Inhibition Against Growth of Glioblastoma Multiforme In Vitro Using Etoposide-Loaded Solid Lipid Nanoparticles with ρ-Aminophenyl-α-D-Manno-Pyranoside and Folic Acid

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 C 2015 Wiley Periodicals, formulation in targeting delivery to the brain and in inhibitory efficacy against tumorous cells for cancer therapy.  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 proliferation of primary and metastatic tumorous cells.1 Among brain cancers, the most commonly encountering malignancy is glioma, which has been categorized into astrocytoma, anaplastic 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-$, are often overexpressed on cancer cells.5 Thus, the high affinity of folic acid (FA) to FR can be employed to promote the drug uptake in GBM and diminish the drug toxicity to normal tissue.6 In addition to tumor targeting, an effective transport of pharmaceuticals across the blood–brain barrier (BBB) is a critical challenge to the drug management in the central Abbreviations used: APMP, p-aminophenyl-"-D-manno-pyranoside; APMP–FA–ETP–SLN, APMP- and FA-grafted ETP–SLN; APMP–ETP– SLN, APMP-grafted ETP–SLN; BBB, blood–brain barrier; ETP, 4 demethylepipodophyllotoxin 9-(4,6-O-ethylidene-$-D-glucopyranoside) (etoposide); 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: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 1804–1814 (2015)

 C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

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nervous system.7,8 The restriction against the BBB penetration was derived mainly from the tight junction and efflux pumps including 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-(4,6O-ethylidene-$-D-glucopyranoside) (etoposide) (ETP) in treating 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 paminophenyl-"-D-manno-pyranoside (APMP) could trigger the GLUT1-mediated influx to the brain via specific conjugation.13 Solid lipid nanoparticles (SLNs), retaining the characteristics of polymer carriers and liposomes, have various advantages including an efficient encapsulation of pharmaceutical-active ingredient, reduction in the chemical toxicity, and preservation 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 antitumor 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 derivative, 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 examined the permeability coefficient for ETP across a BBB, constituted with a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes

Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

RESEARCH ARTICLE – Pharmaceutical Nanotechnology

(HAs). In addition, the anti-GBM efficacy of ETP was evaluated with the viability of U87MG cells using APMP–FA–ETP– SLNs. The targeting of APMP–FA–ETP–SLNs to the expressed GLUT1 and FR was studied by immunochemical staining.

MATERIALS AND METHODS Preparation of APMP–FA–ETP–SLNs 1,3-Ditetradecanoyloxypropan-2-yl tetradecanoate (Dynasan 114; Sigma–Aldrich, St. Louis, Missouri), hexadecanoic acid (palmitic acid; Sigma–Aldrich), and octadecanoic acid (stearic acid; Sigma–Aldrich) with equal weight percentage at 2% (w/v) each were mixed in methanol (J. T. Baker, Phillipsburg, New Jersey) at 400 rpm and 75°C. On the basis of the lipids, 4% (w/v) ETP (Sigma–Aldrich), 1.25% (w/v) 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] [DSPE–PEG(2000)–CA; Avanti Polar Lipid, Alabaster, Alaska], and 3% (w/v) 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[amino(polyethylene glycol)-2000] [DSPE–PEG(2000)–AM; Avanti Polar Lipid] were added to the organic phase. Fluorescent ETP–SLNs were prepared by adding 0.1% (w/v) fluorescein isothiocyanate-conjugated dextran 70000 [FITC-dextran(70000); Sigma–Aldrich] to the organic phase. The aqueous phase comprised 0.5% (w/v) cholesteryl hemisuccinate (Sigma–Aldrich), 0.3% (w/v) taurocholate (Sigma–Aldrich), 0.5% (w/v) L-A-phospatidylcholine type II-S (Sigma–Aldrich), and 2% (v/v) n-butanol (Riedelde Haen, Seelze, Germany) in ultrapure water (Barnstead, Dubuque, Iowa) and was stirred at 400 rpm and 75°C. For emulsification, the organic phase of 0.25 mL was added slowly to 1.25 mL of the aqueous phase at 400 rpm and 75°C for 10 min. The emulsified fluid was added abruptly to ultrapure water of 15 mL at 3°C, stirred at 1000 rpm for 15 min, and filtrated through a cellulosic paper with pores of 1 :m. The filtrate was centrifuged using a centrifuge (AVANTij-25; Beckman Coulter, Palo Alto, California) at 159,000g and 4°C for 10 min. The bottom pellet was resuspended in ultrapure water with 2% (w/v) D-mannitol (Sigma–Aldrich), stored in a freezer (Sanyo, Osaka, Japan) at −80°C for 1 h, and lyophilized using a freeze dryer (Eyela, Tokyo, Japan) at 2–4 torr and −80°C for 24 h. The supernatant of 20 :L containing free ETP was injected into a reverse-phase BDS Hypersil C-18 column (Thermo Hypersil-Keystone, Bellefonte, Pennsylvania) in a high-performance liquid chromatograph (HPLC; Jasco, Tokyo, Japan), warmed using a column heater (Alltech, Derrfield, Illinois) at 30°C, and detected using an ultraviolet (UV) detector (UV-2075 Plus; Jasco) at 284 nm. Two high-pressure pumps (PU-2080 Plus; Jasco) propelled the mobile phase of methanol gradient from 10% to 50% (v/v) at a flow rate of 1 mL/min for 20 min. The entrapment efficiency of ETP in ETP–SLNs was calculated by [(Wt,ETP −Wf,ETP )/Wt,ETP ] × 100%, where Wt,ETP and Wf,ETP are the weight of total ETP in the preparation and the weight of free ETP in the supernatant, respectively. In addition, dehydrated ETP–SLNs of 1 mg were dissolved in methanol of 100 :L to check the accuracy of the entrapment efficiency. Afterward, the resuspended ETP–SLNs were mixed with 0.1% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma–Aldrich) and 0.05% (w/v) N-hydroxysuccinimide (Acros, Morris, New Jersey) at 80 rpm and 25°C for 2 h, reacted with FA (Sigma–Aldrich) of 5, 10, and 15 mg/mL at 150 rpm and 25°C for 3 h and centrifuged at 159,000g DOI 10.1002/jps.24388

