3D systems delivering VEGF to promote angiogenesis for tissue engineering Anne des Rieux a , Bernard Ucakar a , Billy Paul Kaishusha Mupendwa a , Didier Colau b , Olivier Feron c , Peter Carmeliet d , Véronique Préat a, ⁎ a Université catholique de Louvain, Louvain Drug Research Institute, Unité de Pharmacie Galénique, 1200 Brussels, Belgium b Ludwig Institute for Cancer Research, Brussels Branch, 1200 Brussels, Belgium c Université catholique de Louvain, Institut de recherche expérimentale et clinique, Pole of Pharmacology, 1200 Brussels, Belgium d Vesalius Research Center (VRC), K.U. Leuven, VIB, 3000 Leuven, Belgium abstract article info Article history: Received 15 July 2010 Accepted 28 November 2010 Available online 3 December 2010 Keywords: VEGF encapsulation Hydrogel Scaffolds Angiogenesis Tissue engineering In most cases, vascularization is the first requirement to achieve tissue regeneration. The delivery from implants of angiogenic factors, like VEGF, has been widely investigated for establishing a vascular network within the developing tissue. In this report, we investigated if encapsulation of VEGF in nanoparticles could enhance angiogenesis in vivo as compared to free VEGF when incorporated into two different types of 3D matrices: Matrigel™ hydrogels and PLGA scaffolds. Negatively charged nanoparticles encapsulating VEGF were obtained with a high efficiency by complex formation with dextran sulfate and coacervation by chitosan. After 2 weeks, encapsulation reduced VEGF release from hydrogels from 30% to 1% and increased VEGF release from scaffolds from 20% to 30% in comparison with free VEGF. VEGF encapsulation consistently improved angiogenesis in vivo with both type of 3D matrices: up to 7.5- to 3.5-times more endothelial and red blood cells were observed, respectively, into hydrogels and scaffolds. Hence, encapsulation in nanoparticles enhanced VEGF efficiency by protection and controlled release from 3D implants. Encapsulation and incorporation of VEGF into 3D implants that, in addition to sustaining cell infiltration and organization, will stimulate blood vessel are a promising approach for tissue regeneration engineering. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Regardless of the tissue of interest, implants developed for tissue engineering must be biocompatible and biodegradable. They should serve as a three-dimensional template to provide structural support to the newly formed tissue through an interpenetrating network of pores (at least 100 μm wide) to allow cell migration, tissue in-growth and vascularization [1,2]. In addition, further enhancing of the functionality of these matrices by loading them with growth factors, acting on the surrounding tissues at therapeutic concentrations and for an adequate period of time, is highly desirable [3]. The delivery from implants of angiogenic factors, like vascular endothelial growth factor (VEGF), has been widely investigated to promote new blood vessel formation [4,5] which is a basic requirement for establishing a vascular network within the developing tissue [6]. There are different routes for VEGF administration that can be divided into local and systemic delivery [7]. Intravenous delivery of VEGF is problematic. The short circulation half-life, extraneous interactions with multiple binding sites and VEGF degradation are incompatible with the sustained local concentrations of VEGF required for the development of mature blood vessels [8]. Incorporation of VEGF into poly(-lactic- co-glycolic acid) (PLGA) scaffolds or into microspheres has shown the potential to protect and locally deliver VEGF at a more constant rate, leading to site-specific angiogenesis [8]. Still, VEGF released from the implant remains susceptible to degradation. Current techniques to encapsulate and protect VEGF often use harsh organic solvents and submit proteins to mechanical stress, resulting usually in low protein loading [9]. Huang et al. [8] developed a method to efficiently encapsulate VEGF based on the VEGF heparin binding domain affinity for dextran sulfate, and subsequent coacervation by chitosan forming nanoparticular polyelectrolyte complexes. Complexation and encap- sulation preserve VEGF activity, provide a high loading efficiency and allow a controlled release, making this formulation suitable for VEGF incorporation into implants. A wide range of three-dimensional bioactive implants has been developed with potential uses as delivery systems for therapeutic drugs relevant for tissue repair processes [3], among which two main types: hydrogels and porous polymeric scaffolds. Ideally, hydrogels should be injectable in the lesion for a non-destructive delivery, limiting then further damage. Several macromolecules have been used to form injectable hydrogels [10], but Matrigel™ (composed of structural proteins such as laminin, collagen IV, and heparan sulfate proteoglycans [11]) has became the method of choice for many studies involving in vivo testing of angiogenesis [12,13]. Depending on the type of tissue, the use of polymeric scaffolds may be Journal of Controlled Release 150 (2011) 272–278 ⁎ Corresponding author. Université Catholique de Louvain, Louvain Drug Research Institute, Unité de Pharmacie Galénique, Avenue E. Mounier, 73-20, 1200 Brussels, Belgium. Tel.: +32 2 7647320; fax: +32 2 7647398. E-mail address: veronique.preat@uclouvain.be (V. Préat). 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.11.028 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel