Quaternary Ammonium Î²-Cyclodextrin Nanoparticles for Enhancing

Biomacromolecules 2009, 10, 505–516

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Quaternary Ammonium β-Cyclodextrin Nanoparticles for Enhancing Doxorubicin Permeability across the In Vitro Blood-Brain Barrier Eun Seok Gil,†,‡ Jianshu Li,§ Huining Xiao,§ and Tao Lu Lowe*,‡,| Department of Pharmaceutical Sciences, School of Pharmacy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, Departments of Surgery, Pennsylvania State University, Hershey, Pennsylvania 17033, and Department of Chemical Engineering, University of New Brunswick, Fredericton, NB, Canada E3B 5A3 Received September 12, 2008; Revised Manuscript Received December 12, 2008

This study describes novel quaternary ammonium β-cyclodextrin (QAβCD) nanoparticles as drug delivery carriers for doxorubicin (DOX), a hydrophobic anticancer drug, across the blood-brain barrier (BBB). QAβCD nanoparticles show 65-88 nm hydrodynamic radii with controllable cationic properties by adjusting the incorporated amount of quaternary ammonium group in their structure. ATR-FTIR studies confirm the complexation between the QAβCD nanoparticles and DOX. QAβCD nanoparticles are not toxic to bovine brain microvessel endothelial cells (BBMVECs) at concentrations up to 500 µg · mL-1. They also do not change the integrity of BBMVEC monolayers, an in vitro BBB model, including transendothelial electrical resistance value, Lucifer yellow permeability, tight junction protein occludin and ZO-1 expression and morphology, cholesterol extraction, and P-glycoprotein (P-gp) expression and efflux activity, at a concentration of 100 µg · mL-1. Some QAβCD nanoparticles not only are twice as permeable as dextran (Mw ) 4000 g · mol-1) control, but also enhance DOX permeability across BBMVEC monolayers by 2.2 times. Confocal microscopy and flow cytometry measurements imply that the permeability of QAβCD nanoparticles across the in vitro BBB is probably due to endocytosis. DOX/QAβCD complexes kill U87 cells as effectively as DOX alone. However, QAβCD nanoparticles completely protect BBMVECs from cytotoxicity of DOX at 5 and 10 µM after 4 h incubation. The developed QAβCD nanoparticles have great potential in safely and effectively delivering DOX and other therapeutic agents across the BBB.

1. Introduction The blood-brain barrier (BBB) is a dynamic and complex structure, composed principally of specialized capillary endothelial cells jointed by highly restrictive tight junctions with high transendothelial electrical resistance (TER, 1500-2000 Ω · cm2)1 and densely concentrated transferrin receptors.2 It is a physical and metabolic barrier that prevents the passage of therapeutic agents from the bloodstream to the central nervous system.3 To help therapeutic agents penetrate through the BBB, many attempts have been made using a variety of approaches including saturable transport systems,4,5 disruption of the BBB,6-8 bypassing the BBB,9-12 chemical and biochemical modification of therapeutic agents such as conjugation of transport vectors such as receptor-specific transferrin and monoclonal antibody,1,13-15 and drug carriers such as liposomes16-18 and micelles.19 A potential drawback to all these approaches with exception of the receptor-mediated approach is that they involve poorly controlled increase in the BBB permeability, and cause therapeutic agents in the circulating blood to gain access to the brain indiscriminately and even can cause high clinical incidence of hemorrhage, cerebrospinal fluid leak, neurotoxicity, and central nervous system infection. As to the receptor-mediated * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: Department of Biomedical Engineering, Sci. & Tech. Ctr., 4 Colby St., Medford, MA 02155. Phone: (617) 627-0900. E-mail: [email protected]. ‡ Pennsylvania State University. § University of New Brunswick. | Thomas Jefferson University.

method, despite its specificity and affinity, a major problem is its failure to reach the target cells in adequate quantities. In the past decade, polymeric nanoparticles have attracted increasing interest as carriers for transporting therapeutic agents across the BBB.20-26 The reason is that they are superior to liposomes and micelles in terms of prolonged bioavailability, high loading efficiency, low burst effect, and tunable surface chemistry.27 Introduction of cationic property in nanoparticles has been considered as an effective approach to increase the permeability of the nanoparticles across the BBB.19,23,25,26 However, cationic charge with primary amine usually causes high toxicity, and quaternization of primary amine with quaternary ammonium can significantly decrease the cytotoxicity of polymers containing primary amine groups.28 β-Cyclodextrin (βCD)-based polymers have been widely used for drug delivery. The reason is that they contain seven glucose unites that form a hydrophobic central cavity and a hydrophilic outer surface29 so that they can act as host molecules to form inclusion complexes with both hydrophobic and hydrophilic guest molecules.30 Nature (parent) βCD itself usually has limited pharmaceutical applications due to its low water solubility, safety issues in systemic circulation, and nephrotoxicity.31 It has been reported that βCD can extract cholesterol, glycerophospholipids, and proteins from cell membranes and, consequently, can cause hemolytic and morphological changes of red blood cells.32 βCD-cholesterol complexes can accumulate in the kidneys causing renal necrosis.33,34 Therefore, even though parent βCD can be used for drug delivery through oral, topical, buccal, and rectal routes, it is not suitable in medications for

10.1021/bm801026k CCC: $40.75  2009 American Chemical Society Published on Web 02/13/2009

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parenteral administrations including subcutaneous, intraperitoneal, intravenous, and intramuscular administrations.31,35 Methylation of parent βCD can increase the water solubility of parent βCD by breaking its intramolecular hydrogen bonding,36 but this method has no significant effects on the systemic toxicity of βCD.31,37 In comparison, hydroxyalkylation, anionization, branching, and quaternization of βCD can not only increase the water solubility of parent βCD by converting it into amorphous and noncrystallizable derivatives, but also improve hemolytic and renal toxicities of parent and methylated βCDs. As a matter of fact, two of the βCD derivatives 2-hydroxypropyl-βCD and sulfobutylether βCD are currently commercially available for intravenous administration.31,34-40 In the application of drug delivery across the BBB, Tilloy et al. reported that βCD and methylated βCD increased the permeability of anticancer drug doxorubicin across the BBB.41 The authors also explained that the enhanced permeability was due to the extraction of cholesterol from brain capillary endothelial cells induced by βCD and methylated βCD.41 If cholesterol extraction is high, severe neurodamage may occur in the brain because excess cholesterol extraction can lead to strong modulation of P-glycoprotein activity of the BBB to allow brain uptake of many toxic P-gp substrates from blood. Quarternization of βCD with one ammonium group was reported to significantly increase the toxic concentration threshold of βCD to the in vitro BBB.42 Since quaternization of βCD also showed promise to not extract cholesterol,43 in this study we investigate the effects of a series of quaternary ammonium βCD (QAβCD) nanoparticles with different charge density28 on the integrity of bovine brain microvessel endothelial cell (BBMVEC) monolayer, an in vitro BBB model, including TER value, Lucifer yellow permeability, tight junction protein and P-glycoprotein (P-gp) expressions, tight junction morphology, cholesterol extraction, and P-gp efflux activity. We also study the BBB permeability of the QAβCD nanoparticles alone and the effects of QAβCD nanoparticles on the BBB permeability of doxorubicin.

2. Experimental Methods 2.1. Materials. The following materials were obtained from SigmaAldrich, Inc., St. Louis, MO: βCD, epichlorohydrin, choline chloride, phosphate-buffered saline (pH 7.4), Tris-buffered saline, Tween 20, 5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF), 2,5-dihydroxybenzoic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), N,N-dimethylformamide (DMF, HPLC grade), phenyl-methylsulfonyl fuoride, NaCl, Trition X100, sodium deoxycholate, ethylenediamine tetraacetic acid (EDTA), Hepes (pH 7.5), benzamidine, NaVO4, NaF, sodium pyrophosphate, trypsin, fibronectin, MCDB-131 medium, epidermal growth factor (EGF), heparin, antibiotic/antimycotic (penicillin G sodium salt), paraformaldehyde, bovine serum albumin (BSA), dextran (Mw ) 5000 g · mol-1), FITC-dextran (Mw ) 4000 g · mol-1), verapamil, Lucifer yellow CH dilithium salt, rhodamine 123 (R123), and doxorubicin (DOX). Eagle’s Minimal Essential medium with Earle’s BSS and 2 mM L-glutamine (EMEM) was purchased from American type Culture Collection (ATCC), Manassas, VA. Fetal bovine serum (FBS) was purchased from Hyclone, Logan, UT. ENDO GRO was purchased from VEC Technologies, Rensselaer, NY. GIBCO, NuPAGE 4-10% BisTris gels, NuPAGE LDS sample buffer, NuPAGE sample reducing agent, NuPAGE MOPS SDS running buffer, NuPAGE transfer buffer, nitrocellulose membrane, and rabbit antioccludin polyclonal antibody ZYMED were purchased from Invitrogen, Carlsbad, CA. All the chemicals were used as received without further purification. Deionized distilled water was used in all the experiments. The monoclonal rat zonula occludens-1 (ZO-1) antibody was kindly provided by Dr. David A. Antonetti (Departments of Cellular and Molecular Physiology, Penn

Gil et al. Table 1. Hydrodynamic Radii and Zeta Potentials of QAβCD Nanoparticles samplea

hydrodynamic radiusb (Rh)/nm

zeta potential (ζ)c/mV

1-15-0.5 1-15-2 1-15-4 1-15-6

77.3 ( 4.1 81.0 ( 2.5 65.3 ( 4.3 88.0 ( 3.8

-11.8 ( 1.0 1.8 ( 2.3 6.0 ( 2.9 14.0 ( 3.8

a Each QAβCD is denoted as 1-W-N, where W and N denote the feeding molar ratios of choline chloride to β-CD and epichlorohydrin to β-CD, respectively. b Measured by DLS, N ) 5, (SD. c N ) 4, (SD.

State College of Medicine, Hershey, PA). Polyclonal rabbit antioccludin, goat antirabbit immunoglobulin G (IgG)-Cy3, goat-antirat IgG-FITC, and goat-antirat IgG-alkaline phosphatase (AP) were obtained from Zymed Laboratories, South San Francisco, CA. Mouse anti-P-gp antibody C219 and goat antimouse antibody IgG-alkaline phosphatase were obtained from EMD bioscience, Gibbstown, NJ. Goat anti-rabbit IgG-alkaline phosphatase and enhanced chemifluorescence were obtained from Amersham Pharmacia Biotech Inc., Piscataway, NJ. Dialysis membrane (MWCO 1000 Dalton) was purchased from Spectrum Laboratories, Rancho Dominguez, CA. Laboratory-Tek 8 well Permanox chamber slides were purchased from Nalge Nunc International, Rochester, NY. Aqua Poly/Mount was purchased from Polysciences, Inc., Warrington, PA. Polyester Transwells filters (12 mm diameter, 0.4 µm pore size) were purchased from Costar, Cambridge, MA. 2.2. Synthesis of QAβCD Nanoparticles. QAβCD nanoparticles were synthesized by a one-step condensation polymerization according to our previous report.28 Briefly, β-CD (5 mM, 5.675 g) was dissolved in NaOH aqueous solution (20 mL, 0.9 N) with stirring at 25 °C for 24 h. Choline chloride [25 mM (2.313 g), 50 mM (4.626 g), or 75 mM (6.939 g)] was subsequently fed into the solution and epichlorohydrin [2.5 mM (0.349 g), 10 mM (1.396 g), 20 mM (2.792 g), or 30 mM (4.189 g)] was added at a flow rate of about 0.1 mL · min-1. After completion of epichlorohydrin feeding, the reaction was done at 60 °C for 2 h, and stopped by neutralization with an aqueous hydrochloride acid solution (3 N). The final solution was dialyzed against distilled water using a dialysis membrane with MWCO 1000 Dalton for 24 h, and dried at room temperature. The synthesized QAβCD nanoparticles are listed in Table 1. Each QAβCD is denoted as 1-W-N, where W and N denote the feeding molar ratios of choline chloride to β-CD and epichlorohydrin to β-CD, respectively. DTAF-labeled nanoparticles were prepared as follows. DTAF (8.3 mg) and the QAβCD nanoparticles (250 mg) were dissolved in DMSO (0.3 mL) and sodium carbonate buffer (0.1 mol · L-1, 5 mL, pH 9), respectively. The DTAF solution was added dropwise into the nanoparticle solutions with stirring. The reaction was carried out overnight at 4 °C. The final solution was dialyzed with MWCO 1000 Dalton membrane against deionized water while changing outer water every 8 h for 24 h and lyophilized. 2.3. Characterization of QAβCD Nanoparticles. 2.3.1. Chemical Structure. 1H NMR (Bruker Avance 500 MHz NMR spectrometer, Newark, DE) and attenuate total reflection (Pike Technologies, Madison, WI) Fourier transform infrared spectroscope (Thermo Nicolet Avetar 370, Madison, WI; ATR-FTIR) were used to study the chemical structures of the synthesized QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 and the QAβCD nanoparticle 1-15-2/DOX complexes. The QAβCD nanoparticle 1-15-2/DOX complexes were prepared by mixing QAβCD 1-15-2 (5 mg · mL-1, 39 mM of βCD unit) and DOX (2.1 mg · mL-1, 39 mM) in D2O at 1:1 molar ratio of DOX to βCD unit, and storing the mixture at 4 °C for overnight. The QAβCD nanoparticle 1-15-2/DOX complex solution and QAβCD nanoparticles in D2O at approximately 2 mg · mL-1 were used for the 1H NMR and the ATR-FTIR measurements. Tetramethylsilane was used as a reference for the 1H NMR measurements. ATR-FTIR spectra were recorded in the range of 4000-650 cm-1 wavenumber. 2.3.2. Particle Size. The apparent average hydrodynamic radii (Rh) of the synthesized QAβCD nanoparticles in PBS (pH ) 7.4) at 50

Quaternary Ammonium β-Cyclodextrin Nanoparticles mg · mL-1 at room temperature were measured by a dynamic light scattering instrument equipped with an ALV-CGS-8F compact goniometer system, an ALV-5000/EPP multiple tau real time correlator, and an ALV-5000/E/WIN software (ALV, Germany). The light source was JDS Uniphase helium/neon laser (633 nm, 35 mW, Manteca, CA). Autocorrelation functions of the QAβCD nanoparticle solutions at 90° scattering angle were collected. The data were fitted using a Cumulants method to derive apparent hydrodynamic radii of QAβCD nanoparticles. The particle sizes of the QAβCD nanoparticles in PBS (pH 7.4) were also measured by an atomic force microscopy (AFM) equipped with a Molecular Imaging Pico-SPM system and a SPM 100 controller (RHK technology, MI) in tapping mode. The substrates for immobilizing the nanoparticles were silicon wafers pretreated with atmosphere plasma (to introduce an anionic surface). 2.3.3. Zeta Potential. The zeta potential of the QAβCD nanoparticles was measured by a Zeta potentiometer (Coulter Delsa 440 SX, Hialeah, FL). The QAβCD nanoparticles were dissolved in deionized water before measurement. 2.4. Cells and Media. Bovine brain microvessel endothelial cells (BBMVECs, Cell Applications Inc., San Diego, CA) were seeded in bovine fibronectin-coated T25 or T75-flasks at a density of 5000 cells · cm-2, and cultured in MCDB-131 medium containing 10% FBS, 10 ng · mL-1 EGF, 0.2 mg · mL-1 ENDO GRO, 0.9 mg · mL-1 heparin, and antibiotic/antimycotic (penicillin G sodium salt 10 µg · mL-1) at 37 °C with 95% humidity and 5% CO2. The medium was changed every two day. The cells were harvested with trypsin (0.05% trypsin with 0.4 mM EDTA) when they were 80% confluent on the fourth day. U87 human glioblastoma cells (ATCC, Manassas, VA) were seeded in T25 flasks at a density of 8000 cells · cm-2 and cultured in EMEM containing 10% FBS and antibiotic/antimycotic (penicillin G sodium salt 10 µg · mL-1) at 37 °C with 95% humidity and 5% CO2. The media was changed every two day, and the cells were harvested with trypsin (0.05% trypsin with 0.4 mM EDTA) on the seventh day. 2.5. Effects of QAβCD Nanoparticles on BBB Integrity In Vitro. 2.5.1. Cytotoxicity Study of QAβCD Nanoparticles. The cytotoxicity of the QAβCD nanoparticles to BBMVECs was evaluated by MTT assay. BBMVECs were seeded at 50000 cells · cm-2 in bovine fibronectin-coated 96-well plates, and grown in 100 µL culture media for 2 days. The cells were then treated with/without the QAβCD nanoparticles or dextran (Mw ) 4000 g · mol-1) at concentrations of 100, 300, and 500 µg · mL-1 in culture media, and then incubated at 37 °C for 24 h. Afterward, 10 µL MTT/media solution (5 mg · mL-1) was added to each well for 4 h, followed by removal of media from each well, and addition of 100 µL 50% DMF/20% SDS (pH 4.7). After overnight incubation, the absorbance at 570 nm was measured using µQuant microplate reader (Biotek Instruments, Winooski, VT) with background subtraction. Cell viability was calculated by dividing the absorbance of wells containing the QAβCD nanoparticles by the absorbance of wells containing the medium alone (corrected for background). Four replicate wells were used for each sample and control. 2.5.2. Transendothelial Electrical Resistance Test. The effects of the QAβCD nanoparticles on the TER values of BBMVEC monolayers were measured by an Endohm tissue resistance measurement chamber (World Precision Instruments, Sarasota, FL). BBMVECs were seeded at 50000 cells · cm-2 on bovine fibronectin-coated polyester transwells filters (12 mm diameter, 0.4 µm pore size), and then grown to confluence. After 4 h permeability test of QAβCD nanoparticles across the BBB as described in Section 2.6.1, the TER values of the cell monolayers with/without the presence of QAβCD nanoparticles were recorded and expressed in Ω · cm2. 2.5.3. Lucifer Yellow Permeability. The effects of the QAβCD nanoparticles on the BBB tight junction integrity were first studies by measuring the BBB permeability of Lucifer yellow CH dilithium salt (LY), a paracellular permeable marker, in the presence of the QAβCD nanoparticles. BBMVECs harvested from the T-flasks were grown on


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Figure 1. In vitro model for BBB permeability studies.

polyester Transwell filters (6 mm diameter, 0.4 µm pore size) coated with bovine fibronectin at a seeding density of 50000 cells · cm-2. The resulting confluent BBMVEC monolayers were used for permeability studies as an open two-compartment vertical side-by-side dynamic BBB model. Lucifer yellow (100 µM) and QAβCD nanoparticles (100 µg · mL-1) were dissolved in medium and added to the apical chamber of each well (Figure 1). Transport experiments were conducted in the apical to basal direction at 37 °C for 3 h. Thirty µL medium was taken from the basolateral chamber and replaced with 30 µL of fresh medium every 30 min. At 3 h, 30 µL medium was taken from both the apical and basolateral chambers. Aliquots were quantified on a Spectramax EM microplate spectrofluorometer (Molecular Devices Co., Sunnyvale, CA) at 430 nm of excitation and 530 nm of emission. The permeability (P0) of the molecules across the BBMVEC monolayer was calculated by the following formula:44

P0 ) [(FA ⁄ ∆t)VA] ⁄ (FLA)

(1)

where Po, FA, VA, FL and A are the permeability coefficient, the basolateral fluorescence of the solute over ∆t time, the fluid volume of the basolateral chamber, the apical fluorescence of the solute, and the surface area of the filter, respectively. 2.5.4. Western Immunoblotting of Tight Junction Proteins ZO-1, Occludin, and P-gp. The blood-brain barrier regulates the transport of circulating molecules into the brain by tight junctions between brain capillary endothelial cells and P-gp efflux system on the cell membrane. The expression amounts of the tight junction proteins and P-gp are directly related to the integrity of BBB. Therefore, we used western immunoblotting to study the effects of the QAβCD nanoparticles on the expression of two representative tight junction proteins occludin and ZO-1, and P-gp. BBMVECs were seeded at 50000 cells · cm-2 on bovine fibronectin-coated 60 mm polystyrene dishes and then grown to confluence. The plates were treated with/without QAβCD nanoparticles at 100 µg · mL-1 in culture medium (2 mL) for 4 h. After washed twice by ice-cold PBS (pH 7.4) containing phenyl-methylsulfonyl fuoride (200 µM), the BBMEVCs were harvested in 150 µL lysis buffer using a cell lifter. The lysis buffer was formulated with 100 mM NaCl, 1% Trition X100, 0.5% sodium deoxycholate, 0.2% SDS, 2 mM EDTA, 10 mM Hepes (pH 7.5), 1 mM benzamidine, 1 mM NaVO4, 10 mM NaF and 10 mM sodium pyrophosphate with CompleteTM (Roche Diagnostics, Indianapolis, IN), a protease inhibitor cocktail tablet. The lysis was conducted at 4 °C for 15 min with rocking, and cells were pelleted by microcentrifugation at 14000 g for 10 min. Concentration of supernatant was determined using BCA Protein Assay kit and an albumin standard curve. Equal amount of protein (20 µg) was loaded onto NuPAGE 4-10% Bis-Tris gels in NuPAGE LDS sample buffer along with NuPAGE sample reducing agent after heating at 70 °C for 10 min. Gel running was conducted in NuPAGE MOPS SDS running buffer. 120 mA current and 200 V voltage were applied for approximately 55 min. The obtained gels were transferred to nitrocellulose in NuPAGE transfer buffer at 20 V at 4 °C for overnight and then at 30 V at 4 °C for 1 h. The transferred nitrocellulose was blocked with 5% milk in Tris-buffered saline with Tween 20. Rat anti-ZO-1 antibody