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and 4°C for 10 min. The FA quantity in the supernatant was evaluated using an enzyme-linked immunosorbent assay (ELISA) spectrophotometer (Bio-tek, Winooski, Vermont) at 362 nm. The FA grafting efficiency on FA-grafted ETP–SLNs (FA–ETP–SLNs), GFA , was defined as [(Wt,FA −Wf,FA )/Wt,FA ] × 100%, where Wt,FA and Wf,FA are the weight of total FA in the preparation and the weight of free FA in the supernatant, respectively. The grafting of APMP (Sigma–Aldrich) was analogous to that of FA. Briefly, FA–ETP–SLNs were activated with 1% (w/w) genipin (Challenge Bioproducts, Taichung, Taiwan) at 80 rpm and 25°C for 2 h and reacted with APMP at 5, 10, 15, and 20 mM at 80 rpm and 25°C for 12 h. After wash with ultrapure water, APMP–FA–ETP–SLNs were treated with a QuantiPro bicinchoninic acid assay kit (protein concentration from 0.5 to 30 :g/mL; Sigma–Aldrich). The quantity of APMP on APMP–FA–ETP–SLNs was evaluated using the ELISA spectrophotometer at 562 nm. The APMP grafting efficiency on APMP–FA–ETP–SLNs, GAPMP , was defined as (Wp,APMP /Wt,APMP ) × 100%, where Wp,APMP and Wt,APMP are the weight of APMP on the particles and the weight of total APMP in the preparation, respectively. APMP–FA–ETP–SLNs were dehydrated analogously to ETP–SLNs. The entrapment efficiency of ETP in APMP–FA–ETP–SLNs was also investigated by dissolving dehydrated APMP–FA–ETP–SLNs in methanol and analyzed using the HPLC-UV system at 284 nm. In this study, three independent preparations (n = 3) were used for every datum point.

Particle Size, Zeta Potential, SEM Image, and TEM Image of APMP–FA–ETP–SLNs The cumulant Z-average diameter of APMP–FA–ETP–SLNs, D, and zeta potential of APMP–FA–ETP–SLNs, ., were determined using a zetasizer 3000 HSA with a photon correlation spectroscope and a laser Doppler velocimeter (Malvern, Worcestershire, UK) at 25°C. APMP–FA–ETP–SLNs were suspended in 0.1 M tris hydroxymethyl aminomethane (tris; Riedel-de Haen) buffer at pH 7.4 and controlled at a concentration of 2 mg/mL. The tris buffer contains tris+ and Cl− with pKa of 8.08 and the ionic strength of the tris buffer without further addition of electrolyte is about 4 × 10−6 M.18,19 A suspension of 3 mL was slowly added to a quartz tube and was analyzed for 20 s.

Release of ETP from APMP–FA–ETP–SLNs APMP–FA–ETP–SLNs of 1.5 mg/mL were resuspended in Dulbecco’s phosphate-buffered saline (DPBS; Sigma–Aldrich) containing 0.05% (w/v) sodium azide (Sigma–Aldrich) at pH 7.4 and dialyzed with a cellulosic membrane (Spectrum Laboratories, Rancho Dominguez, California) of 3.3 × 20 cm2 and 50 kDa. Suspended APMP–FA–ETP–SLNs of 10 mL were added to the dialysis tube in a flask of 100 mL with DPBS of 50 mL at pH 7.4 and shook in a bath-reciprocal shaker at 50 rpm and 37°C for 48 h. At a specific sampling time point, the dialyzed sample of 100 :L with dissolved ETP was analyzed using the HPLC-UV system at 284 nm. The flask medium was immediately compensated with fresh DPBS of 100 :L. The accumulated percentage of ETP released from APMP–FA–ETP– SLNs, RETP (%), was defined as [(cumulative weight of ETP in DPBS)/(total weight of ETP in APMP–FA–ETP–SLNs)] × 100. Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

