Targeted Antiepidermal Growth Factor Receptor (Cetuximab) Immunoliposomes Enhance Cellular Uptake In Vitro and Exhibit Increased Accumulation in an Intracranial Model of Glioblastoma Multiforme

Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab (α-hEGFR-ILs). The affinity of the α-hEGFR-ILs for the EGF receptor was evaluated in vitro using U87？mg and U251？mg cells and in vivo using an intracranial U87？mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α-hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α-hEGFR-ILs by U87？mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α-hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α-hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery. 1. Introduction Gliomas account for almost all primary tumors in the central nervous system (CNS) among which glioblastoma multiforme (GBM) is the most malignant and invasive. In spite of some therapeutic improvements from neurosurgery, radiation therapy, and pharmacology, the 5-year median survival rate is less than 5%, which clearly justifies attempts to improve treatment. The epidermal growth factor receptor (EFGR) is a transmembrane glycoprotein with its intracellular domain acting as a tyrosine kinase and its extracellular part acting as a receptor with high affinity for EGFR [1]. EGFR is highly expressed in cancer cells in more than 40% of GBM cases, and the mutated form of EGFR, EGFRvIII, is additionally expressed in more than 40% of GBM cases expressing EGFR [2, 3], clearly indicating that EGFR could play a role in GBM pathogenesis. Furthermore, as EGFR and EGFRvIII are substantially expressed by the cancer cells in GBM, these receptors are amendable for targeted therapy [4]. Liposome-based therapeutics are usable for treatment of a variety of cancer types, but current available liposomes for human use are not provided conjugated