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at 1:4 dilution, rabbit antioccludin polyclonal antibody ZYMED at 1:1000 dilution, and the mouse anti-P-gp antibody C219 at 1:100 dilution in the blocking solution were used for primary antibody blotting. The nitrocellulose was blotted with these primary antibodies for 3 h at room temperature and then washed. Afterward, it was blotted with alkaline phosphatase conjugated secondary antibodies (antirat antibody IgG-AP at 1:2000 dilution for ZO-1, antirabbit antibody IgGAP at 1:2000 dilution for occluding, and goat antimouse antibody IgGAP at 1:2500) for 1 h at room temperature, and washed. The blotted antibodies were enhanced with enhanced chemifluorescence and the bands were detected and quantified by Fluorimager 595 with ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA). 2.5.5. Immunofluorescent Staining of Tight Junction Proteins ZO-1 and Occludin. The effects of the QAβCD nanoparticles on the expression of two BBB tight junction proteins, ZO-1 and occludin, were further examined by immunofluorescent staining using confocal microscopy. BBMVECs were seeded at 50000 cells · cm-2 on bovine fibronectin-coated Laboratory-Tek 8 well Permanox chamber slides, grown to confluence, and then treated with/without QAβCD nanoparticles (100 µg · mL-1) with DOX (1 µM) in culture medium for 4 h. After twice washes with PBS (pH 7.4), the cells were fixed in 1% paraformaldehyde for 10 min. Then the cells were permeabilized with PBS (pH 7.4) containing 0.2% Triton X-100 for 10 min, and blocked with PBS (pH 7.4) containing 0.1% Triton X-100 and 10% BSA for 1 h. After incubated with rat anti-ZO-1 antibody at 1:4 dilution or rabbit antioccludin antibody at 1:250 dilution in the blocking solution for 1 h, cells were washed three times with PBS (pH 7.4) containing 0.1% Triton X-100. Then, cells were incubated with second fluorescent antibodies: goat antirat IgG-FITC at 1:200 dilution or goat antirabbit IgG-Cy3 at 1:500 dilution, followed by three times washing with PBS (pH 7.4) containing 0.1% Triton X-100. After the chambers were removed from the Laboratory-Tek slides, glass coverslips were mounted onto the slides with Aqua Poly/Mount. The slides were visualized by a Leica TCS SP2 AOBS confocal microscopy (Leica, Mannheim, Germany) equipped with 488 nm argon and 543 nm He/Ne lasers. The brightness and contrast of all pictures were identically adjusted with Adobe Photoshop. 2.5.6. Cholesterol Extraction. The effects of the QAβCD nanoparticles on the extracellular property of the BBB were assessed by measuring cholesterol extraction of the BBMVEC monolayer in the presence of dextran and the QAβCD nanoparticles. BBMVECs were seeded at 50000 cells · cm-2 on bovine fibronectin-coated 24-well plates and then grown to confluence. The confluent BBMVEC monolayers were incubated in the culture media containing QAβCD nanoparticles at 100 µg · mL-1 for 24 h, or 1 mg · mL-1 for 4 h. After washed twice with PBS (pH 7.4), BBMEVCs were harvested using a cell lifter in 100 µL lysis buffer containing 100 mM NaCl, 1% Trition X100, 0.5% sodium deoxycholate, 0.2% SDS, 2 mM EDTA, and 10 mM Hepes (pH 7.4). The cells were then homogenized by using a 25 5/8-gauge needle. The cholesterol content was determined using an Amplex Red cholesterol assay kit (Molecular Probes, Eugene, OR) following the manufacturer’s instructions. The protein content in each well was determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL) using BSA as a standard. The cholesterol content was normalized by the total protein content. Dextran (Mw ) 5000 g · mol-1) was used as a control. 2.5.7. P-gp Efflux ActiVity Using Rhodamine 123 Efflux Assay. The effects of the QAβCD nanoparticles on the extracellular property of the BBB were also evaluated by measuring the cellular uptake of rhodamine 123 (R123), a fluorescent P-gp efflux activity marker, by BBMVECs. The BBMVECs were seeded onto 24-well plates at a density of 50000 cells/well and grown for 2 days. The cell monolayers were treated with QAβCD nanoparticles (100 or 500 µg/mL) or a P-gp inhibitor, verapamil (100 µM) for 2 h. The media was then removed and the cells were incubated with R123 of 10 µM in media for 2 h. After removing R123 solution, the cell monolayers were washed three times with cold PBS (pH 7.4). The BBMVECs were solubilized using 100 µL of cell culture lysis buffer described above (section 2.5.1). The

Gil et al. cellular content of R123 was determined by fluorescent measurement with R123 standard curve at 480 nm of excitation and 530 nm of emission and normalized per cellular content of protein. The protein content was measured using a Pierce (Rockford, IL) BCA protein assay. 2.6. Effects of QAβCD Nanoparticles on Permeability of Doxorubicin across In Vitro BBB. 2.6.1. Permeability of QAβCD Nanoparticles across the In Vitro BBB. The permeability of QAβCD nanoparticles were measured as described in section 2.5.3. Confluent BBMVEC monolayers on polyester Transwell filters (12 mm diameter, 0.4 µm pore size) coated with bovine fibronectin were used for the permeability test. DTAF-labeled QAβCD nanoparticles or FITC-dextran control (Mw ) 4000 g · mol-1) was dissolved in medium and added to the apical chamber of each well at 100 µg · mL-1. The aliquots from the basolateral chambers at each time interval and the apical chamber at 4 h were used for the quantification of QAβCD nanoparticle permeability. 2.6.2. Cellular Uptake of QAβCD Nanoparticles by BBMVECs. Confocal microscopy and flow cytometry were carried out to examine cellular internalization of the synthesized QAβCD nanoparticles 1-152. BBMVECs were plated at a density of 50000 cells · cm-2 onto on bovine fibronectin-coated Laboratory-Tek 8 well Permanox chamber slides for confocal microscopy experiments. Also, BBMVECs were seeded at a density of 400000 cells/well into 60 mm tissue culture dishes for flow cytometry experiments. After 1-2 days, DTAF-labeled QAβCD nanoparticles 1-15-2 at 100 or 200 µg · mL-1, and FITCDextran (Mw ) 4000 g · mol-1) at 100 µg · mL-1 were added into each well/plate and incubated for 1 h at 37 °C. Subsequently, cell culture medium was gently removed and the plates were washed twice with PBS (pH 7.4). For confocal microscopy study, the cells on the glass coverslips were fixed with 4% paraformaldehyde for 20 min. After twice washing with PBS (pH 7.4), cellular nuclei were stained with 1 mg · mL-1 DAPI for 30 min. Followed by two more washings with PBS (pH 7.4), the middle z-section images of cells were taken by a Leica TCS SP2 AOBS confocal microscopy (Leica, Mannheim, Germany) equipped with 488nm argon and 543nm He/Ne lasers. For flow cytometric study, the treated cells were detached out with trypsin, washed with culture media, centrifuged at 1000 rpm, washed once with PBS (pH 7.4), and centrifuged a second time. The cells were fixed in 4% paraformaldehyde for 20 min and washed twice with PBS (pH 7.4). Subsequently, half of each sample was treated with 0.5% trypan blue for 5 min, an extracellular fluorescencequenching dye in order to differentiate between membrane-bound and internalized QAβCD nanoparticles, followed by two washings with PBS (pH 7.4). The cell uptake of DTAF-labeled nanoparticles and FITC-dextran (Mw ) 4000 g · mol-1) was quantitated using a flow cytometric fluorescenceactivated cell sorter (FACS, Becton Dickinson, San Jose, CA) equipped with an argon-ion laser and 530 nm bandpass filters for emission measurements. Approximate 10000 events were acquired per sample, and the data was analyzed using CellQuest software (Becton Dickinson). Forward and side light scatter gates were normally set to exclude dead cells, debris, and cell aggregate. 2.6.3. In Vitro Permeability of Doxorubicin Complexed with QAβCD Nanoparticles across the BBB. DOX was complexed with the QAβCD nanoparticles by mixing DOX (200 µM) and QAβCD nanoparticles (20 mg · mL-1) in filtered deionized water at 4 °C overnight. The DOX/QAβCD nanoparticle complex stock solutions were diluted and subsequently added to the apical chamber of each Transwell in Figure 1 to obtain the final concentration of DOX/QAβCD nanoparticles at 1 µM/100 µg · mL-1. The permeability of the DOX/ QAβCD complexes across BBMVEC monolayer was measured and quantified by following the above method described in section 2.5.3. DOX (1 µM) alone was used as a control. 2.7. Effects of QAβCD Nanoparticles on Cytotoxicity of Doxorubicin. Cytotoxicity of DOX with/without QAβCD nanoparticles to BBMVECs and U87 human glioblastoma cells was evaluated by MTT assay. BBMVECs were seeded at 50000 cells · cm-2 in fibronectincoated 96-well plates in MCDB-131 medium containing 10% FBS, 10

