TUMOR INHIBITION BY VASCULATURE SPECIFIC DELIVERY OF ...

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siRNA OR DOXORUBICIN USING RGD-TARGETED DRUG CARRIERS ... tumor growth for more than 80% as compared to RGD-nanoparticles delivering.
TUMOR INHIBITION BY VASCULATURE SPECIFIC DELIVERY OF siRNA OR DOXORUBICIN USING RGD-TARGETED DRUG CARRIERS Raymond M. Schiffelers1, Gert Storm1, Timo L.M. ten Hagen2, Adriënne P.C.A. Janssen1, Marcel H.A.M. Fens1, Putthupparampil V. Scaria3, Martin C. Woodle3, Grietje Molema4,* 1. Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht, The Netherlands; 2. Laboratory of Experimental Surgical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands 3. Intradigm Co., Rockville MD 20852, USA; 4. Groningen University Institute for Drug Exploration (GUIDE), Dept. Pathology and Laboratory Medicine, Medical Biology division, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. *[email protected] INTRODUCTION. Cyclic peptides with an Arg-Gly-Asp (RGD) motif have a high affinity for integrins overexpressed on angiogenic endothelial cells (1). We investigated the use of RGD-peptide targeted polyethylene glycol (PEG)-coated nanoscale drug carriers for tumor vasculature specific delivery of siRNA and doxorubicin (Dox) in mice bearing subcutaneous syngeneic tumors (Fig. 1).

PEG

siRNA or Dox RGD

Figure 1. Schematic structure of RGD-targeted drug carriers. RGD is coupled to the distal end of the PEG-coating on the surface of the drug loaded carrier system. The complete system has a size of approximately 100 nm.

METHOD. For delivery of siRNA, RGD-peptide was coupled to poly(ethylene) imine (PEI) via a poly(ethylene) glycol (PEG) spacer. In the presence of siRNA, the RGD-PEG-PEI self-assembles into a nanoparticle with a core formed by an electrostatic complex between siRNA and PEI exposing RGD-peptide on the surface. Incorporated siRNA was specific for silencing of reporter genes or vascular endothelial growth factor receptor 2 (VEGFR2). For delivery of Dox, RGD was coupled to the surface of Dox-filled PEG-liposomes. Both carriers were tested in vitro to determine endothelial cell interaction and processing, and in vivo after tail vein administration to assess tumor vasculature targeting and therapeutic efficacy. 100 nm

RESULTS. Both carriers were shown to be approx. 100 nm in size and having an RGD-PEG-coated surface, as determined by dynamic light scattering and zetapotential measurements, respectively. RGD-PEG-PEI:siRNA nanoparticles as well as RGD-liposomes displayed a high binding affinity for endothelial cells in vitro, whereas carriers lacking RGD-peptide showed poor binding. Binding of both RGD-carriers was inhibited by adding excess of free RGD-peptide. Confocal microscopy analysis of cells showed that binding of carriers was followed by cellular uptake in endosomal vesicles. Delivery of siRNA specific for βgalactosidase with RGD-nanoparticles could knockdown activity of the enzyme in endothelial cells constitutively expressing β-galactosidase. Nanoparticles lacking the RGD-peptide failed to silence the reporter gene. Delivery of Dox with RGDliposomes was shown to result in cytotoxicity in endothelial cells in vitro. In vivo, delivery of FITC-labeled siRNA to tumor tissue after iv administration could be visualized using fluorescence microscopy. Delivery of 3 x 40 µg q 72h of siRNA specific for silencing VEGFR2 using RGD-nanoparticles inhibited tumor growth for more than 80% as compared to RGD-nanoparticles delivering siRNA specific for β-galactosidase or untreated controls. The decrease in tumor growth rate was accompanied by a pronounced loss of vascularization in the tumor region. RGD-liposomes specifically targeted tumor vasculature, as demonstrated by intravital microscopy, while liposomes lacking the RGD-peptide distributed more evenly over the tumor tissue. In a Dox-sensitive tumor model, RGD-targeted liposomal Dox was equally effective to liposomal Dox without targeting peptide. In a Dox-resistant tumor model, RGD-liposomal Dox inhibited tumor growth significantly whereas liposomal Dox failed to suppress tumor growth. DISCUSSION. RGD-targeted drug carriers are able to deliver siRNA or Dox specifically to tumor vasculature leading to therapeutic effects in murine tumor models. The modular design of the drug carriers allows exchange of targeting ligands to support specific delivery via other endothelial cell markers than integrins. This seems especially important in view of the pronounced heterogeneity of angiogenic endothelial cells in human tumor tissue. ACKNOWLEDGMENT. This work was supported by Dutch Cancer Society Grant 2000-2185. REFERENCES 1. Ruoslahti, E., (1996) Annu. Rev. Cell Dev. 12, 697-715. 2. Cao, Y., (2004) Semin. Cancer Biol. 14, 139-145.