In Vitro Assessment of Alkylglyceryl-Functionalized Chitosan

Article pubs.acs.org/Biomac

In Vitro Assessment of Alkylglyceryl-Functionalized Chitosan Nanoparticles as Permeating Vectors for the Blood−Brain Barrier Chun-Fu Lien, Éva Molnár, Petr Toman, John Tsibouklis, Geoffrey J. Pilkington, Dariusz C. Górecki, and Eugen Barbu* School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, PO1 2DT, United Kingdom

ABSTRACT: A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood−brain−barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cellsubstrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood−brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood−brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

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and transient increase in the transport of actives into the brain20,21) could promote increased drug permeability toward the brain, we have reported22 the formulation and physicochemical characterization of nanoparticulate carriers of alkylglyceryl chitosans and alkylglyceryl N,N,N-trimethyl chitosans. For the purpose of assessing toxicity and permeability, we now extend this work to the in vitro characterization of nanoformulations of the same materials and examine their interaction with an in vitro model of BBB that is based on mouse-brain capillary endothelial cells (ECs, bEnd3).

INTRODUCTION While the transport mechanisms of nanoparticles (NPs) through the blood−brain barrier (BBB) are still under debate, it appears that the nanoparticulate dosage forms are potentially useful vehicles for the transport of many biologically active compounds across this barrier.1−6 Among the many biocompatible and biodegradable materials investigated for the preparation of nanoparticulate transporters aimed at drug delivery to the brain, chitosan and its derivatives have received considerable attention.7−16 The potential usefulness of chitosan-derived NPs for such applications has been rationalized in terms of its capability to open tight junctions.17,18 It has been hypothesized that the positive surface charge of chitosan NPs renders the materials transportable across the BBB via adsorptive transcytosis (AMT), which is facilitated by electrostatic interactions with the negative charges of endothelial surfaces.19 Despite recent drug-delivery advances that seem to hold great promise for the treatment of CNS diseases,1 very little is known about the long-term safety of nanomaterials. Rationalized by the hypothesis that the assemblage of a drugcarrier system based on chitosan that had been modified with alkylglycerols (which have been shown to encourage a strong © 2012 American Chemical Society

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EXPERIMENTAL SECTION

General. Low-molecular-weight chitosan (20 000 cps, DDA 75−85%) was obtained from Sigma-Aldrich (Gillingham, U.K.); alkylglyceryl chitosans (where alkyl (OXn) is butyl (OX4), pentyl (OX5), octyl (OX8)) were prepared as described by Molnár et al.22 Polyethersulfone membrane disposable filters (0.2 μm; 25 mm) were purchased from Whatman. NPs were formulated using a magnetic stirring plate IKA RCT and a peristaltic pump Pharmacia Biotech P-1 and separated by centrifugation (1600 g, using a Jouan B4i version Received: December 15, 2011 Revised: March 9, 2012 Published: March 11, 2012 1067

