β‑Cyclodextrin-poly(β-Amino Ester) Nanoparticles for Sustained DrugDelivery across the Blood−Brain BarrierEun Seok Gil,†,§ Linfeng Wu,†,‡ Lichong Xu,† and Tao Lu Lowe*,†,‡

†Department of Surgery, Pennsylvania State University, Hershey, Pennsylvania 17033, United States‡Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States

ABSTRACT: Novel biodegradable polymeric nanoparticles composed of β-cyclo-dextrin and poly(β-amino ester) segments have been developed for sustained drugdelivery across the blood−brain barrier (BBB). The nanoparticles have beensynthesized by cross-linking β-cyclodextrin with poly(β-amino ester) via the Michaeladdition method. The chemical, physical, and degradation properties of thenanoparticles have been characterized by matrix-assisted laser desoption/ionizationtime-of-flight, attenuated total reflectance Fourier transform infrared spectroscopy,nuclear magnetic resonance, dynamic light scattering, and atomic force microscopy techniques. Bovine and human brainmicrovascular endothelial cell monolayers have been constructed as in vitro BBB models. Preliminary results show that thenanoparticles do not affect the integrity of the in vitro BBB models, and the nanoparticles have much higher permeability thandextran control across the in vitro BBB models. Doxorubicin has been loaded into the nanoparticles with a loading efficiency of86%, and can be released from the nanoparticles for at least one month. The developed β-cyclodextrin-poly(β-amino ester)nanoparticles might be useful as drug carriers for transporting drugs across the BBB to treat chronic diseases in the brain.

1. INTRODUCTION

The formidable obstacle faced in brain drug delivery is theblood−brain barrier (BBB).1,2 The BBB is composed ofendothelial cells that line the cerebral microvessels and, inthe meantime, are buttressed by astrocyte and pericyte cells.3 Itfunctions as a physical barrier due to the tight junctionsbetween the adjacent endothelial cells, a selective “transportbarrier” because of the specific transport systems on the luminaland abluminal membranes of these endothelial cells, and a“metabolic barrier” due to the presence of intracellular andextracellular enzymes.1,2 Nonselected substances are extremelydifficult to penetrate into the brain by crossing the BBB, whilenutrients in the bloodstream and waste generated in the braintissues can cross the BBB through different pathways. It hasbeen well-known that 98% of small molecular weight drugs andalmost 100% of large molecular weight drugs cannot cross theBBB by themselves.2 It remains a big challenge how toefficiently deliver drugs into the brain to treat various braindiseases like brain cancers, Parkinson disease and Alzheimer’sdisease. Through the past two decades’ research in this area, ithas been recognized that successful brain drug delivery systemsshould take advantage of the carrier-mediated transport,receptor-mediated transcytosis or adsorptive-mediated trans-cytosis pathways found in the BBB.4−6 In particular, polymericnanoparticles have been designed to exploit these transportpathways for brain drug delivery via systemic administra-tion.7−11

The most extensively studied polymeric nanoparticles fordrug delivery across the BBB are made of biodegradablesynthetic polymers7,8,12−17 [e.g., polyalkylcyanoacrylate, poly-(D,L-lactide-co-glycolide) (PLGA) and poly(ethyleneglycol)−poly(lactide)], polyamines18−25 [e.g., poly(ethylenimine), poly-

(amidoamine), and poly(propylene imine)], and albumin.26,27

The biodegradable nanoparticles (e.g., polyalkylcyanoacrylate,and PLGA) can achieve long-term drug release, but they needspecific antibodies, receptor proteins, cell-penetration peptidesor coating agents such as polysorbate 80 to be conjugated orincorporated into the nanoparticles to enable them to cross theBBB.8,12,13,16,17 Polyamine-based nanoparticles can cross theBBB by themselves, but their toxicity is a general concern.28−30

Albumin-based nanoparticles need to be conjugated withapolipoproteins in order to be able to cross the BBB.27

Moreover, polyamine, and albumin nanoparticles also haveproblems in not being able to achieve sustained drug delivery.In addition to exploring the polymeric nanoparticles mentionedabove, efforts are continuously made to design novel polymericnanoparticles for brain drug delivery to achieve prolongedbioavailability, high loading efficiency, and sustained drugrelease.β-cyclodextrin (β-CD) is cyclic oligosaccharides with seven

glucose unites that form a hydrophobic inner cavity and ahydrophilic outer surface. β-CD cannot be degraded by humanenzymes. It is excreted intact via the kidney after intravenousadministration. β-CD can form inclusion complexes withhydrophobic molecules or large molecules having hydrophobicsegments. β-CD and functionalized β-CD have been extensivelyused in drug formulations such as eye drops, nasal spray, andoral and intravenous solutions to enhance the solubility andstability of hydrophobic drugs such as anandamide, piroxicam,and dexamethasone, and reduce their irritancy.31,32 Moreover,