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Cytotoxicity of APMP–FA–ETP–SLNs to HBMECs, HAs, and U87MG cells HBMECs (Biocompare, South San Francisco, California), HAs (Sciencell, Carlsbad, California), and U87MG cells (Bioresource Collection and Research Center, Hsin-Chu, Taiwan) were propagated and preserved according to the protocols proposed previously.20,21 HBMECs were expanded on a tissue culture flask (T75 flask; 75 cm2 ; Corning Costar, Cambridge, Massachusetts) coated with human fibronectin (Sigma–Aldrich) in endothelial cell medium (ECM; Sciencell), supplemented with 1% (v/v) endothelial cell growth supplement (Sciencell), 5% (v/v) fetal bovine serum (FBS; Life Technologies, Carlsbad, California), and 1% (v/v) penicillinstreptomycin-glutamate (PSG; Gibco, Carlsbad, California), and placed in the humidified CO2 incubator (NuAire, Plymouth, Minnesota) at 37°C. ECM was replaced at the first 6 h and every 2 days afterward. After cultivation for 6 days, HBMECs were washed with DPBS, detached with 0.025% trypsin– 0.5 mM ethylenediaminetetraacetic acid (Sigma–Aldrich) of 0.4 mL, and equally distributed to three dishes. For the passages beyond six, T75 flask coated with gelatin (Sigma–Aldrich) was employed. The culture method for HAs and U87MG cells were analogous to that for HBMECs. The culture medium for HAs was astrocyte medium (Sciencell), supplemented with 1% (v/v) astrocyte growth supplement (Sciencell), 2% (v/v) FBS, and 1% (v/v) PSG. The culture medium for U87MG cells was "-minimum essential medium (Gibco), supplemented with 10% (v/v) FBS and 1% (v/v) PSG. Excess HBMECs, HAs, and U87MG cells were immersed in the culture medium containing 10% (v/v) dimethyl sulfoxide (J. T. Baker) at −80°C for 24 h and cryopreserved in liquid nitrogen. Expanded HBMECs, HAs, and U87MG cells at a density of 7.5 × 103 cells/well were seeded, respectively, on 96-well polystyrene MicroWell plate (Nalge Nunc, Rochester, New York) coated with gelatin (Sigma–Aldrich) in culture medium of 150 :L per well and cultivated in the humidified CO2 incubator at 37°C for 8 h. HBMECs, HAs, and U87MG cells were treated with 0.025% (w/v) APMP–FA–ETP–SLNs in the humidified CO2 incubator at 37°C for 12 h (HBMECs and HAs) and for 6, 12, 24, and 48 h (U87MG cells). For studying the effect of dose, U87MG cells were treated with 0.015%, 0.02%, and 0.03% (w/v) APMP–FA–ETP–SLNs for 24 h. After the treatment, cells were reacted with 2,3-bis-(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; Biological Industries, Beit Haemek, Israel) of 50 :L per well containing 2% (v/v) activation solution, placed in the humidified CO2 incubator at 37°C for 4 h, and analyzed using the ELISA spectrophotometer at 450 nm. The cell viability, PCV (HBMECs and HAs) and PCV,U (U87MG cells), was defined as [(the optical density of cells in XTT after treating with APMP–FA–ETP– SLNs−the optical density of XTT)/(the optical density of cells in XTT−the optical density of XTT)] × 100%. Delivery of ETP Across HBMEC/HA Using APMP–FA–ETP–SLNs A protocol for establishing a model of the BBB comprising a confluent monolayer of HBMECs regulated by HAs (HBMEC/HA) on both sides of a gelatin-coated polyethylene terephthalate (PET) membrane (BD Falcon, Franklin Lakes, New Jersey) was proposed previously.22 HBMECs and HAs at a density of 4 × 105 cells/cm2 were seeded, respectively, on the top and bottom surface of the PET membrane in a Transwell (BD Falcon). The sandwich-like structure of HBMEC–PET–HA was immobile in Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

