Synthesis and Toxicological Evaluation of a Chitosan-l-Leucine

Sep 5, 2014 - Synthesis and Toxicological Evaluation of a Chitosan-l-Leucine Conjugate for Pulmonary Drug Delivery Applications. Mohammad D. A. ...
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Synthesis and Toxicological Evaluation of a Chitosan‑L‑Leucine Conjugate for Pulmonary Drug Delivery Applications Mohammad D. A. Muhsin,†,‡ Graeme George,† Kenneth Beagley,† Vito Ferro,§ Charles Armitage,† and Nazrul Islam*,†,‡ †

Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Brisbane, Queensland 4059, Australia ‡ Pharmacy Discipline, Faculty of Health, Queensland University of Technology, Brisbane, Queensland 4000, Australia § School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Herein are reported the synthesis of a conjugate of chitosan with L-leucine, the preparation of nanoparticles from both chitosan and the conjugate for use in pulmonary drug delivery, and the in vitro evaluation of toxicity and inflammatory effects of both the polymers and their nanoparticles on the bronchial epithelial cell line, BEAS-2B. The nanoparticles, successfully prepared both from chitosan and the conjugate, had a diameter in the range of 10−30 nm. The polymers and their nanoparticles were tested for their effects on cell viability by MTT assay, on trans-epithelial permeability by using sodium fluorescein as a fluid phase marker, and on IL-8 secretion by ELISA. The conjugate nanoparticles had a low overall toxicity (IC50 = 2 mg/mL following 48 h exposure; no induction of IL-8 release at 0.5 mg/mL concentration), suggesting that they may be safe for pulmonary drug delivery applications.

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INTRODUCTION Chitosan is a linear copolymer of β(1→4)-linked D-glucosamine with occasional N-acetyl-D-glucosamine residues. Due to its natural abundance,1,2 biocompatibility,3 and biodegradability,4 chitosan has drawn wide interest in diverse fields including medicine, pharmacy, and biotechnology.5,6 One deterrent to the exploitation of the polymer to its full potential is its poor solubility and processability, and thus there has been much interest in the improvement of its functional applicability by chemical modification.7,8 For many years, there has been a keen interest in the conjugation of amino acids to chitosan for potential utilization in various fields including adsorption of heavy metals,9 removal of low-density lipoproteins,10 and immobilization of lipases.11 Of late, enhanced dispersibility of particles from a chitosanbased dry powder inhaler (DPI) upon addition of the free amino acid, L-leucine to the formulation has been reported.12,13 This effect was attributed to the migration of L-leucine to the surface of chitosan particles during the drying phase owing to its surfactant-like properties giving the particles a pitted appearance and in turn reducing interparticle cohesion.14 It has also been shown for L-leucine and a leucine tripeptide that, at the particle-air interface, the hydrophobic side-chains of these species are projected outward.15 In the current study, the chemical conjugation of chitosan with L-leucine was investigated to determine whether this effect on the dispersibility of chitosan from a DPI could be maintained. Considering the © 2014 American Chemical Society

structural features of L-leucine and the previously reported orientation of the hydrophobic part of L-leucine (added to a particle formulation) at the particle−air interface, it was hypothesized that, upon conjugation, the L-leucine substituent will be oriented around the chitosan backbone with its hydrophobic part projected outward, resulting in a reduced interparticle interaction. It was also hypothesized that the conjugation of L-leucine to chitosan would enhance the dispersibility of the particles from a DPI. Although the physical addition of L-leucine to chitosan has been shown to improve its dispersibility, the poor solubility of chitosan still remains a major obstacle to its utilization. Thus, a second objective behind chemical conjugation of L-leucine to chitosan was to improve its solubility by creating an amphiphilic environment around the chitosan backbone. The respiratory system is an established drug delivery route for lung diseases such as asthma and chronic obstructive pulmonary disease16,17 and is of considerable interest for drug delivery to the systemic circulation.18 However, it is important to ensure the safety of any formulation intended for inhalation. Previous studies on chitosan and its micro- or nanoparticles on pulmonary epithelial cell lines such as Calu-3 and A-549 indicate their low toxicity.19,20 Trimethylchitosan chloride Received: June 11, 2014 Revised: September 4, 2014 Published: September 5, 2014 3596