Received: June 5, 2012Revised: September 4, 2012Published: October 15, 2012

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different β-CD-based polymers have been synthesized andexplored for the delivery of anticancer drugs.32−36 For example,camptothecin has been conjugated to poly(dideoxy-β-cyclo-dextrin-biscysteine-polyethyleneglycol) linear polymers,33,34

and the resulting self-assembled nanoparticles (ca. 30−40nm) have been tested in clinical trial to treat pancreatic cancer,renal cancer, nonsmall cell lung cancer, and ovarian cancer.35

Anticancer drugs erlotinib and suberoylanilide hydroxamic acidwere loaded into β-CD conjugated to dendritic polyaminepolymer for brain cancer treatment. The conjugation of β-CDto dendritic polyamine polymer significantly reduced thecytotoxicity of polyamine.36 Recently, we also developedquaternary ammonium β-cyclodextrin (QAβCD) nanoparticlesfor drug delivery across the BBB.37−39 When doxorubicin(DOX) was loaded into these nanoparticles, these nanoparticlesenhanced the permeability of DOX across the in vitro BBBmodel and in the meantime mask its cytotoxicity to theendothelial cells of the BBB. As biodegradable polymers canachieve sustained drug release and polyamine can enhance drugpermeability across the BBB, in this study we have designednew β-CD-based nanoparticles by introducing poly(β-aminoester), which contains ester bond and amine,40−47 into β-CD.These new β-cyclodextrin-poly(β-amino ester) (CD-p-AE)nanoparticles are cross-linked, hydrolytically degradable, andcationically charged at physiological condition.In this paper, the synthesis of the CD-p-AE nanoparticles was

described and carried out. The chemical, physical, anddegradation properties of the nanoparticles were characterizedby matrix-assisted laser desoption/ionization time-of-flight(MALDI-TOF), attenuated total reflectance Fourier transforminfrared spectroscopy (ATR-FTIR), nuclear magnetic reso-nance (NMR), dynamic light scattering (DLS), and atomicforce microscopy (AFM) techniques. The effects of thenanoparticles on the integrity of the BBB were investigated

using an in vitro model constructed from bovine brainmicrovascular endothelial cells (BBMVECs). In vitro BBBmodels constructed from BBMVECs as well as human brainmicrovascular endothelial cells (HBMVECs) were used toinvestigate the permeability of the nanoparticles across theBBB. DOX was loaded into the nanoparticles as a modelhydrophobic drug. The release of DOX from the CD-p-AEnanoparticles was studied using the dialysis membrane methodin a period of 40 days.

2. EXPERIMENTAL METHODS2.1. Materials. Ether and tetrahydrofuran (THF) were bought

from Fisher Scientific (Fairlawn, NJ). Fetal bovine serum (FBS) wasobtained from Hyclone (Logan, UT), and ENDO GRO was orderedfrom VEC Technologies (Rensselaer, NY). A CSC certified mediumkit including CSC coating solution for ACBRI-376 cells (HBMVEC)was bought from Cell Systems (Kirkland WA). The followingmaterials were obtained from Sigma-Aldrich, Inc., St. Louis, MO: β-CD, phosphate buffered saline (PBS, pH 7.4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate(SDS), N,N-dimethylformamide (DMF, HPLC grade), MCDB-131medium, epidermal growth factor (EGF), heparin, antibiotic/antimycotic (penicillin G sodium salt), bovine serum albumin(BSA), FITC-dextran (Mw = 4,000 g·mol−1), verapamil, Luciferyellow CH dilithium salt, rhodamine 123 (R123), dimethyl sulfoxide-d6 (DMSO-d6, 99.9%), acryloyl chloride (96%), 1,4-butanedioldiacrylate, N,N-dimethylethylenediamine, chloroform, ethyl acetate,anhydrous 1-methyl-2-pyrrolidinone (NMP), acetone, 5-(4,6-dichlor-otriazinyl) aminofluorescein (5-DTAF), and DOX. All these chemicalswere used as received without further purification unless otherwisenoted. Dialysis membrane (molecular weight cuttoff (MWCO)12000−14000 Da) was bought from Spectrum Laboratories. PolyesterTranswell inserts (24 well-plate, 0.4 μm pore size) were obtained fromCostar (Cambridge, MA).

2.2. Synthesis of Acrylated β-CD Macromer. After being driedin an oven at 80 °C for 24 h, 2.27 g of β-CD was weighted and putinto a 50 mL round-bottom flask. Twelve milliliters of anhydrous

Scheme 1. Schematic Illustration of the Synthesis of CD-p-AE Nanoparticles

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NMP (treated with 4A molecular sieves for at least 1 day) was addedto dissolve β-CD under N2 atmosphere and magnetic stirring. After β-CD was completely dissolved, the flask was placed in an ice−waterbath. Acryloyl chloride (1.990 mL) was added dropwise into the flaskunder N2 protection. The flask was kept in the ice−water bath for 1 hand then at room temperature for 48 h. During the reaction, magneticstirring was used and kept at 200 rpm. This synthesis method isdifferent from that reported in the literature, which needed hightemperature (110 °C) and 2,6,-ditert-butyl-4-methylphenol as astabilizer to prevent free radical polymerization.48 The obtainedreaction mixture was slowly dropped into 180 mL deionized (DI)water to precipitate the product, acrylated β-cyclodextrin (acrylatedCD). After centrifuge, the clear liquid was decanted, and theprecipitants were washed using DI water and then filtered, followedby another two washes using DI water in the filtration funnel. Theobtained solids were dried and kept under vacuum. The dried powderwas soluble in acetone, methanol, and THF.2.3. Characterization of Acrylated CD Macromer. MALDI-

TOF, ATR-FTIR, and 1H NMR were used to characterize thechemical structure of the acrylated CD macromer. MALDI-MS spectrawere obtained using a Voyager Biospectrometry (Voyager DE-PROWorkstation, Perseptive Biosystems, Framingham, MA) equipped witha nitrogen laser radiating at 337 nm with 3 ns pulses. Acrylated CDand 2,5-dihydroxybenzoic acid (matrix) were first dissolved separatelyin a cosolvent of THF and water (9:1 v/v) at 10 mg·ml−1. The twoobtained solutions were then mixed at a 1:9 volume ratio (acrylatedCD solution to matrix solution). Thin film samples for MALDI-MSwere prepared by dropping 5 μL of this mixed solution onto thesample holder plate followed by air drying. The acrylated CDmacromer was also characterized using an ATR (Pike Technologies,Madison, WI)-FTIR (Thermo Nicolet Avetar 370, Madison, WI)spectroscope. The macromer solution was prepared by dissolvingabout 1 mg macromer in 50 μL acetone. Five microliters of thesolution was dropped on the ZnSe crystal of the ATR to form a filmafter air drying. FTIR spectra were recorded in the range of 4000−650cm−1. DMSO-d6 (99.9%) was used as solvent to prepare acrylated CDmacromer samples for the 1H NMR (Bruker Avance 500 MHz NMRspectrometer, Newark, DE) studies, and the central DMSO line wasset at 2.50 ppm.49

2.4. Synthesis of CD-p-AE Nanoparticles. CD-p-AE nano-particles were synthesized from acrylated CD macromer with anaverage substitution of 11.5 per β-CD (since one β-CD has 21hydroxyl groups, the maximum substitution is 21 per β-CD), 1,4-butanediol diacrylate, and N,N-dimethylethyldiamine via Michaeladdition40−42 in a cosolvent of ethyl acetate and chloroform (3:7 v/v) at 65 °C. In a typical synthesis, 89.8 mg acrylated CD was dissolvedin 1.5 mL ethyl acetate and 3.5 mL chloroform in a 25 mL round-bottom-flask with magnetic stirring at 600 rpm. 1,4-Butanedioldiacrylate (252 μL) and N,N-dimethylethyldiamine (207 μL) wereadded to the mixture in the flask. After connected to a coolingcondenser, the reaction was carried out in an oil bath under magneticstirring at 600 rpm at 65 °C for 12 h. After solvent was removed byevaporation, the resulting solids were dissolved in chloroform. Thechloroform solution was add into ether, and the obtained CD-p-AEprecipitates was dispersed in DI water, dialysized (MWCO 12 000−14000) against DI water for 6 h in a dialysis tubing, and then lyophilized.During the dialysis, the water was changed every hour. Scheme 1demonstrates the synthesis of the CD-p-AE nanoparticles.2.5. Characterization of CD-p-AE Nanoparticles. 2.5.1. Chem-

ical Structure. ATR-FTIR and 1H NMR were used to characterize thechemical structure of CD-p-AE nanoparticles following the samemethods described above for characterizing the acrylated CDmacromer.2.5.2. Particle Size. The size of CD-p-AE nanoparticles was

determined using DLS and AFM techniques. The hydrodynamic radii(Rh) of CD-p-AE nanoparticles in PBS (pH = 7.4) at 2 mg·ml−1 wasmeasured using a DLS instrument equipped with an ALV-CGS-8Fcompact goniometer system, an ALV-5000/EPP multiple tau real timecorrelator, a JDS Uniphase helium/neon laser (633 nm, 35 mW,Manteca, CA), and an ALV-5000/E/WIN software (ALV, Germany).

Autocorrelation functions of the nanoparticle suspention at 90°scattering angle were collected. The data were fitted using aCumulants method to derive the average hydrodynamic radii Rh ofthe CD-p-AE nanoparticles, and the CONTIN program (a Laplaceinverse program) was used to analyze the Rh distribution of thenanoparticles. The size of the CD-p-AE nanoparticles was alsocharacterized by AFM equipped with a Digital Instruments NanoscopeIIIa controller (Veeco Instruments Inc., Santa Barbara, CA). Driednanoprticles were dispersed in DI water, deposited on a freshly cleavedmica surface, and dried at room temperature. AFM images wereobtained in tapping mode, and analyzed by using Nanoscope imagingprogram. A total of 100 nanoparticles in a 5 × 5 μm area of AFMimages were used in the analysis, and the diameters of thenanoparticles were calculated and reported as mean ± standarddeviation (S.D.).

2.5.3. Hydrolytic Degradation of CD-p-AE Nanoparticles. Thedegradation study of the CD-p-AE nanoparticles was carried out bydispersing the nanoparticles in PBS (pH 7.4) buffer solution at aconcentration of 20 mg·ml−1 in a glass vial and keeping the vial in awater bath at 37 °C for 75 days. At selected time points, 5 μL solutionwas withdrawn from the vial and dropped onto the ZnSe crystal ofATR to prepare a dry thin film, and FTIR spectra were recorded in therange of 4000 to 650 cm−1.

2.6. Cells and Media. BBMVECs were cultured as describedpreviously.37 Briefly, BBMVECs (Cell Applications Inc., San Diego,CA) were seeded in bovine fibronectin-coated T25 or T75 flasks at adensity of 5000 cells·cm−2, and cultured in MCDB-131 mediumcontaining 10% FBS, 10 ng·ml−1 EGF, 0.2 mg·ml−1 ENDO GRO, 0.9mg·ml−1 heparin, and antibiotic/antimycotic (penicillin G sodium salt10 μg·ml−1) at 37 °C with 95% humidity and 5% CO2. The mediumwas changed every 2 days. The cells were harvested with trypsin(0.05% trypsin with 0.4 mM EDTA) when they were 80% confluenton the fourth day.

HBMVECs (ACBRI-376 cells) were cultured according to theinstruction provided by Cell Systems. Briefly, HBMVECs (CellSystems, Kirkland, WA) were seeded in coated T25 or T75 flasks at adensity of 5000 cells·cm−2, and cultured in CSC-certified medium at37 °C with 95% humidity and 5% CO2. The medium was changedevery 2 days. The cells were harvested with trypsin (0.05% trypsin with0.4 mM EDTA) when they were 80% confluent.

2.7. In Vitro Evaluations of Effects of CD-p-AE Nanoparticleson the BBB Integrity. 2.7.1. Cytotoxicity Study of CD-p-AENanoparticles. MTT assay was used to evaluate the cytotoxicity ofCD-p-AE nanoparticles to BBMVECs and HBMVECs. EitherBBMVECs or HBMVECs were seeded at 50 000 cells·cm−2 in bovinefibronectin (for BBMVECs) or CSC coating (for HBMVECs)solution-coated 96-well plates and grown in 100 μL culture mediumfor 2 days. The cells were then incubated with CD-p-AE nanoparticlesat different nanoparticle concentrations in culture medium at 37 °C for24 h. Ten microliters of MTT/medium solution (5 mg·ml−1) wasadded to each well, and 4 h later the medium in each well wascompletely replaced with 100 μL 50% DMF/20% SDS (pH 4.7)aqueous solution. After overnight incubation, the absorbance at 570nm in each well was measured using a μQuant microplate reader(Biotek Instruments, Winooski, VT) with background subtraction.Cell viability was calculated using the following formula: cell viability(%) = (Absorbance of cells treated with nanoparticles − Absorbanceof free medium alone)/(Absorbance of control untreated cells −Absorbance of free medium alone) × 100. The viability of cells leftuntreated was normalized to 100%. Averages from four replicate wellswere used for each sample and control.

2.7.2. Lucifer Yellow Permeability. A paracellular permeablemarker, Lucifer yellow CH dilithium salt, was used to test whetherCD-p-AE nanoparticles have any effects on the BBB tight junctionintegrity.37,50 BBMVECs were seeded at a density of 50 000 cells·cm−2

on 24 well-plate Transwell inserts (polyester, 0.4 μm pore size) coatedwith bovine fibronectin and cultured in MCDB-131 mediumcontaining 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 sodiumsalt 10 μg·ml−1) at 37 °C with 95% humidity and 5% CO2. Culture

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medium in the wells was changed every 2 days. Confluent BBMVECmonolayers were obtained on the 10th day after seeding. The resultingconfluent BBMVEC monolayers were used as an open two-compartment vertical side-by-side dynamic BBB model for perme-ability studies.Lucifer yellow was dissolved in cell culture medium with or without

CD-p-AE nanoparticles, and then added to the apical chamber of eachTranswell. The final concentrations of Lucifer yellow and CD-p-AEnanoparticles in the apical chamber are 100 μM and 100 μg·ml−1,respectively. Transport experiments were conducted in the apical tobasal direction at 37 °C for three and a half hours. Every 30 min, 30 μLmedium was taken from the basolateral chamber and replaced with 30μL of fresh medium. At three and a half hours, 30 μL medium wastaken from both the apical and basolateral chambers. Aliquots werequantified on a Spectramax EM microplate spectrofluorometer(Molecular Devices Co., Sunnyvale, CA) at 430 nm of excitationand 530 nm of emission. The permeability (P0) of Lucifer yellowacross the BBMVEC monolayer was calculated by the followingformula37,51

= ΔP F t V F A[( / ) ]/( )0 A A L (1)

where Po, FA, VA, FL, and A are the permeability coefficient, thebasolateral fluorescence of the solute over Δt time, the fluid volume ofthe basolateral chamber, the apical fluorescence of the solute, and thesurface area of the filter, respectively. Three replicates were performedfor each condition.2.7.3. P-Glyocprotein (P-gp) Efflux Activity Using R123 Efflux

Assay. The cellular uptake of R123, a fluorescent P-gp efflux activitymarker, by BBMVECs was conducted to evaluate the effect of CD-p-AE nanoparticles on the extracellular property of the BBB.37 Generally,BBMVEC were seeded on 24-well plates at a density of 50 000 cells/well and cultured for 2 days. The resulting cell monolayers weretreated with CD-p-AE nanoparticles (100 μg·ml−1) or a P-gp inhibitor,verapamil (100 μM), for 2 h. The medium was then removed, and the

cells were incubated with R123 of 10 μM in culture medium for 2 h.After removing R123 solution, the cell monolayers were washed threetimes with cold PBS (pH 7.4) before BBMVECs were solubilizedusing 100 μL of cell culture lysis buffer. The cellular uptake of R123was determined using fluorescent measurement and normalized percellular content of protein as described in an early paper from ourgroup.37 Pierce (Rockford, IL) BCA protein assay was used todetermine the protein content in each sample.

2.8. In Vitro Study of Permeability of CD-p-AE NanoparticlesAcross the BBB. In order to determine the capability of CD-p-AEnanoparticles to cross the BBB, confluent BBMVEC and HBMVECmonolayers were constructed on coated polyester Transwell inserts(24 well-plate, 0.4 μm pore size). DTAF-labeled CD-p-AE nano-particles (Details about labeling the nanoparticles using DTAF can befound in our previous publication.37), or fluorescein isothiocyanate(FITC)-dextran control (Mw = 4000 g·mol−1) was dispersed ordissolved in culture medium and added to the apical chamber of eachwell at 100 μg·ml−1. Their permeability across the two types ofmonolayers at 37 °C was measured and quantified as described inSection 2.7.2.

2.9. Drug Loading into CD-p-AE Nanoparticles. CD-p-AEnanoparticle aqueous suspension (20 mg·ml−1) was mixed with DOXaqueous solution (1.16 mg·ml−1) at an equal volume ratio for 18 h.The resulting mixture suspension was placed in dialysis membranetubing (MWCO 12 000−14 000 Da), dialyzed against DI waterovernight, and dried by lyophilization. The fluorescence intensity ofthe free DOX in the dialysate after dialysis was measured, and then theamount of the free Dox was calculated using a standard calibrationcurve. The amount of DOX loaded in the nanoparticles was calculatedby subtracting the free DOX amount in the dialysate from the initialfeeding amount. The loading efficiency of DOX in the nanoparticleswas calculated by dividing the amount of DOX loaded in thenanoparticles by the initial DOX feeding amount. The loading contentof DOX in the nanoparticles was calculated by dividing the amount of

Figure 1. MALDI-MS (A), ATR-FTIR (B), and 1H NMR (C) spectra of acrylated CD.

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DOX loaded in the nanoparticles by the total amount of the DOX−nanoparticle system (g DOX/g DOX−nanoparticle).2.10. In Vitro Drug Release from CD-p-AE Nanoparticles.

The release of DOX from CD-p-AE nanoparticles was conducted inPBS (pH 7.4) at 37 °C using the dialysis method. Briefly, drug loadedCD-p-AE nanoparticles were dispersed in PBS (pH 7.4) at 2 mg·ml−1,and then loaded into dialysis membrane tubing (WMCO 12 000−14000). The sealed membrane tubing was immersed into PBS (pH 7.4)release solution in a vial at 37 °C. At each scheduled time point, thefluorescence intensity of the PBS release solution was measured, andthe release medium was completely replaced with fresh PBS releasemedium. Three replicate experiments were performed spontaneously.2.11. Statistical Methods. Data were reported as mean ±

standard deviation (S.D.) from at least three separate experiments forthe size, degradation, toxicity, permeability, and release studies, and atleast 100 particles for the AFM studies. Two-tailed Student’s t test wasused to analyze the differences between treatment groups. Astatistically significant difference was reported if p = 0.05 or less.

