EGFPeEGF1-conjugated nanoparticles for targeting both neovascular and glioma cells in therapy of brain glioma Bo Zhang a,1 , Huafang Wang a,1 , Ziwei Liao b , Yu Wang b , Yue Hu c , Jiarong Yang c , Shun Shen b , Jun Chen b , Heng Mei a , Wei Shi a , Yu Hu a, * , Zhiqing Pang b, ** , Xinguo Jiang b a Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei 430022, PR China b School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery, Ministry of Education, 826 Zhangheng Road, Shanghai 201203, PR China c College of Pharmacy, Jiamusi University, Jiamusi 154007, PR China article info Article history: Received 27 December 2013 Accepted 26 January 2014 Available online 14 February 2014 Keywords: Tissue factor EGFPeEGF1 Nanoparticles Dual-targeting therapy Neovascular cells Brain glioma abstract As neovascular and glioma cells were closely associated and might be mutually promoted in glioma growth, a dual-targeting strategy targeting to both neovascular and glioma cells would be more prom- ising as compared with those targeting one of them. In this study, we reported a drug delivery system where nanoparticles were decorated with EGFPeEGF1 (ENP), a fusion protein derived from factor VII with special affinity for tissue factor (TF) over-expressed in glioma tissues, to facilitate anti-glioma de- livery of paclitaxel (PTX) by targeting both neovascular and glioma cells. In vitro protein binding assay demonstrated that EGFPeEGF1 bound well to C6 cells and perturbed human umbilical vein endothelial cells (HUVEC) with a concentration-dependent manner but not to unperturbed HUVEC. EGFPeEGF1eTF interaction significantly enhanced nanoparticles uptake by perturbed HUVEC and glioma C6 cells as well as nanoparticles penetration in C6 glioma spheroids, and thus improved the cytotoxicity of their payload in both monolayer cells and glioma spheroids models. In vivo imaging of glioma-bearing mice demon- strated the specific accumulation of ENP in glioma tissues. In vivo distribution of nanoparticles intuitively showed ENP mainly sited in both extravascular glioma cells and neovascular cells. Pharmacodynamic results revealed that PTX-loaded ENP (ENPePTX) significantly prolonged the median survival time of glioma-bearing mice compared with that of any other group. TUNEL assay and H&E staining showed that ENPePTX treatment induced significantly more cell apoptosis and tumor necrosis compared with other treatments. In conclusion, the results of this contribution demonstrated the great potential of EGFP eEGF1-functionalized nanoparticles for dual-targeting therapy of brain glioma. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction As the most common primary brain tumor, glioma is also featured by neovascularization and proliferation of glioma cell as other solid tumors [1]. During glioma progression, glioma cells promote neovascular formation to help sustain expanding glioma growth, and thus anti-neovascular therapy has emerged as a mainstream strategy for glioma therapy [2,3]. However, current evidence indicate anti-neovascular mono-therapy may associate with a series of adverse effects, such as drug resistance induction, tumor cells local invasion and distal metastasis, and even tumor recurrence [4]. As an alternative to aim at neovascular alone, the dual-targeting strategy, combining anti-neovascular therapy with glioma cells-oriented therapy, may offer a more promising thera- peutic strategy because it not only chokes the blood supply and starves the glioma cells of nutrients and oxygen but also kills gli- oma cells directly [5,6], holds great potential in reducing those side effects of anti-neovascular mentioned above. However, rare study on the effective combination treatment for glioma has been re- ported [7] and most related research in the field of combined tumor therapy focuses on delivering two agents with different functions [5,7] or aiming at two kinds of target sites [8,9]. And thus a drug delivery system with dual-therapeutic effect for glioma treatment via simple design is still urgently needed. As a transmembrane receptor for an endogenous ligand coag- ulation factor VII (FVII), tissue factor (TF) is aberrantly over- expressed on neovascular cells and almost all the tumor cells [10,11], including glioma cells [12,13], but little expressed on * Corresponding author. Tel.: þ86 27 85726335; fax: þ86 27 85776343. ** Corresponding author. Tel./fax: þ86 21 51980069. E-mail addresses: dr_huyu@126.com (Y. Hu), zqpang@fudan.edu.cn (Z. Pang). 1 Equal contributions to the work. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2014.01.071 Biomaterials 35 (2014) 4133e4145