Effect of PEGylation on the Toxicity and Permeability Enhancement of

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Biomacromolecules 2010, 11, 2854–2865

Effect of PEGylation on the Toxicity and Permeability Enhancement of Chitosan Luca Casettari,†,‡ Driton Vllasaliu,*,‡ Giuseppe Mantovani,‡ Steven M. Howdle,§ Snow Stolnik,‡ and Lisbeth Illum‡,⊥ Drug Delivery and Tissue Engineering Division, School of Pharmacy, and School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, United Kingdom and Critical Pharmaceuticals Limited, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, United Kingdom Received May 13, 2010; Revised Manuscript Received August 16, 2010

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 103 g mol-1) to chitosans (Mw 28.9 and 82.0 × 103 g mol-1) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, 1H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

Introduction Recent advances in the field of biotechnology have resulted in a rapid increase in the appearance of therapeutically active biological macromolecules, including peptides, proteins, and nucleic acids, on the market.1 As oral administration of this class of therapeutics has so far been highly inefficient and with issues associated with the parenteral administration, noninvasive routes of delivery for these molecules with therapeutically acceptable pharmacokinetics are highly desirable. In this respect, nasal delivery has been extensively investigated, mainly due to desirable characteristics of the nasal mucosa such as the relatively large surface area available for absorption, the highly vascularised sub mucosa, and the avoidance of gastric and first pass metabolism.2 However, several biological barriers must be overcome for a macromolecule to translocate from the mucosal lumen into the systemic circulation. These include the mucus layer, the “sweeping effect” of mucociliary clearance, the presence of degradative enzymes (e.g., proteases), and the presence of intercellular tight junctions, which severely limit the transepithelial permeability of macromolecules larger than 1000 Da.3 Efficacious absorption of therapeutic macromolecules across the nasal mucosa therefore in most cases requires coadminis* To whom correspondence should be addressed. E-mail: paxdv@ nottingham.ac.uk. † Present address: Dipartimento di Scienze del Farmaco e della Salute, Universita` degli Studi di Urbino ‘CarloBo’, Urbino, Italy. ‡ School of Pharmacy. § School of Chemistry. ⊥ Critical Pharmaceuticals Limited.

tration of the macromolecule with agents that improve its movement across the mucosal surface, that is, “absorption enhancers”. Various compounds have been investigated for their potential to facilitate the absorption of macromolecules following their nasal administration, with chitosan being a prominent example within this field. The absorption-promoting effect of chitosan has been extensively studied and shown to result from a combination of mucoadhesion and its ability to open the intercellular tight junctions.4-6 Different derivatives of chitosan have been synthesized in an attempt to improve its physical and biological properties, with synthesis of partially quaternized chitosan (TMC), being a particularly dominant option7-9 to increase the solubility of chitosan in biological environments where it is not normally soluble. Other derivatives include thiolated chitosans,10 carboxymethyl-chitosans,11-13 and PEGylated chitosan14-17 or trimethylchitosan (TMC).18 TMC and its PEGylated derivatives have been investigated as absorption enhancers for protein and gene delivery in nasal, lung, transdermal, or colonic delivery, whereby PEGylation of TMC offers a possibility of reducing its cellular toxicity.18,19 A number of synthetic strategies have been applied in PEGylation of chitosan based on different types of activated PEG or monomethoxy-PEG (mPEG). In general, these PEGylating agents target the chitosan primary amine units and they can be divided in two main categories: (a) alkylating agents, such as PEG-aldehyde14,20-24 and PEG-epoxide,14,25 which upon conjugation form secondary amine linkers (still protonable at pH < pKa of the amine), and (b) PEG-activated ester,17,26,27

10.1021/bm100522c  2010 American Chemical Society Published on Web 09/27/2010

Toxicity and Permeability Enhancement of Chitosan

Biomacromolecules, Vol. 11, No. 11, 2010

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Table 1. Synthesis of mPEG-g-chitosans: Reaction Conditions entry

conjugates

CS (g)a

CS NH2 (mmol)b

mPEG-COOH (g)

mPEG-COOH (mmol)

mPEG/CS NH2 (mol/mol)

D.S.%c,d

1 2 3 4 5

mPEG1900-g-MMWC mPEG5000-g-MMWC mPEG5000-g-MMWC mPEG1900-g-LMWC mPEG5000-g-LMWC

0.100 0.100 0.050 0.050 0.050

0.38 0.38 0.19 0.28 0.28

1.00 1.00 1.20 1.00 1.35

0.52 0.27 0.24 0.52 0.27

1.4 0.96 1.3 1.86 0.96

5.4 1.5 6.7 5.3 1.6

a Mass of chitosan (Protasan UP Cl 113 (MMWC) or Ultrasan (LMWC)). b Mmol of chitosan free amino groups, considering an average deacetylation degree of 82% for MMWC and 98% for LMWC. c Measured by SEC-MALLS (using 0.5 M acetic acid/0.1 M NaNO3, as the mobile phase and TSK gel columns. d Degree of substitution expressed as the percentage of chitosan repeating units successfully conjugated with mPEG.

PEG-acid,28-31 and PEG-nitrophenyl carbonate,15,32-34 which afford nonprotonable amide or carbamate linkers. The present work investigates whether modification of chitosan by PEGylation results in improvement of its toxicity at the nasal mucosa, a phenomenon shown in vitro for PEGylated TMC,18 while preserving its ability to open the cellular tight junctions. To this end, in this study a series of mPEG-g-chitosan conjugates were synthesized in which the level of PEG substitution, PEG molecular weight, and chitosan molecular weight were systematically varied and effects of these changes on toxicity and tight junction opening capacity were assessed in mucus producing Calu-3 cells. The use of the human lung (bronchial carcinoma) originating Calu-3 cell line as a model of the nasal epithelium was deemed appropriate as upon specific culture conditions these cells produce a polarized cell layer of a mixed phenotype, including ciliated and secreting cells, with physical and electrical properties comparable to nasal mucosa.35,36 Consequently, Calu-3 cells have previously been employed to study nasal drug absorption.37-41

Experimental Section Materials. Ultrapure chitosan hydrochloride, commercially known as Protasan UP Cl 113, with a weight average molecular weight (Mw) of 50-150 × 103 g mol-1 and a degree of deacetylation (DDA) between 75 and 90%, referred to as medium Mw chitosan (“MMWC”), was purchased from NovaMatrix (FMC Bio-Polymer, Drammen, Norway). Ultrasan (Mw 20-70 × 103 g mol-1 and DDA > 98%, free base form), referred to as low molecular weight chitosan (“LMWC”) was purchased from Biosyntec (Canada). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), glutaric anhydride, paraformaldehyde, and all other reagents were obtained from Sigma-Aldrich at reagent grade or higher and used without further purification. Calu-3 cells (used between passages 22-30) and Eagle’s Minimum Essential Medium (EMEM) were purchased from American Type Culture Collection (ATCC). Nonessential amino acids, L-glutamine, fetal bovine serum (FBS), antibiotic/ antimycotic (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B) solution, trypsin-EDTA solution (2.5 mg/mL trypsin; 0.2 mg/mL EDTA), Hanks’ balanced salt solution (HBSS), fluorescein isothiocyanate (FITC)-labeled dextran Mw 4.4 × 103 g mol-1 (FD4), Mw 10 and 40 × 103 g mol-1 (FD10 and FD40), 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 2-(Nmorpholino)ethanesulfonic acid (MES) were purchased from SigmaAldrich. MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, commercial name CellTiter 96 AQueous One Solution) was purchased from Promega (U.S.A.). DAPI (4′,6-diamidino-2-phenylindole) with mounting media (commercially known as SlowFade Gold) was purchased from Invitrogen. Mouse, anti-human Zonula Occludens-1 (ZO-1; tight junction protein) antibody was purchased from Zymed (part of Invitrogen) and Cy5-labelled goat, anti-mouse IgG was obtained from Invitrogen. Activation of mPEG: Synthesis of mPEG-COOH. Methoxypoly(ethylene)glycol (mPEG) was functionalized at the free hydroxyl

group chain-end by esterification using glutaric anhydride to give a carboxy-terminated mPEG (mPEG-COOH). The method used here is a modification of the protocol described previously by Liu et al.42 Briefly, mPEG (Mn 1.9 × 103 g mol-1, 2.3 g, 1.2 mmol, predried by azeotropic distillation with toluene) was dissolved in anhydrous dichloromethane (15 mL). Glutaric anhydride (0.68 g, 6.0 mmol) was then added to the resulting solution, and after complete dissolution, 0.15 g (1.2 mmol) of DMAP organocatalyst were added. The reaction was stirred under argon atmosphere at room temperature for 48 h. The resulting solution was concentrated and then added to diethyl ether (30 mL) and the mixture cooled at 4 °C for 1 h. The precipitated product was filtered, and the solid filtrate (mPEG-COOH crude product) redissolved in the minimum amount of dichloromethane. The precipitation step in diethyl ether was then repeated, followed by filtration to give an mPEG-COOH crude product as a white powder. This was then dissolved in deionized water and the resulting solution dialyzed against water (membranes tubes MWCO: 1 kDa) for 2 days. The resulting solution was finally freeze-dried to give the desired mPEG-COOH product (1.3 g, 65%) as a light white solid which was stored at -20 °C. mPEG (5 × 103 g mol-1) was converted in its corresponding mPEGCOOH derivative following the same procedure. Synthesis of mPEG-g-chitosans. Functional mPEG-COOH (Mn 1.9 and 5.0 × 103 g mol-1) and chitosans, “MMWC” 53 × 103 g mol-1 and “LMWC” 20 × 103 g mol-1 (number average molecular weights Mn determined by SEC-MALLS analysis of commercially available samples) were used to synthesize mPEG-g-chitosan conjugates with different degrees of substitution and molecular masses, following a modified version of the NHS/EDC coupling approach described previously by Prego et al.43 and Aktas et al.44 Briefly, for MMWC, 100 mg (0.38 mmol of primary amino groups potentially available for conjugation) of chitosan were dissolved in 15 mL of PBS (0.1 M, pH 6.0). LMWC was dissolved in the same buffer, acidified with HCl 0.1 M to facilitate its solubilization, then the pH was adjusted to 6.0 with gradual addition of a concentrated aqueous sodium hydroxide solution. mPEG5000-COOH (1.0 g, 0.52 mmol) was then added to the solutions of chitosan, followed by NHS (0.11 g, 0.94 mmol), and, after 15 min, EDC (0.18 g, 0.94 mmol). The reaction mixture was stirred at ambient temperature for 24 h, then dialyzed against deionized water using dialysis membranes with MWCO of 15 or 50 kDa (Spectrum Laboratories, U.S.A.) for 48 h and finally freeze-dried to give 0.71 g of the desired mPEG5000-g-chitosans conjugate (entry 3, Table 1). Degree of chitosan substitution was calculated using average molecular weight values determined by SEC analysis, as follows:

D.S.% )

Mn(conj) - Mn(csm) 1 × × 100 Mn(mPEG) DP(csm)

where Mn(conj) is the number average molecular weight of the mPEGg-chitosan conjugate, Mn(csm) is the number average molecular weight of the chitosan starting material, Mn(mPEG) is the number average molecular weight of the mPEG starting material, and DP(csm) is a number of chitosan glucosamine repeating units. Similar degrees of substitution were obtained by 1H NMR analysis (see Supporting Information).

2856 Biomacromolecules, Vol. 11, No. 11, 2010 1 H NMR and FT-IR Characterization. mPEG-g-chitosan conjugates and mPEG intermediates were characterized by 1H NMR using a Bruker Advance Ultra Shield 400 MHz spectrometer (Bruker, Germany) with D2O or D2O/DCl as the solvent and by FT-IR using a Nicolet IR200 Spectrometer and KBr discs.

Thermogravimetric (TGA) and Differential Scanning Calorimetry (DSC) Analysis. Thermal weight-loss analysis of chitosans and mPEG-g-chitosans was performed using a thermogravimetric analyzer (Q500 TA Instruments) at a heating rate of 10 °C/min from 0 to 500 °C. DSC measurements were conducted using a Q2000 differential scanning calorimeter from TA Instruments. Approximately 5 mg of each sample were used in these tests. Measurements were conducted at a heating (cooling) rate of 10 °C/min from 0 to 500 °C. SEC-MALLS Analysis of mPEG-g-chitosan Conjugates. The molecular mass distributions of mPEG-g-chitosan conjugates were determined by size exclusion chromatography using a system equipped with multiangle light scattering MALLS detector (SEC-MALLS; Dawn HELOS II, Wyatt), a viscosimeter (ViscoStar, from Wyatt), and a refractive index (RI) detector (Optilab rEX from Wyatt). Analysis was carried out using 0.5 M acetic acid/0.1 M NaNO3 at pH 2.5 as the mobile phase, TSK gel, G6000PW, and G4000PW columns (TOSOH). Sample concentration was 5 mg/mL and the flow rate 0.8 mL/min dn/ dc used for PEG was 0.136 cm3/g, for chitosan 0.181 cm3/g, as described by Beri et al.,45 and 0.19 cm3/g for mPEG-g-chitosan conjugates.26 Solubility of mPEG-g-chitosan Conjugates. The solubility of chitosan and PEGylated chitosan conjugates were determined by measuring solution transparency by UV spectrophotometry, as described recently by Jeong and co-workers.17 Briefly, 5 mg of each chitosan and mPEG-g-chitosans were mixed with 100 µL of deionized water, followed by the addition of 4.9 mL of an aqueous buffer solution (producing a concentration of 1 mg/mL) and vigorous stirring (vortex stirring, 3-4 min). The aqueous buffers employed were 0.1 M carbonate buffer, pH 9.6; 0.1 M phosphate-buffered saline (PBS), pH 7.4; 0.1 M acetate buffer, pH 4.0; and 0.01 M HCl solution, pH 2.0. Analysis of the turbidity was performed using a UV-vis Beckman Coulter DU800 spectrophotometer at l ) 600 nm. Relative solubility of selected mPEGg-chitosans was evaluated by comparing the transmittance (T%) at l ) 600 nm of mixtures of chitosan and mPEG-g-chitosan conjugates in aqueous buffers of different pH values to that of pure water. Cell Culture. Calu-3 cells were cultured to confluence in 75 cm2 flasks at 5% CO2, 37 °C. Once confluent, cells were detached from the flasks by incubating with trypsin/EDTA solution and seeded on Transwell permeable inserts at a density of 105 cells/cm2. Cells were maintained at 5% CO2, 37 °C in EMEM, supplemented with FBS (10% v/v) and antibiotic/antimycotic solution, which was replaced regularly (every 48 h). Air-liquid interface (ALI) was created on day two following seeding of the cells on filters. Cell growth was assessed by TEER measurements, which is a measure of resistance to ion flux across an epithelial layer, reflecting the degree of confluence of the cells (and the formation of cellular tight junctions). Cell monolayers were typically used for TEER and permeability experiments between days 8-10 in culture, as confluent and polarized monolayers (Figure S2, Supporting Information). Cell Toxicity Studies. Cells were seeded on 96-well plates (Costar) at a density of 104 cells/well and incubated overnight at 5% CO2, 37 °C. Toxicity studies were performed by replacing the culture medium with samples consisting of different concentrations of chitosans and PEGylated conjugates, dissolved in HBSS (buffered at pH 6.0 for unmodified chitosan and pH 7.4 for the conjugates). Triton X-100 (0.1% v/v in buffered HBSS) and HBSS (buffered at pH value corresponding to that of the test samples) were used as a positive and negative control, respectively. Cells were incubated with the test samples (and controls) for 2 h and thereafter washed with PBS. The MTS assay, a colorimetric method based on bioreduction of a tetrazolium compound by metabolically active cells, was used to determine the viability of cells and was performed according to the manufacturer’s protocol with at least four

Casettari et al. repeats for each tested sample. The relative cell viability (%) was calculated using the following equation:

relative viability )

S-T × 100 H-T

where S is the absorbance at 492 nm obtained with the tested samples, T is the absorbance observed with Triton X-100 (positive control to induce cell death), and H is the absorbance measured with medium (HBSS). Effect of mPEG-g-chitosans on Calu-3 Cell Monolayer TEER. TEER measurements were employed in this part of the work to determine changes in the tightness of the cellular tight junctions. TEER was measured across the cell monolayers using an EVOM Volthommeter (World Precision Instruments) equipped with a pair of chopstick electrodes. The culture medium was removed from filtercultured Calu-3 monolayers and replaced with warm (37 °C) HBSS (buffered at pH 7.4 with 25 mM HEPES). Cells were incubated with HBSS for approximately 45 min, after which TEER was measured; the recorded value was considered as baseline TEER. Only cell monolayers with a baseline TEER g 500 Ω cm2 were included in the experiment. mPEG-g-chitosans were dissolved in HBSS at pH 7.4 (HEPES-buffered) or pH 6.0 (MES-buffered); unmodified MMWC (used as a positive control) was dissolved in HBSS at pH 6.0 (MESbuffered). HBSS was then removed from the apical side of the cell monolayers and replaced with samples (which were prewarmed to 37 °C). Cell monolayers were transferred to the incubator and TEER measurements were taken at different time intervals following the application of the test samples, consisting of mPEG-g-chitosans or unmodified MMWC at different concentrations. Following an incubation period of 2 h, samples were removed from the apical side of the cell monolayers and replaced with warmed (37 °C) culture medium following a washing step with PBS. Further TEER measurements were conducted 2 and 22 h following sample removal. Background TEER due to the filter was deducted from the measurements. Changes in TEER are reported as % of baseline TEER values. All experiments were performed in triplicates. Permeability Across the Cell Monolayers. In addition to TEER measurements, macromolecular permeability across the cell monolayers was determined as an alternative indication of the status of the tight junctions. Since the permeability of hydrophilic macromolecules larger than approximately 1000 Da is largely limited by the tight junctions,46,47 any increases in the permeability of these compounds potentially suggest tight junctions opening. Similar to TEER studies, only filter-cultured cell monolayers exhibiting a TEER g 500 Ω/cm2 were included in this experiment. Culture medium (EMEM) was first removed and the cells washed with PBS. HBSS (HEPES buffered, pH 7.4) was then applied to the cells and an equilibration (incubation) period of approximately 45 min was allowed. FD4 and FD40 were used as hydrophilic macromolecular model drugs (“permeants”) and HBSS as the transport medium. Test solutions, consisting of mPEG-g-chitosans in combination with a permeant (used at a concentration of 500 µg/ mL), dissolved in HBSS at pH 6.0 or 7.4 (buffered with MES and HEPES, respectively) were applied on the apical side of the cell monolayers; unmodified MMWC (used as a positive control) was also applied in combination with FD4 or FD10 in HBSS at pH 6.0 (MESbuffered). The permeability of FDs was determined by sampling the basolateral solution (100 µL volumes) at regular time intervals (every 30 min) for 3 h. The sampled volumes were replaced with fresh transport medium in order to maintain sink conditions. In-between sampling intervals, cells were incubated at 37 °C, 5% CO2. Basolateral FD was quantified by measurement of fluorescence, using an MFX microtiter plate fluorometer (Dynex Technologies, U.S.A.) through preconstructed calibration curves. After the final sampling, copolymers and FDs were removed from the cells and replaced with the culture medium after a washing step with PBS. Cell monolayers were then incubated overnight at 37 °C, 5% CO2, following which TEER was

Toxicity and Permeability Enhancement of Chitosan Scheme 1. Synthesis of mPEG-g-chitosan Conjugatesa

a Reagents and conditions: mPEG-COOH, EDC, NHS, PBS pH 6.0, 20 °C.

measured in order to ensure an intact cell monolayer integrity during the permeability experiments. Permeability of FDs is expressed as the apparent permeability coefficient (Papp), calculated using this equation:

Papp )

1 × ( ∆Q ∆t ) ( AC ) 0

where Papp is the apparent permeability (cm/s), ∆Q/∆t is the permeability rate (amount of permeant, FD, traversing the cell layers over time), A is diffusion area of the cell layer (cm2), C0 is the concentration of the apically added permeant, FD. The experiment was conducted in triplicates. ZO-1 Distribution Following mPEG-g-chitosan Application. Culture medium (EMEM) was removed from confluent cell monolayers and replaced with HBSS (HEPES buffered, pH 7.4); cells were equilibrated for approximately 45 min. HBSS was then removed from the apical side of the cells and replaced with mPEG5000-g-MMWC, dissolved in HBSS at pH 6.0 (buffered with MES) or 7.4 (buffered with HEPES). Cells were incubated with mPEG5000-g-MMWC, which was applied at a concentration of 0.025% w/v, for 1 h. As a control, cells in the form of a monolayer were also incubated with unmodified MMWC (0.003% w/v in HBSS/MES at pH 6.0). Samples were then removed and cells washed with PBS. Cells were thereafter fixed with paraformaldehyde (4% w/v) for 8-10 min, permeabilized with Triton X-100 (0.1% v/v in PBS) for approximately 10 min and incubated with 1% w/v BSA in PBS for 30 min. Mouse, antihuman ZO-1 antibody (primary antibody), diluted in BSA/PBS to 10 µg/mL, was then applied to the cells for 30 min at room temperature. Thereafter, this solution was removed and cells extensively washed (at least five times) with PBS. Goat, antimouse IgG-Cy5 (secondary antibody), diluted in 1.0% w/v BSA in PBS buffer to 2 µg/mL was then applied and cells incubated with this solution for 30 min. The secondary antibody solution was subsequently removed and cells washed (with PBS) extensively. Cell monolayer-containing filters were excised carefully as to avoid damage of the sample and mounted (using DAPI-containing ProLong Gold antifade/mounting medium) on glass slides for confocal imaging. Cells were imaged using a Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope. Statistical Analysis. Statistical comparisons were performed by Student’s t-test. Values of p < 0.05 were considered statistically significant.