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Alexa Fluor 488 goat anti-rabbit (Fab2 fragment; 4 μg/mL) and nuclear counterstain Hoechst 33342 (Invitrogen Life Technologies; 5 μg/mL). Each incubation step was followed by a 5 min wash step in 1× PBS. ECs were immersed in 1× PBS and visualized using an LSM 710 confocal microscope (Carl Zeiss) equipped with a 40× Aqua immersion lens (fluorescence excitation at 405, 488, and 633 nm; facilitated by a Twin Gate main beam splitter) and collected by a QUASER filter-free spectral detection unit (Carl Zeiss). Images featuring the ZO-1 immunoreactive EC borders were converted to 8-bit format before thresholding positive signal components over the background noise and using the “analyze particles” function (ImageJ software) to delineate morphometric measurements for the perimeter and area of individual EC borders. Subcellular Localization of Chitosan-Based Nanoparticles. ECs were cultured at 2 × 105 cells on glass coverslips that had been placed at the bottom of a six-well plate. Following cell adhesion, the culture medium was changed to a serum-free medium containing EBloaded NPs. After 24 h of incubation with EB-loaded NPs (0.50 mg/mL), ECs were costained with organelle biomarkers following the previously described protocol with the exception that the blocking solution contained primary antibodies (1.25 μg/mL mouse anti-EEA-1 or 1.25 μg/mL mouse anti-Golgin-84 (BD Bioscience)). Detection was by Alexa Fluor 488 goat anti-mouse secondary antibody (4 μg/mL; Invitrogen Life Technologies). For confocal microscopic analysis, ECs were mounted in anti-fade FluorPreserve reagent (Merck). ECIS Monitoring of Endothelial Cell Barrier. ECIS arrays (8W10E; 8-well chambers each with 10 electrodes) had their electrodes stabilized in L-cysteine (10 mM; 15 min), washed in Hank’s balanced salt solution, and coated (after the optimization of the bEnd3 cell response to various barrier enhancers) by exposure (30 min) to Matrigel (1.5 mg/mL; BD Biosciences). After cell culturing, ECs were seeded (2 × 105 cells) into each chamber. After the EC had reached confluence, the growth serum-containing medium was replaced with serum-free medium, and EC barrier integrity was monitored for 120 h over the frequency range 2000 to 32 000 Hz using an ECIS Zθ (Applied Biophysics) instrument. Alkylglyceryl-modified chitosan NPs were added at 24 h and replaced with medium at 48 h; to ascertain the impact of these NPs on the barrier properties of the ECs, changes in electrical resistance were monitored over 96 h. Fluorescein Isothiocyanate-Dextran Permeability Across EC Barrier. ECs were seeded at 2 × 105 cells onto Millicell 24 cell culture filter inserts (1 μM pore sized poly(ethylene terephthalate) (PET) filter; Millipore) and cultured until confluent. ECs were equilibrated in serum-free medium for a day before being treated with a complex of fluorescein isothiocyanate (FITC) and dextran (0.1 mg/mL, 150 kDa; Sigma-Aldrich) and alkylglyceryl-modified chitosan NPs (0.25 mg/mL). By measuring the changes in the concentration of FITC-dextran (FD150), which crosses progressively to the basolateral side of EC and filter inserts, the apparent permeability coefficient (Papp) at each time point was calculated using the formula:25,26 Papp (cm/s) = dQ/dt × VR/(A × C0), where dQ/dt is the flux (μg/sec) of FD150 transported across to the basolateral side, VR is the basolateral volume (0.6 mL), A is the surface area of the filter insert (0.7 cm2), and C0 is the initial concentration of FD150 applied at the apical side (100 μg/mL). Statistical analysis (Origin v.8.1, Origin Lab) was performed using oneway analysis of variance (ANOVA), followed by posthoc Tukey test (unless otherwise stated, p values were set at the 0.05 level). Measurements are presented as mean ± standard error (SE).

