Using catanionic solid lipid nanoparticles with wheat germ agglutinin and lactoferrin for targeted delivery of etoposide to glioblastoma multiforme

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Using catanionic solid lipid nanoparticles with wheat germ agglutinin and lactoferrin for targeted delivery of etoposide to glioblastoma multiforme Yung-Chih Kuo∗, I-Hsin Wang Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 62102, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 10 January 2017 Revised 25 April 2017 Accepted 2 May 2017 Available online xxx Keywords: Blood–brain barrier U87MG cell Encapsulation Release Transendothelial electrical resistance Propidium iodide

a b s t r a c t Catanionic solid lipid nanoparticles (CASLNs) with surface wheat germ agglutinin (WGA) and lactoferrin (Lf) were formulated to entrap and release etoposide (ETP), cross the blood–brain barrier (BBB), and inhibit glioblastoma multiforme (GBM) growth. Microemulsified ETP-CASLNs were modified with WGA and Lf to permeate a cultured monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes and to treat malignant U87MG cells. Experimental evidence revealed that an increase in the weight percentage of ETP from 1% to 4% decreased its encapsulation efficiency about 3444%. In addition, the release rate of ETP from WGA-Lf-ETP-CASLNs decreased with an increase in the concentration of catanionic surfactant from 7.5 μM to 12.5 μM, and WGA-Lf-ETP-CASLNs at 12.5 μM of catanionic surfactant exhibited a feature of sustained release. WGA-Lf-ETP-CASLNs also reduced transendothelial electrical resistance from 245.5 ×cm2 to 191.5 ×cm2 , enhanced the permeability of propidium iodide from 3.62×10−6 cm/s to 5.61×10−6 cm/s, induced a minor cytotoxicity to HBMECs, increased the ability of ETP to cross the BBB by about 5.6 times, and improved the antiproliferative efficacy of U87MG cells. The grafting of WGA and Lf is crucial to control the medicinal property of ETP-CASLNs, and WGALf-ETP-CASLNs can be promising colloidal carriers in GBM management. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Abbreviations BBB BMEC BW CASLN CNS ETP ETP-CASLN HA HBMEC HBMEC/HA Lf Lf-ETP-CASLN LfR PI RME TEER WGA

∗

WGA-ETP-CASLN blood–brain barrier brain-microvascular endothelial cell beeswax catanionic solid lipid nanoparticle central nervous system ETP ETP-loaded catanionic solid lipid nanoparticle human astrocyte human brain-microvascular endothelial cell HBMECs regulated by HAs lactoferrin ETP-loaded catanionic solid lipid nanoparticle with surface lactoferrin lactoferrin receptor propidium iodide receptor-mediated endocytosis transendothelial electrical resistance wheat germ agglutinin

Corresponding author. E-mail address: [email protected] (Y.-C. Kuo).

ETP-loaded catanionic solid lipid nanoparticle with surface wheat germ agglutinin WGA-Lf-ETP-CASLN ETP-loaded catanionic solid lipid nanoparticle with surface wheat germ agglutinin and lactoferrin 1. Introduction Glioma is classified as one of the frequently encountered causes of intracranial carcinoma [1]. Of the four grades of glioma, glioblastoma multiforme (GBM) is the most malignant at grade IV, with patients living an average of 1–1.5 years after diagnosis [2,3]. The major problems in treating GBM are the difficulty of early-stage diagnosis, the location of the GBM next to tissue regulating basic neurophysiology, the aggressiveness of the phenotype in the central nervous system (CNS), angiogenetic propagation, rapid progression, unsatisfactory prognosis, and a high probability of recurrence [4–6]. Therefore, effective GBM management is a crucial challenge to clinical practice in brain pathology. In addition, conventional chemotherapy for GBM can be inefficacious because the ability of antimitotic drugs, such as etoposide (ETP), to cross the blood–brain barrier (BBB) is low [7]. Hence, a promoted BBB permeability is essential to inhibit GBM growth in the CNS. The use of targeting molecules can be a practical response to this

http://dx.doi.org/10.1016/j.jtice.2017.05.003 1876-1070/© 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Y.-C. Kuo, I.-H. Wang, Using catanionic solid lipid nanoparticles with wheat germ agglutinin and lactoferrin for targeted delivery of etoposide to glioblastoma multiforme, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.003

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Nomenclature CLf Cs CWGA D DETP EETP ELf EWGA PBW EWGA PETP,BBB PETP PPI,BBB PV,HBMEC PV,U87MG t

ζ

concentration of Lf for grafting on ETP-CASLNs (μg/mL) concentration of catanionic surfactant (μM) concentration of WGA for grafting on Lf-ETPCASLNs (mg/mL) average diameter of WGA-Lf-ETP-CASLNs (nm) cumulative percentage of ETP dissolved from WGALf-ETP-CASLNs (%) encapsulation efficiency of ETP in CASLNs (%) grafting efficiency of Lf on ETP-CASLNs (%) grafting efficiency of WGA on Lf-ETP-CASLNs (%) weight percentage of beeswax in ETP-CASLNs (%) grafting efficiency of WGA on Lf-ETP-CASLNs (%) ability of ETP to cross the BBB after treatment with WGA-Lf-ETP-CASLNs (cm/s) weight percentage of ETP in ETP-CASLNs (%) ability of PI to cross the BBB after treatment with WGA-Lf-ETP-CASLNs (cm/s) viability of HBMECs after treatment with WGA-LfETP-CASLNs (%) viability of U87MG cells after treatment with WGALf-ETP-CASLNs (%) time consumed in releasing ETP from WGA-Lf-ETPCASLNs (h) zeta potential of WGA-Lf-ETP-CASLNs (mV)

