Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors Kathleen M. McNeeley a , Efstathios Karathanasis a,1 , Ananth V. Annapragada b , Ravi V. Bellamkonda a, * a Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA b School of Health Information Sciences, University of Texas Health Science Center, Houston, TX 77030, USA article info Article history: Received 11 February 2009 Accepted 13 April 2009 Available online 9 May 2009 Keywords: Liposome Folate Glioma Targeting Cleavable PEG Nanocarrier abstract Long-circulating nanocarriers have been extensively studied to deliver chemotherapeutics; however, the inclusion of targeting agents compromises circulation times thereby offsetting the benefits of active targeting. Here, we formulated cysteine-cleavable phospholipid–polyethylene glycol (PEG) to ‘mask’ nanocarrier bound targeting ligands from RES clearance and prolong circulation times of liposomes to allow passive targeting to tumors. This detachable polymer coating can be removed after nanocarrier extravasation to tumor is achieved to expose targeting ligands and promote active targeting to tumor cells. In vivo studies on folate receptor-targeted liposomes demonstrated our ability to prolong circula- tion in the bloodstream using this system thereby verifying the ‘masking’ capacity of cleavable phos- pholipid–PEG 5000 . Controlled modulation of uptake and cytotoxicity of targeted nanocarriers using cleavable phospholipid–PEG was demonstrated through in vitro studies. Finally, studies analyzing uptake by tumor cells in vivo confirmed enhanced intracellular delivery when tumor-inoculated animals received targeted liposomes containing cleavable phospholipid–PEG 5000 followed by a cysteine infusion to expose folate after liposomes had extravasated to tumor. These results indicate that cleavable phos- pholipid–PEG can be used in nanocarrier formulations for controlled exposure of targeting ligands to ensure that circulation times remain uncompromised by the inclusion of targeting agents while enabling active targeting to tumors after removal of the polymer coating. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The ability to specifically target systemically delivered chemo- therapeutics to tumors offers potential advantages over conven- tional non-targeted chemotherapy, most notably a reduction in toxic drug side effects due to decreased delivery to non-target organs [1–3]. Receptor-targeted nanocarriers can be used to package drugs thereby facilitating delivery of high drug payloads to tumors. At the same time, nanocarriers can serve to shield non- target healthy organs from the toxic drug effects and also limit premature degradation of encapsulated drugs by the body [4,5]. Long-circulating nanocarriers, in particular, have been studied extensively as delivery vehicles for chemotherapeutics due to the inherent ability to preferentially accumulate in solid tumors by passive convective transport through leaky endothelium, a process termed extravasation [6,7]. The long blood residence time and repeated passage through the microvascular bed results in high intratumoral concentrations. The efficacy of these passively tar- geted nanocarriers is dependent on the extent of their extravasa- tion to tumors, and the degree of passive accumulation, in turn, is dependent on the nanocarrier circulation time [8]. While passive targeting of nanocarriers results in accumulation of drug at the target site, in vitro studies have shown that uptake by cells is limited unless a targeting agent is utilized to promote active tar- geting to cells [9–13]. Unfortunately, we have recently demonstrated that there is an inherent optimization problem as the properties that confer prolonged nanocarrier circulation times, such as the sphere of hydration made possible by incorporation of polyethylene glycol (PEG), are compromised by the presence of receptor-targeting ligands on the nanocarrier surface [14]. As a result of the incorpo- ration of targeting moieties into nanocarriers, reduced circulation times substantially decrease passive dosing of tumors [14–21]. This consequence partially accounts for the limited success of receptor- targeted nanocarriers in vivo despite the promising results of in vitro experiments [17,22]. Others have investigated methods to achieve a balance between in vivo circulation times and active targeting to tumors by modulating the number of targeting ligands and PEG * Corresponding author. Tel.: þ1 404 385 5038; fax: þ1 404 385 5044. E-mail address: ravi@gatech.edu (R.V. Bellamkonda). 1 Present address: Departments of Biomedical Engineering and Radiology, Case Center for Imaging Research, Case Western Reserve University, Cleveland, OH 44106, USA. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.04.012 Biomaterials 30 (2009) 3986–3995