quaternary ammonium β-cyclodextrin nanoparticles for enhancing doxorubicin permeability across the...

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

1. Introduction

The blood-brain barrier (BBB) is a dynamic and complexstructure, composed principally of specialized capillary endot-helial cells jointed by highly restrictive tight junctions with hightransendothelial electrical resistance (TER, 1500-2000 Ω · cm2)1

and densely concentrated transferrin receptors.2 It is a physicaland metabolic barrier that prevents the passage of therapeuticagents from the bloodstream to the central nervous system.3

To help therapeutic agents penetrate through the BBB, manyattempts have been made using a variety of approaches includingsaturable transport systems,4,5 disruption of the BBB,6-8

bypassing the BBB,9-12 chemical and biochemical modificationof therapeutic agents such as conjugation of transport vectorssuch as receptor-specific transferrin and monoclonal anti-body,1,13-15 and drug carriers such as liposomes16-18 andmicelles.19 A potential drawback to all these approaches withexception of the receptor-mediated approach is that they involvepoorly controlled increase in the BBB permeability, and causetherapeutic agents in the circulating blood to gain access to thebrain indiscriminately and even can cause high clinical incidenceof hemorrhage, cerebrospinal fluid leak, neurotoxicity, andcentral nervous system infection. As to the receptor-mediated

method, despite its specificity and affinity, a major problem isits failure to reach the target cells in adequate quantities. In thepast decade, polymeric nanoparticles have attracted increasinginterest as carriers for transporting therapeutic agents across theBBB.20-26 The reason is that they are superior to liposomesand micelles in terms of prolonged bioavailability, high loadingefficiency, low burst effect, and tunable surface chemistry.27

Introduction of cationic property in nanoparticles has beenconsidered as an effective approach to increase the permeabilityof the nanoparticles across the BBB.19,23,25,26 However, cationiccharge with primary amine usually causes high toxicity, andquaternization of primary amine with quaternary ammoniumcan significantly decrease the cytotoxicity of polymers contain-ing primary amine groups.28

-Cyclodextrin (CD)-based polymers have been widely usedfor drug delivery. The reason is that they contain seven glucoseunites that form a hydrophobic central cavity and a hydrophilicouter surface29 so that they can act as host molecules to forminclusion complexes with both hydrophobic and hydrophilicguest molecules.30 Nature (parent) CD itself usually has limitedpharmaceutical applications due to its low water solubility,safety issues in systemic circulation, and nephrotoxicity.31 Ithas been reported that CD can extract cholesterol, glycero-phospholipids, and proteins from cell membranes and, conse-quently, can cause hemolytic and morphological changes of redblood cells.32 CD-cholesterol complexes can accumulate inthe kidneys causing renal necrosis.33,34 Therefore, even thoughparent CD can be used for drug delivery through oral, topical,buccal, and rectal routes, it is not suitable in medications for

* 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.

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10.1021/bm801026k CCC: $40.75 2009 American Chemical SocietyPublished on Web 02/13/2009

parenteral administrations including subcutaneous, intraperito-neal, intravenous, and intramuscular administrations.31,35 Me-thylation of parent CD can increase the water solubility ofparent CD by breaking its intramolecular hydrogen bonding,36

but this method has no significant effects on the systemic toxicityof CD.31,37 In comparison, hydroxyalkylation, anionization,branching, and quaternization of CD can not only increase thewater solubility of parent CD by converting it into amorphousand noncrystallizable derivatives, but also improve hemolyticand renal toxicities of parent and methylated CDs. As a matterof fact, two of the CD derivatives 2-hydroxypropyl-CD andsulfobutylether CD are currently commercially available forintravenous administration.31,34-40

In the application of drug delivery across the BBB, Tilloy etal. reported that CD and methylated CD increased thepermeability of anticancer drug doxorubicin across the BBB.41

The authors also explained that the enhanced permeability wasdue to the extraction of cholesterol from brain capillaryendothelial cells induced by CD and methylated CD.41 Ifcholesterol extraction is high, severe neurodamage may occurin the brain because excess cholesterol extraction can lead tostrong modulation of P-glycoprotein activity of the BBB to allowbrain uptake of many toxic P-gp substrates from blood.Quarternization of CD with one ammonium group was reportedto significantly increase the toxic concentration threshold of CDto the in vitro BBB.42 Since quaternization of CD also showedpromise to not extract cholesterol,43 in this study we investigatethe effects of a series of quaternary ammonium CD (QACD)nanoparticles with different charge density28 on the integrityof bovine brain microvessel endothelial cell (BBMVEC) mono-layer, an in vitro BBB model, including TER value, Luciferyellow permeability, tight junction protein and P-glycoprotein(P-gp) expressions, tight junction morphology, cholesterolextraction, and P-gp efflux activity. We also study the BBBpermeability of the QACD nanoparticles alone and the effectsof QACD nanoparticles on the BBB permeability of doxorubicin.

2. Experimental Methods

2.1. Materials. The following materials were obtained from Sigma-Aldrich, Inc., St. Louis, MO: CD, epichlorohydrin, choline chloride,phosphate-buffered saline (pH 7.4), Tris-buffered saline, Tween 20,5-(4,6-dichlorotriazinyl) aminofluorescein (5-DTAF), 2,5-dihydroxy-benzoic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT), sodium dodecyl sulfate (SDS), N,N-dimethylformamide(DMF, HPLC grade), phenyl-methylsulfonyl fuoride, NaCl, TritionX100, 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 ) 5000g ·mol-1), FITC-dextran (Mw ) 4000 g ·mol-1), verapamil, Luciferyellow CH dilithium salt, rhodamine 123 (R123), and doxorubicin(DOX). Eagle’s Minimal Essential medium with Earle’s BSS and 2mM L-glutamine (EMEM) was purchased from American type CultureCollection (ATCC), Manassas, VA. Fetal bovine serum (FBS) waspurchased from Hyclone, Logan, UT. ENDO GRO was purchased fromVEC Technologies, Rensselaer, NY. GIBCO, NuPAGE 4-10% Bis-Tris gels, NuPAGE LDS sample buffer, NuPAGE sample reducingagent, NuPAGE MOPS SDS running buffer, NuPAGE transfer buffer,nitrocellulose membrane, and rabbit antioccludin polyclonal antibodyZYMED were purchased from Invitrogen, Carlsbad, CA. All thechemicals were used as received without further purification. Deionizeddistilled water was used in all the experiments. The monoclonal ratzonula occludens-1 (ZO-1) antibody was kindly provided by Dr. DavidA. Antonetti (Departments of Cellular and Molecular Physiology, Penn

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 fromZymed Laboratories, South San Francisco, CA. Mouse anti-P-gpantibody C219 and goat antimouse antibody IgG-alkaline phosphatasewere obtained from EMD bioscience, Gibbstown, NJ. Goat anti-rabbitIgG-alkaline phosphatase and enhanced chemifluorescence were ob-tained from Amersham Pharmacia Biotech Inc., Piscataway, NJ.Dialysis membrane (MWCO 1000 Dalton) was purchased fromSpectrum Laboratories, Rancho Dominguez, CA. Laboratory-Tek 8 wellPermanox chamber slides were purchased from Nalge Nunc Interna-tional, Rochester, NY. Aqua Poly/Mount was purchased from Poly-sciences, Inc., Warrington, PA. Polyester Transwells filters (12 mmdiameter, 0.4 µm pore size) were purchased from Costar, Cambridge,MA.

2.2. Synthesis of QACD Nanoparticles. QACD nanoparticleswere synthesized by a one-step condensation polymerization accordingto our previous report.28 Briefly, -CD (5 mM, 5.675 g) was dissolvedin NaOH aqueous solution (20 mL, 0.9 N) with stirring at 25 °C for24 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. Aftercompletion of epichlorohydrin feeding, the reaction was done at 60 °Cfor 2 h, and stopped by neutralization with an aqueous hydrochlorideacid solution (3 N). The final solution was dialyzed against distilledwater using a dialysis membrane with MWCO 1000 Dalton for 24 h,and dried at room temperature. The synthesized QACD nanoparticlesare listed in Table 1. Each QACD is denoted as 1-W-N, where W andN denote the feeding molar ratios of choline chloride to -CD andepichlorohydrin to -CD, respectively. DTAF-labeled nanoparticleswere prepared as follows. DTAF (8.3 mg) and the QACD nanopar-ticles (250 mg) were dissolved in DMSO (0.3 mL) and sodiumcarbonate buffer (0.1 mol ·L-1, 5 mL, pH 9), respectively. The DTAFsolution was added dropwise into the nanoparticle solutions withstirring. The reaction was carried out overnight at 4 °C. The finalsolution was dialyzed with MWCO 1000 Dalton membrane againstdeionized water while changing outer water every 8 h for 24 h andlyophilized.

2.3. Characterization of QACD Nanoparticles. 2.3.1. Che-mical Structure. 1H NMR (Bruker Avance 500 MHz NMR spectrom-eter, Newark, DE) and attenuate total reflection (Pike Technologies,Madison, WI) Fourier transform infrared spectroscope (Thermo NicoletAvetar 370, Madison, WI; ATR-FTIR) were used to study the chemicalstructures of the synthesized QACD nanoparticles 1-15-0.5, 1-15-2,1-15-4, and 1-15-6 and the QACD nanoparticle 1-15-2/DOX com-plexes. The QACD nanoparticle 1-15-2/DOX complexes were pre-pared by mixing QACD 1-15-2 (5 mg ·mL-1, 39 mM of CD unit)and DOX (2.1 mg ·mL-1, 39 mM) in D2O at 1:1 molar ratio of DOXto CD unit, and storing the mixture at 4 °C for overnight. The QACDnanoparticle 1-15-2/DOX complex solution and QACD nanoparticlesin D2O at approximately 2 mg ·mL-1 were used for the 1H NMR andthe ATR-FTIR measurements. Tetramethylsilane was used as a refer-ence for the 1H NMR measurements. ATR-FTIR spectra were recordedin the range of 4000-650 cm-1 wavenumber.

2.3.2. Particle Size. The apparent average hydrodynamic radii (Rh)of the synthesized QACD nanoparticles in PBS (pH ) 7.4) at 50

Table 1. Hydrodynamic Radii and Zeta Potentials of QACDNanoparticles

samplea hydrodynamic radiusb (Rh)/nm zeta potential ()c/mV

1-15-0.5 77.3 ( 4.1 -11.8 ( 1.01-15-2 81.0 ( 2.5 1.8 ( 2.31-15-4 65.3 ( 4.3 6.0 ( 2.91-15-6 88.0 ( 3.8 14.0 ( 3.8

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

506 Biomacromolecules, Vol. 10, No. 3, 2009 Gil et al.

mg ·mL-1 at room temperature were measured by a dynamic lightscattering instrument equipped with an ALV-CGS-8F compact goni-ometer system, an ALV-5000/EPP multiple tau real time correlator,and an ALV-5000/E/WIN software (ALV, Germany). The light sourcewas JDS Uniphase helium/neon laser (633 nm, 35 mW, Manteca, CA).Autocorrelation functions of the QACD nanoparticle solutions at 90°scattering angle were collected. The data were fitted using a Cumulantsmethod to derive apparent hydrodynamic radii of QACD nanoparticles.

The particle sizes of the QACD nanoparticles in PBS (pH 7.4)were also measured by an atomic force microscopy (AFM) equippedwith a Molecular Imaging Pico-SPM system and a SPM 100 controller(RHK technology, MI) in tapping mode. The substrates for immobiliz-ing the nanoparticles were silicon wafers pretreated with atmosphereplasma (to introduce an anionic surface).

2.3.3. Zeta Potential. The zeta potential of the QACD nanoparticleswas measured by a Zeta potentiometer (Coulter Delsa 440 SX, Hialeah,FL). The QACD nanoparticles were dissolved in deionized waterbefore measurement.

2.4. Cells and Media. Bovine brain microvessel endothelial cells(BBMVECs, Cell Applications Inc., San Diego, CA) were seeded inbovine fibronectin-coated T25 or T75-flasks at a density of 5000cells · 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) at37 °C with 95% humidity and 5% CO2. The medium was changedevery two day. The cells were harvested with trypsin (0.05% trypsinwith 0.4 mM EDTA) when they were 80% confluent on the fourthday.

U87 human glioblastoma cells (ATCC, Manassas, VA) were seededin T25 flasks at a density of 8000 cells · cm-2 and cultured in EMEMcontaining 10% FBS and antibiotic/antimycotic (penicillin G sodiumsalt 10 µg ·mL-1) at 37 °C with 95% humidity and 5% CO2. The mediawas 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 QACD Nanoparticles on BBB Integrity InVitro. 2.5.1. Cytotoxicity Study of QACD Nanoparticles. The cyto-toxicity of the QACD nanoparticles to BBMVECs was evaluated byMTT assay. BBMVECs were seeded at 50000 cells · cm-2 in bovinefibronectin-coated 96-well plates, and grown in 100 µL culture mediafor 2 days. The cells were then treated with/without the QACDnanoparticles or dextran (Mw ) 4000 g ·mol-1) at concentrations of100, 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) wasadded to each well for 4 h, followed by removal of media from eachwell, and addition of 100 µL 50% DMF/20% SDS (pH 4.7). Afterovernight incubation, the absorbance at 570 nm was measured usingµQuant microplate reader (Biotek Instruments, Winooski, VT) withbackground subtraction. Cell viability was calculated by dividing theabsorbance of wells containing the QACD nanoparticles by theabsorbance of wells containing the medium alone (corrected forbackground). Four replicate wells were used for each sample andcontrol.

2.5.2. Transendothelial Electrical Resistance Test. The effects ofthe QACD nanoparticles on the TER values of BBMVEC monolayerswere measured by an Endohm tissue resistance measurement chamber(World Precision Instruments, Sarasota, FL). BBMVECs were seededat 50000 cells · cm-2 on bovine fibronectin-coated polyester transwellsfilters (12 mm diameter, 0.4 µm pore size), and then grown toconfluence. After 4 h permeability test of QACD nanoparticles acrossthe BBB as described in Section 2.6.1, the TER values of the cellmonolayers with/without the presence of QACD nanoparticles wererecorded and expressed in Ω · cm2.

2.5.3. Lucifer Yellow Permeability. The effects of the QACDnanoparticles on the BBB tight junction integrity were first studies bymeasuring the BBB permeability of Lucifer yellow CH dilithium salt(LY), a paracellular permeable marker, in the presence of the QACDnanoparticles. BBMVECs harvested from the T-flasks were grown on

polyester Transwell filters (6 mm diameter, 0.4 µm pore size) coatedwith bovine fibronectin at a seeding density of 50000 cells · cm-2. Theresulting confluent BBMVEC monolayers were used for permeabilitystudies as an open two-compartment vertical side-by-side dynamic BBBmodel.

Lucifer yellow (100 µM) and QACD nanoparticles (100 µg ·mL-1)were dissolved in medium and added to the apical chamber of eachwell (Figure 1). Transport experiments were conducted in the apicalto basal direction at 37 °C for 3 h. Thirty µL medium was taken fromthe basolateral chamber and replaced with 30 µL of fresh medium every30 min. At 3 h, 30 µL medium was taken from both the apical andbasolateral chambers. Aliquots were quantified on a Spectramax EMmicroplate 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 calculatedby the following formula:44

P0 ) [(FA ⁄ ∆t)VA] ⁄ (FLA) (1)

where Po, FA, VA, FL and A are the permeability coefficient, thebasolateral fluorescence of the solute over ∆t time, the fluid volume ofthe basolateral chamber, the apical fluorescence of the solute, and thesurface 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 transportof circulating molecules into the brain by tight junctions between braincapillary endothelial cells and P-gp efflux system on the cell membrane.The expression amounts of the tight junction proteins and P-gp aredirectly related to the integrity of BBB. Therefore, we used westernimmunoblotting to study the effects of the QACD nanoparticles onthe expression of two representative tight junction proteins occludinand ZO-1, and P-gp. BBMVECs were seeded at 50000 cells · cm-2 onbovine fibronectin-coated 60 mm polystyrene dishes and then grownto confluence. The plates were treated with/without QACD nanopar-ticles at 100 µg ·mL-1 in culture medium (2 mL) for 4 h. After washedtwice by ice-cold PBS (pH 7.4) containing phenyl-methylsulfonylfuoride (200 µM), the BBMEVCs were harvested in 150 µL lysis bufferusing 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 mMNaF and 10 mM sodium pyrophosphate with CompleteTM (RocheDiagnostics, Indianapolis, IN), a protease inhibitor cocktail tablet. Thelysis was conducted at 4 °C for 15 min with rocking, and cells werepelleted by microcentrifugation at 14000 g for 10 min. Concentrationof supernatant was determined using BCA Protein Assay kit and analbumin standard curve. Equal amount of protein (20 µg) was loadedonto NuPAGE 4-10% Bis-Tris gels in NuPAGE LDS sample bufferalong with NuPAGE sample reducing agent after heating at 70 °C for10 min. Gel running was conducted in NuPAGE MOPS SDS runningbuffer. 120 mA current and 200 V voltage were applied for ap-proximately 55 min. The obtained gels were transferred to nitrocellulosein NuPAGE transfer buffer at 20 V at 4 °C for overnight and then at30 V at 4 °C for 1 h. The transferred nitrocellulose was blocked with5% milk in Tris-buffered saline with Tween 20. Rat anti-ZO-1 antibody

Figure 1. In vitro model for BBB permeability studies.

Quaternary Ammonium -Cyclodextrin Nanoparticles Biomacromolecules, Vol. 10, No. 3, 2009 507

at 1:4 dilution, rabbit antioccludin polyclonal antibody ZYMED at1:1000 dilution, and the mouse anti-P-gp antibody C219 at 1:100dilution in the blocking solution were used for primary antibodyblotting. The nitrocellulose was blotted with these primary antibodiesfor 3 h at room temperature and then washed. Afterward, it was blottedwith alkaline phosphatase conjugated secondary antibodies (antiratantibody IgG-AP at 1:2000 dilution for ZO-1, antirabbit antibody IgG-AP at 1:2000 dilution for occluding, and goat antimouse antibody IgG-AP at 1:2500) for 1 h at room temperature, and washed. The blottedantibodies were enhanced with enhanced chemifluorescence and thebands were detected and quantified by Fluorimager 595 with Im-ageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA).

2.5.5. Immunofluorescent Staining of Tight Junction ProteinsZO-1 and Occludin. The effects of the QACD nanoparticles on theexpression of two BBB tight junction proteins, ZO-1 and occludin,were further examined by immunofluorescent staining using confocalmicroscopy. BBMVECs were seeded at 50000 cells · cm-2 on bovinefibronectin-coated Laboratory-Tek 8 well Permanox chamber slides,grown to confluence, and then treated with/without QACD nanopar-ticles (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 withPBS (pH 7.4) containing 0.2% Triton X-100 for 10 min, and blockedwith PBS (pH 7.4) containing 0.1% Triton X-100 and 10% BSA for1 h. After incubated with rat anti-ZO-1 antibody at 1:4 dilution or rabbitantioccludin 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% TritonX-100. Then, cells were incubated with second fluorescent antibodies:goat antirat IgG-FITC at 1:200 dilution or goat antirabbit IgG-Cy3 at1:500 dilution, followed by three times washing with PBS (pH 7.4)containing 0.1% Triton X-100. After the chambers were removed fromthe Laboratory-Tek slides, glass coverslips were mounted onto the slideswith Aqua Poly/Mount. The slides were visualized by a Leica TCSSP2 AOBS confocal microscopy (Leica, Mannheim, Germany) equippedwith 488 nm argon and 543 nm He/Ne lasers. The brightness andcontrast of all pictures were identically adjusted with Adobe Photoshop.

2.5.6. Cholesterol Extraction. The effects of the QACD nanopar-ticles on the extracellular property of the BBB were assessed bymeasuring cholesterol extraction of the BBMVEC monolayer in thepresence of dextran and the QACD nanoparticles. BBMVECs wereseeded at 50000 cells · cm-2 on bovine fibronectin-coated 24-well platesand then grown to confluence. The confluent BBMVEC monolayerswere incubated in the culture media containing QACD nanoparticlesat 100 µg ·mL-1 for 24 h, or 1 mg ·mL-1 for 4 h. After washed twicewith PBS (pH 7.4), BBMEVCs were harvested using a cell lifter in100 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-gaugeneedle. The cholesterol content was determined using an Amplex Redcholesterol assay kit (Molecular Probes, Eugene, OR) following themanufacturer’s instructions. The protein content in each well wasdetermined using a bicinchoninic acid assay kit (Pierce, Rockford, IL)using BSA as a standard. The cholesterol content was normalized bythe total protein content. Dextran (Mw ) 5000 g ·mol-1) was used asa control.

2.5.7. P-gp Efflux ActiVity Using Rhodamine 123 Efflux Assay. Theeffects of the QACD nanoparticles on the extracellular property ofthe BBB were also evaluated by measuring the cellular uptake ofrhodamine 123 (R123), a fluorescent P-gp efflux activity marker, byBBMVECs. The BBMVECs were seeded onto 24-well plates at adensity of 50000 cells/well and grown for 2 days. The cell monolayerswere treated with QACD nanoparticles (100 or 500 µg/mL) or a P-gpinhibitor, verapamil (100 µM) for 2 h. The media was then removedand the cells were incubated with R123 of 10 µM in media for 2 h.After removing R123 solution, the cell monolayers were washed threetimes with cold PBS (pH 7.4). The BBMVECs were solubilized using100 µL of cell culture lysis buffer described above (section 2.5.1). The

cellular content of R123 was determined by fluorescent measurementwith R123 standard curve at 480 nm of excitation and 530 nm ofemission and normalized per cellular content of protein. The proteincontent was measured using a Pierce (Rockford, IL) BCA protein assay.

2.6. Effects of QACD Nanoparticles on Permeability of Doxo-rubicin across In Vitro BBB. 2.6.1. Permeability of QACDNanoparticles across the In Vitro BBB. The permeability of QACDnanoparticles were measured as described in section 2.5.3. ConfluentBBMVEC monolayers on polyester Transwell filters (12 mm diameter,0.4 µm pore size) coated with bovine fibronectin were used for thepermeability test. DTAF-labeled QACD nanoparticles or FITC-dextrancontrol (Mw ) 4000 g ·mol-1) was dissolved in medium and added tothe apical chamber of each well at 100 µg ·mL-1. The aliquots fromthe basolateral chambers at each time interval and the apical chamberat 4 h were used for the quantification of QACD nanoparticlepermeability.

2.6.2. Cellular Uptake of QACD Nanoparticles by BBMVECs.Confocal microscopy and flow cytometry were carried out to examinecellular internalization of the synthesized QACD nanoparticles 1-15-2. BBMVECs were plated at a density of 50000 cells · cm-2 onto onbovine fibronectin-coated Laboratory-Tek 8 well Permanox chamberslides for confocal microscopy experiments. Also, BBMVECs wereseeded at a density of 400000 cells/well into 60 mm tissue culture dishesfor flow cytometry experiments. After 1-2 days, DTAF-labeledQACD nanoparticles 1-15-2 at 100 or 200 µg ·mL-1, and FITC-Dextran (Mw ) 4000 g ·mol-1) at 100 µg ·mL-1 were added into eachwell/plate and incubated for 1 h at 37 °C. Subsequently, cell culturemedium was gently removed and the plates were washed twice withPBS (pH 7.4). For confocal microscopy study, the cells on the glasscoverslips were fixed with 4% paraformaldehyde for 20 min. After twicewashing with PBS (pH 7.4), cellular nuclei were stained with 1mg ·mL-1 DAPI for 30 min. Followed by two more washings withPBS (pH 7.4), the middle z-section images of cells were taken by aLeica 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 withtrypsin, washed with culture media, centrifuged at 1000 rpm, washedonce with PBS (pH 7.4), and centrifuged a second time. The cells werefixed 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 inorder to differentiate between membrane-bound and internalizedQACD 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 fluorescence-activated cell sorter (FACS, Becton Dickinson, San Jose, CA) equippedwith an argon-ion laser and 530 nm bandpass filters for emissionmeasurements. 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 deadcells, debris, and cell aggregate.

2.6.3. In Vitro Permeability of Doxorubicin Complexed withQACD Nanoparticles across the BBB. DOX was complexed with theQACD nanoparticles by mixing DOX (200 µM) and QACDnanoparticles (20 mg ·mL-1) in filtered deionized water at 4 °Covernight. The DOX/QACD nanoparticle complex stock solutionswere diluted and subsequently added to the apical chamber of eachTranswell in Figure 1 to obtain the final concentration of DOX/QACDnanoparticles at 1 µM/100 µg ·mL-1. The permeability of the DOX/QACD complexes across BBMVEC monolayer was measured andquantified by following the above method described in section 2.5.3.DOX (1 µM) alone was used as a control.

2.7. Effects of QACD Nanoparticles on Cytotoxicity ofDoxorubicin. Cytotoxicity of DOX with/without QACD nanoparticlesto BBMVECs and U87 human glioblastoma cells was evaluated byMTT assay. BBMVECs were seeded at 50000 cells · cm-2 in fibronectin-coated 96-well plates in MCDB-131 medium containing 10% FBS, 10


ng ·mL-1 EGF and 0.2 mg ·mL-1 ENDO GRO. Two days later theBBMVECs were treated with DOX alone and DOX/QACD complexesin the culture medium at 37 °C for 4 h. The studied concentrations ofDOX were 1, 2, 5, and 10 µM and that of the QACD nanoparticleswas 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 dayslater the U87 cells were treated with DOX alone and DOX/QACDcomplexes in the culture medium at 37 °C for 4, 24, and 60 h. Thestudied concentration of DOX and QACD nanoparticles were 1 µMand 100 µg ·mL-1, respectively. Subsequently, the cytotoxicity of DOXwith/without QACD nanoparticles to both BBMVECs and U87 wasevaluated using the MTT assay following the method described insection 2.5.1.

2.8. Statistical Methods. Differences between treatment groupswere statistically analyzed using one-way analysis of variance (ANO-VA). A statistically significant difference was reported if p < 0.05 orless. Data is reported at the mean ( standard deviation (SD) from atleast three separate experiments.

3. Results

3.1. Physicochemical Properties of QACDs. In the currentstudy, we synthesize QACDs with different amount ofquaternary ammonium groups (Figure 2A,B). In 1H NMRspectra of QACDs (Figure 2C), the peak assigned to the protonof quaternary ammonium group occurs at around 3.1 ppm. Dueto the structural irregularity of QACDs, the glucose protonpeaks are split up to 5 peaks between 3.4∼5.2 ppm. The peaksassigned to the protons of methyl segments of epichlorohydrinand choline chloride are overlapped by the proton peaks inglucose units. As expected, the peak intensity assigned to choline

chloride at 3.1 ppm increases with increasing the feeding ratioof choline chloride to CD. More precisely, the ratios of thechemical shift peak areas of choline chloride at 3.1 ppm to CDat 5.0 (c1) ppm, increase proportionally with increasing thecholine chloride/CD feeding ratio from 0.5 to 4 and then moredramatically at choline chloride/CD feeding ratio ) 6 (Figure2D).

ATR-FTIR and 1H NMR were used to determine theformation of DOX/QACD complexes. In ATR-FTIR study,QACD 1-15-2 shows bands at 1012 and 1059 cm-1 due tocoupled C-C and C-O stretching vibrations, and band at 1130cm-1 due to C-O-C glycosidic bridge antisymmetric stretchingvibration (Figure 3A). DOX shows characteristic IR bands:stretching vibration of carbonyl group at 13-keto position at1726 cm-1, in-plane bending of NH2 at 1593 cm-1, stretchingvibration of two carbonyl groups of anthracene ring at 1577cm-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 QACD 1-15-2 is complexed with DOX,many intrinsic IR bands of QACDs 1-15-2 and DOX shift theirwavenumber positions, for examples, the IR bands of QACD1-15-2 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 characteristicIR bands also have significant changes after the complexation,for examples, the peak at 1705 cm-1 becomes stronger whilethe peaks at 1589, 1568, and 1284 cm-1 become much weaker.The results strongly suggest that there exist interaction be-tween the QACD 1-15-2 and doxorubicin probably due to the

Figure 2. Structures and 1H NMR spectra of QACDs. (A) Schematic structure of QACDs. (B) Chemical structure of QACDs. (C) 1H NMRspectra 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 CD unit in QACDs at chemical shifts 5.0 (cl)and at 3.1 ppm (a), respectively.


binding/association between the nitrogen of the quaternary amineof QACD 1-15-2 and the oxygen of the 13-keto of DOX, andmaybe also the binding/association between the oxygen of thehydroxyl group of QACD 1-15-2 and the nitrogen of the 3′-amino group of DOX.45 The 1H NMR peaks of the QACDsafter DOX complexation are shifted to upfield, but the shiftsare much minor (data not shown).

Figure 4 and Table 1 exhibit the particle size and morphologyof QACD nanoparticles. The hydrodynamic radii Rh ofQACD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 inPBS (pH 7.4) measured by dynamic light scattering techniqueat 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 ofQACD nanoparticles 1-15-2 is representatively shown inFigure 4A. AFM measurements (Figure 4B-D) further revealthat QACD nanoparticles 1-15-2 are very soft on anionicsurface in wet state with 200∼500 nm in width and 15∼25 nmin height. By considering the volume as a half of oblate spheroidvolume, the calculated volume of the spread QACD nanopar-ticles 1-15-2 in the AFM pictures is 0.3∼3.3 × 10-3 µm3, whichmatches well with the sphere volume 2.2 × 10-3 µm3 calculatedby the Rh (81 nm).

Because surface charge of nanoparticles plays an importantrole in nanoparticle-cell interactions, we measured the zeta-potential values of the QACD nanoparticles. Table 1 showsthat the zeta-potential values of the four nanoparticles increasewith increasing the feeding ratio of [choline chloride]/[CD].The zeta-potential values of QACD 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 thatQACD nanoparticles 1-15-2, 1-15-4, and 1-15-6 have positivezeta potential values, whereas QACD nanoparticles 1-15-0.5have negative zeta potential value. In general, parent CD hasintrinsic negative charge property.

3.2. Effects of QACD Nanoparticles on BBB IntegrityIn Vitro. MTT assay demonstrates that QACD nanoparticles1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 are not toxic to BBMVECsat concentrations 100, 300, and 500 µg ·mL-1 over a period of24 h, similar to dextran control (Figure 5).

TER test displays that the four types of QACD nanoparticles1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 at 100 µg ·mL-1 do notchange the TER value of BBMVEC monolayers after 4 hincubation (Figure 6A). To further evaluate the effects of thefour QACD nanoparticles on the function of the BBB tightjunction, studies were performed on the permeability of Luciferyellow, a paracellular permeable marker,46 across a confluentBBMVEC monolayer, an in vitro BBB model (Figure 6B), in

the presence of the QACD nanoparticles. The permeabilitycoefficients of Lucifer yellow in the presence of the fourQACD nanoparticles at 100 µg ·mL-1 (even 500 µg ·mL-1 forQACD nanoparticles 1-15-2) are either similar or lower thanthat of Lucifer yellow alone, indicating that the tightness of theBBB junctions between cells is not destroyed by QACDnanoparticles.

Western immunoblotting analysis illustrates that QACDnanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg ·mL-1 do

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

Figure 4. (A) Hydrodynamic size distribution of QACD nanoparticle1-15-2 measured by dynamic light scattering. The cumulated averagehydrodynamic radius (Rh) of QACD nanoparticles 1-15-2 in PBS (pH7.4) is 81.0 ( 2.5 nm (N ) 5, mean ( S.D.). (B) Topographic and(C) phase (in tapping) atomic force microscopy (AFM) images ofQACD nanoparticles 1-15-2. (D) AFM dimension of QACD nano-particle 1-15-2 is 200∼500 nm in width and 15∼25 nm in height.

Figure 5. Effects of QACD 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. Resultsrepresent the mean ( SD of four measurements.


not change the contents of two tight junction proteins ZO-1 andoccludin of BBMVEC monolayers after 4 h incubation (Figure7A-C). Immunofluorescent staining analysis using conforcalmicroscopy further confirms that QACD nanoparticles 1-15-2do not disrupt the expression patterns of ZO-1 and occludinafter 4 h incubation, even in the presence of 1 µM DOX (Figure7D-G).

Cholesterol extraction has been considered as the initial stepof CD to damage cell membranes.31 Cholesterol contentquantification reveals that QACD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg ·mL-1 do not cause any cholesterolextraction from the BBMVECs even after 24 h incubation(Figure 8). Western blotting results show that the three QACDnanoparticles at 100 µg ·mL-1 have no effects on the P-gpexpression of BBMVEC monolayers after 2 h incubation (Figure9A,B). The further evaluation of the cellular uptake of R123, aP-gp efflux activity marker (Figure 9C), by BBMVECs dem-onstrate that QACD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6at 100 µg ·mL-1 and even 500 µg ·mL-1 do not affect the cellularuptake value of R123; however, verapamil, a well-known P-gpinhibitor, increases the cellular uptake value of R123 by a factorof 2.

3.3. Effects of QACD Nanoparticles on Permeabilityof Doxorubicin across In Vitro BBB. Before investigating onthe effects of QACD nanoparticles on the permeability ofdoxorubicin across the BBB, we first tested the permeability ofQACD nanoparticles alone across an in vitro BBB model,

confluent BBMVEC monolayers. The permeability study wascarried out by examining the transport of DTAF-labeled QACDnanoparticles at 100 µg ·mL-1 from apical to basolateral sideof the BBMVEC monolayer for 4 h (Figure 1). The permeabilitycoefficient of QACD nanoparticles 1-15-0.5 is 8.1 ( 0.9 ×10-6 cm · s-1, which is similar to that of very permeable 4000Da FITC-dextran control (8.2 ( 0.7 × 10-6 cm · s-1) (Figure10). With increasing the [choline chloride]/[CD] feeding molarratio in the QACD nanoparticles to two or higher, thepermeability coefficient of the nanoparticles can be even higherthan that of FITC-dextran control. For example, the permeabilitycoefficients of QACD nanoparticles 1-15-2, 1-15-4, and 1-15-6are 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 ofQACD nanoparticle 1-15-0.5 and FITC-dextran control.

Figure 6. Effects of QACD 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 BBMVECmonolayers (B). TER values are quantified by Endohm tissueresistance measurements after 4 h incubation. The TER value of aninherent BBMVEC monolayer was 156.4 ( 0.65 Ω ·cm2. Y-axis valuesare the TER values normalized to that of the cell monolayers withoutQACD nanoparticles. Results represent the mean ( SD of threemeasurements.

Figure 7. Effects of QACD nanoparticle 1-15-0.5, 1-15-2, and 1-15-6at 100 µg ·mL-1 on the expression of tight junction proteins ZO-1 andoccludin of BBMVEC monolayers after 4 h incubation, quantified bywestern immunoblotting analysis (A∼C) and visualized by confocalmicroscopy after immunofluorescent staining (D∼G). Y-axis valuesin (B) and (C) are the occludin or ZO-1 content normalized to that ofthe cells without QACD 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 monolayerstreated without and with 100 µg ·mL-1 QACD nanoparticles 1-15-2complexed with 1 µM doxorubicin, respectively.


To understand the permeation of QACD nanoparticles acrossthe BBMVEC monolayers, we studied the cellular uptake ofQACD nanoparticles 1-15-2 by BBMVECs using confocalmicroscopy and flow cytometry. Z-section of middle part ofcells by confocal laser scanning microscopy clearly shows thatQACD nanoparticles 1-15-2 at 100 and 200 µg ·mL-1 are takenup 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 QACD nano-

particles display punctate staining that may indicate the nano-particles accumulate within endocytotic vesicles.

Further analysis by flow cytometry shows that 7, 87, and 99%of BBMVECs are associated with FITC-dextran (Mw ) 4000g ·mol-1) at 200 µg ·mL-1, and DTAF-QACD nanoparticles1-15-2 at 100 and 200 µg ·mL-1, respectively (Figure 12A). Toconfirm that the DTAF-labeled QACD nanoparticles wereinternalized and not merely attached to the cell surface, we useda trypan blue quenching dye to quench any extracellularfluorescence, leaving the internal fluorescence protected.47 Withtrypan blue treatment, the percentages of BBMVECs associatedwith FITC-dextran at 200 µg ·mL-1, and DTAF-QACDnanoparticles at 100 and 200 µg ·mL-1, decrease to 2, 40, and78%, respectively (Figure 12A). By dividing the fluorescenceafter trypan blue treatment by that before trypan blue treatment,we calculated that nearly 47 and 78% DTAF-QACD nano-particles at 100 and 200 µg ·mL-1, respectively, are internalizedinto BBMVECs (Figure 12B).

After the above understanding of the permeability of QACDnanoparticles across the BBB, we evaluated the effects ofQACD nanoparticles on the permeability of doxorubicin acrossthe BBB. The results show that when 1 µM DOX is complexedwith 100 µg ·mL-1 QACD nanoparticles 1-15-0.5, 1-15-2, and

Figure 8. Effects of QACD nanoparticles 1-15-0.5, 1-15-2, and1-15-6 on the cholesterol content of BBMVEC monolayers at 100µg ·mL-1 after 24 h incubation, quantified by Amplex Red cholesterolassay. Y-axis values are the cholesterol content normalized to thatof the cell monolayers without QACD nanoparticles, which was 21.83( 1.43 µg ·mL-1 total protein (Control, N ) 12). Dextran was usedas a control. Results represent the mean ( SD of four measurements.

Figure 9. Effects of QACD nanoparticles 1-15-0.5, 1-15-2, and1-15-6 on the expression and efflux activity of P-gp of BBMVECmonolayers. (A) Western immunoblotting analysis after 4 h incubationat 100 µg ·mL-1. (B) P-gp content is normalized to that of the cellswithout QACD nanoparticles. Results represent the mean ( SD ofthree measurements. (C) P-gp efflux activity with/without QACDnanoparticles at 100 (solid) and 500 (blank) µg ·mL-1 using R123assay (10 µM). Verapmil (100 µM) was used as a P-gp inhibitor fora negative control. Results represent the mean ( SD of fourmeasurements. **p < 0.001.

Figure 10. Permeability of DTAF-labeled QACD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 at 100 µg ·mL-1 across BBMVECmonolayers at 37 °C for 4 h. Results represent the mean ( SD ofthree measurements. **P < 0.001.

Figure 11. Accumulation of FITC-labeled dextran (Mw ) 4000g ·mol-1) at 100 µg ·mL-1 (A) and DTAF-labeled QACD nanoparticles1-15-2 at 100 (B) and 200 (C) µg ·mL-1 in BBMVECs cells observedby confocol microscopy. Z-section images are taken from middle partof cells to visualize the internal section of the cells. Contrast andbrightness of all images are equally adjusted to visualize weakly DAPI-bounded cell shape as well as DAPI-stained nuclei (Blue). FITC-labeled dextran and DTAF-labeled are visualized as green color. Allimages are taken using 60× optical magnification lens. Scale barsrepresent 10 µm.


1-15-6, its permeability across the BBMVEC monolayer isenhanced by a factor of 1.8, 2.2, and 2.2 times, respectively(Figure 13).

3.4. Effects of QACD Nanoparticles on Cytotoxicityof Doxorubicin. We first investigated the effect of QACDnanoparticles on the cytotoxicity of DOX to BBMVECs at 37°C. DOX alone displays a significant dose-dependent cytotox-icity to BBMVECs after 4 h incubation (Figure 14A). It is notsignificantly toxic to BBMVECs at 1 and 2 µM, but decreasesBBMVEC viability to 85 ( 5.2 and 79 ( 5.7% at 5 and 10µM, respectively. When DOX is complexed with QACDnanoparticles 1-15-0.5 and 1-15-2 (100 µg ·mL-1), the BBM-VEC cell viability increases to 100% at 5 and 10 µM DOX.We next investigated the effect of QACD nanoparticles onthe efficacy of DOX in killing U87 human glioblastoma cellsat 37 °C. DOX (1 µM)/QACD nanoparticle (100 µg ·mL-1)complexes display a significant time-dependent inhibition ofU87 cell viability similar to the free DOX (Figure 14B). TheU87 cell viability is 100% at 4 h, decreases to about 80% at24 h and about 30% at 60 h.

4. Discussion

CD and its derivatives have been widely studied as deliverysystems to improve drugs ’ solubility, chemical stability,dissolution, and bioavailability across the dermal, nasal andintestinal barriers.30,31 However, they have been only recentlyexplored for drug delivery across the BBB.42,48,49 To developa new class of CD that will not only effectively enhance drugs’

permeability across the BBB, but also will not change theintegrity of the BBB, in this study we incorporate quaternaryammonium groups into CD using choline chloride andepichlorohydrin through one-step condensation polymerization.The reason for the introduction of quaternary ammonium groupsis that quaternary ammonium is cationic and much less toxicthan primary amine28 and monoquaternized CDs are less toxicthan parent CD to the brain capillary endothelial cells42 anddo not extract cholesterol.43

In this study, we first characterized the physicochemical anddrug complexation properties of the newly developed quaternaryammonium CD nanoparticles. In the design of QACDnanoparticles, the feeding ratio of epichlorohydrin to CD was

Figure 12. Quantitative uptake of QACD nanoparticles 1-15-2 by BBMVECs analysized by flow cytometry. (A) Percentage of cells associatedwith the nanoparticles before (solid bar) and after (stripe bar) trypan blue dye was used to quench membrane bound nanoparticles. (B) Percentageof total cell-associated nanoparticles that were internalized in BBMVECs. The concentration of the nanoparticles for each study was 100 and200 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 QACD nanoparticles 1-15-0.5, 1-15-2, and1-15-6 on the permeability of DOX across BBMEC monolayer at 37°C for 4 h. The concentration of the DOX/QACD nanoparticlecomplexes was 1 µM /100 µg ·mL-1. Results represent the mean (SD of three measurements. *p < 0.005, **P < 0.001.

Figure 14. Effects of QACD nanoparticles 1-15-0.5 and 1-15-2 onthe cytotoxicity of DOX to BBMVECs and U87 human glioblastomacells, evaluated by MTT assay. (A) BBMVECs are incubated withDOX/QACD nanoparticle complexes at 1 µM/100 µg ·mL-1 (blankbar), 2 µM/100 µg ·mL-1 (striped bar), 5 µM/100 µg ·mL-1 (checkedbar), and 10 µM/100 µg ·mL-1 (solid bar) for 4 h. (B), U87 humanglioblastoma cells are incubated with DOX/QACD nanoparticlecomplexes 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 fourmeasurements. *p < 0.05, **p < 0.01.


kept constant at 15 and the feeding ratios of choline chloride toCD were varied from 0.5 to 6. The increase of the ratios ofthe 1H NMR chemical shift peak areas of choline chloride at3.1 ppm to CD at 5.0 (c1) ppm with increasing the cholinechloride/CD feeding ratio from 0.5 to 6 (Figure 2) clearlydemonstrates that quaternary ammonium group can be success-fully chemically incorporated into CD with tunable amount.The increase of zeta-potential values of the QACD nanopar-ticles with increasing the feeding ratio of [choline chloride]/[CD] (Table 1) further confirms the success of the synthesis.The QACDs form nanosized particles of 65∼88 nm hydro-dynamic radii with soft morphology on anionic surface in PBS(pH 7.4) at 37 °C (Table 1 and Figure 4), implying that manywater molecules are bounded to the nanoparticles. ATR-FTIRanalysis reveals that QACD and DOX form complexation bydecreasing the relative intensities or shifting the positions oftheir respective characteristic bands (Figure 3).

With the above understandings of the physicochemical andDOX complexation properties of QACD nanoparticles, we nextinvestigated the effects of the physicochemical properties of thequaternized CD nanoparticles on the in vitro BBB cytotoxicityand integrity. It is well-known that materials can be toxic tocells by inhibiting the capability of cells to reduce MTT toformazon in mitochondria/endosomes. Thus, we used MTTassay to study the cytotoxicity of four designed QACDnanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 to BBM-VECs, and find that all of them are not toxic to BBMVECs atconcentration up to 500 µg ·mL-1 after 24 h incubation (Figure5). Of course, materials may not cause mitochondrial/endosomaldysfunction, but can disrupt the BBB integrity by (1) regulatingtight junction function or protein expression to increase para-celluar permeability, and (2) releasing biological membranecomponent cholesterol and thus reducing P-gp (a membranebound efflux transporter) expression or its activity.41,50,51

Therefore, we employed EndohmTM tissue resistance measure-ment, permeability study of a paracellular permeable markerusing Lucifer yellow, western immunoblotting, immunofluo-rescent staining, Amplex Red cholesterol assay, and uptake ofR123 assay to investigate the effects of the four designedQACD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 onthe integrity of an in vitro BBB model, BBMVEC monolayer,at 100 µg ·mL-1 after 4 h incubation (24 h incubation for thecholesterol extraction studies). The results reveal that all thefour QACD nanoparticles do not affect TER value and Luciferyellow permeability and thus BBMVEC monolayer tight junc-tion function (Figure 6), and tight junction proteins occludinand ZO-1 expression quantified by Western immunoblotting andvisualized by confocal microscopy after immunofluorescentstaining (Figure 7). Cholesterol and P-gp are observed in thecaveolae of BBMVECs.52 The unchanged cholesterol and P-gpcontents as well as uptake of R123 (Figures 8 and 9) suggestthat the four nanoparticles do not affect the cholesterol extractionand P-gp expression and efflux activity and thus the stabilityof BBMVEC membranes. Taken together, all the above resultsdemonstrate that the four QACD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 do not affect the integrity of the in vitroBBB model, despite their differences in physicochemicalproperties (Table 1 and Figures 2-34).

In the next step, we studiedthe permeability of QACDnanoparticles alone across BBMVEC monolayers, an in vitroBBB model, at 100 µg ·mL-1 (the concentration at which allthe four QACD nanoparticles do not affect the BBB integrityparameters). Figure 10 demonstrates that the permeabilitycoefficients of QACD nanoparticles 1-15-2, 1-15-4, and 1-15-6

are about twice those of QACD nanoparticle 1-15-0.5 andFITC-dextran (Mw ) 4000 g ·mol-1) control. It is well-knownthat introduction of cationic charges to nanoparticles can increasethe BBB permeability of nanoparticles in vitro and in vivo dueto adsorptive-mediated endocytosis.19,53,54 Thus, our data furthersuggest that when cationic quaternary ammonium is introducedto CD to make the resulting nanoparticles to have non-negativezeta potential values (Table 1), significantly high permeabilityof the resulting nanoparticles across the BBMVEC monolayercan be achieved. The maximum permeability coefficient isobtained when the feeding molar ratio of [choline chloride]/[CD] is 2 (Figure 10). This may indicate that there is limitationof the number of quaternary ammonium groups of QACDnanoparticles that can maximally interact with BBMVECmembranes.