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Biomedical Sciences and Pharmacy, The University of Newcastle, Australia). Pyridine was stored over 4 Å molecular sieves to prevent moisture absorption. Glacial acetic acid was diluted to a 2% aqueous solution for dissolution of chitosan. Trypsin-EDTA was diluted to 0.01% in PBS prior to use for splitting cells. All other reagents were used as received without any further purification or dilution. Synthesis. N-Phthaloylchitosan (2). N-Phthaloylchitosan (2) was synthesized according to the method of Holappa et al.30 with some modifications. Phthalic anhydride (2.43 g, 16.4 mM) was added to a dispersion of chitosan (1) obtained by overnight stirring of 1 (1 g, 5.48 mM free −NH2 group) in DMF (20 mL) containing 5% (v/v) water. The mixture was heated with stirring at 130 °C for 8 h, cooled to room temperature, and poured into ice water (500 mL). The precipitate was collected by filtration, washed with copious amounts of methanol, and dried to give 1.56 g (95%) of a pale tan powder. Degree of substitution (DS): 0.91. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 2980−2830 (C−H stretch, pyranose), 1774, 1703 (CO stretch, imide), 1612 (amide I, N-acetyl), 1547 (N−H bend overlapping amide II), 1468 (asymmetric C−H bend in CH2), 1385 (CC, phth), 1200−800 (C−O stretch, pyranose), 718 cm−1 (arom, phth). Calcd: C, 53.73; H, 5.00; N, 4.63. Found: C, 51.25; H, 4.87; N, 4.44. N-Phthaloyl-3,6-di-O-acetylchitosan (2a). N-Phthaloyl-3,6-di-Oacetylchitosan (2a) was synthesized following the method described by Nishimura et al.31 with some modifications. Acetic anhydride (10 mL, 105.8 mmol) was added to a suspension of 2 (100 mg, 0.33 mmol) in pyridine (20 mL) and heated with stirring at 130 °C overnight. The resulting homogeneous mixture was cooled to room temperature and precipitated in ice−water (60 mL). The precipitate was washed successively with ethanol and ether and dried to give 124 mg of 2a (97%). DS: 1.97. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 2980−2830 (C−H stretch, pyranose), 1777 (CO stretch, imide), 1743 (CO stretch, O-acetyl ester), 1711 (CO stretch, imide), 1613 (amide I, N-acetyl), 1543 (N−H bend overlapping amide II), 1469 (asymmetric C−H bend in CH2), 1430 (asymmetric C−H bend in CH3), 1385 (CC, phth), 1371 (symmetric C−H bend in CH3), 1219 (C−O stretch, O-acetyl), 1200−800 (C−O stretch, pyranose), 722 cm−1 (arom, phth). 1H NMR (CDCl3): δ 1.63−2.30 (O- and N-acetyl), 2.80−4.90 (pyranose), 5.18 (bs), 5.51 (bd, J = 8.4 Hz, H1), 7.60−7.80 (Ar). 13 C NMR (CDCl3): δ 20.4−20.8 (O- and N-acetyl), 55.3 (C-2), 62.1 (C-6), 70.2 (C-3), 72.4 (C-5), 75.3 (C-4), 97.2 (C-1), 124.0, 131.3, 134.6 (Ar), 167.7−170.2 ppm (CO). Calcd: C, 54.47; H, 4.96; N, 3.63. Found: C, 54.48; H, 4.88; N, 3.66. N-Phthaloyl-6-O-tritylchitosan (3). N-Phthaloyl-6-O-tritylchitosan (3) was synthesized from N-phthaloylchitosan (2) according to the method of Zhang et al.32 with some modifications. A suspension of 2 (1 g, 3.32 mmol free −OH) in pyridine (47 mL) was treated with trityl chloride (9.23 g, 33.1 mmol) and stirred with heating at reflux (∼115 °C) for 24 h under argon. After cooling to room temperature, the reaction mixture was poured into EtOH. The precipitate was collected by filtration and washed successively with EtOH and Et2O. The yield of the product was 1.45 g (80%). DS: 1.06. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 3100−3000 (C−H, trityl), 2980−2830 (C−H, pyranose and CH3), 1776, 1712 (CO, imide), 1611 (amide I, N-acetyl), 1591 (N−H bend overlapping amide II), 1490 (CC, trityl), 1468 (asymmetric C−H bend in CH2), 1448 (CC, trityl), 1384 (CC, phth), 1200−800 (C−O, pyranose), 764, 746 (arom, trityl), 719 (arom, phth), 699 cm−1 (arom, trityl). Calcd: C, 71.72; H, 5.38; N, 2.57. Found: C, 71.91; H, 5.27; N, 2.50. N-Phthaloyl-3-O-acetyl-6-O-tritylchitosan (3a). N-Phthaloyl-3-Oacetyl-6-O-tritylchitosan (3a) was synthesized following the same procedure as described above for 2a. Acetic anhydride (10 mL, 105.8 mM) was added to a suspension of 3 (100 mg, 0.18 mM) in pyridine (20 mL) and heated overnight at reflux (130 °C). The resulting solution was cooled to room temperature and precipitated by pouring into ice−water (60 mL). The precipitate was collected by filtration, washed successively with ethanol and ether, and dried to give a yield of 90 mg. This was 83% of the theoretical yield assuming that 100% substitution has taken place. The microanalysis results were not

(TMC)a water-soluble derivative of chitosanhas, however, been reported to be more toxic than the precursor chitosan.21,22 The safety of respiratory formulations can best be evaluated in vitro by a combination of toxicological tests on respiratory epithelial cell lines. Cell viability assays, e.g., the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, are widely used to evaluate the safety of inhaled materials.23,24 Another indicator of adverse effects of inhaled agents on epithelial cell layers is the change in the permeability of the epithelial barrier. This can be assessed by measuring the permeability of a fluid phase marker such as sodium fluorescein (Na Flu) across a confluent cell monolayer.20 Another important concern is to rule out any inflammatory response of the respiratory epithelium to the inhaled product. This can be accomplished by monitoring the secretion of chemokines such as interleukin 8 (IL-8) by enzyme-linked immunosorbent assay (ELISA).25 IL-8 is a chemokine whose secretion is often associated with initiation of inflammatory processes in lung tissue.26 The respiratory epithelial cell models, used in previous toxicological investigations of chitosan and TMC (e.g., Calu-3 and A-549), are derived from human lung tumor. BEAS-2B27 is a nonmalignant cell line derived from normal human bronchial epithelial cells immortalized by a Simian Virus (SV) 40/ adenovirus-12 hybrid virus.28 It has been used widely as an in vitro bronchial epithelial cell model.29 However, no studies have so far been reported to investigate the toxicity of chitosan or any of its derivatives using this cell line. This study describes the synthesis of an L-leucine conjugate of chitosan and its subsequent use for fabrication of nanoparticles suitable for inhalation as a DPI formulation. The L-leucine conjugate was characterized by Fourier transform infrared spectroscopy (FT-IR), 1H, 13C and 2D 1H−13C gradient-enhanced heteronuclear single quantum correlation (ge-HSQC) nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, and X-ray photoelectron spectroscopy (XPS). The safety of chitosan, its L-leucine conjugate, and their nanoparticles for pulmonary delivery was evaluated in terms of three well-recognized toxicity indicators (cell viability, trans-epithelial permeability and IL-8 secretion) using the BEAS-2B cell line as an in vitro model.

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MATERIALS AND METHODS

Materials. Low molecular weight chitosan (degree of deacetylation (DDA), 92%; molecular weight (MW), 50−190 kDa), acetic anhydride, trityl chloride, aqueous hydrazine hydrate (50−60%), Boc-L-leucine succinimide (Boc-Leu-OSu), 4 M HCl in 1, 4-dioxane, N,N-dimethylformamide (DMF), pyridine, chloroform-d (CDCl3, 99.8 atom % D), pyridine-d5 (99.5 atom % D, 0.03% v/v tetramethylsilane, TMS), deuterium oxide (D2O, 99.99 atom % D), MTT, dimethyl sulfoxide (DMSO, spectrophotometric grade) and Na Flu were obtained from Sigma-Aldrich Pty Ltd. (Australia). Diethyl ether, glacial acetic acid, hexane and petroleum ether (40−60) were purchased from Merck Pty. Ltd. (Australia). RPMI 1640 (Gibco), fetal bovine serum (Lonza), L-glutamine (Gibco), penicillin-streptomycin (Gibco), and 0.5% trypsin-ethylenediaminetetracetic acid (EDTA) (Gibco) were supplied by Life Technologies Australia Pty Ltd. Phthalic anhydride was obtained from Merck Schuchardt (Germany), methanol and paraffin oil heavy 68 (viscosity: 66.0−70.0 cST @ 40 °C) were from Chem-Supply (Australia), ethanol was from Recochem (Australia), span 80 was from PCCA (Australia), phosphate-buffered saline (PBS) tablets (pH 7.3 ± 0.2 when dissolved in prescribed volume of H2O) were from Oxoid Ltd. (England), and the human IL-8 ELISA MAX Deluxe kit was from Biolegend (USA). The bronchial epithelial cell line, BEAS-2B was a gift from Prof. Philip Hansbro (School of 3597

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anlaytical data indicated that 91% of the protecting Boc and triytl groups had been removed. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 2980−2830 (C−H, pyranose & L-leucine), 1672 and 1628 (amide I, N-acetyl overlapping N-L-leucine), 1558 and 1512 (amide II overlapping symmetric N−H bend in NH3+), 1390 and 1372 (isopropyl, leu), 1322 (amide III), 1200−800 cm−1 (C−O, pyranose). 1H NMR: δ 0.98 (H-δ1&2, leu), 1.78 (H-δ2 and H-β of leu with some overlapping with H-δ1 and H-γ), 2.07 (N-acetyl and H-δ1, leu), 2.80 (H-γ, leu), 3.19 (H-2, pyranose), 3.30−4.34 (H-2 to H-6, pyranose overlapped with H-α and H-β of leu), 4.50−5.54 ppm (H-1, pyranose), 13C NMR (CDCl3): δ 20.7 (CH3, N-acetyl), 21.7 and 23.5 (C-δ1&2, leu), 25.0 (C-γ, leu), 40.0 (C-β, leu), 52.0 (C-α, leu), 55.6 (C2), 59.8 (C-6), 69.8−76.1 (C-3, C-5, C-4), 97.3−101.1 (C-1), 170.8 (CO, N-leu), 174.4 ppm (CO, N-acetyl). Calcd: C, 43.81; H, 7.62; N, 8.40. Found: C, 36.84; H, 7.14; N, 6.93. XPS Analysis (Atom %): Chitosan: O, 28.59; C, 63.02; N, 7.34, Cl 1.05. L-leucine: O, 19.40; C, 69.44; N, 11.55. Chitosan-N-L-leucine·HCl: O, 25.68; C, 60.99; N, 9.31; F, 0.64; Cl, 3.38. Preparation of Chitosan and Chitosan-L-leucine Conjugate Nanoparticles. Chitosan and conjugate nanoparticles were prepared by a water-in-oil (W/O) emulsion-solvent evaporation technique modified from that reported previously for fabrication of chitosan microparticles.33,34 For fabricating chitosan nanoparticles, 2.5 mL of a 2% solution of chitosan in 2% acetic acid was added dropwise at 60 °C to 100 g heavy paraffin oil containing 1 mL span 80 and homogenized into a W/O emulsion at 11,000 rpm. The emulsion was stirred overnight at 5000 rpm and 60 °C using an IKA Eurostar Digital overhead stirrer (IKA Works (Asia), Inc.) to remove the internal water phase and harden the droplets into nanoparticles. The nanoparticles were then separated from the oil phase by centrifugation overnight at 5000g using an Allegra X15R benchtop centrifuge (Beckman-Coulter, Inc. USA), washed three times with hexane to remove excess oil, filtered and dried under vacuum at 60 °C. For making chitosan-Lleucine conjugate nanoparticles, the same protocol was followed except that no acetic acid was added for preparing the solution of the conjugate in water. The yields of nanoparticles were quantified as the percentage of anticipated yields. The nanoparticles obtained were weighed and the percentage yield was calculated using the following formula: production yield (%) = (weight of the dried particle/sum of the dry weight of starting material) × 100.35 Measurement was done in triplicate (n = 3). Characterization. FT-IR spectra of the chitosan, conjugates, and intermediates were recorded on a Nicolet 5700 FT-IR attenuated total reflectance (ATR) spectrophotometer (Thermo Electron Corporation, Madison, Wisconsin, USA) equipped with a Smart Endurance diamond internal reflection element under dry air at ambient temperature (25 °C). The spectra were collected by using standard spectral collection techniques and the software Omnic 32 over the wavenumber range 4000−650 cm−1 using 64 scans at a resolution of 4 cm−1. 1 H, 13C and 2D 1H−13C ge-HSQC NMR spectra were recorded on a Bruker AVANCE 400 high-resolution spectrometer (Bruker BioSpin GmbH, Germany) using a quad nucleus probe (QNP; tunable for 1H, 19 F, 31P, 13C) under a static magnetic field of 400 and 100 MHz, respectively, at 298 K. Spectra of 16384 data points were recorded over a spectral width of 12.0144 ppm using the accumulation of 32 transients and an exponential line broadening (LB) of 0.30 Hz was applied prior to Fourier transformation for 1H NMR. 13C NMR spectra were recorded over a spectral window of 219.8552 ppm with 16384 data points and 6144 transients using proton decoupling. Prior to Fourier transformation, 2.00 Hz LB was applied. The 1H−13C geHSQC experiments were performed in phase-sensitive mode using Echo/Antiecho-time proportional phase incrementation (TPPI) gradient selection. The data matrix was 256 × 2048 with spectral widths of 4807.69 Hz for proton and 18 115.94 Hz for carbon. The evolution time was set to 1/(8JCH) = 0.89 ms. Four gradient pulses were applied along the z axis with ratios of 80, 20.1, 11 and −5, respectively, and a duration of 1 ms. Eighteen transients were accumulated for each increment. A square sine window function was

satisfactory and so could not be used for calculating the actual DS. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 3100−3000 (C−H, trityl), 2980−2830 (C−H stretch, pyranose and CH3), 1777 (CO stretch, imide), 1745 (CO stretch, O-acetyl ester), 1714 (CO stretch, imide), 1612 (amide I, N-acetyl), 1595 (N−H bend), 1547 (amide II, N-acetyl), 1529, 1491 (CC, trityl), 1468 (asymmetric C−H bend in CH2), 1449 (CC, trityl), 1385 (CC, phth), 1220 (C−O stretch, O-acetyl), 1200−800 (C−O stretch, pyranose), 765, 747 (arom, trityl), 721 (arom, phth), 704 cm−1 (arom, trityl). 1H NMR (CDCl3): δ 1.63−2.30 (O- and N-acetyl), 3.00−5.00 (pyranose), 5.19 (bs), 5.50 (bs), 6.89−7.80 ((Ar). 13C NMR (CDCl3): δ 20.4−20.8 (O- and N-acetyl), 55.3 (C-2), 62.1 (C6), 70.2 (C-3), 72.4 (C-5), 75.3 (C-4), 97.2 (C-1), 123.9−147.0 (Ar), 167.8−170.3 (CO,). Calcd: C, 70.68; H, 5.33; N, 2.39. Found: C, 61.65; H, 4.89; N, 3.05. 6-O-Tritylchitosan (4). Deprotection of the phthaloyl group from 3 to produce 6-O-tritylchitosan (4) was performed according to the literature31,32 with some modifications. A suspension of N-phthaloyl-6O-trityl-chitosan 3 (1 g, 1.84 mmol −NH2 group equivalent) in hydrazine hydrate (50−60%, 50 mL) was stirred with heating at reflux (∼110 °C) for 18 h under an argon atmosphere. The reaction mixture was cooled to room temperature and poured into water (500 mL). The precipitate was filtered off and washed with water (2 × 500 mL) and finally with ethanol and ether. The yield of the product was 0.75 g (96%). Microanalytical data was unsatisfactory to calculate the actual degree of dephthaloylation. However, FT-IR and 1H NMR spectra indicated complete removal of the phthaloyl group. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 3100−3000 (C−H, trityl), 2980−2830 (C−H, pyranose and CH3), 1661 (amide I, N-acetyl), 1596 (amide II overlapping N−H bend), 1490, 1448 (C C, trityl), 1374 (symmetric C−H bend in CH3), 1318 (amide III), 1200−800 (C−O, pyranose), 763, 746, 697 cm−1 (arom, trityl). 1H NMR (pyridine-d5): δ 1.67−2.13 (CH3, N-acetyl), 3.00−5.79 (pyranose), 7.28−7.80 (Ar). Calcd: C, 71.13; H, 6.44; N, 3.30. Found: C, 71.14; H, 6.13; N, 3.54. N-(Boc-L-leucine)-6-O-tritylchitosan- (5). A solution of Boc-LeuOSu (1.16 g, 3.53 mmol) in pyridine (15 mL) was added dropwise to 4 (500 mg, 1.18 mmol −NH2 group equivalent) at 0 °C over a period of 1 h under an argon atmosphere, and the reaction mixture was then stirred for an additional 3 h at 0 °C. An additional 773 mg (2.35 mmol) of Boc-Leu-OSu in pyridine (10 mL) was then added dropwise to the reaction mixture and the reaction was continued at room temperature under argon for a further 21 h until a clear solution was obtained. Finally, the product was precipitated by pouring the reaction mixture into diethyl ether (250 mL). The precipitate was washed with diethyl ether (× 3) and dried to give 485 mg (70%) of (5) as a solid. DS: 0.74. IR (ATR): ν 3700−3100 (O−H stretch overlapping N−H stretch), 3100−3000 (C−H, trityl), 2980−2830 (C−H, pyranose and Boc-Leucine), 1683 (amide I, N-acetyl, N-L-leucine, and N-carbamate), 1597 (amide II overlapping N−H bend), 1491, 1448 (CC, trityl), 1390 and 1367 (Boc t-butyl and L-leucine isopropyl), 1319 (amide III), 1200−800 (C−O, pyranose), 764, 747, 702 cm−1 (arom, trityl). 1 H NMR: δ 0.90 (H-δ1, leu), (H-γ, leu overlapped with t-butyl, Boc), 1.44 (t-butyl, Boc overlapped with CH3, N-acetyl at 1.66 ppm), 1.92 (H-β, leu overlapped with H-δ1), 2.15 (CH3, N-acetyl), 2.65 (H-δ2, leu), 3.00−5.5 (pyranose protons overlapped with H-α, leu at 4.51), 7.10−8.00 (Ar). 13C NMR (CDCl3): δ 20.7−23.8 (CH3, N-acetyl), 25.3, 26.4 (C-δ1&2, leu), 28.9 (t-butyl, Boc), 30.3 (C- γ, leu), 43.0 (C-β, leu), 54.5 (C-α, leu), 56.3 (C-2), 58.4 (C-6), 72.6 (C-3), 75.8 (C-5), 77.4 (C-4), 79.2 (C(CH3)3), 87.1 (C-Ph3), 97.4, 101.0, 102.0, 104.5, 104.8 (C-1), 127.8, 128.7, 129.9, 145.0 (Ar), 156.9 (CO, carbamate), 173.6 (CO, N-leu), 176.3 ppm (CO, N-acetyl). Calcd: C, 68.23; H, 7.25; N, 4.33. Found: C, 67.98; H, 6.78; N, 4.14. Chitosan-N-L-leucine·HCl (6). 4 M HCl in 1,4-dioxane (4 mL) was added dropwise to 5 (200 mg, 343 μmol) under an argon atmosphere and stirred for 15 min at this temperature at 0 °C. Stirring was continued at room temperature under argon for 24 h. The reaction mixture was poured into diethyl ether (40 mL), the precipitate filtered off, washed with diethyl ether (×3) and dried to give the product, chitosan-N-L-Leucine·HCl 6 (80 mg, 66%) as a solid. The micro3598

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applied in both dimensions prior to Fourier transformation. CDCl3, pyridine-d5, and D2O were the solvents used for preparing NMR solutions of the intermediates and the final product according to their solubility. A concentration of approximately 1 mM was used for preparing the NMR samples. After adding the sample to the solvent in a vial, it was stirred for several hours on a magnetic stirrer to thoroughly mix and dissolve it, and finally centrifuged to settle down any undissolved solids. Approximately 0.6 mL of the sample solution was transferred to a 5 mm NMR tube, which was inserted in the magnet to perform the experiment. Chemical shifts were recorded in parts per million (ppm) and were referenced to solvent peaks (at 7.24 and 77.23 ppm, for example, for 1H and 13C NMR in CDCl3). Elemental analyses were performed by the Microanalytical Service, School of Chemistry and Molecular Biosciences, the University of Queensland. The samples were extensively (ca. 24 h) dried in vacuo at 60 °C. The C/N ratio determined with elemental analysis was used to evaluate the DS of the intermediates and the final product. The XPS analysis of chitosan, L-leucine and chitosan-N-L-leucine· HCl was performed with a Kratos Axis Ultra Spectrophotometer (Kratos Analytical, Manchester, UK) equipped with monochromatized aluminum X-ray source (powered at 10 mA and 15 kV), 165 mm radius hemispherical analyzer and 8-channel electron multiplier (channeltron) detection system. Samples were mounted onto stainless steel sample holders using double-sided adhesive tape and the spectra were collected using an analysis area of 700 × 300 μm. Low-resolution wide scans (survey spectrum) in the binding energy scale (0−1200 eV with 1.0 eV steps) were collected at a constant analyzer pass energy of 160 eV to identify the elements present. High-resolution narrow scans (multiplex spectra) were collected at 20 eV with 0.05 eV steps for O 1s, N 1s, C 1s, and Cl 2p to identify their chemical status. The data were processed using Casa XPS software Version 2.3.14. Binding energies (BE) of the various elements were referenced to the C 1s line at 285.0 eV. The scanning electron microscopy (SEM) imaging of chitosan and chitosan-L-leucine conjugate nanoparticles was done on an ultrahigh resolution scanning electron microscope, JEOL 7001F (JEOL Australasia Pty Ltd., Frenchs Forest, Australia). A tiny drop (5 μL) of nanoparticle suspension in hexane (100 μg/mL) was placed on carbon adhesive tape adhered onto a properly labeled aluminum stub. The specimen stubs were then sputter coated with a thin layer of gold with a Leica EM SCD005 sputter coater (Leica Microsystems, North Ryde, Australia) at 30 mA for 1.5 min using an argon gas purge. The specimens were examined with the SEM under high vacuum using a spot size of 10 with an accelerating voltage of 20.0 kV and a working distance of 10 mm. Several photomicrographs (secondary electron images) of the samples were recorded at different magnifications. The transmission electron microscopy (TEM) analysis was conducted using a high resolution transmission electron microscope, JEOL 1010 (JEOL Australasia Pty Ltd., Frenchs Forest, Australia). For analysis, each nanoparticle sample was suspended in ethanol at a concentration of 0.05−0.1 mg/mL. A tiny drop (2 μL) of the suspension was placed on strong carbon film on a 400-mesh copper grid and allowed to sit for 1−2 min until it became nearly dry. Then the sample was negatively stained with a drop (2 μL) of 1% uranyl acetate solution (AJAX Univar, Ajax Finechem Pty Ltd., Australia) for another 2−3 min, and any excess uranyl acetate was removed with filter paper. The specimens were then examined on the TEM machine under high vacuum using an accelerating voltage of 100 kV and a spot size of 6. The images of samples were taken at different magnifications. The size distribution and polydispersity index (PDI) of chitosan and chitosan-L-leucine conjugate nanoparticles were determined by dynamic light scattering using a ZetaSizer Nano S instrument (Malvern Instruments Ltd., UK) equipped with a 4 mW He−Ne laser operating at a wavelength of 633 nm. The analysis was performed at 25 °C with a detection angle of 173°. For analysis, the particles were diluted to an appropriate concentration (ca. 0.1%) with heavy mineral oil containing 1% span 80 as the dispersant and placed in a glass cuvette with square aperture (Sarstedt AG & Co., Nümbrecht, Germany). The measurement was done in triplicate and averaged. The reported size is the Z-average (or “cumulants mean”) for the particle

hydrodynamic diameters calculated from the intensity of scattered light, and the PDI is a width parameter, which was also calculated from the cumulants analysis of the signal intensity. Data acquisition and analysis was done by Malvern Zetasizer Software version 6.32. The viscosity and refractive index values of the dispersant, heavy paraffin oil with 1% span 80 (used for data analysis) were determined to be 72.7 cps (at 25 °C) and 1.482, respectively, by a Cannon-Fenske Routine Viscometer (Poulten, Self-e and Lee, Ltd. England), and an Abbe Refractometer (Atago Co., Ltd., Tokyo, Japan), respectively. Cell Culture. For in vitro toxicological and inflammatory evaluation of chitosan, its L-leucine conjugate and their nanoparticles, the bronchial epithelial cell line, BEAS-2B (ATCC# CRL-9609) was used. Cell cultures were grown using 75 cm2 flasks in a humidified incubator in an atmosphere of 5% CO2/95% air at 37 °C. The same conditions were used for cell cultivation in subsequent experiments. The cell culture medium (CCM) used consisted of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin sulfate. In Vitro Evaluation of Cytotoxicity by MTT Assay. The MTT assay was performed according to previous reports36 with some modifications. BEAS-2B cells were seeded at 5 × 104 cells/well in a 96well plate and grown overnight at 37 °C. Cells were then incubated with fresh medium containing chitosan or the conjugate or their nanoparticles at a range of concentrations (viz., 0.125, 0.25, 0.375, 0.50, 1.0, 2.0, 4.0, 8.0, 12.0, and 16.0 mg/mL). After 12, 24, or 48 h, 50 μL of MTT solution (0.5 mg/mL in PBS) was added to each well. The cells were incubated for an additional 4 h, and the medium was removed. The formazan crystals generated by the cells were solubilized with 100 μL DMSO and the absorbance of each well was measured by an xMark Microplate Spectrophotometer (Bio-Rad Laboratories, USA) at 550 nm. Relative cell viability was determined from the equation: viability (%) = (absorbance of test−absorbance of blank)/ (absorbance of untreated cells−absorbance of blank) × 100. The concentration causing 50% inhibition of cell growth (IC50) was determined by regression analysis using the points on the steep portion of the concentration effect curve according to Adamson et al.37 Effect on Na Flu Transport across BEAS-2B Cell Monolayers. The effect of different concentrations of chitosan, the conjugate and their nanoparticles on the permeability of polarized BEAS-2B cell monolayers was investigated using Na Flu as the permeability marker according to the method described by Grenha et al.19 with some modifications. 1 ×105 BEAS-2B cells in a volume of 200 μL medium were seeded onto collagen-coated 0.4 μm polytetrafluoroethylene (PTFE) Transwell inserts (Corning Incorporated, USA) and grown in 24-well plates. Transepithelial electrical resistance (TEER) was measured daily using an EVOM electrode (Millipore Australia Pty Ltd.). Fresh media were applied every second day. After 5 days when the TEER reached a stable maximum value, 200 μL of CCM containing 0.2 mg/mL of Na Flu was added to the apical chamber in combination with test substances at increasing concentrations (viz., 0, 0.5, 1, 2, and 4 mg/mL). Cells were then incubated at 37 °C and basolateral samples taken at 0.5, 1, 2, 4, 6, 12, 24, and 48 h. Passive transport of Na Flu across epithelial layers was determined from a standard curve using a POLARstar OPTIMA fluorescent spectrophotometer (BMG Labtech Inc., USA). The apparent permeability coefficient (Papp) of Na Flu was determined from the equation: Papp (cm/s) = (the cumulative amount of Na Flu transported over time) × (1/(transwell surface area (cm2) × the initial apical concentration of Na Flu (mg/mL). Immunocytochemistry. BEAS-2B cells were seeded at 5 × 104 cells/0.4 μm transwell inserts and grown in 24 well transwell chambers for 6 days at 37 °C in 5% CO2, changing media every second day. On day six, cells were washed twice in PBS, and then fixed with ice cold 100% MeOH for five min at 4 °C. Cells were washed twice with PBS, and then blocked in 10% FCS in PBS + Tween 20 (PBST) (0.5% v/v) for 1 h at room temperature. Cells were then probed with primary antibody, rabbit antizona occludins (ZO)-1 antibody (Invitrogen, AUS) in blocking buffer (1:1000) for 1 h at room temperature. Cells were washed three times with PBST and secondary antibody, goat antirabbit IgG-AlexaFluor 488 (Invitrogen, AUS) (1:2000), and 4,63599

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Figure 1. FT-IR spectra of (A) chitosan, (B) N-phthaloyl-chitosan (2), (C) N-phthaloyl-3,6-di-O-acetylchitosan (2a), (D) N-phthaloyl-6-Otritylchitosan (3), (E) N-phthaloyl-3-O-acetyl-6-O-tritylchitosan (3a), (F) 6-O-tritylchitosan (4), (G) N-(Boc-L-leucine)-6-O-tritylchtiosan (5), and (H) chitosan-N-L-leucine·HCl (6). as mean ± standard deviation (SD). The statistical significance of the differences was assessed by one-way and two-way analysis of variance (ANOVA) with posthoc (Tukey-HSD) analysis; differences with a pvalue of 0.05 or less were considered significant.