Results and Discussion Synthesis and Characterization of mPEG-g-chitosan Conjugates. mPEG-g-chitosan copolymers were prepared via a two-step procedure: (1) conversion of mPEG into carboxylic acid-terminated mPEG-COOH and (2) grafting of mPEG-COOH to chitosan to give the desired mPEG-g-chitosan conjugates (Scheme 1). Methoxy PEGs (mPEGs) were employed here instead of the uncapped versions in order to avoid unwanted


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cross-linking side-reactions in the conjugation step due to the presence of difunctional PEGylating agents. mPEG-COOH derivatives can be readily obtained by treatment of commercially available mPEG with a cyclic anhydride and an organocatalyst (i.e., DMAP). In the present study glutaric anhydride was chosen because of the superior hydrolytic stability of the linker obtained by reaction of the corresponding mPEG-COOH with primary amines, compared with those obtained from other cyclic anhydrides, such as maleic, malonic, or succinic anhydrides.48 The mechanism of the grafting process involves EDCmediated conversion of mPEG-COOH into its corresponding N-hydroxysuccinimide (NHS) activated ester, which then reacts in situ with the amino groups of chitosan forming a stable amido linkage. The mPEG-COOH which does not react with chitosan presumably undergoes NHS ester hydrolysis, a side-reaction which always occurs for these derivatives under aqueous conditions. In general, an increase in the pH results in faster NHS ester hydrolysis; on the other hand, with nucleophiles such as organic amines the higher the pH, the faster is the conjugation reaction, due to a higher proportion of nonprotonated amino groups available for the conjugation step. In general, the choice of the pH at which pegylation with mPEG NHS ester derivatives is conducted depends on a number of factors. In our case, here, pH 6.0 at 20 °C was a good compromise between the need of using a pH low enough to maintain native chitosan soluble in aqueous conditions and that of having a pH still high enough to ensure fast conjugation reaction. This synthetic strategy allowed for the preparation of a library of PEGylated chitosans obtained from two different mPEGs (Mw 1.9 and 5.0 × 103 g mol-1) and two chitosans, MMWC (Mw(SEC) 82.0 × 103 g mol-1) and LMWC (Mw(SEC) 28.9 × 103 g mol-1), with degrees of substitution, expressed as the proportion of chitosan amino groups conjugated with mPEG, ranging from 1.5 to 6.7% (Table 1). Under these experimental conditions the conjugation reactions were found to have good interbatch reproducibility. SEC analysis of the mPEG-g-chitosan conjugates (see Determination of the Molecular Weight of mPEG-g-chitosan Conjugates paragraph below for more details on the average molecular weight of both chitosan starting materials and mPEG-g-chitosan conjugates) showed that for both MMWC and LMWC no significant difference in the degree of substitution was observed when the molecular weight of the conjugating polymer mPEG-COOH was increased from 1.9 to 5.0 × 103 g mol-1 (Table 1; entry 1 vs 3). This could be explained in terms of the relatively low degrees of substitution that were targeted in this study, which would make phenomena like the decrease of the rate of conjugation that occurs during the pegylation reaction as the number of mPEG chains grafted onto the chitosan backbone increase (due to steric effects), less important. No significant difference in terms of reactivity was observed in the two chitosan samples, MMWC and LMWC, employed in this study (Table 1; entry 1 vs 4 and 2 vs 5). It should be noted that the reactions used here for preparation of the PEGylated chitosans use neither a metal catalyst, that is, CuI complexes employed for click chemistry PEGylation of azido-containing chitosan with PEG-alkyne derivatives49 nor a metal-based coupling system, that is, Ag2O/PEG-iodide PEGylating systems,50 which consequently reduces the potential toxicity of the final product presented in this work. 1 H NMR and FT-IR Characterization of mPEG-g-chitosan Conjugates. The FT-IR spectra of mPEG-g-MMWC (see Supporting Information) showed absorption bands associated

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Casettari et al.

Figure 1. Partial 1H NMR spectra of (1) mPEG1900-COOH in D2O, (2) mPEG1900-g-MMWC in D2O, and (3) MMWC in D2O/DCl.

Figure 2. TGA of MMWC (point2-stroke -••-••), mPEG5000 (unbroken line) and mPEG-g-MMWCs (mPEG1900-g-MMWC 5.4% D.S. (point3stroke3, ---•••---) and mPEG5000-g-MMWC 6.7% D.S. broken line, ---).

with the mPEG backbone at 842, 960, and 2915 cm-1, in addition to two bands related to ester and amide functionalities at around 1732 and 1651 cm-1, respectively, corresponding to the ester and amide groups present in the linker between mPEG and chitosan. The 1H NMR spectrum of mPEG1900-g-MMWC (Figure 1) shows the characteristic peaks for chitosan at 1.9-2.0, 3.1, and 3.5-3.9 ppm. The presence of mPEG in the conjugates is confirmed by the fact that methoxy group of mPEG is clearly visible in the spectrum at 3.3 ppm, its oxyethylene repeating unit at 3.6-3.7 ppm and a typical C(O)OCH2 signal at 4.2 ppm. FT-IR and 1H NMR results therefore strongly suggest successful conjugation of mPEG-COOH to chitosan. Thermogravimetric Analysis (TGA). TGA analysis of unmodified chitosan exhibits two sigmoids, one in the 35-130 °C range and the second one from 210 to 350 °C (Figure 2). The first sigmoid is associated with approximately 12% loss in weight, which may correspond to the loss of adsorbed water. From 210 to 350 °C, an approximately 50% reduction in weight resulting from degradation of chitosan was observed. PEGgrafted conjugates display a different TGA pattern, with three observed weight loss intervals. The first interval ranges between

40 and 80 °C, the second from 200 to 240 °C, possibly resulting from decomposition of the chitosan backbone, and the third stage of weight loss is observed from 320 to 380 °C, which could be ascribed to the decomposition of PEG units (or a combination of the latter and that of the chitosan backbone). Our results appear to be in agreement with the TGA data reported by Deng showing that mPEG-g-chitosan was more thermally stable than the unmodified chitosan,51 which led the authors to suggest that the grafting of mPEG on the chitosan backbone reduced thermal decomposition, due to destruction of part of the hydrogen bonds between the chitosan chains. Determination of the Molecular Weight of mPEG-g-chitosan Conjugates. SEC-MALLS analysis of unmodified MMWC chitosan and mPEG-g-chitosan conjugates showed that upon conjugation with mPEG-COOH the measured molecular mass of chitosan increased (Figure 3). It was estimated that 1.5 to 6.7% of available NH2 groups on the chitosan polymer molecule were conjugated by the mPEG. The melting temperatures (Tm) of mPEG and PEGylated chitosans (shown in Table 2) were obtained from DSC analysis using the second run method, in analogy to what was reported



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and Ultrasan LMWC) have decreased solubility in buffers pH 7.4 and 9.6. On the contrary, all the PEGylated conjugates were fully soluble in aqueous buffers from pH 2.0 up to pH 9.6. In other words, our data indicates that the substitution with PEG in the weight percentage ranging from 28 to 69% (Table 2) results in the conjugates that are soluble in aqueous buffers in neutral or basic pH. Similar to our study (entries 6 and 8 in Table 2), the conjugation of PEG2000 at the level of substitution of 4.6 mol % (35% weight %) to 10 × 103 g mol-1 chitosan, a somewhat smaller chitosan than in the present study, afforded soluble conjugates at pH above neutral.51 It has been reported for PEG2000 conjugates of 210 × 103 g mol-1 chitosan, a somewhat higher molecular weight chitosan than in the present study, that 15 to 42% level of (presumably) weight substitution produced conjugates with a complete solubility up to pH 10.0.52 For a higher molecular weight chitosan, 400 × 103 g mol-1, it has been reported that a weight substitution with mPEG550 at a level of 44-55% and mPEG5000 at 75-87% (degrees determined by 1 H NMR, expressed as a weight ratio of mPEG in the graft copolymer) did not result in fully soluble conjugates at pH 7.0.18 The need for a relatively high degree of substitution to achieve solubility in neutral and basic media was also highlighted by Du et al.,54 who for chitosans of molecular weight of 137, 190, and 400 × 103 g mol-1 conjugated with mPEGs 550, 2000, and 5000 g mol-1 reported that substitution of more than 50% (weight) was required. Effect of mPEG-g-MMWC Conjugates on Cell Viability. Figure 5 summarizes the effect of PEGylation on the cytotoxicity of MMWC toward Calu-3 cells. It is apparent from the figure that, following a two-hour incubation of cells with unmodified chitosan (in HBSS/MES at pH 6.0) there is a dose-dependent reduction in relative cell viability. The extent of this reduction ranged from 17% for the lowest applied concentration (0.003%) to 82% for the highest concentration (0.025% w/v) used in the experiment. With the exception of the lowest applied concentra-

Figure 3. SEC-MALLS profile of mPEG1900-g-MMWC (bold line) and MMWC (normal line) using 0.5 M acetic acid/0.1 M NaNO3 at pH 2.5 as the mobile phase.

by Deng et al.51 Such an analysis allowed the elimination of the effects of water, which is easily adsorbed by chitosan, on the Tm. DSC results shown in Table 2 reveal that (a) Tm could not be determined for unmodified chitosan (entries 1 and 2) and (b) Tm of mPEG-g-chitosan conjugates was lower than that of the corresponding mPEG starting materials (entry 3 vs 5 and 8; entry 4 vs 6, 7, and 9). Solubility of Chitosan and mPEG-g-chitosan Conjugates in Aqueous Buffers. Grafting of PEG onto chitosan in an attempt to increase its solubility in water under neutral pH and basic conditions and organic solvents has been extensively investigated,17,20,29,52,53 whereby the effect is dependent on the degree of substitution, the size of chitosan and the length of PEG chains.52 In the present work the solubilities of mPEG-g-chitosan conjugates and that of unmodified chitosans, both in their hydrochloride and free-base forms, were compared by a qualitative turbidimetric assay, in analogy to that reported by Jeong and co-workers.51 The data obtained (Figure 4) clearly display that both unmodified chitosans (Protasan Cl 113 MMWC Table 2. SEC-MALLS and DSC Analysis of Chitosan and Its Conjugates

1 2 3 4 5 6 7 8 9

polymer

Mwa,b

Mna,b

PDia

Protasan Cl 113 “MMWC” Ultrasan 20-70 “LMWC” mPEG 1.9 kDa mPEG 5 kDa mPEG1900-g-MMWC mPEG5000-g-MMWC mPEG5000-g-MMWC mPEG1900-g-LMWC mPEG5000-g-LMWC

82.0 28.9 1.9 5.0 136.7 142.2 261.5 42.3 40.9

53.5 19.6 1.8 4.7 90.9 81.5 174.3 30.2 27.2

1.53 1.48 1.06 1.06 1.5 1.7 1.5 1.4 1.5

D.S.%a,c

5.4 1.5 6.7 5.3 1.6

mPEG (wt %)a,d

41.2 34.4 69.3 35.1 28.0

ne

Tm (°C)f

21 6 26 6 1.6

55 61 46 52 56 42 50

a Measured by SEC-MALLS (using 0.5 M acetic acid/0.1 M NaNO3 as the mobile phase). b Mw and Mn are expressed in g mol-1 · 10-3 (i.e., entry 1: Mw 82.0 ≡ 82.0 · 103 g mol-1). c Degree of substitution expressed as the percentage of chitosan repeating units successfully conjugated with mPEG-COOH. d mPEG weight percentage in the mPEG-g-chitosan conjugates. e Average number of mPEG chains/molecule of chitosan. f Determined by DSC.

Figure 4. Relative solubility (%T) of chitosans and PEGylated chitosans in aqueous buffers at different pH values determined by turbidimetric assay.

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Figure 5. Effect of MMWC and its PEGylated derivatives (with different Mw PEG) on relative viability of Calu-3 cells. MMWC, mPEG1900-g-MMWC 5.4% D.S., and mPEG5000-g-MMWC 6.7% D.S. were applied to cells in HBSS (MES-buffered at pH 6.0 and HEPESbuffered at pH 7.4 for MMWC and the conjugates, respectively) at four concentrations (expressed as % w/v). Results expressed as cell viability relative to HBSS (pH 6.0 for MMWC and pH 7.4 for conjugates) and Triton X-100 (0.1% v/v in HBSS). Results represent the mean ( SD (n ) 4; * denotes a statistical significance between the conjugates and unmodified chitosan; † signifies a statistical difference between the two conjugates).

tion (0.003% w/v), where unmodified chitosan also showed low cell toxicity, the two PEG-conjugates exhibited lower reduction (statistically significant) in cell viability compared to the unmodified MMWC at concentrations of 0.006, 0.0125, and 0.025% w/v, with the effect seemingly not dependent on the applied concentration. The obvious conclusion that can be drawn from Figure 5 is that PEGylation of MMWC dramatically reduces its toxicity. This phenomenon could be attributed to the steric effect of the conjugates’ PEG chains shielding positive charges on the chitosan molecule, the latter thought to interact with cell membrane components resulting in cytotoxicity.18 The extent of toxicity is considered to be related to the charge density and the spatial arrangement of the cationic residues on the chitosan molecule.55 That PEGylation improves the cytotoxicity profile of chitosan is in agreement with a previously published report by Mao18 who found that PEGylation of TMC decreased its cytotoxicity, with the extent of this reduction being dependent on the level of PEG substitution, Mw of TMC and Mw of PEG. It could be argued that the difference in effect on cell viability caused by unmodified MMWC and its PEGylated conjugates may arise due to a lower overall MMWC content in the conjugates compared to the unmodified MMWC. However, it must be pointed out that at concentration of 0.025% w/v the MMWC content in the conjugates is equivalent to applying 0.015% of MMWC for mPEG1900-g-MMWC and 0.008% for mPEG5000-g-MMWC; unmodified MMWC at concentrations lower than these caused a significant decrease in cell viability (by 79% at 0.0125% w/v and by 54% at 0.006% w/v), suggesting that PEGylation indeed improves toxicity profile of the conjugates. Additionally, comparing the effect of PEG chain size at the similar level of substitution, MMWC conjugated to higher Mw PEG (5 × 103 g mol-1) showed a significantly lower toxicity compared to the conjugate with a lower Mw PEG of 1.9 × 103 g mol-1, at the highest two concentrations applied (the difference in effect is statistically significant at 0.0125% w/v and 0.025% w/v). As mPEG5000-g-MMWC conjugate at the relatively high level of substitution of 6.7% did not demonstrate significant cell toxicity, a conjugate with a reduced substitution level was synthesized and its cytotoxicity assessed.

Casettari et al.

Figure 6. Effect of mPEG5000-g-MMWC 1.5% D.S. applied at four different concentrations (expressed as % w/v) on the relative viability of Calu-3 cells. Results represent the mean ( SD (n ) 4).

Figure 7. Effect of LMWC and mPEG5000-g-LMWC 1.6% D.S. applied at four different concentrations (expressed as % w/v) on the viability of Calu-3 cells. Results represent the mean ( SD (n ) 4).

Effect of Conjugate with Low Levels of mPEG Substitution on Cell Viability. As Figure 6 demonstrates and contrary to MMWC with 6.7% D.S., MMWC with a lower level of PEGylation of 1.5% D.S. (i.e., mPEG5000-g-MMWC 1.5% D.S.) resulted in a significant, concentration-dependent toxicity. The relative cell viability decreased between 58 and 87% as the applied concentration increases. This clearly indicates the importance of the extent of chitosan PEGylation on the cytotoxicity, as discussed previously.18 It is not clear at the moment why the toxicity of this particular conjugate composition appears to reduce cell viability more than unmodified chitosan. Effect of mPEG-g-LMWC Conjugate on Cell Viability. Considering LMWC, Figure 7 displays a clear and pronounced difference between the effects on cell viability obtained with unmodified LMWC and its PEGylated derivative. The addition of unmodified LMWC at a concentration range of 0.003 to 0.025% w/v was associated with a remarkable decrease in cell viability, ranging from 73% (at a concentration of 0.003% w/v) to 93% (observed with 0.0125% w/v). This decrease appears to be concentration-dependent (albeit for three out of four concentrations tested). mPEG5000-g-LMWC 1.6% D.S. on the other hand had a significantly lower adverse effect on relative cell viability compared to the corresponding concentrations of the non-PEGylated LMWC for three out of four concentrations tested (0.003-0.0125% w/v). The reduction in cell viability with mPEG5000-g-LMWC 1.6% D.S. was a dose-dependent event, similar to the toxicity profile for MMWCs with a low level of substitution (mPEG5000-g-MMWC 1.5% D.S.) and unlike the profile for MMWC with a high level of substitution (mPEG5000g-MMWC 6.7% D.S.). An acceptable level of cytotoxicity (which is associated with a reversible effect on TEER, as will