size of human mononuclear U-937 macrophages and their mitochondrial membrane potential [27]. It was found that CASLNs carrying carmustine could significantly reduce the malignancy of GBM [28]. Thus, CASLNs can be qualified carriers in encapsulating hydrophobic ETP and in controlling its delivery. The aim of this study was to develop ETP-loaded CASLNs (ETPCASLNs) for sustained release of ETP, a chemotherapeutic agent, and to develop WGA- and Lf-grafted ETP-CASLNs (WGA-Lf-ETPCASLNs) for targeting delivery to CNS tumors. Since catanionic surfactant can mediate charge behavior on the particle surface, and WGA and Lf can be functional in brain-targeting transport, it would be important to investigate WGA-Lf-ETP-CASLNs as a colloidal delivery system for GBM therapy. In addition, anticancer drugs are generally lipophilic with poor stability. They are usually eliminated in the blood and generate side responses in the human body, and their brain uptake is limited by the BBB [29,30]. In this study, the potential of CASLNs, acting as ETP carriers, to prolong the efficacy of ETP in the circulation system and reduce cytotoxicity was investigated. CASLNs grafted with WGA and Lf may have a dual ability to cross the BBB and conjugate tumor cells for better GBM management. We examined the particle size distribution, surface charge, particle structure, and encapsulation efficiency of ETP, the dissolution kinetics of ETP, the integrity of the BBB after treatment with WGA-Lf-ETP-CASLNs, the ability of ETP to cross the BBB, toxicity to human BMECs (HBMECs), and efficacy against propagation of U87MG cells. 2. Materials and methods 2.1. Materials

problem. For example, wheat germ agglutinin (WGA) on poly(ethylene glycol)-poly (lactic acid) nanoparticles could be effective in delivering a drug across the BBB through its immobilized carbohydrate-binding pockets [8]. It is worthy of note that the substances in wheat can be important natural bioresources in medicine and agriculture [9–14]. WGA was also capable of interacting with an in vitro BBB model composed of human epithelial ECV304 [15]. Moreover, lactoferrin receptor (LfR) on brain-microvascular endothelial cells (BMECs) could facilitate the infiltration of lactoferrin (Lf) across the BBB via receptor-mediated endocytosis (RME) [16]. In addition to BMECs, it was reported that GBM cells also expressed LfR [17]. Catanionic solid lipid nanoparticles (CASLNs) can be stable particles because they have a unique microstructure with a composite interface of an amphoteric charge. In colloidal science, CASLNs evolved from emulsion of vesicles or micelles that contained zwitterionic surfactant [18]. Cancellation of the opposite charge of polar groups and conjugation of hydrocarbon chains could decrease the energy barrier and lead to a high affinity between catanionic micelles in an isometric cluster formation [19]. In addition, the physicochemical behavior of catanionic surfactant could be explained by considering three major components (oil, water, and alcohol) of microemulsion [20]. In fact, a transition enthalpy study indicated that catanionic surfactants could be spontaneously selfassembled into aggregates [21]. Smoluchowski’s theory of Brownian flocculation also revealed that in a non-equilibrium state, disklike bilayer fusion could be faster than catanionic vesicle coalescence [22]. Moreover, an emulsified system of catanionic surfactant could produce wormlike micelles [23]. In pharmaceutical formulation, these complicated colloids are important carriers for drug loading and dissolution [24]. Catanionic aggregates, for instance, were regarded as an effective dosage form for prolonged release [25]. Furthermore, a cytotoxicity study reported that catanionic aggregates could injure cell membrane during fusional internalization and induce the apoptotic pathway of 3T6 and HeLa cells [26]. Positively charged catanionic vesicles were also able to vary the cell

Beeswax (BW), behenic acid (BA; docosanoic acid), D-mannitol, Dulbecco’s phosphate-buffered saline (DPBS), ETP, ethylenediaminetetraacetic acid (EDTA), phosphotungstic acid (PTA), sodium dodecylsulfate (SDS), sodium azide, stearic acid (SA; octadecanoic acid), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), bicinchoninic acid (BCA) assay kit, propidium iodide (PI), and 4 ,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO). Ethanol was obtained from Tedia (Fairfield, OH). 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[carboxy(polyethylene glycol)-20 0 0] (DSPE-PEG(20 0 0)-CA) was purchased from Avanti Polar Lipid (Alabaster, AL). N,N-di-(bstearoylethyl)-N,N-dimethyl-ammonium chloride (esterquat 1; EQ 1) was obtained from Gerbu Biotechnik (Gaiberg, Germany). N-hydroxysuccinimide (NHS) and Triton-X-100 were purchased from Acros (Morris, NJ). Tris hydroxymethyl aminomethane (Tris) was purchased from Riedel-de Haen (Seelze, Germany). HBMECs were obtained from Biocompare (South San Francisco, CA). Human astrocytes (HAs) were purchased from Sciencell (Corte Del Cedro Carlsbad, CA). U87MG cells were obtained from Bioresource Collection and Research Center (Hsin-Chu, Taiwan). 2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was purchased from Biological Industries (Beit Haemek, Israel). Transwell and polyethylene terephthalate (PET) membrane were obtained from BD Falcon (Franklin Lakes, NJ). Anti-LfR monoclonal antibody, anti-O-linked N-acetylglucosamine antibody, and goat polyclonal secondary antibody to mouse immunoglobulin G (heavy and light) (IgG (H+L)) with rhodamine were obtained from Abcam (Cambridge, MA). 2.2. Preparation of WGA-Lf-ETP-CASLNs BW, BA, SA, DSPE-PEG(20 0 0)-CA and ETP were homogenized at 150 rpm and 75 °C for 10 min. The weight percentage of BW was 25%, 50%, or 75% in the lipid phase, and that of ETP was 1%, 2%, or 4%. The weight percentage of DSPE-PEG(20 0 0)-CA was 1.25% in the

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lipid phase. An equal weight of BA and SA comprised the remaining lipid phase. Surfactants, composed of an equal molar percentage of EQ 1 and SDS, 2% (w/v) Tween 80, and 2.5% (w/v) ethanol, were mixed in ultrapure water at 150 rpm and 75 °C. The lipid phase was emulsified with surfactants at 550 rpm and 75 °C for 30 min. Solid products of ETP-CASLNs were prepared by cooling, filtration, centrifugation, resuspension, and lyophilization, as described in our previous study [31]. 2.3. Encapsulation of ETP in ETP-CASLNs The quantity of unloaded ETP was obtained using a highperformance liquid chromatograph (HPLC; Jasco, Tokyo, Japan) and detected using an ultraviolet (UV)–visible detector (Jasco) at 284 nm. The column and mobile phase were described previously [32]. The encapsulation efficiency of ETP in CASLNs, EETP , was defined as [(Wt,ETP – Wu,ETP )/Wt, ETP ] × 100%, where Wt,ETP and Wu,ETP are, respectively, the weight of total ETP in the preparation and the weight of unloaded ETP in the supernatant. 2.4. Conjugation of WGA and Lf on ETP-CASLNs The method for conjugating Lf and WGA using EDC and NHS and evaluating unloaded Lf and WGA with a BCA assay kit using an enzyme-linked immunosorbent assay (ELISA) spectrofluorometer (Bio-tek, Winooski, VT) was described previously [33]. The grafting efficiency of Lf on ETP-CASLNs, ELf , was defined as [(Wt,Lf – Wu,Lf )/Wt,Lf ] × 100%, where Wt,Lf and Wu,Lf are, respectively, the weight of total Lf in the preparation and the weight of unloaded Lf in the supernatant. The grafting efficiency of WGA on Lf-ETP-CASLNs, EWGA , was defined analogously to that of Lf on ETPCASLNs. 2.5. Average diameter and zeta potential of WGA-Lf-ETP-CASLNs The cumulant Z-average diameter, D, and zeta potential, ζ , of WGA-Lf-ETP-CASLNs were obtained using a zetasizer 30 0 0 HSA (Malvern, Worcestershire, UK). The sample volume was 2 mL of 0.1 M Tris buffer at pH 7.4 and the concentration of WGA-Lf-ETPCASLNs in the sample was 1 mg/mL.

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2.9. Cytotoxicity of WGA-Lf-ETP-CASLNs to HBMECs and U87MG cells The culture and seeding of HBMECs, HAs, and U87MG cells in a humidified CO2 incubator (NuAire, Plymouth, MN) were described previously [35,36]. HBMECs were treated with 0.02% (w/v) WGALf-ETP-CASLNs for 6 h and U87MG cells were treated with culture medium containing WGA-Lf-ETP-CASLNs after permeation of HBMEC/HA for 12 h. HBMECs and U87MG cells at a density of 7.5 × 103 cells/well were reacted in a 50 μL/well of solution containing 2% (v/v) XTT for 4 h, and the optical density (OD) of the solution was evaluated using an ELISA spectrofluorometer at 450 nm [37]. The viability of HBMECs, PV,HBMEC , was defined as [(ODHBEMC,t – ODXTT )/(ODHBMEC,0 – ODXTT )] × 100%, where ODHBEMC,t , ODXTT , and ODHBMEC,0 are the OD of HBMECs with WGA-Lf-ETP-CASLNs, the OD of XTT, and the OD of the original HBMECs, respectively. The viability of U87MG cells, PV,U87MG , was defined analogously to that of PV,HBMEC . 2.10. Integrity of the BBB after being affected by WGA-Lf-ETP-CASLNs A BBB model comprising HBMECs regulated by HAs (HBMEC/HA) on PET membrane in a transwell was established in our previous study [38]. 0.02% (w/v) WGA-Lf-ETP-CASLNs was added to the transwell for 4 h. The transendothelial electrical resistance (TEER) of the BBB (×cm2 ) was evaluated using a Millicell electrical resistance system (Millipore, Billerica, MA) and defined as (RHBMEC/HA,PET – RPET ) × APET , where RHBMEC/HA,PET , RPET , and APET are, respectively, the electrical resistance of PET membrane including HBMEC/HA, the electrical resistance of PET membrane, and the surface area of PET membrane. In addition, 0.02% (w/v) PI was added to the transwell after being affected by WGA-Lf-ETPCASLNs for 5 h. The transported PI was determined using an ELISA spectrofluorometer at an excitation of 485 nm and emission of 590 nm. The ability of PI to cross the BBB, PPI,BBB , was defined as PPI,BBB = (1/PPI,e – 1/PPI,m )−1 , where PPI,e and PPI,m are, respectively, the ability of PI to cross PET membrane including HBMEC/HA and the ability of PI to cross PET membrane [39]. 2.11. Delivery of WGA-Lf-ETP-CASLNs across the BBB

The surface morphology of WGA-Lf-ETP-CASLNs was examined using a field emission scanning electron microscope (FE-SEM; JSM6330 TF, Jeol, Tokyo, Japan). 10 μL of the suspension containing 2 mg/mL of WGA-Lf-ETP-CASLNs in Tris buffer were used.

0.02% (w/v) WGA-Lf-ETP-CASLNs was added to the transwell for penetration of HBMEC/HA for 5 h. 20 μL of culture medium was sampled for 2 h, 4 h and 5 h. 20 μL of fresh culture medium was added immediately for volume balance at the sampling point. The delivery of ETP was determined using an HPLC-UV system at 284 nm. The definition of the ability of ETP to cross the BBB, PETP,BBB , was analogous to that of PI.

2.7. TEM imaging of WGA-Lf-ETP-CASLNs

2.12. Staining of HBMECs and U87MG cells with WGA-Lf-ETP-CASLNs

The structure of WGA-Lf-ETP-CASLNs with negative staining of PTA was examined using a transmission electron microscope (TEM; JEM-1400, Jeol, Tokyo, Japan). The sample volume and concentration were the same as those for SEM imaging.

HBMECs or U87MG cells were seeded on a microscope coverslip at a density of 1×105 cells/cm2 in a 24-well plate containing 1 mL of culture medium for 8 h. 0.02% (w/v) fluorescent WGALf-ETP-CASLNs was added to the culture of HBMECs and incubated for 3 h. U87MG cells were treated with culture medium containing WGA-Lf-ETP-CASLNs to permeate HBMEC/HA for 3 h. The pretreatment steps have been described previously [40]. The sample was reacted with 0.5 μg/mL of anti-LfR monoclonal antibody and/or 5 μg/mL of anti-O-linked N-acetylglucosamine antibody at 4 °C for 12 h, stained with goat polyclonal antibody secondary to mouse IgG (H+L) with rhodamine (1:300) at 25 °C for 1 h, counterstained with 0.5% (w/v) DAPI in 0.1% (v/v) Triton-X-100 at 25°C for 3 min, and incubated with aqueous mounting medium. The fluorescence of staining images was visualized using a confocal laser scanning microscope (LSM 510, Zeiss, Oberkochen, Germany) associated with

2.6. SEM imaging of WGA-Lf-ETP-CASLNs

2.8. Dissolution of ETP from WGA-Lf-ETP-CASLNs The method for the release of ETP and the analysis of dissolved ETP was described previously [34]. 1.5 mg/mL of suspended WGALf-ETP-CASLNs were placed in a cellulose membrane bag of 3.3 cm × 20 cm and shaken at 70 rpm. The cumulative percentage of ETP dissolved from WGA-Lf-ETP-CASLNs, DETP (%), was defined as (Wd,ETP /W0,ETP ) × 100%, where Wd,ETP and W0,ETP are the cumulative dissolved quantity of ETP and the quantity of ETP before dissolution, respectively.

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Fig. 1. (A) Influence of catanionic surfactant on the average particle diameter of WGA-Lf-ETP-CASLNs. PETP = 2%; (◦) PBW = 25%; () PBW = 50%; () PBW = 75%; n = 3. (B) Influence of catanionic surfactant on the zeta potential of WGA-Lf-ETP-CASLNs. PETP = 2%; (◦) PBW = 25%; () PBW = 50%; () PBW = 75%; n = 3. (C) Influence of catanionic surfactant on the encapsulation efficiency of ETP in CASLNs. PBW = 25%; () PETP = 1%; (◦) PETP = 2%; () PETP = 4%; n = 3. (D) Morphology of WGA-Lf-ETP-CASLNs from SEM and TEM imaging. PETP = 2%; PBW = 25%; (a) Cs = 7.5 μM; (b) Cs = 10 μM; (c) Cs = 12.5 μM.

an argon laser source at an excitation of 350 nm, 490 nm, and 540 nm, and an emission of 475 nm, 520 nm, and 565 nm. 3. Results and discussion 3.1. Physicochemical property of WGA-Lf-ETP-CASLNs 3.1.1. Average diameter Fig. 1(A) shows the effect of catanionic surfactant and BW on the particle size of WGA-Lf-ETP-CASLNs. As seen in this figure, an

increase in the concentration of catanionic surfactant from 5 μM to 7.5 μM reduced the particle size. When the concentration of catanionic surfactant increased from 7.5 μM to 12.5 μM, the particle size increased, yielding a minimal diameter of WGA-Lf-ETPCASLNs at 7.5 μM of catanionic surfactant. The reasons for this behavior are as follows. First, the emulsified efficiency at a low concentration of catanionic surfactant was low because the quantity of catanionic surfactant attached to the WGA-Lf-ETP-CASLNs was small. This led to unstable suspended particles and their aggregation. Second, when the concentration of catanionic surfactant