the Transwell and separated the donor chamber from the receiver chamber, where HBMECs and HAs faced the donor and receiver chamber, respectively. This Transwell was cultured in the humidified CO2 incubator at 37°C for 14 days. The upper and lower chambers of the Transwell were filled, respectively, with fresh culture medium for HBMECs and HAs at the same height to balance the hydrostatics. The culture mediums were replaced at a rate of every 2 days. After cultivation, the donor chamber was added with 0.025% (w/v) APMP–FA–ETP–SLNs in ECM (Sciencell) and astrocyte medium (Sciencell) was used in the receiver chamber. The Transwell system was placed in a 24-well tissue culture microtiter plate (flat bottom, polystyrene; Midwest Scientific, St. Louis, Missouri) and cultured in the humidified CO2 incubator at 37°C for 4 h. To evaluate the transendothelial electrical resistance (TEER), the two mediums were replaced with fresh mediums. A Millicell electrical resistance system (Millipore, Billerica, Massachusetts) was employed to determine the electrical resistance of PET membrane with and without HBMEC/HA. The TEER of HBMEC/HA was defined as (electrical resistance of PET membrane with HBMEC/HA−electrical resistance of PET membrane) × surface area of PET membrane. To evaluate the permeability for propidium iodide (PI; Sigma–Aldrich), the medium in the donor chamber was replaced with the ECM containing PI of 0.25 mg/mL after treating with 0.025% (w/v) APMP–FA–ETP– SLNs and placed in the CO2 incubator at 37°C for 5 h. The quantity of PI in the receiver chamber was assessed using a microplate fluorescent reader (Synergy HT; Bio-tek) at excitation of 485 nm and emission of 590 nm. The permeability coefficient for PI across the BBB, PPI , was defined as 1/PPI = 1/Pe,PI −1/Pm,PI , where Pe,PI and Pm,PI are the permeability coefficient for PI across PET membrane with HBMEC/HA and the permeability coefficient for PI across PET membrane, respectively. Pe,PI and Pm,PI were evaluated by J/C = Vr (dCr /dt)/(A·C), where J, C, Vr , Cr , t, and A are the flux of PI from the donor to receiver chamber, the difference in the concentration of PI between the donor and receiver chamber, the volume of receiver chamber, the concentration of PI in the receiver chamber, the time, and the surface area of PET membrane, respectively. The Transwell system, where the donor chamber containing 0.025% (w/v) APMP–FA–ETP–SLNs in culture medium for HBMECs, was applied to the delivery of ETP across HBMEC/HA and placed in the humidified CO2 incubator at 37°C for 5 h. For studying the competition effect of blood sugar, free glucose of 1.4 mg/mL was added to the donor chamber. The fluid of 20 :L in the receiver chamber was sampled every 2 h and was immediately compensated with fresh astrocyte medium of 20 :L. The quantity of ETP infiltrated to the receiver chamber was determined using the HPLC-UV at 284 nm. The permeability coefficient for ETP across the BBB, PETP , was evaluated by 1/PETP = 1/Pe,ETP −1/Pm,ETP , where Pe,ETP and Pm,ETP are the permeability coefficient for ETP across PET membrane with HBMEC/HA and the permeability coefficient for ETP across PET membrane, respectively. Immunochemical Staining of GLUT1 on HBMECs and FR on U87MG Cells with APMP–FA–ETP–SLNs HBMECs at a density of 1 × 105 cells/cm2 on a gelatincoated microscope cover glass (Marienfeld GmbH, LaudaKoenigshofen, Germany) in 24-well tissue culture plate were DOI 10.1002/jps.24388

RESEARCH ARTICLE – Pharmaceutical Nanotechnology

cultured in ECM of 1 mL and placed in the humidified CO2 incubator for 8 h. HBMECs were treated with 0.025% (w/v) fluorescent APMP–FA–ETP–SLNs in the humidified CO2 incubator for 3 h, fixed with 10% (v/v) formalin (Sigma–Aldrich) of 0.5 mL at 25°C for 10 min, immersed in 0.5% (v/v) TritonX-100 at 25°C for 10 min, reacted in serum blocking solution (Zymed, South San Francisco, California) at 25°C for 30 min, incubated with mouse monoclonal antibody to GLUT1 (1:100; Abcam, Cambridge, Massachusetts) at 4°C for 12 h, treated with goat polyclonal secondary antibody to mouse IgG (H and L) (Abcam) at 25°C for 1 h in darkness, counterstained with 0.5% (w/v) 4 ,6-diamidino-2-phenylindole (Sigma–Aldrich) in 0.1% (v/v) Triton-X-100 at 25°C for 3 min in darkness, and immersed in aqueous mounting medium (Bio SB, Santa Barbara, California). The experimental procedures for identifying FR expressed by U87MG cells during an impact of APMP– FA–ETP–SLNs were similar to those for staining of GLUT1 on HBMECs. The only difference between the two was the immunochemistry of monoclonal antihuman folate binding protein antibody (1:200; Abcam) as the primary antibody for FR expression. Blue nuclei, green APMP–FA–ETP–SLNs, and red GLUT1 and FR were visualized using a confocal laser scanning microscope (LSM 510; Zeiss, Oberkochen, Germany) with excitation at 350, 490, and 540 nm and emission at 475, 520, and 565 nm. Statistics Data shown are mean ± SD. Datum groups for statistical significance were compared using a one-way analysis of variance (ANOVA) followed by Tukey’s HSD test.