diamidino-2-phenylindole (DAPI) (125 ng/mL) for 1 h at room temperature in the dark. Inserts were washed three times with PBST, and then the transwell membrane was removed with a scalpel blade, placed on a slide and mounted with a coverslip in Prolong Gold (Invitrogen, AUS) overnight at room temperature in the dark. ZO-1 expression and DNA was observed using a TCS SP5 X confocal microscope (Leica Microsystems, AUS). Effect on IL-8 Secretion. The proinflammatory effect of the test materials was evaluated by measuring IL-8 secretion by BEAS-2B cells according to the method reported38,39 with some modifications. Briefly, 5 × 104 BEAS-2B cells were seeded into a 96-well plate and grown at 37 °C until 80−90% confluent. Fresh media containing increasing concentrations of test substances (0, 0.5, 1, 2, and 4 mg/ mL) were applied. After 24 h at 37 °C, supernatants were collected and secreted IL-8 quantified using an IL-8 ELISA MAX kit (Biolegend Inc., USA) as per manufacturer’s instructions.40 Statistical Analysis. All the pharmaceutical and cell line experiments were performed in triplicate, and the results are expressed

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RESULTS AND DISCUSSION Conjugation of L-Leucine with Chitosan. The free amino group at C-2 of chitosan 1 was selected as the preferred site for chemoselective conjugation of L-leucine. For controlled functionalization at C-2 and to confer sufficient organosolubility,30,41 the C-6 position was required to be protected as the trityl ether. Since chitosan per se is not sufficiently organosoluble to allow its reaction with trityl chloride, it was first reacted with phthalic anhydride to give N-phthaloylchitosan 2.31,42 After tritylation at C-6, the phthaloyl group was removed by treatment with aqueous hydrazine to give 6-O3600

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tritylchitosan 4. L-Leucine was conjugated to the amino group at C-2 by reaction with Boc-leu-OSu to give N-(Boc-L-leucine)6-O-trityl-chitosan 5. Finally, both trityl and Boc groups were deprotected simultaneously by treatment with 4 M HCl in dioxane to give the target conjugate, chitosan-N-L-leucine 6 as the hydrochloride salt. The intermediates and the final product, chitosan-N-L-leucine·HCl 6 were characterized by a combination of FT-IR, 1H and 13C NMR spectroscopy, and elemental analysis. Compounds 5 and 6 were further characterized by 2D 1 H−13C ge-HSQC spectroscopy. The FT-IR spectra of the unreacted chitosan, the intermediates, and the final product 6 in the synthetic pathway are presented in Figure 1. In accordance with previously published data,30,32,43 the spectra showed characteristic absorptions for N-phthaloyl-chitosan 2 (phthaloyl CC 1703 and 1774 cm−1, phthaloyl aromatic 718 cm−1), Nphthaloyl-6-O-trityl-chitosan 3 (trityl CC 1448 and 1490 cm−1, trityl aromatics 699, 746, and 764 cm−1 in addition to phthaloyl signals in place), N-(Boc-L-leucine)-6-O-trityl-chitosan 5 (intensified amide-I signal at 1683 cm−1, Boc t-butyl/Lleucine isopropyl at 1367 and 1390 cm−1 in addition to trityl signals in place) and chitosan-N-L-leucine·HCl 6 (amide-I 1628 and 1672 cm−1, L-leucine isopropyl 1372 and 1390 cm−1). The IR spectrum of 6-O-trityl-chitosan 4 confirmed the complete removal of the phthaloyl group (disappearance of the signals at 1712 and 1776 cm−1). The spectrum of chitosan-N-L-leucine· HCl confirmed the removal of the trityl group (disappearance of signals at 702, 747, and 764 cm−1), and a reduction in the intensity of the doublet at 1372 and 1390 cm−1 suggested that the Boc group had been removed with the signal arising from Lleucine isopropyl only. The 1H and 13C NMR spectra of the final product 6 are shown in Figure 2. The 1H and 13C NMR spectra of the intermediates and their derivatives and the 2D 1H−13C geHSQC spectra of the final two products 5 and 6 are also provided in the Supporting Information (Figures S-1 to S-4). The solubility of 2 and 3 in common NMR solvents was not sufficient to obtain useful 1H and 13C NMR spectra and so, following the strategy of Nishimura et al.,44 they were acetylated (Scheme 1B) to improve their solubility for NMR characterization. The NMR spectra of 2 and 3 in DMSO-d6 and pyridine-d5 have been reported previously.45,46 This difference in solubility probably arises from differences in the molecular weight and DDA of the chitosan used as the starting material. The 1H NMR spectrum of 4 in pyridine-d5 was in accord with the IR data, confirming the selective removal of the phthaloyl groups. However, a 13C NMR spectrum of the compound could not be obtained due to poor solubility. The 1H and 13C NMR spectra of 5 in pyridine-d5 showed signals at δH 1.44 ppm and δC 28.9 ppm, respectively, for Boc t-butyl in addition to signals for trityl aromatic, pyranose, and L-leucine protons and carbons at their expected chemical shifts, thereby confirming conjugation of Boc-L-leucine to 4. NMR signals were also observed for CO of carbamate (156.9 ppm) and amide (Lleucine and N-acetyl) at 173.6 and 176.3 ppm, respectively, as well as for the quaternary carbons of Boc and trityl groups at 79.2 and 87.1 ppm, respectively. The final product chitosan-NL-leucine·HCl 6 showed adequate solubility in water to give more intense proton and carbon NMR signals in solution in D2O; however, while the 1H NMR signals of the compound showed considerable overlap, many carbon signals, especially those from pyranose carbons, were doubled up. Apparently, this was due to the presence of three different kinds of monomeric

Figure 2. 1H (A) and 13C (B) NMR spectra of chitosan-N-L-Leucine· HCl 6 (in D2O).

units in the product, viz., unsubstituted, N-acetyl-substituted, and L-leucine-substituted glucosamine residues. This kind of multiplicity of carbon peaks in chitosan derivatives has also been reported previously.47 As Boc t-butyl and L-leucine isopropyl had IR absorptions at the same positions (a doublet at around 1366 and 1390 cm−1), the NMR spectra were important to definitively ascertain the conjugation of L-leucine and ultimate deprotection of the Boc group. The 2D 1H−13C ge-HSQC spectra of 5 and 6 showed clear cross peaks correlating the carbon signals to proton signals (Figures S-3 and S-4). The 2D data of 5 was helpful in confirming the chemical shifts of some carbons that did not give very welldefined peaks in the 1-D 13C NMR spectrum (viz. pyranose carbons). The 2D data were also useful in assigning the signals of different protons in the 1H NMR spectrum that showed a lot of overlap. One limitation of the 2D spectrum of this compound was the absence of peaks corresponding to the anomeric carbon, but the presence of corresponding peaks in the 2D spectrum of 6 indicated that their absence from the spectrum of the former was simply due to lack of solubility. Similarly to 5, the 2D spectrum of 6 was also able to resolve the overlappings of 1H NMR signals. The final product 6 was also subjected to XPS analysis (see Supporting Information, Figure S-6). The high resolution XPS scan of N (Figure S-5A) showed a reduction in the intensity of NH signal from 5.94% (in 1) to 0.68% (in 6) and elevation in the intensity of N+ (from 0.78% to 2.99%) and Namide (from 0.72% to 5.14%), as expected. Moreover, a high-resolution scan of C (Figure S-5 B) also showed the expected elevation in the C−C carbon signal from 12.39% (in 1) to 19.14% (in 6). The 3601