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was high, tiny micelles might be generated spontaneously without enclosing lipids and ETP. This phenomenon could be relevant to the critical micellization concentration of the catanionic surfactant because micellization depleted the catanionic surfactant [41]. Hence, the formation of micelles consumed catanionic surfactant that could adhere to the surface of WGA-Lf-ETP-CASLNs and decreased colloidal stability. It is worthy of note that an increase in the catanionic surfactant from 7.5 μM to 12.5 μM at 25% BW significantly increased the particle size. In addition, an increase in the weight percentage of BW from 25% to 75% enlarged WGA-Lf-ETPCASLNs, as indicated in Fig. 1(A). This was because the multiple components of BW might increase the mobility of lipids in emulsified particles and expand the size of WGA-Lf-ETP-CASLNs. In this study, the average diameter of WGA-Lf-ETP-CASLNs was relatively small because the lipids had long hydrocarbon chains, which organized a compact structure and decreased the size of the internal core. Moreover, the catanionic surfactant could reduce surface tension, diminish its size for enclosing lipids, and produce small WGA-Lf-ETP-CASLNs. As a result, WGA-Lf-ETP-CASLNs as colloidal carriers had an average particle diameter of about 70–230 nm and could be suitable for drug delivery. 3.1.2. Zeta potential Fig. 1(B) shows the zeta potential of WGA-Lf-ETP-CASLNs when the level of catanionic surfactant and BW varied. WGA-Lf-ETPCASLNs were negatively charged. This indicated that the attachment of SDS to the surface of the lipid core was stronger than that of EQ 1. An increase in the concentration of catanionic surfactant from 5 to 15 μM increased the absolute value of the zeta potential of WGA-Lf-ETP-CASLNs (Fig. 1(B)). This was because the difference in the quantity of SDS and EQ 1 on ETP-CASLNs increased with increasing concentrations of catanionic surfactant. The increment of change as a function of the concentration of catanionic surfactant was approximately linear, suggesting that the affinity of catanionic surfactant to lipids could be constant. In addition, the adsorption of catanionic surfactant could alter the surface free energy of ETPCASLNs and enhance their physical stability [42]. Moreover, an increase in the weight percentage of BW from 25% to 75% slightly enhanced the absolute value of the zeta potential of WGA-Lf-ETPCASLNs, as indicated in Fig. 1(B). This was because the catanionic surfactant might be prone to binding to BW, although the linkage force between them was hydrophobic attraction. In contrast to cationic or anionic SLNs, the catanionic surfactant produced a low zeta potential of WGA-Lf-ETP-CASLNs, rendering a weak electrical repulsion between the particles [43,44]. Since WGA-Lf-ETP-CASLNs prepared at 25% BW had appropriate surface charge and particle size, 25% BW was used as the lipid level in the following study. 3.1.3. Encapsulation efficiency of ETP in CASLNs Fig. 1(C) shows the effect of catanionic surfactant and ETP on the encapsulation efficiency of ETP in CASLNs. As seen in this figure, a variation between 5 μM and 15 μM in the concentration of catanionic surfactant yielded a maximal encapsulation efficiency of ETP at 7.5 μM of catanionic surfactant. The rationale behind this maximal encapsulation can be described as follows. First, the hydrocarbon tails of EQ 1 and SDS could help capture hydrophobic ETP [45,46]. Second, during the formation of small emulsified colloids, the possibility of interaction between lipids and ETP was high [47]. This was because lipids of small particles were prone to collide with ETP via hydrophobic attraction during emulsification, and improve the solubility of ETP in lipids. However, large CASLNs might have less solubility of ETP than small CASLNs [48]. This was because large particles restrained sufficient interaction between ETP and lipids and reduced the encapsulation efficiency of ETP. Therefore, the effect of catanionic surfactant on the encapsulation efficiency of ETP was the opposite of that on the parti-