centrifuge equipped with a rotor type S40) and further by ultracentrifugation (Beckman Optima Ultracentrifuge equipped with a 70.1 Ti rotor at 146550 g). An ultrasonic bath (FS 400 Decon Ultrasonics, England) was used for the redispersion of NPs in saline phosphate buffer (PBS; pH 7.4, containing 12 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, and 3 mM KCl). Zeta potential, hydrodynamic diameter (expressed as Z-average), and polydispersity index (PDI) were all recorded at 25 °C, following an equilibration time of 5 min using a MALVERN Zetasizer Nano ZS instrument (DLS) equipped with a 633 nm He−Ne laser. For size determinations, a backscattering detection angle of 173° was employed; the measurement of zeta potential was by means of laser Doppler electrophoresis using the Smoluchowski model; each value was obtained as the average of a total of nine measurements (three samples, each being measured three times). Complementary size determinations were conducted using a Nanosight instrument (NTA) equipped with a thermostatted LM-14 unit, a 532 nm laser and NTA 2.2 analytical software. Formulation and Characterization of Nanoparticles. To a solution of modified chitosan (25 mL, 0.15% w/v) in aqueous acetic acid (0.35% w/v) was added dropwise (1 mL/min, by means of a peristaltic pump; magnetic stirring) the TPP solution (15 mL, 0.063% w/v); all solutions had been filtered through a 0.2 μm PES filter prior to use. The NPs that formed were centrifuged (1600 g; 15 min) to remove any large aggregates, and the size of the NPs was determined using dynamic light scattering and NP tracking technology (Malvern and Nanosight, respectively). The NPs were isolated by centrifugation (146550g, 20 °C, 30 min) before being suspended in serum-free media at a specified concentration. Control NPs were prepared from commercially available chitosan (CS/TPP) following an identical procedure. The same procedure was also followed for the preparation of Evans Blue (EB)-loaded NPs (CS-EB/TPP; CS-OXn-EB/TPP), employing the following mixing ratios: chitosan solution (10 mL, 0.28% w/v), EB aqueous solution (8.2 mL, 0.001% w/v), and TPP solution (7.3 mL, 0.063% w/v). Cell Culture. Mouse brain ECs, bEnd3 (previously evaluated as a BBB model),23,24 were obtained from Health Protection Agency Culture Collections. ECs were cultured in DMEM supplemented with fetal bovine serum (10% v/v), nonessential amino acids (1% v/v), L-glutamine (2 mM), sodium pyruvate (1 mM), β-mercapthoethanol (5 μM), and antibiotics (penicillin (100 U/mL) and streptomycin (0.1 mg/mL)). For culturing, ECs were maintained under controlled humidity, at 37 °C and 5% CO2. For this study, ECs were dissociated with TrypLE Express (Invitrogen Life Technologies) and used at passages 31−35. Optimal growth was achieved by seeding EC onto surfaces that had been coated following exposure to Matrigel (BD Biosciences; 1.5 mg/mL, 30 min). ECs were switched to the serumfree medium a day prior to treatments with NPs. Cell Viability Assay. Using a 96-well plate, ECs were seeded (1 × 104 cells per well) in serum-free medium (100 μL) and grown for 24 h. ECs were then exposed (24 or 48 h) to suspensions of alkylglycerylmodified chitosan NPs at specified concentrations. Cytotoxic effects from the NPs were assessed using the PrestoBlue cell viability reagent (Invitrogen Life Technologies). ECs were incubated (37 °C, 30 min) in 1× PrestoBlue solution, and the fluorescence (Ex/Em at 540/ 590 nM) was measured using a PolarStar Optima (BMG Labtech) Fluorimeter. (This colorimetric-cum-fluorescent resazurin-based assay measures the amount of byproduct resorufin within live EC.) The mean percentage of NPs-treated live ECs relative to that of untreated cells is reported as the mean from three independent experiments. Immunocytochemistry and Morphometric Analysis of Zonula Occludens 1 (ZO-1) in Endothelial Cells. ECs were seeded (1 × 106 cell) onto six-well plates. The confluent EC layer had its serum-containing medium replaced, and cells were allowed to equilibrate in fresh serum-free medium for a day prior to treatment with EB-loaded NPs for 24 h. For visualization of tight junctions, ECs were fixed in paraformaldehyde (4% w/v; 10 min, 4 °C), blocked (20 min) in normal goat serum (5% v/v) and Triton X-100 (0.1% v/v), and incubated (40 min, room temperature (RT)) in blocking solution that had been supplemented with rabbit anti-ZO-1 (Zymed; 1 μg/mL). ECs were next incubated (20 min, RT) in blocking solution containing

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RESULTS AND DISCUSSION Alkylglyceryl chitosans (butyl, pentyl, and octyl; Figure 1) have been prepared by the selective grafting of these moieties onto the primary hydroxy functionalities of chitosan; this was achieved via N-phthaloylation, followed by treatment with the corresponding oxirane and subsequent deprotection with hydrazine, as previously reported.22 NPs (Table 1) have been prepared by ionotropic gelation with pentasodium tripolyphosphate (TPP); following purification, these were redispersed in media as required. NPs incorporating the readily detectable fluorescent 1068

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and at prolonged exposure, chitosan-derived NPs were seen to induce cell death to ECs, as also reported by Schütz et al.30 Dose-response experiments using the butylglyceryl-derived NPs have demonstrated that these are less toxic than those prepared from unmodified chitosan (Figure 2A); this effect becomes statistically significant at concentrations >500 μg/mL (Anova, p = 0.05). Varying the length of the alkylglyceryl chain does not appear to have a marked effect upon toxicity (Figure 2B). A similar viability profile has been observed in corresponding 48 h experiments. Consequent to these findings, further work was limited to NPs in the concentration range 0.10 to 0.50 mg/mL. A model barrier consisting of bEnd3 cells has been employed in an effort to simulate the effects of alkyglyceryl-modified chitosan NPs on the BBB. Electric cell-substrate impedance sensing (ECIS) provided the means of assessing the functional responses and morphological changes and hence integrity of the modeled barrier.31 Monitored over 96 h, all NPs were seen to effect decreases in transcellular resistance, but the magnitude of this decrease was sensitive to the structure of the alkylglyceryl chitosan used to prepare these NPs (Figure 3A). ECIS resistance plots (recorded at frequencies ranging from 2000 to 32 000 Hz) have shown that NP formulations prepared from alkylglyceryl chitosans having a high degree of substitution induce a marked drop in the electrical resistance of the bEnd3 cell layers, which in turn suggests an effect at the tight junction level. With most formulations, the removal of NPs at 48 h effected a recovery in the resistance values, but the extent of this recovery appeared highly dependent on NP formulation. Cells treated with a dose of 0.50 mg/mL of NPs prepared from pentyglyceryl chitosan (CS-OX5(67)) did not show any sign of recovery, whereas those treated with control NPs (prepared from unmodified chitosan; CS/TPP) appeared to recover completely. Statistical analysis of the data presented in Table 2 demonstrate the significance of differences at the 48 and 72 h time points (Anova, p = 0.05) between alkylglyceryl-based materials and unmodified chitosan or the control, pointing to a potentially opened state of the EC barrier following treatment with NPs prepared from alkylglyceryl chitosans (especially CSOX5(67)/TPP and CS-OX8(127)/TPP). Whereas there was some indication of alkylglyceryl chain length-induced differences, these were not of statistical significance (Anova; p = 0.05). NPs prepared from butylglyceryl chitosan with a low degree of substitution (CS-OX4(15)) had no significant effect on the

Figure 1. (A) Alkylglyceryl-modified chitosan and (B) SEM images (17 000×) of nanoparticles formulated from normal chitosan (B1) and from butylglyceryl chitosan (CSOX4(95); B2) (scale bars: 1 μm).

Table 1. Characteristics (Size and Zeta Potential) of Nanoparticles dynamic light scattering NP formulation CS/TPP CS-OX4(25) /TPP CS-OX4(95) /TPP CS-OX5(67) /TPP CS-OX8(127) /TPP

NP tracking analysis

Z-av ± SD (nm)

PDI

diam. ± SD (mV)

zeta potential (mV)

143 ± 2 133 ± 3

0.308 0.284

153 ± 75 128 ± 54

40.1 ± 1 36.0 ± 1

151 ± 2

0.200

124 ± 55

36.9 ± 1

100 ± 10

0.227

100 ± 20

39.0 ± 6

188 ± 50

0.367

125 ± 54

42.7 ± 3

anionic dye EB, which does not cross the BBB,27,28 were also prepared for the purpose of visualizing their interactions with cells; EB was selected as a marker for chitosan NPs because release studies (48 h, PBS; UV−Vis detection) had shown that this dye is well-retained within the alkylglyceryl chitosan NPs. In accord with comments from Zaki et al.,29 a study (24 h) of the influence of the degree of butylglyceryl substitution on the cytotoxicity of chitosan to ECs, Figure 2, has shown that the 50% inhibitory concentration (IC50) for both normal chitosan and alkylglyceryl chitosan NPs is ≥0.50 mg/mL. At large doses

Figure 2. (A) Effect of alkylglyceryl-modified chitosan nanoparticles on bEnd3 cell viability. The bEnd3 cells were exposed for 24 h to (A) CS/TPP (◆), CS-OX4(15)/TPP (■), or CS-OX4(95)/TPP (▲) and (B) CS/TPP (◆), CS-OX4(95)/TPP (▲), CS-OX5(67)/TPP (pentagon), or CSOX8(127)/TPP (●) at doses ranging from 1 to 1000 μg and the percentage of viable cells quantified. Data are presented as mean ± standard errors (nine wells per condition). 1069

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Table 2. Electrical Resistance (ohm; mean ± SE) Recorded by ECIS in the bEnd3 Cells at 48 and 72 h (the Respective Time Points Just Before and Just After the Removal of Nanoparticles)a resistance ± SD (Ω) at 48 h

NP formulation control − no NP CS/TPP CS-OX4(95)/TPP CS-OX5(67)/TPP CS-OX8(127) /TPP

759.95 720.75 735.00 525.95 529.45

± ± ± ± ±

45.45 3.05 39.20 11.65* 0.35*

resistance ± SD (Ω) at 72 h 725.95 735.70 639.75 406.15 548.40

± ± ± ± ±

26.85 3.50 97.05 14.65 20.72

a

Data were analyzed via one-way ANOVA with post-hoc Tukey test *p < 0.05; n = 3.

with barrier integrity in a dose-dependent fashion (Figure 3C). CS-OX4(95)/TPP NPs, which effected only minor changes to the morphology of the bEnd3 cell layer, provided the focus of further investigations. Whereas the resistance values showed signs of recovery toward baseline levels after the removal of the NPs, this recovery was not sustainable, especially when larger doses (0.25 to 0.50 mg/mL) of NPs were employed (Figure 3C). Similar results have been obtained for octylglyceryl chitosan NPs. Adding to previous studies concerned with the interaction between glial cells and nanoparticulate formulations,32,33 our ECIS experiments using confluent cultures of primary mouse glial cells (which are components of a functional BBB and participate in the formation of tight junctions)34 have demonstrated that treatment with NPs does not induce changes to the electrical resistance of confluent glial cell structures (derived from brain cerebral cortex of postnatal day 0−1 C57BL/6 mice; data not shown), which in turn implies that the observed variations in electrical resistance are highly specific to ECs. In view of the capability of chitosan NPs to open cellular tight junctions18 and to assess paracellular NP transport, a further evaluation of the influence of butylglyceryl chitosan NPs on the integrity of the adopted BBB model was undertaken by means of an established procedure that monitors the permeability of dextran (150 kDa molecular weight; FD150) that is labeled with the tracer FITC. The capacity of the synthesized NPs to promote the translocation of FITC-dextran through the bEnd3 cell layer is illustrated in Figure 4. Incubation with CS/TPP, CS-OX4(15)/TPP, and CS-OX4(95)/TPP induced increased permeation of FITC through the bEnd3 cell layer, which correlates well with the observed decline in the electrical resistance measured for ECs that had been exposed to CS-OX4(95)/ TPP. Statistical analysis of the time dependence of the transport enhancement ratios associated with the CS-OX4(95) formulation (Figure 4C) pointed to differences being of significance only at the 6 h time point (for alpha 0.1; p = 0,088; Anova one-way, Excel); however, the observed directional effects may be of biological or of clinical importance. Because the permeation of FD150 was evident even at 24 h, it is assumed that NPs prepared from CS-OX4(95)/TPP cause the irreversible disruption in the adopted EC BBB model. Cell viability was indicated by microscopy, but the possibility of the NPs causing adverse effects upon the physiological functions of cells cannot be excluded. Because the permeation enhancing potential of chitosan NPs has been linked both to particle size and to its inherent, concentration-dependent, toxicity,13 all internalization experiments were performed using NPs at concentrations below IC5029 such that any effects due to intrinsic toxicity would be minimized.

Figure 3. Normalized ECIS plot (% relative resistance vs time) of TEER development (2000 Hz) on bEnd3 endothelial monolayers following exposure to and then removal of alkylglyceryl-modified nanoparticles. Treatment of bEnd3 cells with NP prepared from: (A) CS and highly substituted alkylglyceryl chitosans (0.5 mg/mL); (B) butylglyceryl chitosan with a low degree of substitution (CSOX4(15)/TPP at 0.25 or at 0.5 mg/mL; CS/TPP at 0.5 mg/mL); and (C) butylglyceryl chitosan with a high degree of substitution (CSOX4(95)/TPP at 0.1, at 0.25, or at 0.5 mg/mL). Nanoformulations added to bEnd3 cells cultured (24 h) on ECIS electrodes were removed at 48 h, after which the bEnd3 cells were allowed to recover for a further 48 h. Data are presented as means of at least three ECIS electrodes (n = 3) for each condition of a representative measurement (±SE). All relative resistance values (in %) were normalized relative to untreated (no NP) controls.