RESULTS AND DISCUSSION Average Diameter of APMP–FA–ETP–SLNs Figure 1 shows the average diameter of APMP–FA–ETP–SLNs. As indicated in this figure, an increase in the FA concentration enlarged the particles. This was because FA linking to DSPE– PEG(2000)–CA was conjugated on the surface layer and expanded the particle exterior. As revealed in Figure 1, the order in the average diameter was APMP–FA–ETP–SLNs > FA– ETP–SLNs > FA–SLNs. The rationales behind this order were explained as follows. First, encapsulated ETP in the internal lipid phase extended the core region. Second, surface APMP and FA stretched the periphery of the particles.23 Third, the steric stabilization of APMP and FA could prevent APMP–FA– ETP–SLNs from flocculation.24 In this study, the entrapment efficiency of ETP was 82.0 ± 4.7% from subtracting the ETP quantity in supernatant and 78.2 ± 5.1% from direct evaluating the ETP quantity in ETP–SLNs. No significant difference (p > 0.05) was found between the two data. In addition, the entrapment efficiency of ETP was 79.8 ± 3.4% after grafting APMP and FA on ETP–SLNs. No significant difference in the entrapment efficiency of ETP was found between APMP- and FA-grafted and bare particles. This was because the majority of ETP was loaded in the lipid matrix via hydrophobic grasping. Thus, modifications of the external surface did not significantly vary the stability of ETP in APMP–FA–ETP–SLNs. It has been observed that the dehydration, thermal transition, and decomposition temperatures of ETP were 85°C–110°C, 198°C–206°C, and above 250°C, respectively.25,26 Moreover, the residual percentage of ETP was 82.54% after a thermal treatment at 75% DOI 10.1002/jps.24388

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Figure 1. Effect of FA, ETP, and APMP on the average diameter of APMP–FA–ETP–SLNs. (◦) FA–SLNs; () FA–ETP–SLNs; () APMP– FA–ETP–SLNs, CAPMP = 20 mM. Key: lipid phase: 2% (w/v) Dynasan 114, 2% (w/v) palmitic acid, 2% (w/v) stearic acid, 0.24% (w/v) ETP; 0.075% (w/v) DSPE–PEG(2000)–CA; 0.18% (w/v) DSPE–PEG(2000)– AM, aqueous phase: 0.5% (w/v) cholesteryl hemisuccinate; 0.3% (w/v) taurocholate; 0.5% (w/v) L-A-phospatidylcholine type II-S; 2% (v/v) nbutanol. n = 3.

relative humidity and 80°C for 8 h.27 It has also been found that DSPE–PEG(2000) micelles did not show any endothermic and exothermic signal, such as melting transition peak, between 15°C–85°C.28 Therefore, the thermal stability of ETP and phospholipids was sufficient in the current microemulsion at 75°C. Zeta potential of APMP–FA–ETP–SLNs Figure 2 shows the zeta potential of APMP–FA–ETP–SLNs. As indicated in this figure, APMP–FA–ETP–SLNs carried negative charge. In addition, an increase in the FA concentration increased the absolute value of zeta potential. This was because FA was negatively charged in the grafting medium. When bare SLNs were grafted with FA of 15 mg/mL, the absolute value of mean zeta potential increased from 22.3 to 38.2 mV. The difference of 16 mV in the zeta potential suggested that ample FA molecules were grafted on the particle surface. As revealed in Figure 2, the order in the absolute value of zeta potential was FA–SLNs > FA–ETP–SLNs > APMP–FA–ETP–SLNs. This order could be explained by the following three reasons. First, an incorporation of ETP in lipids enlarged the particles (data shown in Fig. 1), decreased the specific surface area of ETP– SLNs, and hindered the conjugation of FA. Second, APMP was positively charged in the grafting medium. Thus, negatively charged FA–ETP–SLNs could preferentially attract APMP to the particle vicinity via electrostatic interaction. Third, the expanded surface layer of APMP on APMP–FA–ETP–SLNs intensified the shielding effect on the charge, lengthened the distance between the shear plane and the particle surface, and decreased the zeta potential.29–32 When APMP of 20 mM was used, the difference of 2–7 mV in the zeta potential suggested Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

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Figure 2. Effect of FA, ETP, and APMP on the zeta potential of APMP–FA–ETP–SLNs. (◦) FA–SLNs; () FA–ETP–SLNs; () APMP– FA–ETP–SLNs, CAPMP = 20 mM. Key: same as Figure 1. n = 3.

Figure 3. Effect of APMP and FA on the dissolution profile of ETP from APMP–FA–ETP–SLNs. () Free ETP; (◦) ETP–SLNs; () FA– ETP–SLNs, CFA = 15 mg/mL; () APMP–FA–ETP–SLNs, CFA = 15 mg/mL, CAPMP = 20 mM. Key: same as Figure 1. n = 3.

Table 1. APMP and FA Grafting Efficiency on APMP-FA-ETP-SLNs CFA (mg/mL)

GFA (%)

5 10 15

76.3 ± 3.2 66.1 ± 4.8 52.4 ± 4.1

CAPMP (mM)

GAPMP (%)

5 10 15 20

71.7 ± 4.7 63.1 ± 5.2 53.6 ± 4.3 41.5 ± 6.8 n = 3.

that a considerable amount of APMP molecules adhered to FA– ETP–SLNs. Grafting Efficiency of APMP and FA Table 1 shows the grafting efficiency of APMP and FA. As indicated in this table, an increase in the FA concentration reduced the grafting efficiency of FA on FA–ETP–SLNs. This was because FA molecules competed with each other for the reaction sites on the surface of ETP–SLNs. Although FA and ETP–SLNs were negatively charged (data shown in Fig. 2), the grafting efficiency of FA was sufficiently high. As revealed in Table 1, an increase in the APMP concentration reduced the grafting efficiency of APMP on APMP–FA–ETP–SLNs. This could also be explained by the fact that the intensity of competition among APMP molecules was promoted for cross-linking with DSPE– PEG(2000)–AM. Dissolution of ETP from APMP–FA–ETP–SLNs Figure 3 shows the kinetics of ETP released from APMP–FA– ETP–SLNs. This figure also displayed the dissolution of free Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

