Elsevier

Biomaterials

Volume 35, Issue 22, July 2014, Pages 5897-5907
Biomaterials

Nanoparticles functionalized with Pep-1 as potential glioma targeting delivery system via interleukin 13 receptor α2-mediated endocytosis

https://doi.org/10.1016/j.biomaterials.2014.03.068Get rights and content

Abstract

The treatment for glioma is one of the most challenging problems and therapeutic effect of glioma is often limited due to poor penetration into the tumor tissue. Interleukin 13 receptor α2 (IL-13Rα2) is over-expressed on tumor including established glioma cell lines and primary glioblastoma cell cultures. However, it will not cause activation of its signaling pathways. So it could be served as a promising targeted moiety for anti-glioma drug delivery. Pep-1, one specific ligand of IL-13Rα2, was identified to exhibit excellent capacity of crossing the blood tumor barrier (BTB) and homing to giloma. In this study, based on the IL-13Rα2-mediated endocytosis, Pep-1 was exploited as a potential ligand for effective glioma-targeting delivery. Pep-1 was functionalized to the surface of PEG-PLGA nanoparticles (Pep-NP) to evaluate its glioma homing, by taking advantage of the excessive expression of the IL-13Rα2 on the surface of glioma cells. Compared with non-targeting nanoparticles, Pep-NP exhibited a significantly enhanced cellular association in rat C6 glioma cells and improved penetration in 3D avascular C6 glioma spheroids. Following intravenous administration, Pep-NP could facilitate the distribution of the coumarin-6 in vivo glioma region, 2.21 times higher than that of NP for quantitative analysis. In conclusion, the Pep-NP could precisely target to the brain glioma, which was a potential targeting drug delivery system for glioma treatment.

Introduction

At present, glioblastoma multiforme (GBM) is the most frequent primary central nervous system tumor in human [1]. Because of its diffuse invasion of the surrounding normal brain tissue, it is impossible to eradicate the peripheral infiltrating part by surgery [2]. Therefore, chemotherapy seems necessary in the treatment of glioma. However, the currently available therapeutics by chemotherapy have less than optimal usefulness for GBM, mainly owing to delivery problems to tumor, including the low permeability of drug across blood tumor barrier (BTB) and poor glioma targeting of the chemotherapeutics [3], [4].

It was showed that if the local drug concentration was elevated by twofold, the efficacy to kill the brain tumor cells could be increased by tenfold [5]. Therefore, efficient delivery of drugs to glioma cells is crucial for effective chemotherapy of glioma. Many efforts have been made to enhance the penetration across the BTB [6], [7], [8], [9]. Nanoparticulate drug delivery systems have been attracted increasing attentions in recent years. The major advantages of nanoparticles include their sustained release property, high drug loading and the property of passive targeting by the enhanced permeability and retention (EPR) effects of tumor [10]. Furthermore, based on the receptor-mediated endocytosis, nanoparticles could exhibit active targeting effect after functionalization with cell recognizable targeting ligands, such as monoclonal antibodies [11], endogenous targeting peptides [12], and low molecular-weight compounds, such as folate [13].

Receptor-mediated endocytosis is one of the mechanisms through which nanoparticulate carriers can overcome the obstacle of BTB. The interleukin 13 receptor α2 (IL-13Rα2), one of the subunits of the interleukin-13 receptor, is encoded for a 65 kDa receptor protein. It is over-expressed on human tumors including established glioma cell lines and primary glioblastoma cell cultures [14], [15], [16], [17], [18], which makes IL-13Rα2 an attractive target. It has been reported that IL-13Rα2 can undergo internalization after binding to ligand without causing activation of its signaling pathways [19]. This property indicates that internalization of IL-13Rα2 is signal independent and IL-13Rα2 can be exploited for receptor-directed cancer therapy [19]. Based on the high affinity of IL-13Rα2 with IL13, IL13-PE38QQR, a conjugation complex of human IL-13 with a mutated form of Pseudomonas exotoxin, could effectively target tumor cell-specific recognition domains [20], [21], [22], [23] and show remarkable antitumor activity in animal models of several human cancers [24]. So it could be served as a promising targeted moiety for anti-glioma drug delivery [25].

Pep-1, a linear peptide with 9 amino acid residues (CGEMGWVRC), was isolated by a cyclic disulphide-constrained heptapeptide phages display library [26]. As a specific ligand of IL-13Rα2, Pep-1 could bind to IL-13Rα2 with high affinity and specificity, which was capable of crossing the BTB and homing to glioma [26]. As a new ligand, there is no previous study about Pep-1 conjugation to the drug delivery system which confers glioma targeting property through IL-13Rα2-mediated endocytosis by the glioma cells.

Based on IL-13Rα2 over-expression on glioma cells and the high affinity with Pep-1, Pep-1 can be used for enhancing delivery across the BTB and homing to glioma. In this study, Pep-1 was used as a targeting ligand to develop glioma targeting drug delivery system through chemical bonding with PEG-PLGA nanoparticle. In order to evaluate the in vitro and in vivo targeting efficacy of Pep-1 functionalized PEG-PLGA copolymer nanoparticle (Pep-NP), coumarin-6 was used as fluorescence probe to trace the targeting nanoparticles. The cellular uptake of Pep-NP by C6 glioma cells and the penetration ability into avascular C6 glioma spheroids were investigated. Furthermore, the in vivo biodistribution and brain targeting efficiency of Pep-NP was evaluated by intracranial glioma mice model.

Section snippets

Materials

Methoxyl poly(ethylene glycol)-co-poly(d,l-lactic-co-glycolic acid) copolymer (MePEG-PLGA, 40 KDa) and Maleimidyl-poly(ethylene glycol)-co-poly(d,l-lactic- co-glycolic acid) copolymer (Male-PEG-PLGA, 41.5 KDa) were synthesized by the ring opening polymerization as described before [27]. BCA kit, TritonX-100 and LysoTracker Red were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Penicillin-streptomycin, RPMI 1640 medium, fetal bovine serum (FBS) and 0.25% (w/v) trypsin

Synthesis of Pep-PEG-PLGA

The structure of Male-PEG-PLGA and Pep-PEG-PLGA were determined by 1H NMR spectroscopy. The solvent peak of CDCl3 was present at 7.26 ppm. In the spectrum of Male-PEG-PLGA, the maleimide group had the characteristic peak at 6.7 ppm (Fig. 1A), whereas disappeared after reaction with Pep-1 in the spectrum of Pep-PEG-PLGA (Fig. 1B), suggesting that Pep-1 was conjugated with Male-PEG-PLGA copolymer [31].

The FTIR spectra of Male-PEG-PLGA and Pep-PEG-PLGA were shown in Fig. 2. The spectrum of

Discussion

GBM is the most malignant form of primary astrocytic brain tumors in adults with a median overall survival (OS) of three months without standard treatment [32]. Several factors concur to make GBM treatment notoriously difficult. First, brain has a limited ability to repair itself, any damage may be irreversible. In addition, due to the aggressive growth of glioma, it is hard to completely remove the tumor by surgery. Last but not least, adequate penetration across the BTB by chemotherapeutics

Conclusion

In this study, we proposed PEG-PLGA nanoparticles modified with Pep-1 for glioma drug delivery via IL-13Rα2 mediated endocytosis. The Pep-1 peptide was conjugated to the surface of PEG-PLGA nanoparticles via a maleimide-thiol coupling reaction with the particle size of 94.25 ± 3.32 nm. Cellular experiments showed that Pep-NP significantly enhanced cellular uptake than that of unmodified NP and the internalization of Pep-NP was concentration-dependent, time-dependent and energy-dependent. More

Acknowledgments

This work was supported from the National Natural Science Foundation of China (81302710, 81273457), Natural Science Foundation of Jiangsu Province (BK2012445, BK2012843) and the ordinary university natural science research project of Jiangsu Province (13KJB350004). The authors also acknowledge the support from School of Pharmacy, Fudan University (SDD2012-4) & the Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China (SDD2012-4) and Science and

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