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Scheme 1. (A) Synthetic Pathway for Conjugation of L-Leucine to Chitosan (1) and (B) Acetylation of N-Phthaloylchitosan (2) and N-phthaloyl-6-O-tritylchitosan (3)

elemental analysis of most intermediates and the final product 6 provided satisfactory results (see Materials and Methods). Chitosan and Conjugate Nanoparticles. Nanoparticles of chitosan and the conjugate were prepared by a W/O emulsion solvent evaporation technique. Being freely soluble in water, in contrast to chitosan, no acidification was needed for dissolving the conjugate when preparing the conjugate nanoparticles. To our knowledge, this is the first report for preparing particles of chitosan or any of its derivatives in nanoscale by this technique using paraffin oil as the external phase. The morphologies of nanoparticles were examined by SEM, and almost all particles appeared to be oblate, rather than perfect spheres (Figure 3). The particles showed a tendency to aggregate, although there appeared to be some isolated single particles as well. In addition, we studied the morphological properties of nanoparticles using TEM, which further revealed that those nanoparticles are nonspherical with unsmooth surfaces (Figure 4). The SEM analysis suggested that the nanoparticles had diameters predominantly in the range of 10− 30 nm (Figure 3); however, SEM is not an appropriate method for determining the particle size. Using a zetasizer, particle size and size distributions of nanoparticles were analyzed, and both the chitosan and conjugate nanoparticles showed a unimodal size distribution with average diameters (Z-averages) of 134.4 ± 2.8 and 153.2 ± 1.2 nm, respectively (Figure 5A,B) and the corresponding polydispersity indices were 0.58 ± 0.26 and 0.35 ± 0.08, respectively. The Z-average of conjugate nanoparticles was

Figure 3. Scanning electron micrographs of (A) chitosan and (B) conjugate nanoparticles. [Note: Dimension bar is shown under each micrograph.]

larger than that of chitosan nanoparticles. The larger size of particles observed by te zetasizer probably suggests the presence of nanoagglomerates arising from cohesion of very small individual particles. This is a common phenomenon with micro- and nanosized particles, which results from high surface area and associated surface free energy of particles in this size range. Similar observations have been reported previously for various polymeric and nonpolymeric microparticles.48,49 The average particle sizes measured by the zetasizer were much larger compared with the sizes of the particles observed by SEM. The actual reason behind this is unclear; however, the smaller sizes revealed by the SEM could be due to shrinkage during dehydration under the high vacuum conditions, thus 3602

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icals have been reported to be categorized as IC50 < 1 mg/L very high toxicity; IC50 = 1−10 mg/L - high toxicity; and IC50 >100 mg/L - low toxicity.51 Therefore, the outcomes of our studies (i.e., 2 mg/mL) belong to the low level of toxicity. Drugs administered by inhalation (e.g., beta agonists and corticosteroids) are generally potent with a dose size not exceeding a few hundred micrograms. Therefore, the conjugate nanoparticles could be considered promising as a matrix for controlled release delivery through the pulmonary route. The increased toxicity of conjugate and its nanoparticles appears to be reasonable if compared with TMC, another water-soluble derivative that has been reported to be more toxic than chitosan.22 This has been attributed to its increased solubility that allows a better interaction with anionic cell surface components. Previous literature suggests that nondegradable additives, such as surfactants (e.g., (Pluronic P-105) or polymers (e.g., poly(vinyl alcohol)),52 or residues from organic solvents53 used in the emulsion method may contribute to the toxicity of the prepared particles. The increased toxicity of the particulate forms compared to the neat polymers observed in the this study might be related to any remaining organic chemicals like span 80 or hexane used in the preparation and purification of the nanoparticles. Nanoparticles have also been reported to be internalized by cellular uptake into pulmonary epithelial cells.54 This kind of possible uptake of the nanoparticles might have also contributed to the toxicity by allowing the particles to interact with subcellular organelles in addition to the cell surface. The IL-8 release caused by conjugate nanoparticles at concentrations of 1 and 2 mg/mL was 3783 ± 738 and 6062 ± 533 pg/mL, respectively, which were 5- to 8-fold higher than the basal level of IL-8 release (768 ± 133 pg/mL) and so raises some concern about their safety for respiratory delivery. However, there was no significant induction of IL-8 at a concentration of 0.5 mg/mL. Previous literature reported a wide range of constitutive IL-8 secretion from as low as 274 pg/mL20 to as high as 1000−10 000 pg/ mL55 at different time-points of incubation from various pulmonary epithelial cell lines. So, it is difficult to predict what could be the biologically relevant concentration of IL-8 secretion for initiating pro-inflammatory effect; however, considering that there was no significant IL-8 induction compared with the control at a conjugate nanoparticle concentration of 0.5 mg/mL, they could be considered adequate for respiratory delivery of potent drugs with small dose sizes. The trans-epithelial permeability study was performed using sodium fluorescein as a fluid phase marker (Figures 8 and S-6). The results indicated that chitosan and its nanoparticles do not cause any significant change in permeability. On the other

Figure 4. Transmission electron micrographs (TEM), (A) chitosan and (B) conjugate nanoparticles. [Note: Dimension bar is shown under each micrograph.]

underestimating nanoparticle size. Therefore, the particle sizes measured by the hydrodynamic technique (zetasizer) are larger and more accurate. It is noteworthy that the principles of the two instruments are different in determining particle sizes and SEM has limited efficiency in determining particle size accurately. Toxicological Evaluation of Chitosan, the Conjugate, and Their Nanoparticles. The polymers were assessed, both in neat and particulate forms, for their safety for pulmonary delivery using cytotoxicity, trans-epithelial permeability and IL8 secretion as the indicators of their toxicity and inflammatory effects on the bronchial epithelial cell line, BEAS-2B. The MTT assay assessed effects of increasing concentrations (0.125 to 16 mg/mL) of chitosan, the conjugate, and their nanoparticles on cell viability over three time periods, viz., 12, 24, and 48 h. The 4 highest concentrations at which most of the test samples showed a percent survival above 50% and beyond which it was found to fall abruptly (viz. 0.5, 1, 2, and 4 mg/ mL), were chosen to perform the subsequent sodium fluorescein transport and IL-8 secretion studies. The results of the MTT assay (Figure 6) and IL-8 induction study (Figure 7) indicated that particulate forms of the polymers were more cytotoxic and inflammatory than neat polymers, with the conjugate nanoparticles appearing to be the most cytotoxic and inflammatory. However, the IC50 value of the conjugate nanoparticles following the longest exposure tested (48 h) was determined to be 2 mg/mL. Although chitosan is biocompatible, due to its broad molecular weight range and heterogeneity, it produces different toxicological profiles in varieties of cell lines. In addition, the toxicity of chitosan nanoparticles in different cell lines is concentration dependent.50 Therefore, actual toxicity profiles (low or high level) of chitosan nanoparticles prepared by different methodologies are largely unclear. The toxicity levels of pharmaceut-