ETP from the cellulosic membrane. As demonstrated in the dissolution profile of free ETP, ETP could rapidly reach the equilibrium concentration, suggesting that the dialysis tube was not a limiting factor for transferring ETP. Two reasons could explain this behavior. First, the diffusion coefficient of free ETP is 4.2 × 10−6 cm2 /s, which enabled a quick migration of ETP across the dialysis membrane.33 Second, the dissolution system was agitated for convection, yielding a faster rate in equilibrating the concentration between fluids separated by the membrane than the release rate of ETP from APMP–FA–ETP–SLNs. As revealed in Figure 3, the release rate of ETP decelerated with time. These dissolution profiles were explained as follows. At the beginning, the dissolution of ETP was comparatively fast for 9 h. This was because a portion of ETP encapsulated in surface layer was prone to escape from the surfactant hydrocarbon chains. In the period of 9–32 h, the dissolution of ETP was stable. This was because the continued agitation of buffer during drug dissolution partially deteriorated the external layer of APMP–FA–ETP–SLNs. The buffer gradually penetrated into the inner lipids, loosened ETP from hydrophobic linkage, and triggered the release and migration of ETP from the internal core. Afterward, the gradual depletion of ETP in the nanocarriers led to the final stage of slow dissolution from 32 to 48 h. As displayed in Figure 3, the accumulated percentage of released ETP was in the order of ETP–SLNs > FA–ETP–SLNs > APMP–FA–ETP–SLNs. This suggested that surface APMP and FA retarded the rapid dissolution of ETP from APMP– FA–ETP–SLNs. Two reasons could contribute to this behavior. First, surface APMP and FA could physically obstruct the diffusion of ETP. Second, the order in migration length for releasing ETP was ETP–SLNs < FA–ETP–SLNs < APMP–FA– ETP–SLNs. Thus, the surface layer of APMP and FA hindered the initial burst of ETP and mediated the subsequent stable dissolution. DOI 10.1002/jps.24388

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Figure 4. Effect of SLN, FA, and APMP on the TEER and permeability for PI across HBMEC/HA after treating with APMP–FA–ETP– SLNs. (◦) TEER, CFA = 15 mg/mL; () TEER, CFA = 0 :g/mL; (•) permeability for PI, CFA = 15 mg/mL; () permeability for PI, CFA = 0 mg/mL. (◦) And () use left axis; (•) and () use right axis; short dash (- – -): TEER of the control group (no carriers); long dash (– – –): permeability for PI of the control group. Key: same as Figure 1. n = 3.

Permeability for ETP Across the BBB Using APMP–FA–ETP–SLNs Figure 4 shows the TEER and permeability coefficient for PI across the BBB. As indicated in this figure, a treatment with APMP–FA–ETP–SLNs decreased the TEER and increased the permeability coefficient for PI from the value of the control group (the intact BBB). This was because an assault of APMP–FA–ETP–SLNs might temporarily deteriorate the tight junction.34,35 As revealed in Figure 4, an increase in the APMP and FA concentration reduced the TEER and enhanced the permeability coefficient for PI across the BBB. This was because the bioactive APMP and FA could enhance the internalization of APMP–FA–ETP–SLNs by HBMECs and weaken the structure of the BBB.36,37 The TEER and permeability coefficient for PI were, respectively, a physical and chemical index of the intercellular tightness among endothelia.38,39 In fact, a satisfactory BBB model for delivering drug to the brain required a high TEER and low permeability coefficient for PI.40 Figure 4 suggested that APMP–FA–ETP–SLNs were proper to permeate the current BBB without inducing a serious damage to the cell configuration. Figure 5 shows the permeability coefficient for ETP across the BBB using APMP–FA–ETP–SLNs. As indicated in this figure, an increase in the APMP and FA concentration enhanced the permeability for ETP. This could be explained by the following three reasons. First, the positively charged APMP neutralized the surface charge, reduced the electrical repulsion between APMP–FA–ETP–SLNs and HBMECs, and advanced the uptake (data shown in Fig. 2; images in Fig. 6). Second, the ligands of APMP could target GLUT1 on HBMECs, accelerate the binding and endocytosis of APMP–FA–ETP–SLNs, and enhance the transport of ETP across HBMEC/HA.41 Third, a strong affinity between FA on APMP–FA–ETP–SLNs and FR DOI 10.1002/jps.24388