electrical resistance of the bEnd3 cell layer over the range of concentrations considered (Figure 3B), whereas those prepared from the highly substituted material were observed to interfere 1070

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Figure 5. Immunolocalization of Evans Blue-loaded alkylglycerylmodified nanoparticles within the organelles of bEnd3 endothelial cells. bEnd3 cells were incubated with 0.5 mg/mL of CS/TPP-EB or of CS-OX4(95)-EB/TPP (visualized in red) for 24 h before staining with the respective biomarkers for the endosomal and the Golgi compartments, EEA-1 (A) and Golgin-84 (B; arrows). Scale bars: 20 μm.

perinuclear localization (consistent with association with the Golgi apparatus), which further points to a potential endocytotic uptake pathway. EB-loaded butylglyceryl chitosan NPs, which had partially colocalized with the early endosome marker EEA-1, exhibited an intracellular punctuate appearance (Figure 5A). Golgin-84 immunopositive costaining (Figure 5B) confirmed the accumulation of NPs within the Golgi apparatus. Experiments performed in ECs incubated at 4 °C for 1 h revealed that no NPs had been taken up by cell organelles, suggesting that the uptake of CS/TPP-EB and that of CS-OX4(95)/TPP-EB are pH/temperature-sensitive endocytotic processes. To assess the effects of butylglyceryl chitosan NPs on the subcellular distribution of tight junctions, we visualized ZO-1 protein localization by immunofluorescence microscopy following 24 h of incubation of bEnd3 cells with EB-loaded NPs of CS, CS-OX4(15), and CS-OX4(95). Incubation with 0.50 mg/mL chitosan NPs effected continuous staining of ZO-1 at the intercellular interface (Figure 6B), as was also observed for EC interfaces that had not been subjected to treatment with nanoparticulate formulations (Figure 6A). By contrast, incubation with 0.50 mg/mL butylglyceryl chitosan NPs resulted in serrated staining of ZO1 at some cell borders (Figure 6C,D), indicating some degree of rearrangement at tight junctions that had been subjected to this treatment. Some agglomeration was observed

Figure 4. FITC-dextran translocation through a bEnd3 cell layer that had been treated with nanoparticles prepared from CS or from butylglyceryl chitosans with different degrees of substitution: (A) cumulative FITC-dextran (FD-150) transport over time; (B) variation in the apparent permeability coefficients Papp; and (C) variation in transport enhancement ratios (calculated as the ratio of the permeability coefficients of nanoformulations vs that of the control). Data are presented as mean ± standard errors (n = 3).

FITC-dextran translocation experiments using bEnd3 cell layers that had been treated with NPs prepared from chitosan or alkylglyceryl chitosans (butyl, pentyl, octyl) did not unmask any statistically significant chain-length-related effects. The loading of butylglyceryl chitosan NPs with EB allowed a cellular-level study of nanoparticle-cell interactions: following 24 h of incubation, all NPs considered were observed to have been uptaken by bEnd3 cells (Figure 5). Initial uptake into the cell cytoplasm was fairly rapid, taking place within 4 h, but the EB signal continued to intensify over 24 h, as is consistent with the progressive accumulation of this dye. Figure 5 shows a 1071

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Table 3. Area, Perimeter, and Ratio of Area/Perimeter from bEnd3 Cells after Application of Alkylglyceryl-Modified NPs (n = 3; Data Are Normalized against Control and Represent Mean ± Standard Error) area ± SD (μM2)

NP formulation control − no NP CS/TPP CS-OX4(15)/TPP CS-OX4(95)/TPP

1.00 0.74 0.85 0.85

± ± ± ±

6.53 6.08 6.32 5.82

perimeter ± SD (μM) 1.00 0.98 1.01 1.01

± ± ± ±

2.32 2.43 2.61 2.30

area/perimeter ± SD 1.00 0.99 0.98 0.97

± ± ± ±

0.03 0.02 0.03 0.02

Figure 7. Assessment of morphology or intercellular membrane organization of bEnd3 endothelial cells following the application of (A) CS/TPP and (B) CS-OX4(95)/TPP (0.5 mg/mL; 24 h of treatment). ImageJ software identified cell borders (visualized in white) based on the results of the ZO-1 immunoreaction (visualized in green). Scale bars: 50 μm.