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cle diameter (data shown in Fig. 1A). Third, low electrostatic stabilization of catanionic surfactant might yield a flocculated system, and a concentration of catanionic surfactant higher than the critical micellization concentration might also yield micelles and hinder the formation of ETP-CASLNs [49,50]. In addition, an increase in the weight percentage of ETP from 1% to 4% reduced the encapsulation efficiency, as revealed in Fig. 1(C). This was because an increase in the ETP level enhanced the competition among ETP molecules for void space in the internal core [51]. The encapsulation efficiency of ETP in CASLNs decreased by about 6%–15% in response to an increase in the weight percentage of ETP from 1% to 2%. However, when the weight percentage of ETP increased from 2% to 4%, the encapsulation efficiency of ETP was slashed by about 26%–33%. This suggested that when 2% ETP was used, ETP approached its saturation state in CASLNs with a few excess drug molecules unloaded. Moreover, the high encapsulation efficiency of ETP in CASLNs at 1% and 2% ETP could be attributed to a complicated lipid core with disordered structure, which provided random vacancies and lattice defects of internal ingredients to confine ETP [52,53]. Furthermore, a high shear stress was not suitable for preparing WGA-Lf-ETP-CASLNs because a large drag might detach ETP from the lipids and reduce drug encapsulation [54,55]. It was also reported that an encapsulation of all-trans retinoic acid in SLNs could enhance its bioavailability [56]. An increase in the concentration of catanionic surfactant from 7.5 μM to 15 μM increased the particle size and the absolute value of the zeta potential, but reduced the encapsulation efficiency of ETP, as seen in Fig. 1(A)–(C). This suggested that the catanionic surfactant could produce an appropriate emulsified microenvironment, attract hydrophobic ingredients to form lipid cores, stabilize ETP-CASLNs, attach onto the colloids to accumulate charge, alter interaction between ETP and lipids, and accelerate the development of the ETP-lipid complex in the internal core. In addition, an increase in the weight percentage of BW from 25% to 75% increased both the particle size and the absolute value of the zeta potential. This revealed that the affinity between BW and the catanionic surfactant was relatively high, rendering large ETPCASLNs and increases in the quantity of imbalanced charges from SDS and EQ 1. 3.1.4. Morphology Fig. 1(D) shows the structure of WGA-Lf-ETP-CASLNs. In this figure, WGA-Lf-ETP-CASLNs revealed a sphere-like morphology. The particle diameters in Fig. 1(D) a, b, and c were about 70 nm, 110 nm, and 155 nm, respectively. These diameters were slightly smaller than those shown in Fig. 1(A) because the drying step before SEM imaging could remove surface water and shrink particles [57,58]. In addition, the WGA-Lf-ETP-CASLNs revealed an integral structure with a smooth particle contour. This suggested that the catanionic surfactant could stabilize the colloids and maintain the morphology of WGA-Lf-ETP-CASLNs containing assembled lipids. The black zone in Fig. 1(D) d–f resulted from negative stains of PTA and created a contrast between WGA-Lf-ETP-CASLNs and the background. WGA-Lf-ETP-CASLNs displayed a lighter exterior layer of catanionic surfactant, WGA, and Lf, and a darker interior region of solid lipids, suggesting loosened surface moieties attached to a rigid core. Moreover, the periphery of WGA-Lf-ETP-CASLNs in Fig. 1(D) d–f exhibited an extended structure of minor irregularity. However, the particles were completely consolidated and the geometry deviated slightly from a sphere, showing a diameter of 70–160 nm. 3.2. Grafting efficiency of WGA and Lf on WGA-Lf-ETP-CASLNs Fig. 2 shows the grafting efficiency of Lf on ETP-CASLNs and WGA on Lf-ETP-CASLNs. As seen in this figure, an increase in

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Fig. 3. Release kinetics of ETP from WGA-Lf-ETP-CASLNs. PBW = 25%; PETP = 2%; () Cs = 7.5 μM; () Cs = 10 μM; (◦) Cs = 12.5 μM; n = 3.

Fig. 2. Grafting efficiency of WGA and Lf. PBW = 25%; Cs = 12.5 μM; PETP = 2%; (◦) Lf on ETP-CASLNs; () WGA on Lf-ETP-CASLNs; n = 3.

the concentration of Lf decreased the grafting efficiency of Lf. This was due to the intensified competition among Lf molecules for crosslinking sites on ETP-CASLNs when the quantity of Lf increased. An increase in the concentration of WGA also reduced the grafting efficiency of WGA. This reduction was similarly due to the competition among WGA for grafting. It is worthy of note that the crosslinking could decrease the leakage of WGA and Lf from the surface of WGA-Lf-ETP-CASLNs. As seen in Fig. 2, the grafting efficiency of WGA was lower than that of Lf, suggesting that the steric repulsion between WGA and Lf-ETP-CASLNs could be stronger than that between Lf and ETP-CASLNs.

this decelerated release can be explained as follows. First, the surface catanionic surfactant could obstruct the escape of ETP from the interior core of the WGA-Lf-ETP-CASLNs. Second, a high concentration of catanionic surfactant (12.5 μM) enlarged the particles, reduced the specific surface area, and lengthened the migration path of ETP from the WGA-Lf-ETP-CASLNs. Furthermore, the release behavior of ETP from WGA-Lf-ETP-CASLNs was comparable to the delivery of hydrophobic drug from bio-conjugated nanoparticles [60]. Therefore, the dissolution kinetics suggested that WGALf-ETP-CASLNs prepared at 25% BW, 2% ETP, and 12.5 μM of catanionic surfactant could be a possible formulation of ETP for sustained release.

3.3. Profile of ETP dissolved from WGA-Lf-ETP-CASLNs

3.4. Variation of TEER and ability of PI to cross the BBB after treatment with WGA-Lf-ETP-CASLNs

Fig. 3 shows the release profile of ETP from WGA-Lf-ETPCASLNs. As seen in this figure, the dissolution rate of ETP was relatively slow for the first 2 h, especially in the case of 12.5 μM catanionic surfactant. This suggested no initial burst of ETP from the partly loosened surface layer of catanionic surfactant. In addition, the dissolution buffer experienced difficulty in invading the lipid core. Thus, the major mechanism of release for the first 2 h was the diffusion of ETP from the hydrophobic tails of catanionic surfactant. The dissolution of ETP from WGA-Lf-ETP-CASLNs accelerated from 6 h to 15 h. During this period, the shear stress could detach ETP from the catanionic surfactant and slowly disintegrate WGA-Lf-ETP-CASLNs by fluid penetration [59]. Thus, the binding of catanionic surfactant and lipids was destroyed little by little, rendering a rapid migration and expediting the dissociation of ETP. Although hydrophobic interaction of ETP and lipids could avoid the exhaustion of ETP within 48 h, continuous agitation and collision among WGA-Lf-ETP-CASLNs yielded a retarded release from 24 h to 48 h, and a steady decomposition of the interior structure led to a stable dissolution of ETP. Moreover, an increase in the concentration of catanionic surfactant prolonged the release of ETP from WGA-Lf-ETP-CASLNs, as revealed in Fig. 3. The reasons for

Fig. 4(A) shows the effect of WGA-Lf-ETP-CASLNs on the TEER and ability of PI to cross the BBB. As seen in this figure, the variation in the TEER and ability of PI to cross the BBB was negligible when the concentration of Lf increased. In addition, an increase in the concentration of WGA from 0.2 to 0.6 mg/mL insignificantly altered the TEER and ability of PI to cross the BBB. However, when 0.8 mg/mL of WGA was used, the TEER was reduced and the ability of PI to cross the BBB increased substantially. The rationale behind these results can be described as follows. First, after treatment with WGA-Lf-ETP-CASLNs, the TEER and ability of PI to cross the BBB were, respectively, the physical and chemical index of BBB integrity. Second, negatively charged CASLNs and HBMECs might repel each other electrically. Third, WGA-Lf-ETP-CASLNs could be harmful to the cytoskeleton of HBMECs due to the slight cytotoxicity of CASLNs and dissolved ETP. Fourth, HBMECs could recognize WGA and Lf on WGA-Lf-ETP-CASLNs due to specific binding to Olinked N-acetylglucosamine and LfR, respectively. Fifth, the ability of WGA-Lf-ETP-CASLNs to target the BBB was higher than that of Lf-ETP-CASLNs. In Fig. 4(A), a high TEER (190-230 ×cm2 ) and low ability of PI to cross the BBB (4.1–5.6 cm/s) were obtained after treatment with WGA-Lf-ETP-CASLNs. This suggested a reasonable

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Fig. 4. (A) TEER and ability of PI to cross the BBB after treatment with WGA-Lf-ETP-CASLNs. PBW = 25%; Cs = 12.5 μM; PETP = 2%; (◦) effect of Lf on TEER using Lf-ETP-CASLNs; (•) effect of WGA on TEER using WGA-Lf-ETP-CASLNs; () effect of Lf on ability of PI to cross the BBB using Lf-ETP-CASLNs; () effect of WGA on ability of PI to cross the BBB using WGA-Lf-ETP-CASLNs; (- - -): control of TEER (no nanocarriers used); (– – –): control of ability of PI to cross the BBB; n = 3. (B) Ability of ETP to cross the BBB using WGA-Lf-ETP-CASLNs. PBW = 25%; Cs = 12.5 μM; PETP = 2%; (◦) effect of Lf using Lf-ETP-CASLNs; () effect of WGA using WGA-Lf-ETP-CASLNs; n = 3.

density of the tight junction and sufficient compactness of the BBB to avoid paracellular transport [61,62]. 3.5. Ability of ETP to cross the BBB using WGA-Lf-ETP-CASLNs Fig. 4(B) shows the effect of WGA-Lf-ETP-CASLNs on the ability of ETP to cross the BBB. As seen in this figure, when the concentration of Lf increased from 2.5 to 7.5 μg/mL and the concentration of WGA increased from 2.5 to 5 μg/mL, enhancement of the ability of ETP to cross the BBB was minor. However, 10 μg/mL of Lf promoted this ability of ETP using Lf-ETP-CASLNs, and 5 and 10 μg/mL of WGA could considerably improve the ability of ETP to cross the BBB using WGA-Lf-ETP-CASLNs. The reasons for these results are explained as follows. First, lipophilic ETP, having a molecular weight of 588.56 g/mol, could not sufficiently infiltrate the BBB [63]. Second, membrane fusion of ETP-CASLNs could be nonspecific for permeation of the BBB [64]. Third, Lf-ETP-CASLNs triggered the mechanism of RME via the ligand of LfR on HBMECs. In fact, LfR could actuate the ability to cross the BBB and facilitate brain uptake [65]. Fourth, WGA and Lf on WGA-Lf-ETP-CASLNs could recognize their counterpart on HBMECs. It was reported that the affinity of WGA to porcine capillary endothelial cells was high [66]. 3.6. Viability of HBMECs and U87MG cells after treatment with WGA-Lf-ETP-CASLNs Fig. 5 shows the cytotoxic effect of WGA-Lf-ETP-CASLNs on HBMECs and U87MG cells. As seen in this figure, the order in the viability of HBMECs was ETP-CASLNs ∼ = Lf-ETP-CASLNs ∼ = WGA-Lf-ETPCASLNs > ETP. This order suggested that the lipids and surfactants

Fig. 5. Viability of HBMECs and U87MG cells after treatment with WGA-Lf-ETPCASLNs. PBW = 25%; Cs = 12.5 μM; PETP = 2%; CWGA = 0.8 mg/mL; CLf = 10 μg/mL; n = 3.