Quaternary Ammonium β-Cyclodextrin Nanoparticles


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Figure 2. Structures and 1H NMR spectra of QAβCDs. (A) Schematic structure of QAβCDs. (B) Chemical structure of QAβCDs. (C) 1H NMR spectra of 1-15-0.5 (i), 1-15-2 (ii), 1-15-4 (iii), and 1-15-6 (iv). (D) Ratios of choline chloride to βCD unit in QAβCDs at chemical shifts 5.0 (cl) and at 3.1 ppm (a), respectively.

ng · mL-1 EGF and 0.2 mg · mL-1 ENDO GRO. Two days later the BBMVECs were treated with DOX alone and DOX/QAβCD complexes in the culture medium at 37 °C for 4 h. The studied concentrations of DOX were 1, 2, 5, and 10 µM and that of the QAβCD nanoparticles was set at 100 µg · mL-1. U87 cells were seeded at 50000 cells · cm-2 in 96-well plates in EMEM medium containing 10% FBS. Two days later the U87 cells were treated with DOX alone and DOX/QAβCD complexes in the culture medium at 37 °C for 4, 24, and 60 h. The studied concentration of DOX and QAβCD nanoparticles were 1 µM and 100 µg · mL-1, respectively. Subsequently, the cytotoxicity of DOX with/without QAβCD nanoparticles to both BBMVECs and U87 was evaluated using the MTT assay following the method described in section 2.5.1. 2.8. Statistical Methods. Differences between treatment groups were statistically analyzed using one-way analysis of variance (ANOVA). A statistically significant difference was reported if p < 0.05 or less. Data is reported at the mean ( standard deviation (SD) from at least three separate experiments.

3. Results 3.1. Physicochemical Properties of QAβCDs. In the current study, we synthesize QAβCDs with different amount of quaternary ammonium groups (Figure 2A,B). In 1H NMR spectra of QAβCDs (Figure 2C), the peak assigned to the proton of quaternary ammonium group occurs at around 3.1 ppm. Due to the structural irregularity of QAβCDs, the glucose proton peaks are split up to 5 peaks between 3.4∼5.2 ppm. The peaks assigned to the protons of methyl segments of epichlorohydrin and choline chloride are overlapped by the proton peaks in glucose units. As expected, the peak intensity assigned to choline

chloride at 3.1 ppm increases with increasing the feeding ratio of choline chloride to βCD. More precisely, the ratios of the chemical shift peak areas of choline chloride at 3.1 ppm to βCD at 5.0 (c1) ppm, increase proportionally with increasing the choline chloride/βCD feeding ratio from 0.5 to 4 and then more dramatically at choline chloride/βCD feeding ratio ) 6 (Figure 2D). ATR-FTIR and 1H NMR were used to determine the formation of DOX/QAβCD complexes. In ATR-FTIR study, QAβCD 1-15-2 shows bands at 1012 and 1059 cm-1 due to coupled C-C and C-O stretching vibrations, and band at 1130 cm-1 due to C-O-C glycosidic bridge antisymmetric stretching vibration (Figure 3A). DOX shows characteristic IR bands: stretching vibration of carbonyl group at 13-keto position at 1726 cm-1, in-plane bending of NH2 at 1593 cm-1, stretching vibration of two carbonyl groups of anthracene ring at 1577 cm-1, skeleton vibration of aromatic backbone at 1280 cm-1, and stretching vibration of C-O bonds at 1113 and 1070 cm-1 (Figure 3B). After QAβCD 1-15-2 is complexed with DOX, many intrinsic IR bands of QAβCDs 1-15-2 and DOX shift their wavenumber positions, for examples, the IR bands of QAβCD 1-15-2 at 1130 and 1012 cm-1 shift to 1149 and 1028 cm-1, and the IR bands of DOX at 1726, 1593, 1577, and 1280 cm-1 shift to 1703, 1589, 1568, and 1284 cm-1 (Figure 3C). Furthermore, the transmission intensities of the characteristic IR bands also have significant changes after the complexation, for examples, the peak at 1705 cm-1 becomes stronger while the peaks at 1589, 1568, and 1284 cm-1 become much weaker. The results strongly suggest that there exist interaction between the QAβCD 1-15-2 and doxorubicin probably due to the

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Figure 3. ATR-FTIR spectra of (A) QAβCD 1-15-2, (B) DOX, and (C) DOX/QAβCD 1-15-2 complex.

binding/association between the nitrogen of the quaternary amine of QAβCD 1-15-2 and the oxygen of the 13-keto of DOX, and maybe also the binding/association between the oxygen of the hydroxyl group of QAβCD 1-15-2 and the nitrogen of the 3′amino group of DOX.45 The 1H NMR peaks of the QAβCDs after DOX complexation are shifted to upfield, but the shifts are much minor (data not shown). Figure 4 and Table 1 exhibit the particle size and morphology of QAβCD nanoparticles. The hydrodynamic radii Rh of QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 in PBS (pH 7.4) measured by dynamic light scattering technique at 90° angle were 77.3 ( 4.1, 81.0 ( 2.5, 65.3 ( 4.3, and 88.0 ( 3.8, respectively. The hydrodynamic size distribution of QAβCD nanoparticles 1-15-2 is representatively shown in Figure 4A. AFM measurements (Figure 4B-D) further reveal that QAβCD nanoparticles 1-15-2 are very soft on anionic surface in wet state with 200∼500 nm in width and 15∼25 nm in height. By considering the volume as a half of oblate spheroid volume, the calculated volume of the spread QAβCD nanoparticles 1-15-2 in the AFM pictures is 0.3∼3.3 × 10-3 µm3, which matches well with the sphere volume 2.2 × 10-3 µm3 calculated by the Rh (81 nm). Because surface charge of nanoparticles plays an important role in nanoparticle-cell interactions, we measured the zetapotential values of the QAβCD nanoparticles. Table 1 shows that the zeta-potential values of the four nanoparticles increase with increasing the feeding ratio of [choline chloride]/[βCD]. The zeta-potential values of QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 are -11.8 ( 1.0, 1.8 ( 2.3, 6.0 ( 2.9, and 14.0 ( 3.8 mV, respectively. It is noteworthy that QAβCD nanoparticles 1-15-2, 1-15-4, and 1-15-6 have positive zeta potential values, whereas QAβCD nanoparticles 1-15-0.5 have negative zeta potential value. In general, parent βCD has intrinsic negative charge property. 3.2. Effects of QAβCD Nanoparticles on BBB Integrity In Vitro. MTT assay demonstrates that QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 are not toxic to BBMVECs at concentrations 100, 300, and 500 µg · mL-1 over a period of 24 h, similar to dextran control (Figure 5). TER test displays that the four types of QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 at 100 µg · mL-1 do not change the TER value of BBMVEC monolayers after 4 h incubation (Figure 6A). To further evaluate the effects of the four QAβCD nanoparticles on the function of the BBB tight junction, studies were performed on the permeability of Lucifer yellow, a paracellular permeable marker,46 across a confluent BBMVEC monolayer, an in vitro BBB model (Figure 6B), in

Figure 4. (A) Hydrodynamic size distribution of QAβCD nanoparticle 1-15-2 measured by dynamic light scattering. The cumulated average hydrodynamic radius (Rh) of QAβCD nanoparticles 1-15-2 in PBS (pH 7.4) is 81.0 ( 2.5 nm (N ) 5, mean ( S.D.). (B) Topographic and (C) phase (in tapping) atomic force microscopy (AFM) images of QAβCD nanoparticles 1-15-2. (D) AFM dimension of QAβCD nanoparticle 1-15-2 is 200∼500 nm in width and 15∼25 nm in height.

