation efficiency (EE) of NLC-rhEGF was significantly greater than obtained with SLN-rhEGF. The in vitro experiments demonstrated that gamma sterilisation is a suitable process for the final sterilisation because no loss in activity was observed after the sterilisation process. In addition, the proliferation assays revealed that the bioactivity of the nanoformulations was even higher than that of free rhEGF. Finally, the effectiveness of the rhEGF-loaded lipid nanoparticles was assayed in a full-thickness wound model in db/db mice. The data demon- strated that four topical administrations of SLN-rhEGF and NLC-rhEGF significantly improved healing in terms of wound closure, restoration of the inflammatory process, and re-epithelisation grade. In addition, the data did not reveal any differences in the in vivo effectiveness between the different rhEGF-loaded lipid nanoparticles. Overall, these findings demonstrate the promising potential of rhEGF-loaded lipid nanoparticles, particularly NLC-rhEGF, for the promotion of faster and more effective healing and suggest their future application for the treatment of chronic wounds. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Since their first description in the1990s, lipid nanoparticles, mainly solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), have become potent drug delivery systems that have attracted much attention and interest as efficient and non-toxic carriers for various active compounds [1,2]. Both lipid nanoparticles are suitable for the protection of drugs against degradation, enhance drug stability against light, oxidation, or hydrolysis, and provide the sustained release of active compounds [3]. The main difference between SLN and NLC is the use of a liquid lipid (oil) for the preparation of NLC which, as de- scribed by several authors, increases the loading capacity and reduces leakage of the encapsulated drug during storage [1,2,4]. Lipid nano- particles are widely used through different routes of administration (such as parenteral, oral, ocular, and pulmonary administration) due to their excellent tolerability, biodegradability, and low toxicity. These nanoparticles are also suitable delivery systems for the topical treat- ment of skin diseases because they provide high drug concentrations in the treated skin area. In addition, systemic site effects may be reduced compared with the oral or parenteral administration routes as a result of the lower systemic drug bioavailability provided by topical administra- tion [5,6]. Moreover, their small particle size and lipidic composition ensure close contact between the nanoparticles and the skin, the release of the encapsulated drugs in a controlled manner, and an increase in their residence time in the skin [7]. These particles also show occlusive properties that increase skin hydration and enhance drug penetration Journal of Controlled Release 185 (2014) 51–61 ⁎ Corresponding author at: Laboratory of Pharmaceutics, University of the Basque Country, School of Pharmacy, Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain. Tel.: +34 945013875; fax: +34 945013040. E-mail address: manoli.igartua@ehu.es (M. Igartua). http://dx.doi.org/10.1016/j.jconrel.2014.04.032 0168-3659/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel