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Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers Huitong Ruan, Xishan Chen, Cao Xie, Beibei Li, Man Ying, Yu Liu, Mingfei Zhang, Xuesai Zhang, Changyou Zhan, Wuyuan Lu, and Weiyue Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017
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ACS Applied Materials & Interfaces
Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers Huitong Ruan,a,‡ Xishan Chen,a,‡ Cao Xie,a Beibei Li,a Man Ying, Yu Liu,a Mingfei Zhang,a Xuesai Zhang,a Changyou Zhan,c Wuyuan Lu,b Weiyue Lu*,a,d,e a
Key Laboratory of Smart Drug Delivery of the Ministry of Education (Fudan University), &
Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, P.R. China. b
Institute of Human Virology & Department of Biochemistry and Molecular Biology, University
of Maryland School of Medicine, Baltimore, Maryland 21201, United States. c
Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai
200032, China d
State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai
200433, P.R. China. e
State Key Laboratory of Medical Neurobiology & the Collaborative Innovation Center for Brain
Science, Fudan University, Shanghai200032, P.R. China.
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ABSTRACT
Malignant glioma, the most frequent and aggressive central nervous system (CNS) tumor, severely threats human health. One reason for its poor prognosis and short survival is the presence of the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB), restricting the penetration of therapeutics into the brain at different stages of glioma. Herein, inspired by the peptide stapling technique, we designed a cyclic RGD ligand via an all-hydrocarbon staple (stapled RGD, sRGD) to facilitate BBB penetration while remaining the capacity of BBTB penetration and targeting ability to glioma cells. As expected, sRGD-modified micelles were able to penetrate the in vitro BBB model while remaining the glioma targeted capability. The results of the in vivo imaging studies further revealed that this nanocarrier could not only efficiently transverse the intact BBB of normal mice, but specifically target glioma cells of intracranial glioma-bearing nude mice. Furthermore, Paclitaxel-loaded sRGD-modified micelles exhibited improved anti-glioma efficacy in vitro and significantly prolonged survival time of gliomabearing nude mice. Overall, this stapled RGD peptide showed potency for glioma-targeted drug delivery by overcoming multiple barriers.
KEYWORDS: glioma • BBB • stapled RGD • tumor-targeted • drug delivery systems
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1、 、INTRODUCTION Malignant tumors of the central nervous system (CNS) severely threat human health because of their poor prognosis and short survival. For instance, patients with glioblastoma multiforme (GBM), one of the most frequent and aggressive CNS cancers, have an average median survival of less than only 15 months and a low rate of 5-year overall survival (below 4%).1-3 One reason for the poor prognosis and survival is the highly diffuse, invasive properties of GBM, which makes it nearly impossible to completely remove tumor-infiltrated tissue by surgery without affecting normal functions of the brain.4-5 In addition, traditional therapeutic paradigms work poorly in treating GBM due to the radioresistance of glioma cells and the hindrance to drug permeability caused by the two barriers unique to the brain: blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB).6-8 The delivery of most small and large therapeutics into the brain tumor is restricted by the BBB/BBTB. Therefore, there is a great need of multifunctional drug delivery systems to penetrate the BBB/BBTB barriers and deliver drugs to the tumor region effectively. In recent decades, numerous endogenous receptors overexpressed on the BBB or BBTB are utilized to mediate the intracerebral delivery of their corresponding ligands-modified drug delivery vehicles.9-16 Among them, integrin αvβ3 are well studied and utilized to enable RGDmodified drug delivery systems to overcome the BBTB and target glioma cells.17-19 The RGD peptide motif was designed to be a penta cyclic peptide (cyclic RGDyK or RGDfK, cRGD), which could recognize integrin αvβ3 overexpressed on neovasculature and glioma cells.20-21 However, as evidenced in Figure 1, bEnd.3 cells, murine brain endothelial cell lines, which are widely used as a model for mimicking brain capillary endothelial cells showed low expression level of integrin αvβ3. At the early stage of glioma, RGD-modified drug delivery strategy for
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glioma treatment is severely constrained since the BBB remains intact.22-23 Even though the BBB could become compromised in GBM, it still partially exists at the edge of tumor because of the infiltrating property of glioma.24-25 Therefore, it would be of great significance if RGD peptide could be reconstructed for effectively overcoming the BBB, while remaining BBTB permeability and glioma targeted capacity. As is reported, a hydrophobic 14-carbon moiety of mytistic acid enabled peptides or gene carriers to achieve the efficient brain target.26-28 Moreover, our laboratory found out that the alkyl chain length of fatty acids influenced the gene transfection in the brain, and the longer ones (C14,C16 and C18) exhibited better brain uptake compared with the shorter ones (C8,C12).29 Recently, a chemical strategy, termed hydrocarbon peptide stapling, was developed to generate α-helical peptide with all-hydrocarbon tethers which mostly attributed to high-level cell penetration.18, 30-34All-hydrocarbon staple was firstly reported by Verdine et al,35 who employed α,α-disubstituted unnatural amino acids bearing all-hydrocarbon macrocyclic cross-link via the ring-closing olefin metathesis (RCM), namely, α,α-disubstitution and macrocyclic bridge formation. Though RGD peptide does not possess the α-helical structure, it is reported that the increased α-helix structure may not generally correlate with the enhanced cell permeability of stapled peptides.34 Therefore, inspired by the stapling-peptide technique, we introduced two N-αFmoc-protected cross-linking amino acids R8, S5 respectively at ends of linear RGD, and synthesized a cyclic RGD (stapled RGD, sRGD) ligand by RCM chemical strategy, expecting to endow it with the ability of BBB penetration (Scheme 1). Polymeric micelles have been widely applied as nanoscale carriers to encapsulate various hydrophobic drugs. It has been verified that micelles may have better deep tumor penetrating ability because of their controlled particle size (