Figure 5. Effects of QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 on the viability of BBMVECs at 100 (blank bars), 300 (striped bars), and 500 (solid bars) µg · mL-1 after a 1 day incubation, evaluated by MTT assay. Dextran was used as a control. Results represent the mean ( SD of four measurements.

the presence of the QAβCD nanoparticles. The permeability coefficients of Lucifer yellow in the presence of the four QAβCD nanoparticles at 100 µg · mL-1 (even 500 µg · mL-1 for QAβCD nanoparticles 1-15-2) are either similar or lower than that of Lucifer yellow alone, indicating that the tightness of the BBB junctions between cells is not destroyed by QAβCD nanoparticles. Western immunoblotting analysis illustrates that QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg · mL-1 do



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Figure 6. Effects of QAβCD nanoparticles 1-15-0.5, 1-15--2, 1-15-4, and 1-15-6 at 100 µg · mL-1 on TER values of BBMVEC monolayers (A) and Lucifer yellow (100 µM) permeability across BBMVEC monolayers (B). TER values are quantified by Endohm tissue resistance measurements after 4 h incubation. The TER value of an inherent BBMVEC monolayer was 156.4 ( 0.65 Ω · cm2. Y-axis values are the TER values normalized to that of the cell monolayers without QAβCD nanoparticles. Results represent the mean ( SD of three measurements.

not change the contents of two tight junction proteins ZO-1 and occludin of BBMVEC monolayers after 4 h incubation (Figure 7A-C). Immunofluorescent staining analysis using conforcal microscopy further confirms that QAβCD nanoparticles 1-15-2 do not disrupt the expression patterns of ZO-1 and occludin after 4 h incubation, even in the presence of 1 µM DOX (Figure 7D-G). Cholesterol extraction has been considered as the initial step of βCD to damage cell membranes.31 Cholesterol content quantification reveals that QAβCD nanoparticles 1-15-0.5, 1-152, and 1-15-6 at 100 µg · mL-1 do not cause any cholesterol extraction from the BBMVECs even after 24 h incubation (Figure 8). Western blotting results show that the three QAβCD nanoparticles at 100 µg · mL-1 have no effects on the P-gp expression of BBMVEC monolayers after 2 h incubation (Figure 9A,B). The further evaluation of the cellular uptake of R123, a P-gp efflux activity marker (Figure 9C), by BBMVECs demonstrate that QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg · mL-1 and even 500 µg · mL-1 do not affect the cellular uptake value of R123; however, verapamil, a well-known P-gp inhibitor, increases the cellular uptake value of R123 by a factor of 2. 3.3. Effects of QAβCD Nanoparticles on Permeability of Doxorubicin across In Vitro BBB. Before investigating on the effects of QAβCD nanoparticles on the permeability of doxorubicin across the BBB, we first tested the permeability of QAβCD nanoparticles alone across an in vitro BBB model,

Figure 7. Effects of QAβCD nanoparticle 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg · mL-1 on the expression of tight junction proteins ZO-1 and occludin of BBMVEC monolayers after 4 h incubation, quantified by western immunoblotting analysis (A∼C) and visualized by confocal microscopy after immunofluorescent staining (D∼G). Y-axis values in (B) and (C) are the occludin or ZO-1 content normalized to that of the cells without QAβCD nanoparticles. Results represent the mean ( SD of three measurements. (D) and (F) are ZO-1 staining (green), and (E) and (G) are occludin staining (red) of BBMVEC monolayers treated without and with 100 µg · mL-1 QAβCD nanoparticles 1-15-2 complexed with 1 µM doxorubicin, respectively.

confluent BBMVEC monolayers. The permeability study was carried out by examining the transport of DTAF-labeled QAβCD nanoparticles at 100 µg · mL-1 from apical to basolateral side of the BBMVEC monolayer for 4 h (Figure 1). The permeability coefficient of QAβCD nanoparticles 1-15-0.5 is 8.1 ( 0.9 × 10-6 cm · s-1, which is similar to that of very permeable 4000 Da FITC-dextran control (8.2 ( 0.7 × 10-6 cm · s-1) (Figure 10). With increasing the [choline chloride]/[βCD] feeding molar ratio in the QAβCD nanoparticles to two or higher, the permeability coefficient of the nanoparticles can be even higher than that of FITC-dextran control. For example, the permeability coefficients of QAβCD nanoparticles 1-15-2, 1-15-4, and 1-15-6 are 17.1 ( 0.7 × 10-6, 16.8 ( 1.7 × 10-6, and 16.5 ( 1.4 × 10-6 cm · s-1, respectively, which are about twice those of QAβCD nanoparticle 1-15-0.5 and FITC-dextran control.

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Figure 8. Effects of QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 on the cholesterol content of BBMVEC monolayers at 100 µg · mL-1 after 24 h incubation, quantified by Amplex Red cholesterol assay. Y-axis values are the cholesterol content normalized to that of the cell monolayers without QAβCD nanoparticles, which was 21.83 ( 1.43 µg · mL-1 total protein (Control, N ) 12). Dextran was used as a control. Results represent the mean ( SD of four measurements.

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Figure 10. Permeability of DTAF-labeled QAβCD nanoparticles 1-150.5, 1-15-2, 1-15-4, and 1-15-6 at 100 µg · mL-1 across BBMVEC monolayers at 37 °C for 4 h. Results represent the mean ( SD of three measurements. **P < 0.001.

Figure 11. Accumulation of FITC-labeled dextran (Mw ) 4000 g · mol-1) at 100 µg · mL-1 (A) and DTAF-labeled QAβCD nanoparticles 1-15-2 at 100 (B) and 200 (C) µg · mL-1 in BBMVECs cells observed by confocol microscopy. Z-section images are taken from middle part of cells to visualize the internal section of the cells. Contrast and brightness of all images are equally adjusted to visualize weakly DAPIbounded cell shape as well as DAPI-stained nuclei (Blue). FITClabeled dextran and DTAF-labeled are visualized as green color. All images are taken using 60× optical magnification lens. Scale bars represent 10 µm.

Figure 9. Effects of QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 on the expression and efflux activity of P-gp of BBMVEC monolayers. (A) Western immunoblotting analysis after 4 h incubation at 100 µg · mL-1. (B) P-gp content is normalized to that of the cells without QAβCD nanoparticles. Results represent the mean ( SD of three measurements. (C) P-gp efflux activity with/without QAβCD nanoparticles at 100 (solid) and 500 (blank) µg · mL-1 using R123 assay (10 µM). Verapmil (100 µM) was used as a P-gp inhibitor for a negative control. Results represent the mean ( SD of four measurements. **p < 0.001.

To understand the permeation of QAβCD nanoparticles across the BBMVEC monolayers, we studied the cellular uptake of QAβCD nanoparticles 1-15-2 by BBMVECs using confocal microscopy and flow cytometry. Z-section of middle part of cells by confocal laser scanning microscopy clearly shows that QAβCD nanoparticles 1-15-2 at 100 and 200 µg · mL-1 are taken up by BBMVECs after 2 h incubation (Figure 11B,C). However, accumulation of FITC-dextan in BBMVECs was not observed (Figure 11A). The images of the internalized QAβCD nano-

particles display punctate staining that may indicate the nanoparticles accumulate within endocytotic vesicles. Further analysis by flow cytometry shows that 7, 87, and 99% of BBMVECs are associated with FITC-dextran (Mw ) 4000 g · mol-1) at 200 µg · mL-1, and DTAF-QAβCD nanoparticles 1-15-2 at 100 and 200 µg · mL-1, respectively (Figure 12A). To confirm that the DTAF-labeled QAβCD nanoparticles were internalized and not merely attached to the cell surface, we used a trypan blue quenching dye to quench any extracellular fluorescence, leaving the internal fluorescence protected.47 With trypan blue treatment, the percentages of BBMVECs associated with FITC-dextran at 200 µg · mL-1, and DTAF-QAβCD nanoparticles at 100 and 200 µg · mL-1, decrease to 2, 40, and 78%, respectively (Figure 12A). By dividing the fluorescence after trypan blue treatment by that before trypan blue treatment, we calculated that nearly 47 and 78% DTAF-QAβCD nanoparticles at 100 and 200 µg · mL-1, respectively, are internalized into BBMVECs (Figure 12B). After the above understanding of the permeability of QAβCD nanoparticles across the BBB, we evaluated the effects of QAβCD nanoparticles on the permeability of doxorubicin across the BBB. The results show that when 1 µM DOX is complexed with 100 µg · mL-1 QAβCD nanoparticles 1-15-0.5, 1-15-2, and



