RESEARCH ARTICLE – Pharmaceutical Nanotechnology Inhibition Against Growth of Glioblastoma Multiforme In Vitro Using Etoposide-Loaded Solid Lipid Nanoparticles with p-Aminophenyl--D-Manno-Pyranoside and Folic Acid YUNG-CHIH KUO, CHIA-HAO LEE Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, Republic of China Received 17 December 2014; revised 14 January 2015; accepted 23 January 2015 Published online 18 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24388 ABSTRACT: Solid lipid nanoparticles (SLNs) grafted with p-aminophenyl--D-manno-pyranoside (APMP) and folic acid (FA) (APMP– FA–SLNs) were applied to encapsulate 4 ′ -demethylepipodophyllotoxin 9-(4,6-O-ethylidene--D-glucopyranoside) (etoposide) (ETP) for promoting the antiproliferation of malignant glioblastoma multiforme. ETP-loaded APMP–FA–SLNs (APMP–FA–ETP–SLNs) were used to penetrate the blood–brain barrier (BBB) and retard the propagation of U87MG cells. An incorporation of APMP and FA increased the particle size, the cytotoxicity to U87MG cells, and the permeability coefficient for propidium iodide and ETP across the BBB. In addition, an increase in the APMP and FA concentration reduced the zeta potential, the grafting efficiency of APMP and FA, the dissolution rate of ETP, and the transendothelial electrical resistance. Immunochemical staining images evidenced that APMP–FA–ETP–SLNs could infiltrate the BBB via glucose transporter 1 and recognize U87MG cells via folate receptor. APMP–FA–ETP–SLNs can be an effective pharmacotherapeutic formulation in targeting delivery to the brain and in inhibitory efficacy against tumorous cells for cancer therapy. C  2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1804–1814, 2015 Keywords: glioblastoma multiforme; blood–brain barrier; solid lipid nanoparticle; etoposide; p-aminophenyl--D-manno-pyranoside; folic acid; targeted drug delivery; cancer chemotherapy; drug targeting; microemulsions INTRODUCTION Brain cancer, a solid neoplasm in cranium and central spinal canal, is derived from abnormal and uncontrollable prolif- eration of primary and metastatic tumorous cells. 1 Among brain cancers, the most commonly encountering malignancy is glioma, which has been categorized into astrocytoma, anaplas- tic astrocytoma, and glioblastoma multiforme (GBM). 2 GBM usually propagates fast in an undesirable manner and is one of the most detrimental cancers with an incidence above 50% in primary brain tumor and a mean survival time below 1.5 years. 3 To manage GBM in chemotherapy, typical receptors on cell membrane can be used for targeting delivery. 4 For example, folate receptor (FR), a membrane glycoprotein, is appropriate for antiglioma medication because its subfamily members, FR- " and FR-$, are often overexpressed on cancer cells. 5 Thus, the high affinity of folic acid (FA) to FR can be employed to promote the drug uptake in GBM and diminish the drug toxicity to nor- mal tissue. 6 In addition to tumor targeting, an effective trans- port of pharmaceuticals across the blood–brain barrier (BBB) is a critical challenge to the drug management in the central Abbreviations used: APMP, p-aminophenyl-"-D-manno-pyranoside; APMP–FA–ETP–SLN, APMP- and FA-grafted ETP–SLN; APMP–ETP– SLN, APMP-grafted ETP–SLN; BBB, blood–brain barrier; ETP, 4 ′ - demethylepipodophyllotoxin 9-(4,6-O-ethylidene-$-D-glucopyranoside) (etopo- side); ETP–SLN, ETP-loaded SLN; FA, folic acid; FA–ETP–SLN, FA-grafted ETP–SLN; FR, folate receptor; GBM, glioblastoma multiforme; GLUT1, glucose transporter 1; HA, human astrocyte; HBMEC, human brain-microvascular endothelial cell; HBMEC/HA, HBMECs regulated by HAs; PI, propidium iodide; RMT, receptor-mediated transcytosis; SLN, solid lipid nanoparticle; TEER, transendothelial electrical resistance. Correspondence to: Yung-Chih Kuo (Telephone: +886-5-272-0411, x33459; Fax: +886-5-272-1206; E-mail: chmyck@ccu.edu.tw) Journal of Pharmaceutical Sciences, Vol. 104, 1804–1814 (2015) C  2015 Wiley Periodicals, Inc. and the American Pharmacists Association nervous system. 7,8 The restriction against the BBB penetration was derived mainly from the tight junction and efflux pumps in- cluding P-glycoprotein and multidrug resistance proteins. 9,10 In a preclinical study, it has been concluded that the BBB strongly hampered the efficacy of 4 ′ -demethylepipodophyllotoxin 9-(4,6- O-ethylidene-$-D-glucopyranoside) (etoposide) (ETP) in treat- ing recurrent brain tumors. 11 To facilitate the permeability across the BBB, the membrane transporter such as glucose transporter 1 (GLUT1) can be applied to the administration of anticancer preparations. 12 It has been observed that p- aminophenyl-"-D-manno-pyranoside (APMP) could trigger the GLUT1-mediated influx to the brain via specific conjugation. 13 Solid lipid nanoparticles (SLNs), retaining the characteris- tics of polymer carriers and liposomes, have various advantages including an efficient encapsulation of pharmaceutical-active ingredient, reduction in the chemical toxicity, and preserva- tion in the solid structure when use. 14,15 In a study on cancer therapy, SLNs extended the residence time of camptothecin in mouse brain after intravenous injection. 16 SLNs could also decrease the elimination and increase the internalization of an- titumor 3 ′ ,5 ′ -dioctanoyl-5-fluoro-2 ′ -deoxyuridine in the brain. 17 The aim of this study was to investigate the antipropagation capacity of ETP-loaded SLNs (ETP–SLNs) with grafted APMP and FA for GBM pharmacotherapy. ETP, a glycoside deriva- tive, has a crucial pharmacological trait of cellular toxin and P-glycoprotein substrate. APMP and FA favor, respectively, the linking to GLUT1 for the BBB penetration and binding to FR for the GBM infiltration. Therefore, a dual targeting involving APMP- and FA-grafted ETP–SLNs (APMP–FA–ETP–SLNs) is a feasible strategy to meliorate the GBM treatment. We ex- amined the permeability coefficient for ETP across a BBB, constituted with a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes 1804 Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015