3. RESULTS AND DISCUSSION

3.1. Synthesis and Characterization of Acrylated CDMacromer. β-CD was first converted to acrylated CDmacromers by reacting it with acryloyl chloride in NMP(Scheme 1A). Acrylated CD macromers with different averagesubstitution can be obtained by adjusting the molar ratio ofacryloyl chloride to β-CD. The successful synthesis of acrylatedCD was confirmed by MALDI-MS, FTIR, and 1H NMRspectra. In the MALDI-MS spectrum, peaks appeared in therange of 1587 to 2021 m/z for the acrylated CD macromers(Figure 1A). Therefore, the substitution varied from 8 to 16 perβ-CD for these acrylated CD macromers, with an averagesubstitution of 11.5 ± 0.5. Figure 1B shows typical FTIRspectra for unmodified β-CD and acrylated CD macromers.Two characteristic peaks for unmodified β-CD, 1153 cm−1

(vC−O−C in the glycosidic bridges) and 1026 cm−1 (coupled

vC−C and vC−O),52 were still observed in the spectrum foracrylated CD macromers with a little shift in positions if any,while one characteristic peak 1080 cm−1 (vC−O near theprimary hydroxyl groups) for unmodified β-CD was reduced/disappeared after the reaction due to the formation of esterbonds in acrylated CD macromers. The peaks 1633 cm−1

(vCC), 1412 cm−1 (vH−CCH2), and 808 cm−1 (vC−H) were the characteristic peaks for the vinyl groups inacrylated CD macromers. Although we also observed a verybroad peak at 1637 cm−1 for unmodified β-CD due to absorbedmoisture in β-CD, the peak at 1633 cm−1 for acrylated CD wasvery sharp and distinguished from the peak 1637 cm−1 sinceacrylated CD is hydrophobic and not soluble in water. Foracrylated CD, the peak at 1724 cm−1 was due to the vibrationof CO bonds in the ester groups with unsaturated α−βcarbons, while peaks 1188 cm−1 and 1296 cm−1 were due to thevibration of C−O in the ester groups with unsaturated α−βcarbons. In the 1H NMR spectrum (Figure 1C) for acrylatedCD, the three proton signals at 6.32, 6.13, and 5.88 ppm couldbe assigned to the hydrogen atoms of −CHCH2 groups inacrylated CD. The residual protons from β-CD appearedbetween 2.85 and 5.07 ppm. The average substitutioncalculated from this spectrum was consistent with the degreedetermined from the MALDI-MS spectra.

3.2. Synthesis and Characterization of CD-p-AENanoparticles. CD-p-AE nanoparticles were synthesized viaMichael addition according to Scheme 1. Macromers with anaverage substitution of about 11.5 per β-CD was used for thesynthesis of CD-p-AE nanoparticles. CD-p-AE nanoparticleswere synthesized in chloroform/ethylacetate cosolvent from theMichael addition of acrylated CD/1,4-butanediol diacrylate andN,N-dimethylethylenediamine. Acrylated CD also serves as the

Figure 2. ATR-FTIR spectrum (A), 1H NMR spectrum (B), hydrodynamic size distribution (C), and AFM image (D) of CD-p-AE nanoparticles.

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cross-linker due to the multiacryloyl groups per β-CD asillustrated in Scheme 1.The FTIR spectrum (Figure 2A) for CD-p-AE nanoparticles

clearly demonstrates the existence of β-CD segments in thenanoparticles since peaks at 1153 cm−1 (vC−O) and 1030 cm−1

(coupled vC−C and vC−O) are characteristic peaks for β-CD.After Michael addition, peaks 1633 cm−1 (vCC) and 808cm−1 (vC−H) observed for acrylated CD disappeared, andpeaks due to the ester group shifted from 1724 cm−1 to 1728cm−1 for vibration of CO, from 1296 cm−1 to 1257 cm−1 forvibration of C−O, and disappeared at 1188 cm−1. The peak1412 cm−1 due to vH−CCH2 of the acrylated CDmacromers disappeared, and a new peak appeared at 1462

cm−1 due to the formation of −CH2− groups after CD-p-AEnanoparticles were synthesized by the Michael additionmethod.

1H NMR result (Figure 2B) showed that the protons from β-CD segments appeared in the range of 2.8 to 5.0 ppm. Thedistinguish proton signals (Hi and Hj) confirmed the chemicalincorporation of 1,4-butanediol diacrylate into the nano-particles. The proton peaks (Hp, Hm, Hn) confirmed thechemical incorporation of N,N-dimethylethyldiamine into thenanoparticles.The sizes of the synthesized CD-p-AE nanoparticles were

characterized using DLS and AFM. The average hydrodynamicradius Rh of the CD-p-AE nanoparticles was 77.8 ± 2.8 nm in

Figure 3. (A) ATR-FTIR peaks at 1728 cm−1 (vCO of ester group) and 1153 cm−1 (vC−O−C of glycosidic bridges) of CD-p-AE nanoparticlesafter the nanoparticles degrade in PBS (pH 7.4) at 20 mg·ml−1 for 0, 5, 15, and 64 days. (B) ATR-FTIR peak intensities at 1728 cm−1 normalized bythose at 1153 cm−1 are plotted as a function of time when CD-p-AE nanoparticles degrade in PBS (pH 7.4) at 20 mg·ml−1 for a period of 75 days.