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Figure 12. Quantitative uptake of QAβCD nanoparticles 1-15-2 by BBMVECs analysized by flow cytometry. (A) Percentage of cells associated with the nanoparticles before (solid bar) and after (stripe bar) trypan blue dye was used to quench membrane bound nanoparticles. (B) Percentage of total cell-associated nanoparticles that were internalized in BBMVECs. The concentration of the nanoparticles for each study was 100 and 200 µg · mL-1 and the incubation time of the nanoparticles with BBMVECs was 1 h. *p < 0.01, **p < 0.001.

Figure 13. Effects of QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 on the permeability of DOX across BBMEC monolayer at 37 °C for 4 h. The concentration of the DOX/QAβCD nanoparticle complexes was 1 µM /100 µg · mL-1. Results represent the mean ( SD of three measurements. *p < 0.005, **P < 0.001.

1-15-6, its permeability across the BBMVEC monolayer is enhanced by a factor of 1.8, 2.2, and 2.2 times, respectively (Figure 13). 3.4. Effects of QAβCD Nanoparticles on Cytotoxicity of Doxorubicin. We first investigated the effect of QAβCD nanoparticles on the cytotoxicity of DOX to BBMVECs at 37 °C. DOX alone displays a significant dose-dependent cytotoxicity to BBMVECs after 4 h incubation (Figure 14A). It is not significantly toxic to BBMVECs at 1 and 2 µM, but decreases BBMVEC viability to 85 ( 5.2 and 79 ( 5.7% at 5 and 10 µM, respectively. When DOX is complexed with QAβCD nanoparticles 1-15-0.5 and 1-15-2 (100 µg · mL-1), the BBMVEC cell viability increases to 100% at 5 and 10 µM DOX. We next investigated the effect of QAβCD nanoparticles on the efficacy of DOX in killing U87 human glioblastoma cells at 37 °C. DOX (1 µM)/QAβCD nanoparticle (100 µg · mL-1) complexes display a significant time-dependent inhibition of U87 cell viability similar to the free DOX (Figure 14B). The U87 cell viability is 100% at 4 h, decreases to about 80% at 24 h and about 30% at 60 h.

4. Discussion CD and its derivatives have been widely studied as delivery systems to improve drugs ’ solubility, chemical stability, dissolution, and bioavailability across the dermal, nasal and intestinal barriers.30,31 However, they have been only recently explored for drug delivery across the BBB.42,48,49 To develop a new class of CD that will not only effectively enhance drugs’

Figure 14. Effects of QAβCD nanoparticles 1-15-0.5 and 1-15-2 on the cytotoxicity of DOX to BBMVECs and U87 human glioblastoma cells, evaluated by MTT assay. (A) BBMVECs are incubated with DOX/QAβCD nanoparticle complexes at 1 µM/100 µg · mL-1 (blank bar), 2 µM/100 µg · mL-1 (striped bar), 5 µM/100 µg · mL-1 (checked bar), and 10 µM/100 µg · mL-1 (solid bar) for 4 h. (B), U87 human glioblastoma cells are incubated with DOX/QAβCD nanoparticle complexes at 1 µM/100 µg · mL-1 for 4 (blank bar), 24 (striped bar), and 60 h (solid bar). Results represent the mean ( SD of four measurements. *p < 0.05, **p < 0.01.

permeability across the BBB, but also will not change the integrity of the BBB, in this study we incorporate quaternary ammonium groups into βCD using choline chloride and epichlorohydrin through one-step condensation polymerization. The reason for the introduction of quaternary ammonium groups is that quaternary ammonium is cationic and much less toxic than primary amine28 and monoquaternized βCDs are less toxic than parent βCD to the brain capillary endothelial cells42 and do not extract cholesterol.43 In this study, we first characterized the physicochemical and drug complexation properties of the newly developed quaternary ammonium βCD nanoparticles. In the design of QAβCD nanoparticles, the feeding ratio of epichlorohydrin to βCD was

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kept constant at 15 and the feeding ratios of choline chloride to βCD were varied from 0.5 to 6. The increase of the ratios of the 1H NMR chemical shift peak areas of choline chloride at 3.1 ppm to βCD at 5.0 (c1) ppm with increasing the choline chloride/βCD feeding ratio from 0.5 to 6 (Figure 2) clearly demonstrates that quaternary ammonium group can be successfully chemically incorporated into βCD with tunable amount. The increase of zeta-potential values of the QAβCD nanoparticles with increasing the feeding ratio of [choline chloride]/ [βCD] (Table 1) further confirms the success of the synthesis. The QAβCDs form nanosized particles of 65∼88 nm hydrodynamic radii with soft morphology on anionic surface in PBS (pH 7.4) at 37 °C (Table 1 and Figure 4), implying that many water molecules are bounded to the nanoparticles. ATR-FTIR analysis reveals that QAβCD and DOX form complexation by decreasing the relative intensities or shifting the positions of their respective characteristic bands (Figure 3). With the above understandings of the physicochemical and DOX complexation properties of QAβCD nanoparticles, we next investigated the effects of the physicochemical properties of the quaternized βCD nanoparticles on the in vitro BBB cytotoxicity and integrity. It is well-known that materials can be toxic to cells by inhibiting the capability of cells to reduce MTT to formazon in mitochondria/endosomes. Thus, we used MTT assay to study the cytotoxicity of four designed QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 to BBMVECs, and find that all of them are not toxic to BBMVECs at concentration up to 500 µg · mL-1 after 24 h incubation (Figure 5). Of course, materials may not cause mitochondrial/endosomal dysfunction, but can disrupt the BBB integrity by (1) regulating tight junction function or protein expression to increase paracelluar permeability, and (2) releasing biological membrane component cholesterol and thus reducing P-gp (a membrane bound efflux transporter) expression or its activity.41,50,51 Therefore, we employed EndohmTM tissue resistance measurement, permeability study of a paracellular permeable marker using Lucifer yellow, western immunoblotting, immunofluorescent staining, Amplex Red cholesterol assay, and uptake of R123 assay to investigate the effects of the four designed QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 on the integrity of an in vitro BBB model, BBMVEC monolayer, at 100 µg · mL-1 after 4 h incubation (24 h incubation for the cholesterol extraction studies). The results reveal that all the four QAβCD nanoparticles do not affect TER value and Lucifer yellow permeability and thus BBMVEC monolayer tight junction function (Figure 6), and tight junction proteins occludin and ZO-1 expression quantified by Western immunoblotting and visualized by confocal microscopy after immunofluorescent staining (Figure 7). Cholesterol and P-gp are observed in the caveolae of BBMVECs.52 The unchanged cholesterol and P-gp contents as well as uptake of R123 (Figures 8 and 9) suggest that the four nanoparticles do not affect the cholesterol extraction and P-gp expression and efflux activity and thus the stability of BBMVEC membranes. Taken together, all the above results demonstrate that the four QAβCD nanoparticles 1-15-0.5, 1-152, 1-15-4, and 1-15-6 do not affect the integrity of the in vitro BBB model, despite their differences in physicochemical properties (Table 1 and Figures 2-34). In the next step, we studiedthe permeability of QAβCD nanoparticles alone across BBMVEC monolayers, an in vitro BBB model, at 100 µg · mL-1 (the concentration at which all the four QAβCD nanoparticles do not affect the BBB integrity parameters). Figure 10 demonstrates that the permeability coefficients of QAβCD nanoparticles 1-15-2, 1-15-4, and 1-15-6