Because the QACD nanoparticles have hydrodynamic radiiof 65∼88 nm in PBS (pH 7.4) at 37 °C (Table 1), which ismuch bigger than the pore size of the BBB junction (less than3 nm),55,56 and the permeability of the nanoparticles isindependent of their particle sizes (Table 1), it is more thanunlikely that the nanoparticles cross the BBMVEC monolayersby paracellular pathway. To investigate the mechanisms for theQACD nanoparticles to cross the BBMVEC monolayers, weused confocal microscopy and flow cytometry to study theinteractions between the QACD nanoparticles and BBMVECs.The results show that more than 85% of BBMVECs areassociated with representative QACD nanoparticles 1-15-2 and47% or higher percentage of the nanoparticles are internalizedinto the endocytotic vesicles of BBMVECs when the concentra-tion of the nanoparticles is 100 µg ·mL-1 or higher; while lessthan 10% of BBMVECs are associated with dextran controland less than 20% of dextran is internalized into the BBMVECsat 100 µg ·mL-1 (Figures 11 and 12). These results suggest thatthe QACD nanoparticles can cross the BBB probably byendocytosis, due to their cationic property generated by thequaternary ammonium groups of the nanoparticles.47,57 Morethorough studies on the endocytosis mechanism are underinvestigation.

After understanding that the designed QACD nanoparticlesare easy to cross the BBMVEC monolayer without disruptingthe monolayer integrity at 100 µg ·mL-1, we used DOX as amodel drug and investigated the effect of QACD nanoparticleson the permeability of DOX across the BBMVEC monolayerin vitro. DOX is a very potent anticancer drug to treat braintumor. However, DOX has been known to show a restrictedtransport to brain due to the P-gp efflux activity as well as tightjunction of brain capillary endotherial cells.58 CDs and CDbased drug carriers have been known to make 1:1 complexationwith many drugs including DOX,28,41,45 and our ATR-FTIRmeasurements also confirm the complexation between theQACD nanoparticles and DOX (Figure 3). Our further studiesdemonstrate that QACD nanoparticles 1-15-0.5, 1-15-2, and1-15-6 at 100 µg ·mL-1 enhance DOX (1 µM)’s permeabilityby a factor of 1.8, 2.2, and 2.2, respectively (Figure 13). In theliterature, it was reported that methylated-CD (MeCD) at 1mM also enhanced DOX (1 µM) permeability across the in vitroBBB by a factor of 2.41 However, the enhanced DOX perme-ability was due to the decreased P-gp activity of BBMVECsthrough cholesterol extraction induced by MeCD.41 In ourstudies, we previously demonstrated that our four QACDnanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 at 100µg ·mL-1 did not affect the integrity of the in vitro BBB modelincluding cholesterol extraction and P-gp expression and effluxactivity (Figures 8 and 9) under the same condition as we carried


out the permeability test. Therefore, the mechanisms for theenhanced DOX permeability induced by our QACD nanopar-ticles are fundamentally different from those induced byMeCD. As the permeability of DOX in the presence of theQACD nanoparticles increases in the order of 1-15-0.5 <1-15-2 = 1-15-6 (Figure 13), which matches well with theincreasing order of the permeability of the corresponding threenanoparticles (Figure 10), the enhanced DOX permeability isprobably controlled by the permeability of the QACDnanoparticles.

Finally, we investigated the effects of QACD nanoparticleson the cytotoxicity of DOX to BBMVECs and U87 humanglioblastoma cells. QACD nanoparticles 1-15-0.5 and 1-15-2(100 µg ·mL-1) protect BBMVECs from the toxicity of DOXat 5 and 10 µM after 4 h incubation (Figure 14A), but do notreduce the efficacy of DOX on killing U87 human glioblastomacells (Figure 14B).

5. Conclusions

A series of quaternary ammonium CD (QACD) nanopar-ticles with different charge density were synthesized by a one-step condensation polymerization of -CD, choline chloride,and epichlorohydrin. DLS and AFM data illustrate that thesynthesized QACDs form nanosized soft particles with Rh

around about 65∼88 nm. NMR and zeta potential measurementssuggest that the charge density of the nanoparticles is tunableby changing the feeding molar ratio of [choline chloride]/[CD].FTIR mesurements confirm the complexation between doxo-rubicin and the CD units of QACD nanoparticles.

The studied QACD nanoparticles at 100 µg ·mL-1 do notaffect the TER value, Lucifer yellow permeability, tight junctionand P-gp protein expression, tight junction morphology, cho-lesterol content, and P-gp efflux activity of BBMVEC mono-layers, analysized by Endohm tissue resistance test, in vitropermeability assay, western immunoblotting, immunofluorescentstaining, Amplex Red cholesterol assay, and R123 efflux assay,respectively. All the synthesized QACD nanoparticles 1-15-0.5, 1-15-2, 1-15-4, and 1-15-6 are not toxic to BBMVECs atconcentration up to 500 µg ·mL-1 tested by MTT assay.

QACD nanoparticles are very permeable across BBMVECmonolayers at 100 µg ·mL-1 with permeability coefficients equalto or twice higher than that of FITC-dextran control (Mw )4000 g ·mol-1). The permeability of QACD nanoparticlesacross BBMVEC monolayer increases with increasing thenumber of quaternary ammonium groups (NMR and zetapotential value) and reaches maximum when the feeding molarratio of quaternary ammonium groups (choline chloride)/CDis 2 (zeta potential value becomes non-negative). The mecha-nism for the permeability of the QACD nanoparticles acrossthe BBB is probably due to endocytosis, based on preliminaryinternalization results obtained from confocal microscopy andflow cytometry measurements. The QACD nanoparticles 1-15-0.5, 1-15-2, and 1-15-6 at 100 µg ·mL-1 enhance the perme-ability of DOX (1 µM) across BBMVEC monolayers by a factorof 1.8, 2.2, and 2.2, respectively.

The QACD nanoparticles 1-15-0.5 and 1-15-2 preserve thecell death effect of DOX on U87 human glioblastoma cells at1 µM, while protect BBMVECs from the cytotoxicity effectsof DOX by increasing BBMVEC viability from 85 ( 5.2 and79 ( 5.7%, at 5 and 10 µM DOX concentration, respectively,to 100% after 4 h incubation. Therefore, the designed QACDnanoparticles are promising carriers for delivering DOX and

other therapeutics across the BBB to treat brain disorders. Futurework should include validation of the in vitro results in animalmodels.

Acknowledgment. This work was supported by the WallaceH. Coulter Foundation and the National Institute of Health. Theauthors would like to thank Dr. Seong H. Kim, Department ofChemical Engineering, Pennsylvania State University, forhelping with the AFM measurements, and Dr. David A.Antonetti, Department of Cellular and Molecular Physiology,Pennsylvania State University, for kindly providing ZO-1antibody.

References and Notes(1) Pardridge, W. M. AdV. Drug DeliVery ReV. 1999, 36, 299–321.(2) Jefferies, W. A.; Brandon, M. R.; Hunt, S. V.; Williams, A. F.; Gatter,

K. C.; Mason, D. Y. Nature 1984, 312, 162–163.(3) Huber, J. D.; Egleton, R. D.; Davis, T. P. Trends Neurosci. 2001, 24,

719–725.(4) Pan, W.; Banks, W. A.; Kastin, A. J. Brain Res. 1998, 788, 87–94.(5) Plotkin, S. R.; Banks, W. A.; Kastin, A. J. J. Neuroimmunol. 1996,

67, 41–47.(6) Tomas-Camardiel, M.; Venero, J. L.; Herrera, A. J.; De Pablos, R. M.;

Pintor-Toro, J. A.; Machado, A.; Cano, J. J. Neurosci. Res. 2005, 80,235–246.