Figure 4. Effect of CD-p-AE nanoparticles on the cell viability of BBMVEC (A) and HBMVEC (B) after 24 h incubation. Effect of CD-p-AEnanoparticles at 100 μg·ml−1 on the permeability of Lucifer yellow (100 μM) across BBMVEC monolayer (C); and P-gp efflux activity to R123 withor without CD-p-AE nanoparticles (D). Results represent the mean ± SD of three measurements. *: p < 0.05.

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PBS (pH 7.4) determined by a DLS Cumulants method. TheRh distribution of the nanoparticles was analyzed by a DLSCONTIN program and is demonstrated in Figure 2C. The sizeof the CD-p-AE nanoparticles was calculated to be 43.3 ± 7.5nm in diameter by using 100 nanoparticles captured in AFMimages. The representative AFM image of the nanoparticles isshown in Figure 2D. The reason why the size measured by DLSwas higher than that measured by AFM could be possiblyattributed to the fact that CD-p-AE nanoparticles were dry inthe AFM measurement but fully hydrated in the DLSmeasurement.We monitored the hydrolytic degradation of CD-p-AE

nanoparticles as a function of time in PBS (pH 7.4) at 20mg·ml−1 using ATR-FTIR, as shown in Figure 3. In Figure 3A,it can be clearly seen that the intensity of ester CO stretchingat 1728 cm−1 decreased with time. Figure 3B shows that theFTIR peak intensities at 1728 cm−1 normalized by those at1153 cm−1 (vC−O−C of glycosidic bridges) continuouslydecreased for 2 months. These results suggested that the estergroups incorporated in CD-p-AE nanoparticles were continu-ously hydrolyzed, implying the nanoparticles are degraded withtime at physiological condition. Furthermore, the curve inFigure 3B shows three modes of decrease of the FTIR intensitywith time: fast during the first day (50% decrease), mediumfrom day 1 to 30 (45% decrease), and slow from day 30 to 60(5% decrease). These results indicated that the nanoparticles’degradation rate was very fast initially during the first day,became moderately fast during the first month, and then sloweddown during the second month.3.3. In Vitro Study of Effects of Nanoparticles on the

Integrity of the BBB. 3.3.1. In vitro Study of Cytotoxicity ofCD-p-AE Nanoparticles. For new nanoparticles designed forbrain drug delivery, it is important to know the cytotoxicity ofthese nanoparticles to brain microvascular endothelial cells.MTT assay is an extensively used method to evaluate the cellviability in vitro. The basic principal underlying MTT assay isthat metabolic dysfunction in the cells caused by toxic materialsleads to decreased activity of enzymes, which in turn reducesthe transformation of MTT to formazan in mitochondria. Thecytotoxicity studies showed that CD-p-AE nanoparticles did notdecrease the cell viability of BBMVEC (Figure 4A) andHBMVEC (Figure 4B) measured by MTT assay atconcentration up to 100 μg·ml−1 after 24 h incubation. Onthe basis of these results, we used a concentration of 100μg·ml−1 for CD-p-AE nanoparticles in the following perme-ability and P-gp efflux activity studies.3.3.2. Lucifer Yellow Permeability. Materials may disrupt

the integrity of the BBB without causing any toxicity to thebrain microvascular endothelial cells. Lucifer yellow is aparacellularly permeable marker and has been used to testthe integrity of tight junctions in in vitro BBB monolayers. Wechecked whether CD-p-AE nanoparticles disrupted the tightjunctions in the in vitro BBB by measuring the permeability ofLucifer yellow with or without the presence of CD-p-AEnanoparticles. For this purpose, we constructed an in vitro BBBusing BBMVEC. The results (Figure 4C) showed that in thepresence of CD-p-AE nanoparticles at 100 μg·ml−1, thepermeability of Lucifer yellow did not statistically increase,indicating that the tight junctions in the BBMVEC monolayerkept a good shape in the presence of CD-p-AE nanoparticles.3.3.3. P-gp Efflux Activity Using R123 Efflux Assay. The P-

gp efflux activity in the BBMVEC was studied in the presenceof CD-p-AE nanoparticles or the P-gp inhibitor verapamil. The

results (Figure 4D) showed that the up-take of R123 (asubstrate for P-gp) by BBBMVEC remained essentially at thesame level in the presence of CD-p-AE nanoparticles, but wasdoubled in the presence of P-gp inhibitor verapamil, comparedto the control. These preliminary results suggest that CD-p-AEnanoparticles are generally safe for brain drug delivery withoutdisrupting the BBB at the in vitro level.

3.4. In Vitro Study of Permeability of CD-p-AENanoparticles across the BBB. CD-p-AE nanoparticleswere designed as drug carriers to deliver drugs across theBBB without impairing the integrity of the BBB. Therefore, thepermeability of these nanoparticles alone to the BBB is of greatinterest. To evaluate the permeability of CD-p-AE nano-particles across the BBB, we constructed two in vitro BBBmodels using BBMVEC and HBMVEC monolayers, respec-tively. The permeability study was conducted by examining thetransport of DTAF-labeled CD-p-AE nanoparticles from theapical to the basolateral side of the in vitro BBB monolayers.FITC-labeled dextran with a molecular weight of 4k Da wasused as a control since this dextran is highly permeable to theBBB. The dextran tested here had a permeability of 4.2 ± 0.5 ×10−6 cm·s−1 and 5.6 ± 1.1 × 10−6 cm·s−1 across the in vitroBBMVEC and HBMVEC monolayers, respectively (Figure 5).