Figure 6. Confocal analysis of Zonula Occludens 1 staining (ZO-1; green) in bEnd3 endothelial monolayers treated with 0.5 mg/mL of Evans Blue-loaded nanoparticles (red): untreated bEnd3 cells (no NP) (A), CS-EB/TPP (B), CS-OX4(15)-EB/TPP (C), and CS-OX4(95)EB/TPP (D). Nanoparticles uptaken following 24 h of incubation were detectable within specific cell organelles (yellow arrowheads); no signal could be found in untreated bEnd3 cells. Both types of alkylglyceryl-modified nanoparticles (CS-OX4(15)-EB/TPP and CSOX4(95)-EB/TPP) appear to cause subtle changes to ZO-1 immunoreactivity (white arrows). bEnd3 cell bodies shown in differential interference contrast (DIC). Scale bars: 50 μm.

morphology may be the result of subtle, but as yet unspecified, effects on the integrity of the barrier following exposure to NPs.36

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CONCLUSIONS In vitro tests using a mouse-brain EC model have indicated the efficient cellular uptake of NPs prepared from either normal chitosan or from alkylglyceryl-modified chitosans. NPs appeared to have been uptaken via an endocytotic pathway because they had been observed to exhibit perinuclear localization and association with the Golgi apparatus. At large doses, unmodified chitosan NPs impacted negatively upon the viability of bEnd3 cells; this effect was less pronounced with butylglyceryl chain-functionalized chitosan NPs. NPs formulated from butylglyceryl chitosans induced a degree of rearrangement at the tight junction level that seemed to increase with the degree of alkylglyceryl chain substitution. The same NPs appeared to effect an increase in FITC translocation through the bEnd3 cell layer, suggesting increased permeability.

with most NPs, with those prepared from highly substituted butylglyceryl-chitosan (CS-OX4(95) exhibiting the highest tendency to aggregate; this became most apparent at 24 h. Attempts to address this issue through coformulation with surfactants (Polysorbate-80, cetyl trimethylammonium bromide, or poly(ethylene glycol) (2000 Da); 1% w/v) or by using alternative media (phosphate buffer; phosphate buffer saline; DMEM with or without serum) were unsuccessful. Consistent with literature reports,35 the long-term stability issues of chitosan-based colloids may limit their usefulness for certain biomedical applications. Whereas the quantification of cell area and perimeter (>50 cells per condition; 24 h after application of 0.50 mg/mL NPs) seemed to indicate a decrease in the area and in the area/ perimeter ratio of the cells that had been treated with butylglyceryl chitosan NPs as compared with the control or with those that had been treated with unmodified chitosan NPs (Table 3), morphometric analysis of the confocal images (ImageJ software; Figure 7) has revealed that these differences were not of statistical significance (Anova with posthoc Tukey test, p = 0.05); very similar results have been obtained by processing the data from the 48 h treatment. Changes in EC

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of BBSRC, U.K. (grant No BB/F011865/1) and IBBS and thank Mr. Anthony 1072

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(31) Wegener, J.; Keese, C. R.; Giaever, I. Exp. Cell Res. 2000, 259, 158−166. (32) Au, C.; Mutkus, L.; Dobson, A.; Riffle, J.; Lalli, J.; Aschner, M. Biol. Trace Elem. Res. 2007, 120, 248−256. (33) Seil, J. T.; Webster, T. J. Int. J. Nanomed. 2008, 3, 523−531. (34) Abbott, N. J.; Patabendige, A. A. K.; Dolman, D. E. M.; Yusof, S. R.; Begley, D. J. Neurobiol. Dis. 2010, 37, 13−25. (35) Fan, W.; Yan, W.; Xu, Z.; Ni, H. Colloids Surf., B 2012, 90, 21−27. (36) Rempe, R.; Cramer, S.; Hüwel, S.; Galla, H.-J. Biochem. Biophys. Res. Commun. 2011, 406, 64−69.

Sinadinos for help with the ImageJ software. D.G. and G.P. acknowledge partial support from TC2N EU Interreg and Brain Tumour Research.

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dx.doi.org/10.1021/bm201790s | Biomacromolecules 2012, 13, 1067−1073