(7) Gumerlock, M. K.; Belshe, B. D.; Madsen, R.; Watts, C. J. Neurooncol.1992, 12, 33–46.

(8) Rapoport, S. I. Expert. Opin. InVest. Drugs 2001, 10, 1809–1818.(9) Born, J.; Lange, T.; Kern, W.; McGregor, G. P.; Bickel, U.; Fehm,

H. L. Nat. Neurosci. 2002, 5, 514–516.(10) Chamberlain, M. C. J. Neurooncol. 1998, 38, 135–140.(11) Kusuhara, H.; Sugiyama, Y. Drug DiscoVery Today 2001, 6, 150–

156.(12) Kemper, E. M.; Boogerd, W.; Thuis, I.; Beijnen, J. H.; van Tellingen,

O. Cancer Treat. ReV. 2004, 30, 415–423.(13) Smith, M. W.; Gumbleton, M. J. Drug Targeting 2006, 14, 191–214.(14) Pardridge, W. M. Nat. ReV. Drug DiscoVery 2002, 1, 131–139.(15) Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Pharmacol. ReV. 2002, 54, 561–

587.(16) Thole, M.; Nobmanna, S.; Huwyler, J.; Bartmann, A.; Fricker, G. J.

Drug Targeting 2002, 10, 337–344.(17) Huwyler, J.; Yang, J.; Pardridge, W. M. J. Pharmacol. Exp. Ther.

1997, 282, 1541–1546.(18) Shi, N. Y.; Pardridge, W. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,

7567–7572.(19) Gao, Z. G.; Lee, D. H.; Kim, D. I.; Bae, Y. H. J. Drug Targeting

2005, 13, 391–397.(20) Lockman, P. R.; Mumper, R. J.; Khan, M. A.; Allen, D. D. Drug

DeV. Ind. Pharm. 2002, 28, 1–13.(21) Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski,

W. E. J. Controlled Release 2001, 70, 1–20.(22) Kreuter, J. AdV. Drug DeliVery ReV. 2001, 47, 65–81.(23) Vinogradov, S. V.; Batrakova, E. V.; Kabanov, A. V. Bioconjugate

Chem. 2004, 15, 50–60.(24) Lockman, P. R.; Koziara, J. M.; Mumper, R. J.; Allen, D. D. J. Drug

Targeting 2004, 12, 635–641.(25) Gulyaev, A. E.; Gelperina, S. E.; Skidan, I. N.; Antropov, A. S.;

Kivman, G. Y.; Kreuter, J. Pharm. Res. 1999, 16, 1564–1569.(26) Kreuter, J. J. Nanosci. Nanotechnol. 2004, 4, 484–488.(27) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. ReV. 2001,

53, 283–318.(28) Brownlie, A.; Uchegbu, I. F.; Schatzlein, A. G. Int. J. Pharm. 2004,

274, 41–52.(29) Szejtli, J. Chem. ReV. 1998, 98, 1743–1753.(30) Hirayama, F.; Uekama, K. AdV. Drug DeliVery ReV. 1999, 36, 125–

141.(31) Irie, T.; Uekama, K. J. Pharm. Sci. 1997, 86, 147–162.(32) Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Pitha, J Biochem.

Eur. J. 1989, 186, 17–22.(33) Perrin, J. H.; Field, F. P.; Hansen, D. A.; Mufson, R. A.; Torosian, G.

Res. Commun. Chem. Pathol. Pharmacol. 1978, 19, 373–6.(34) Brewster, M. E.; Loftsson, T. AdV. Drug DeliVery ReV. 2007, 59, 645–

66.(35) Davis, M. E.; Brewster, M. E. Nat. ReV. Drug DiscoVery 2004, 3,

1023–1035.(36) Szente, L.; Szejtli, J. AdV. Drug DeliVery ReV. 1999, 36, 17–28.


(37) Leroylechat, F.; Wouessidjewe, D.; Andreux, J. P.; Puisieux, F.;Duchene, D. Int. J. Pharm. 1994, 101, 97–103.

(38) Frijlink, H. W.; Visser, J.; Hefting, N. R.; Oosting, R.; Meijer, D. K. F.;Lerk, C. F. Pharm. Res. 1990, 7, 1248.

(39) Rajewski, R. A.; Traiger, G.; Bresnahan, J.; Jaberaboansari, P.; Stella,V. J.; Thompson, D. O. J. Pharm. Sci. 1995, 84, 927–932.

(40) Li, J. S.; Xiao, H. N.; Li, J. H.; Zhong, Y. P. Int. J. Pharm. 2004,278, 329–342.

(41) Tilloy, S.; Monnaert, V.; Fenart, L.; Bricout, H.; Cecchelli, R.;Monflier, E. Bioorg. Med. Chem. Lett. 2006, 16, 2154–2157.

(42) Binkowski-Machut, C.; Hapiot, F.; Martin, P.; Cecchelli, R.; Monflier,E. Bioorg. Med. Chem. Lett. 2006, 16, 1784–1787.

(43) Zhong, N.; Ohvo-Rekila, H.; Ramstedt, B.; Slotte, J. P.; Bittman, R.Langmuir 2001, 17, 5319–5323.

(44) Chang, Y. S.; Munn, L. L.; Hillsley, M. V.; Dull, R. O.; Yuan, J.;Lakshminarayanan, S.; Gardner, T. W.; Jain, R. K.; Tarbell, J. M.MicroVasc. Res. 2000, 59, 265–277.

(45) Husain, N.; Ndou, T. T.; Delapena, A. M.; Warner, I. M. Appl.Spectrosc. 1992, 46, 652–658.

(46) Zheng, Y.; Zuo, Z.; Chow, A. H. L. Int. J. Pharm. 2006, 309, 123–128.

(47) Stover, T. C.; Kim, Y. S.; Lowe, T. L.; Kester, M. Biomaterials 2008,29, 359–369.

(48) Monnaert, V.; Betbeder, D.; Fenart, L.; Bricout, H.; Lenfant, A. M.;Landry, C.; Cecchelli, R.; Monflier, E.; Tilloy, S. J. Pharmacol. Exp.Ther. 2004, 311, 1115–1120.

(49) Machut-Binkowski, C.; Hapiot, F.; Cecchelli, R.; Martin, P.; Monflier,E. J. Inclusion Phenom. Macrocyclic Chem. 2007, 57, 567–572.

(50) Monnaert, V.; Tilloy, S.; Bricout, H.; Fenart, L.; Cecchelli, R.;Monflier, E. J. Pharmacol. Exp. Ther. 2004, 310, 745–751.

(51) Arima, H.; Yunomae, K.; Morikawa, T.; Hirayama, F.; Uekama, K.Pharm. Res. 2004, 21, 625–634.

(52) Jodoin, J.; Demeule, M.; Fenart, L.; Cecchelli, R.; Farmer, S.; Linton,K. J.; Higgins, C. F.; Beliveau, R. J. Neurochem. 2003, 87, 1010–1023.

(53) Lu, W.; Wan, J.; She, Z.; Jiang, X. J. Controlled Release 2007, 118,38–53.

(54) Fenart, L.; Casanova, A.; Dehouck, B.; Duhem, C.; Slupek, S.;Cecchelli, R.; Betbeder, D. J. Pharmacol. Exp. Ther. 1999, 291, 1017–1022.

(55) Karakotchian, M.; Fraser, I. S. Micron 2007, 38, 632–636.(56) Burns, E. M.; Dobben, G. D.; Kruckeberg, T. W.; Gaetano, P. K. AdV.

Neurol. 1981, 30, 159–165.(57) Lu, W.; Tan, Y. Z.; Hu, K. L.; Jiang, X. G. Int. J. Pharm. 2005, 295,

247–260.(58) Tsuji, A. Ther. Drug Monit. 1998, 20, 588–590.

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