Figure 5. Particle size (diameter, d (nm)) distribution of (A) chitosan and (B) conjugate nanoparticles (n = 3). 3603

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Figure 6. Effect of chitosan (A), the conjugate (C), and their nanoparticles (B and D) on the viability of BEAS-2B cell line. The % survival of BEAS2B cells was determined by MTT assay after 12, 24 and 48 h exposure to increasing concentrations (viz. 0.125, 0.25, 0.375, 0.50, 1.0, 2.0, 4.0, 8.0, 12.0, and 16.0 mg/mL) of suspension/solution of chitosan, the conjugate and their nanoparticles in cell culture medium. * = The concentration at which no significant difference was observed in % survival with control. Results presented as % survival relative to controls and expressed as the mean ± SD (n = 3). p ≤ 0.05 was considered significant.

transports of 0.011458, 0.011842, and 0.011617 mg at 0.5, 1, and 2 mg/mL concentrations vs 0.010738 mg at 0 mg/mL concentration (control) as per ANOVA), while the conjugate nanoparticles caused an overt reduction in permeability. Increased permeability with the conjugate is consistent with reports on the permeability enhancing effect of TMC.56,57 It is worth noting that cellular death causing gaps in the epithelial layer might have contributed to the increased Na Flu permeability. Further studies are warranted to investigate the permeability effect of the highest nanoparticle concentration. Using immunocytochemistry, the ZO-1 staining (Figure S-7) confirmed the presence of tight junctions under the culture conditions used, consistent with previous studies.58 Thus, we believe that the culture conditions used were appropriate to determine transport across the cell monolayer. The reduction in the permeability shown by the conjugate nanoparticles does not fit with the reports on the micro-/ nanoparticles of TMC that have been found to induce permeation enhancement similar to neat TMC.59,60 Further studies are required to understand this unexpected effect produced by the conjugate nanoparticles. Except for neat chitosan, the highest dose of 4 mg/mL applied in the experiments showed a different trend in causing increase/decrease in permeability compared with the lower doses. This may be related to the high level of toxicity/ cell death encountered at this dose; further studies are warranted to explore the exact mechanism involved.

Figure 7. Effect of chitosan, the conjugate, and their nanoparticles on IL-8 secretion by BEAS-2B cells. The IL-8 levels in culture supernatants were determined by ELISA 24 h after exposure of the cells with increasing concentrations (0, 0.5, 1, 2, and 4 mg/mL) of the test samples suspended/dissolved in the cell culture medium. The IL-8 levels were normalized to the number of cells by dividing the measured values by the % viability of cells estimated by MTT assay. Results expressed as the mean ± SD (n = 3) and p ≤ 0.05 was considered significant.

hand, the conjugate showed a mild but significant (p < 0.05) increase in permeability (estimated mean cumulative Na Flu 3604

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Figure 8. Effect of chitosan (A), the conjugate (C), and their nanoparticles (B and D) on Na Flu transport across the BEAS-2B cell monolayer. Cumulative amounts of Na Flu transported to the basolateral chambers of transwells containing polarized cell monolayers were determined by spectrofluorometry at 0.5, 1, 2 4, 6, 12, 24, and 48 h time-points after apical treatment with increasing concentrations (viz., 0, 0.5, 1, 2, and 4 mg/mL) of suspension/solution of chitosan, the conjugate, and their nanoparticles in cell culture medium containing Na Flu (0.2 mg/mL). Results expressed as the mean ± SD (n = 3) and p ≤ 0.05 was considered significant.

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CONCLUSIONS L-Leucine has been successfully conjugated to chitosan, giving a novel compound with improved solubility and thus paving the way for better utilization of chitosan in drug delivery applications. The work also established a method based on emulsion-solvent evaporation technique for preparing nanoparticles of both chitosan and its conjugate suitable for deposition to the deeper regions of the respiratory airways. The conjugate and its nanoparticles appeared to be relatively more toxic and inflammatory than chitosan and its nanoparticles; however, the level of toxicity (IC50 2 mg/mL following 48 h exposure) and inflammatory effect (no induction of IL-8 at 0.5 mg/mL) can still enable its utilization for pulmonary drug delivery unless the dose size of the drug to be incorporated is high. The leucine-conjugated nanoparticles require further investigation to evaluate appropriate toxicity levels at wider concentration ranges using different cell lines at different exposure times. More conclusive inferences can, however, be drawn only after in vivo trials in animal models.

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for nitrogen and carbon and comparison of the effect of chitosan, the conjugate, and their nanoparticles on Na Flu transport across the BEAS-2B cell monolayer; and confocal images of ZO-1 expression in BEAS-2B cells grown on 0.4 μm transwell inserts for 6 days. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +61 7 3138 1899. Fax: +61 7 3138 1534. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Prof Philip Hansbro, School of Biomedical Sciences and Pharmacy, The University of Newcastle, Australia, for providing the BEAS-2B cell line. This research was supported by an International Postgraduate Research Scholarship (IPRS) to M.D.A.M. granted by the Australian Government through Queensland University of Technology.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of synthetic intermediates and the final product 6; 2D 1H−13C ge-HSQC data of the final two products 5 and 6 in the synthetic pathway; XPS multiplex spectra of chitosan 1, L-leucine, and chitosan-N-L-leucine·HCl 6 3605

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ABBREVIATIONS ATCC, American type culture collection; ATR, attenuated total reflectance; Boc-Leu-OSu, t-butyloxycarbonyl-L-leucine-succinimide; CCM, cell culture medium; DDA, degree of Ndeacetylation; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DPI, dry powder inhaler; DS, degree of substitution; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FT-IR, Fourier transform infrared; IC50, median inhibitory concentration; ge-HSQC, gradient-enhanced heteronuclear single quantum correlation; IL-8, inerleukin-8; LB, line broadening; MTT, 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MW, molecular weight; Na Flu, sodium fluorescein; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PDI, polydispesity index; SEM, scanning electron microscopy; SV40, Simian virus 40; TEER, trans-epithelial electrical resistance; TMC, trimethlychitosan chloride; XPS, X-ray photoelectron spectroscopy

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