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Figure 5. Effect of APMP and FA on the permeability for ETP across HBMEC/HA using APMP–FA–ETP–SLNs. (◦) APMP–FA–ETP–SLNs, CFA = 15 mg/mL, use Y1 axis; () APMP–ETP–SLNs, use Y1 axis; (•) APMP–FA–ETP–SLNs, CFA = 15 mg/mL, CGLU = 1.4 mg/mL, use Y2 axis; () APMP–ETP–SLNs, CGLU = 1.4 mg/mL, use Y2 axis. Key: same as Figure 1. n = 3.

on HBMECs activated the pathway of receptor-mediated transcytosis (RMT).42 Although surface APMP and FA decelerated the release rate of ETP (data shown in Fig. 3), Figure 5 demonstrated the advantages of surface charge, FA, and APMP on APMP–FA–ETP–SLNs for penetrating the BBB. As revealed in Figure 5, an excess of free glucose in the medium reduced the permeability coefficient for ETP across the BBB, when APMP was involved. The glucose concentration used in this study was equivalent to the mean blood sugar concentration of adult after meal and denoted the critical value of impaired glucose homeostasis.43 The reduced ability of APMP–FA–ETP–SLNs resulted mainly from the competition between surface APMP and medium glucose for GLUT1 on HBMECs. However, surface APMP with an interference of free glucose could still assist the delivery of ETP across the BBB. In addition, the medium glucose did not diminish the capability of FA for transporting ETP across HBMEC/HA. Thus, APMP–FA–ETP–SLNs could be a practical formulation for penetrating the BBB in the presence of free glucose. Images of APMP–FA–ETP–SLNs and GLUT1 on HBMECs Figure 6 shows the immunochemical staining for identifying the uptake of APMP–FA–ETP–SLNs by HBMECs. As indicated in this figure, an aggregation of APMP–FA–ETP–SLNs in HBMECs was prevalent. In addition, the order in the green intensity in cytoplasm and nucleus of HBMECs was Figure 6d > 6c > 6b > 6a. This order was explained as following. First, HBMECs could internalize ETP–SLNs without modified moiety, as revealed in Figure 6a. Second, when Figure 6b was compared with Figure 6a, it could be drawn that surface FA was effective in infiltrating HBMECs and favoring the transport of FA–ETP–SLNs across the BBB. Third, the Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

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Figure 6. Fluorescent images of HBMECs after treating with APMP–FA–ETP–SLNs. CFA = 15 mg/mL, CAPMP = 20 mM. (a-1, a-2, a-3, a-4) ETP–SLNs; (b-1, b-2, b-3, b-4) FA–ETP–SLNs; (c-1, c-2, c-3, c-4) APMP–ETP–SLNs; (d-1, d-2, d-3, d-4) APMP–FA–ETP–SLNs; (a-1, b-1, c-1, d-1) red channel; (a-2, b-2, c-2, d-2) green channel; (a-3, b-3, c-3, d-3) blue channel; (a-4, b-4, c-4, d-4) merged image. Key: 0.1% (w/v) FITC-dextran(70000) and the other components are the same as Figure 1.

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els with the cell viability higher than 88%. As indicated in Figure 7a, the multiplication capacity of U87MG cells was reduced with the time of treatment. In addition, an incorporation of APMP and FA decreased the viability of U87MG cells. Four reasons could explain this outcome. First, free ETP had no controlled release characteristics and no targeting ability (data shown in Fig. 3).44 Second, APMP on APMP–ETP–SLNs could be a competent graft to intensify endocytosis by HBMECs through GLUT1 (data shown in Fig. 5; images in Figs. 6c–6d). Third, in addition to FR expressed by HBMECs, FA on FA– ETP–SLNs could dock FR on U87MG cells (images shown in Fig. 8).45 Fourth, APMP and FA on APMP–FA–ETP–SLNs revealed a trait of sustained release and improved the efficacy of ETP (data shown in Fig. 3). As a result, APMP–FA–ETP– SLNs could substantially reduce the effective dosage of toxic ETP for antitumor treatment. As revealed in Figure 7b, an increase in the SLN weight percentage decreased the viability of U87MG cells. This was because an increase in the dose promoted the cytotoxic effect of ETP on U87MG cells. On the basis of the interpolated data in Figure 7b, the estimated half maximal inhibitory concentration of ETP–SLNs, APMP–ETP– SLNs, FA–ETP–SLNs, and APMP–FA–ETP–SLNs for 24 h was 0.022%, 0.024%, 0.025%, and 0.027% (w/v), respectively. In regard to conjugated APMP and FA in binding their receptors, several reasons were elaborated. First, APMP is a derivative of D-glucose, which recognizes GLUT1.46,47 When conjugated on FA–ETP–SLNs, APMP could sustain its characteristics.48 As the primary amine of APMP was used for cross-linking, the D-glucose portion of APMP could be predominantly exposed on the exterior of APMP–FA–ETP–SLNs to target GLUT1. Second, the orientation of the immobilized ligands of APMP on AMPM–FA–ETP–SLNs might slightly alter its activity.49 Third, in addition to APMP, the steric effect could hinder the association of FA ligands, including pterin, aminobenzoate, and glutamate, with FR.50,51 The primary amine of pterin was principally linked to ETP–SLNs and lost its possibility of forming hydrogen bond with carboxylic acid (D81) of FR.52 Fourth, the secondary amines of FA in the current formulation could still target FR via hydrogen bonding. Images of APMP–FA–ETP–SLNs and FR on U87MG Cells After Crossing the BBB Figure 7. (a) Effect of SLN, FA, and APMP on the viability of U87MG cells. CFA = 15 mg/mL, CAPMP = 20 mM. Key: same as Figure 1. n = 3. (b) Effect of SLN weight percentage on the viability of U87MG cells. () ETP–SLNs; () APMP–ETP–SLNs; (◦) FA–ETP–SLNs; () APMP–FA–ETP–SLNs. CFA = 15 mg/mL, CAPMP = 20 mM. Key: same as Figure 1. n = 3.