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are about twice those of QAβCD nanoparticle 1-15-0.5 and FITC-dextran (Mw ) 4000 g · mol-1) control. It is well-known that introduction of cationic charges to nanoparticles can increase the BBB permeability of nanoparticles in vitro and in vivo due to adsorptive-mediated endocytosis.19,53,54 Thus, our data further suggest that when cationic quaternary ammonium is introduced to βCD to make the resulting nanoparticles to have non-negative zeta potential values (Table 1), significantly high permeability of the resulting nanoparticles across the BBMVEC monolayer can be achieved. The maximum permeability coefficient is obtained when the feeding molar ratio of [choline chloride]/ [βCD] is 2 (Figure 10). This may indicate that there is limitation of the number of quaternary ammonium groups of QAβCD nanoparticles that can maximally interact with BBMVEC membranes. Because the QAβCD nanoparticles have hydrodynamic radii of 65∼88 nm in PBS (pH 7.4) at 37 °C (Table 1), which is much bigger than the pore size of the BBB junction (less than 3 nm),55,56 and the permeability of the nanoparticles is independent of their particle sizes (Table 1), it is more than unlikely that the nanoparticles cross the BBMVEC monolayers by paracellular pathway. To investigate the mechanisms for the QAβCD nanoparticles to cross the BBMVEC monolayers, we used confocal microscopy and flow cytometry to study the interactions between the QAβCD nanoparticles and BBMVECs. The results show that more than 85% of BBMVECs are associated with representative QAβCD nanoparticles 1-15-2 and 47% or higher percentage of the nanoparticles are internalized into the endocytotic vesicles of BBMVECs when the concentration of the nanoparticles is 100 µg · mL-1 or higher; while less than 10% of BBMVECs are associated with dextran control and less than 20% of dextran is internalized into the BBMVECs at 100 µg · mL-1 (Figures 11 and 12). These results suggest that the QAβCD nanoparticles can cross the BBB probably by endocytosis, due to their cationic property generated by the quaternary ammonium groups of the nanoparticles.47,57 More thorough studies on the endocytosis mechanism are under investigation. After understanding that the designed QAβCD nanoparticles are easy to cross the BBMVEC monolayer without disrupting the monolayer integrity at 100 µg · mL-1, we used DOX as a model drug and investigated the effect of QAβCD nanoparticles on the permeability of DOX across the BBMVEC monolayer in vitro. DOX is a very potent anticancer drug to treat brain tumor. However, DOX has been known to show a restricted transport to brain due to the P-gp efflux activity as well as tight junction of brain capillary endotherial cells.58 βCDs and βCD based drug carriers have been known to make 1:1 complexation with many drugs including DOX,28,41,45 and our ATR-FTIR measurements also confirm the complexation between the QAβCD nanoparticles and DOX (Figure 3). Our further studies demonstrate that QAβCD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg · mL-1 enhance DOX (1 µM)’s permeability by a factor of 1.8, 2.2, and 2.2, respectively (Figure 13). In the literature, it was reported that methylated-βCD (MeβCD) at 1 mM also enhanced DOX (1 µM) permeability across the in vitro BBB by a factor of 2.41 However, the enhanced DOX permeability was due to the decreased P-gp activity of BBMVECs through cholesterol extraction induced by MeβCD.41 In our studies, we previously demonstrated that our four QAβCD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 at 100 µg · mL-1 did not affect the integrity of the in vitro BBB model including cholesterol extraction and P-gp expression and efflux activity (Figures 8 and 9) under the same condition as we carried


out the permeability test. Therefore, the mechanisms for the enhanced DOX permeability induced by our QAβCD nanoparticles are fundamentally different from those induced by MeβCD. As the permeability of DOX in the presence of the QAβCD nanoparticles increases in the order of 1-15-0.5 < 1-15-2 = 1-15-6 (Figure 13), which matches well with the increasing order of the permeability of the corresponding three nanoparticles (Figure 10), the enhanced DOX permeability is probably controlled by the permeability of the QAβCD nanoparticles. Finally, we investigated the effects of QAβCD nanoparticles on the cytotoxicity of DOX to BBMVECs and U87 human glioblastoma cells. QAβCD nanoparticles 1-15-0.5 and 1-15-2 (100 µg · mL-1) protect BBMVECs from the toxicity of DOX at 5 and 10 µM after 4 h incubation (Figure 14A), but do not reduce the efficacy of DOX on killing U87 human glioblastoma cells (Figure 14B).

5. Conclusions A series of quaternary ammonium βCD (QAβCD) nanoparticles with different charge density were synthesized by a onestep condensation polymerization of β-CD, choline chloride, and epichlorohydrin. DLS and AFM data illustrate that the synthesized QAβCDs form nanosized soft particles with Rh around about 65∼88 nm. NMR and zeta potential measurements suggest that the charge density of the nanoparticles is tunable by changing the feeding molar ratio of [choline chloride]/[βCD]. FTIR mesurements confirm the complexation between doxorubicin and the βCD units of QAβCD nanoparticles. The studied QAβCD nanoparticles at 100 µg · mL-1 do not affect the TER value, Lucifer yellow permeability, tight junction and P-gp protein expression, tight junction morphology, cholesterol content, and P-gp efflux activity of BBMVEC monolayers, analysized by Endohm tissue resistance test, in vitro permeability assay, western immunoblotting, immunofluorescent staining, Amplex Red cholesterol assay, and R123 efflux assay, respectively. All the synthesized QAβCD nanoparticles 1-150.5, 1-15-2, 1-15-4, and 1-15-6 are not toxic to BBMVECs at concentration up to 500 µg · mL-1 tested by MTT assay. QAβCD nanoparticles are very permeable across BBMVEC monolayers at 100 µg · mL-1 with permeability coefficients equal to or twice higher than that of FITC-dextran control (Mw ) 4000 g · mol-1). The permeability of QAβCD nanoparticles across BBMVEC monolayer increases with increasing the number of quaternary ammonium groups (NMR and zeta potential value) and reaches maximum when the feeding molar ratio of quaternary ammonium groups (choline chloride)/βCD is 2 (zeta potential value becomes non-negative). The mechanism for the permeability of the QAβCD nanoparticles across the BBB is probably due to endocytosis, based on preliminary internalization results obtained from confocal microscopy and flow cytometry measurements. The QAβCD nanoparticles 1-150.5, 1-15-2, and 1-15-6 at 100 µg · mL-1 enhance the permeability of DOX (1 µM) across BBMVEC monolayers by a factor of 1.8, 2.2, and 2.2, respectively. The QAβCD nanoparticles 1-15-0.5 and 1-15-2 preserve the cell death effect of DOX on U87 human glioblastoma cells at 1 µM, while protect BBMVECs from the cytotoxicity effects of DOX by increasing BBMVEC viability from 85 ( 5.2 and 79 ( 5.7%, at 5 and 10 µM DOX concentration, respectively, to 100% after 4 h incubation. Therefore, the designed QAβCD nanoparticles are promising carriers for delivering DOX and


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other therapeutics across the BBB to treat brain disorders. Future work should include validation of the in vitro results in animal models. Acknowledgment. This work was supported by the Wallace H. Coulter Foundation and the National Institute of Health. The authors would like to thank Dr. Seong H. Kim, Department of Chemical Engineering, Pennsylvania State University, for helping with the AFM measurements, and Dr. David A. Antonetti, Department of Cellular and Molecular Physiology, Pennsylvania State University, for kindly providing ZO-1 antibody.

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