These permeability coefficients are consistent with thosereported in the literature.37 CD-p-AE nanoparticles had apermeability coefficient of 8.4 ± 0.4 × 10−6 cm·s−1 and 9.1 ±0.4 × 10−6 cm·s−1, which is 100% and 60% higher than that ofthe dextran control, across the in vitro BBMVEC andHBMVEC monolayers, respectively (Figure 5). These resultssuggest that the designed CD-p-AE nanoparticles are highlypermeable to the two in vitro BBB models. Moreover, in termsof the permeability across the BBMVEC monolayer, these CD-p-AE nanoparticles are even slightly better than the previouslyreported QAβCD nanoparticles.37 The pore size of the BBBjunction is generally believed to be less than 3 nm.37,53 SinceCD-p-AE nanoparticles have an average hydrodynamic radiusof 77.8 nm in PBS (pH 7.4), it is more than unlikely that thenanoparticles cross the in vitro BBB models by a paracellularpathway. On the other hand, it is well known that introductionof cationic charges to nanoparticles can increase the BBBpermeability of nanoparticles in vitro and in vivo due toadsorptive-mediated endocytosis.5 Therefore, the high perme-

Figure 5. Permeability of DTAF-labeled CD-p-AE nanoparticles at100 μg·ml−1 across BBMVEC (left stripe) and HBMVEC (rightstripe) monolayers at 37 °C for 3.5 h. Results represent the mean ±SD of three measurements.

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ability across the in vitro BBB for these CD-p-AE nanoparticlescould be partially attributed to the tertiary amine groupsincorporated, which can be positively charged at thephysiological condition. The exact mechanism for CD-p-AEnanoparticles across the BBB is currently under investigation.3.5. In vitro Release of DOX from CD-p-AE Nano-

particles. DOX was used as a model hydrophobic drug andloaded into CD-p-AE nanoparticles with a loading efficiency of86%. The loading content of DOX in the nanoparticles was4.8% (g DOX/g DOX−nanoparticle). Figure 6 shows that the

release of DOX from the CD-p-AE nanoparticles lasted for atleast 4 weeks. The degradation study showed that the CD-p-AEnanoparticles gradually degraded by cleaving the ester bonds inthe backbone for more than 1 month (Figure 3). This longtime degradation might contribute to the long release time forDOX. Moreover, as DOX is hydrophobic and less soluble inwater at pH 7.4, the hydrophobic interaction between DOXand CD-p-AE nanoparticles might be strong enough to limitthe dissociation of DOX from the nanoparticles contributing tothe sustained DOX release.

4. CONCLUSIONS

Novel biodegradable CD-p-AE nanoparticles were synthesizedby cross-linking β-CD with p-AE via the Michael additionmethod. DLS and AFM data illustrated that the synthesizednanoparticles had an average hydrodynamic radius of 77.8 ±2.8 nm in PBS (pH 7.4) and ca. 43.3 ± 7.5 nm in diameter inthe dry state, respectively. The nanoparticles were hydrolyti-cally degradable through the mechanism of the cleavage of theirester bonds in the cross-linked backbones. They were notcytotoxic to HBMVEC and BBMVEC cells at concentration upto at least 100 μg·ml−1 tested by MTT assay. They did notimpair the integrity of the in vitro BBB at 100 μg·ml−1 by notincreasing Lucifer yellow permeability across the BBMVECmonolayer and P-gp efflux activity to R123 in the BBMVECmonolayer. The nanoparticles were very permeable to the invitro BBB at 100 μg·ml−1 with permeability coefficients 100%and 60% higher than that of the dextran control across the invitro BBMVEC and HBMVEC monolayers, respectively. Theycould sustain release of DOX for 1 month. In summary, theseresults suggested that the developed biodegradable CD-p-AEnanoparticles might be useful as drug carriers for transportingdrug across the BBB to treat chronic diseases in the brain.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: tlowe4@uthsc.edu.

Present Address§Department of Biomedical Engineering, Sci & Tech Ctr-4Colby St., Medford, MA 02155. Phone: (617) 627−0900. E-mail Address: Eun.Gil@tufts.edu.

NotesThe authors declare no competing financial interest.To better interpret Figures 1A, 1B and 2A, color graphs areavailable online.

■ ACKNOWLEDGMENTS

This work was financially supported by the National Institute ofHealth, the Wallace H. Coulter Foundation, and the JuvenileDiabetes Research Foundation International.

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Figure 6. Cumulative release of DOX from CD-p-AE nanoparticles inPBS (pH 7.4) AT 37 °C. Results represent the mean ± SD of threemeasurements.

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