plentiful green dots attached to red patches near blue stains in Figures 6c–6d demonstrated that GLUT1 expressed by HBMECs could recognize APMP–FA–ETP–SLNs for specifically delivering ETP to HBMECs. Toxicity to U87MG Cells After Treating with APMP–FA–ETP–SLNs Figure 7 shows the viability of U87MG cells after treating with APMP–FA–ETP–SLNs. The cytotoxicity of APMP– FA–ETP–SLNs to HBMECs and HAs was at acceptable lev-

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Figure 8 shows the fluorescent micrographs of APMP–FA– ETP–SLNs interacting with FR on U87MG cells. As indicated in this figure, FR was regularly expressed by U87MG cells, which internalized APMP–FA–ETP–SLNs. In addition, the order in the green intensity was Figure 8d > 8b > 8c > 8a. The reasons for this order were explained as follows. A very weak signal of green ETP–SLNs emerged around U87MG cells, as revealed in Figure 8a. This was because the interactions between ETP–SLNs and U87MG cells were nonspecific. As displayed in Figure 8b, the uptake of FA–ETP–SLNs could be attributed to the RMT pathways via the binding to FR on U87MG cells. Figures 6c and 8c suggested that APMP could only promote transcytosis of APMP–ETP–SLNs across the BBB, however, could not target U87MG cells. As demonstrated in Figures 6d and 8d, APMP–FA–ETP–SLNs invoked both GLUT1 and FR on HBMECs and targeted FR on U87MG cells, yielding the highest delivery efficiency of ETP to malignant U87MG cells.

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Figure 8. Fluorescent images of U87MG cells after treating with APMP–FA–ETP–SLNs across HBMEC/HA. CFA = 15 mg/mL, CAPMP = 20 mM. (a-1, a-2, a-3, and a-4) ETP–SLNs; (b-1, b-2, b-3, and b-4) FA–ETP–SLNs; (c-1, c-2, c-3, and c-4) APMP–ETP–SLNs; (d-1, d-2, d-3, and d-4) APMP–FA–ETP–SLNs; (a-1, b-1, c-1, and d-1) red channel; (a-2, b-2, c-2, and d-2) green channel; (a-3, b-3, c-3, and d-3) blue channel; (a-4, b-4, c-4, and d-4) merged image. Key: same as Figure 6.

CONCLUSIONS Innovated pharmaceutical formulations for ETP in SLNs with surface APMP and FA were fabricated to permeate the BBB and target GBM. The experimental results revealed a high Kuo and Lee, JOURNAL OF PHARMACEUTICAL SCIENCES 104:1804–1814, 2015

entrapment efficiency of ETP in SLNs and characteristics of sustained release of ETP from APMP–FA–ETP–SLNs. In addition, a conjugation of APMP and FA enhanced the permeability for ETP across the BBB and reduced the viability of U87MG cells. Although APMP–FA–ETP–SLNs might lead to DOI 10.1002/jps.24388

RESEARCH ARTICLE – Pharmaceutical Nanotechnology

preparation complications and slightly endanger the BBB safety, these evidences suggested that APMP–FA–ETP–SLNs could significantly improve the efficiency in delivering ETP to the brain and targeting the malignant tumor.

ACKNOWLEDGMENT This work is supported by the Ministry of Science and Technology of the Republic of China.

NOMENCLATURE CAPMP CETP CFA CGLU D GAPMP GFA PCV PCV,U PETP PPI PSLN RETP t .

APMP concentration for surface grafting (mM/mL) ETP concentration for encapsulation (mg/mL) FA concentration for surface grafting (mg/mL) glucose concentration in the medium (mg/mL) average diameter of APMP–FA–ETP–SLNs (nm) APMP grafting efficiency on APMP–FA–ETP–SLNs (%) FA grafting efficiency on FA–ETP–SLNs (%) viability of HBMECs or HAs after treating with APMP–FA–ETP–SLNs (%) viability of U87MG cells after treating with APMP– FA–ETP–SLNs (%) permeability coefficient for ETP across the BBB (cm/s) permeability coefficient for PI across the BBB (cm/s) SLN weight percentage in medium (%) accumulated percentage of ETP released from APMP– FA–ETP–SLNs (%) time for releasing ETP from APMP–FA–ETP–SLNs (h) zeta potential of APMP–FA–ETP–SLNs (mV).

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