of ETP-CASLNs did not obviously imperil the BBB, and CASLNs decreased the cytotoxicity of ETP to HBMECs. In addition, the surface modification of WGA and/or Lf did not increase their toxicity to HBMECs. It was reported that Lf could accelerate the proliferation of endothelia and stimulate angiogenesis [67]. In contrast to Lf, a conjugation of WGA on polymer nanoparticles induced a slight

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Fig. 6. Fluorescent WGA-Lf-ETP-CASLNs with HBMECs and U87MG cells. PBW = 25%; Cs = 12.5 μM; PETP = 2%; CWGA = 0.8 mg/mL; CLf = 10 μg/mL; (a), (b), (c) for HBMECs; (d), (e), (f) for U87MG cells; (a) and (d) ETP-CASLNs; (b) and (e) Lf-ETP-CASLNs; (c) and (f) WGA-Lf-ETP-CASLNs; (a-1, b-1, c-1, d-1, e-1, f-1), (a-2, b-2, c-2, d-2, e-2, f-2), (a-3, b-3, c-3, d-3, e-3, f-3), and (a-4, b-4, c-4, d-4, e-4, f-4) are red (against O-linked N-acetylglucosamine and LfR; excitation at 540 nm), green (nanocarriers; excitation at 490 nm), blue (nuclei; excitation at 350 nm), and merged channels, respectively. (a-4) is a merged image from (a-1) to (a-3); the difference between (a-1) and (a-4) is the same for b, c, d, e and f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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toxicity in rats [68]. Fig. 5 reveals that the order in the efficacy of inhibiting U87MG cells was WGA-Lf-ETP-CASLNs > Lf-ETP-CASLNs > ETP-CASLNs > ETP. The rationale behind this order can be described as follows. First, ETP had an antiproliferative capacity to inhibit the growth of U87MG cells. Second, the continuous release of ETP from WGA-Lf-ETP-CASLNs extended the inhibitory effect of ETP on U87MG cells. Third, the targeting ability of Lf-ETP-CASLNs enhanced the likelihood of ETP inhibiting U87MG cells. Fourth, WGALf-ETP-CASLNs had a strong tendency to permeate the BBB and could also be recognized by U87MG cells. 3.7. Fluorescent images of HBMECs and U87MG cells with WGA-Lf-ETP-CASLNs Fig. 6 shows the staining of WGA-Lf-ETP-CASLNs internalized by HBMECs or U87MG cells. As seen in Fig. 6a, a very feeble green intensity appeared, suggesting that the affinity of ETP-CASLNs to HBMECs was weak. Fig. 6b shows several Lf-ETP-CASLNs around HBMECs, supporting the recognition of Lf-ETP-CASLNs. In fact, surface Lf could activate internalization of Lf-ETP-CASLNs by HBMECs via RME [69]. Fig. 6c reveals a stronger green intensity than Fig. 6b. This suggested that the uptake probability of WGA-Lf-ETP-CASLNs by HBMECs via O-linked N-acetylglucosamine and LfR was higher than that of Lf-ETP-CASLNs. Thus, WGA and Lf on WGA-Lf-ETPCASLNs could considerably improve the delivery of ETP to HBMECs. In addition, the order in the green intensity in Fig. 6d–f was WGALf-ETP-CASLNs > Lf-ETP-CASLNs > ETP-CASLNs. The reasons for this order can be explained as follows. First, ETP-CASLNs had only nonspecific interaction with HBMECs and were inefficient in transporting ETP to U87MG cells. Second, U87MG cells could express LfR to conjugate Lf on WGA-Lf-ETP-CASLNs. Third, the quantity of WGA-Lf-ETP-CASLNs crossing the BBB was higher than that of LfETP-CASLNs, rendering a stronger green intensity of the former in Fig. 6f. 4. Conclusions ETP-CASLNs were stabilized with EQ 1 and SDS in an emulsified system and modified with WGA and Lf for targeted delivery of ETP. We found that an increase in the content of BW enlarged WGALf-ETP-CASLNs and slightly enhanced their negative charge. In addition, the absolute value of the zeta potential increased with increasing concentrations of catanionic surfactant. Particle morphology showed an extended surface layer on WGA-Lf-ETP-CASLNs, indicating an attachment of loosened catanionic surfactant, WGA, and Lf on the lipid core. Moreover, a minimal particle size and maximal encapsulation efficiency of ETP occurred at 7.5 μM of catanionic surfactant. The dissolution study showed that WGA-LfETP-CASLNs fabricated at 12.5 μM of catanionic surfactant could be a possible preparation of ETP for prolonged release. Furthermore, WGA-Lf-ETP-CASLNs did not imperil the BBB and considerably promoted the ability of ETP to cross the BBB. The order in the inhibitory efficacy of U87MG cells was WGA-Lf-ETP-CASLNs > LfETP-CASLNs > ETP-CASLNs > ETP. Therefore, WGA-Lf-ETP-CASLNs have proper biomedical characteristics to act as a potential ETP delivery system for GBM pharmacotherapy. This paper showed the function of WGA-Lf-ETP-CASLNs in permeating HBMEC/HA and inhibiting U87MG cells via O-linked N-acetylglucosamine and LfR. The BBB model in vitro may include human brain vascular pericytes and this factor can be considered in future investigations. Acknowledgments This work is supported by the Ministry of Science and Technology of the Republic of China.

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