Figure 8. Effect of unmodified and PEGylated MMWC (mPEG5000g-MMWC 6.7% D.S. and mPEG1900-g-MMWC 5.4% D.S.) on TEER. Unmodified MMWC was applied to cells in HBSS (MES-buffered) at pH 6.0, whereas the conjugates were applied in HBSS (HEPESbuffered) at pH 7.4. Cells were incubated with the samples for 2 h. Arrow indicates removal of the materials from the cell monolayers and replacement with culture medium. Results are expressed as TEER relative to the baseline value and presented as the mean ( SD (n ) 3).

be shown later) could be achieved only with the lowest applied concentration of the conjugate (0.003% w/v). It should be noted that the data in Figures 6 and 8 indicate higher relative toxicity of unmodified LMWC when compared to unmodified MMWC at the corresponding concentrations. To this end, different relationships between molecular weight and toxicity of chitosans have been reported.56,57 In summary, the cell viability data demonstrate that PEGylation with 1.9 and 5 × 103 g mol-1 mPEG improves the cytotoxicity profile of both chitosan hydrochloride salts used in the synthesis (113 × 103 g mol-1 “MMWC” and 20-70 × 103 g mol-1 “LMWC”), whereby the extent of this reduction in toxicity is clearly dependent on the molecular weight of introduced PEG and on the extent of substitution. It is difficult to directly compare these data with the literature as, to the best of our knowledge, the reports on toxicity of PEG-chitosan conjugates applied to cells as a solution are missing, the literature mainly reporting on complexes or nanoparticles based on PEGylated chitosans or TMC.58,43 Effect of PEGylated MMWC Conjugates on Calu-3 Monolayer TEER. Effect at pH 7.4. Application of mPEG5000g-MMWC and mPEG1900-g-MMWC conjugates with similar level of substitution (6.7 and 5.4% D.S., respectively), to Calu-3 monolayers was associated with a decrease in TEER, as illustrated in Figure 9. Both solutions were applied at the concentration (0.025% w/v) that did not show significant cytotoxicity (Figure 6) in HBSS at pH 7.4. As a comparison, unmodified chitosan solution was applied to cell monolayers at the highest tolerable concentration of 0.003% w/v in HBSS/ MES at pH 6.0. The most pronounced reduction in TEER for both PEGylated conjugates was observed 30 min following the application (Figure 8), with the effect being more prominent for PEG1900-g-MMWC (33% of the baseline TEER) compared to mPEG5000-g-MMWC (64% of baseline TEER). However, in comparison to unmodified MMWC chitosan, which was associated with a reduction in TEER to approximately 6-7% of the baseline value, TEER effects of the conjugates were “moderate” in both the magnitude and the duration. For both PEGylated MMWCs, the initial decline in TEER was followed by its reversal, which occurred within the 2 h of the incubation period despite the fact that the conjugates were still present in the apical medium. In contrast, TEER remained low (6-7% of


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Figure 9. Effect of two PEGylated MMWCs applied at pH 6.0, on cell monolayer TEER. mPEG-g-MMWC conjugates, mPEG1900-gMMWC 5.4% D.S. and mPEG5000-g-MMWC 6.7% D.S., were applied to cell monolayers at two concentrations, 0.025 and 0.05% w/v. Unmodified chitosan was applied at 0.003 and 0.006% w/v. All materials were applied to cells in HBSS (MES-buffered, pH 6.0) and cells were incubated with the compounds for 2 h. Arrow indicates removal of the materials from the cell monolayers and replacement with culture medium. Results are expressed as TEER relative to the baseline value and presented as the mean ( SD (n ) 3).

the baseline figure) for unmodified MMWC. Twenty-four hours after the application of the samples (and 22 h following their removal from apical medium) the TEER was reversible for all samples, reaching values close to the baseline figures; 123% for mPEG5000-g-MMWC, 88% for mPEG1900-g-MMWC, and 84% for unmodified chitosan. It should be noted that under the experimental conditions employed (Figure 8) unmodified MMWC was applied at pH 6.0 (due to the solubility issue), while the conjugates were applied at pH 7.4. This consequently implies that the degree of ionization of chitosan/chitosan moiety of the conjugates was different in the two scenarios. As the tight junction modulating effect of chitosan is believed to be mediated by its cationic charges and enhancement in permeability achieved only with the protonated form of chitosan in acidic environments,56,59,60 in the next set of experiments the TEER and permeability effects of PEGylated chitosan conjugates were assessed at pH 6.0. Effect at pH 6.0. In this experiment the PEGylated MMWCs conjugates, mPEG5000-g-MMWC and mPEG1900-g-MMWC, with a similar level of PEG substitution (6.7 and 5.4%, respectively) were applied to the cell monolayers at pH 6.0 and at two different concentrations. Application of the conjugates under such experimental conditions resulted in a sharp decrease in TEER (Figure 9) to levels similar to those observed for unmodified chitosan. The decrease in TEER was somewhat more pronounced for the PEG conjugate with smaller PEG, i.e., mPEG1900-g-MMWC (3-4% of the baseline value), as compared to mPEG5000-g-MMWC (4-13% of the baseline), for both applied concentrations. Following the removal of the samples, a reversal in TEER was already observed with both PEGylated MMWCs at both applied concentrations at the 4 h time point (i.e., 2 h following removal). At 24 h following sample application (i.e., 22 h following the sample removal), TEER reversal to levels similar to the baseline value was seen, amounting to 82 and 102% of the baseline for 0.025 and 0.05% w/v mPEG1900-g-MMWC, respectively, and 103 and 85% of the baseline for 0.025 and 0.05% w/v mPEG5000-g-MMWC, respectively. A 24 h TEER reversal for unmodified chitosan was observed for the lower tested concentration of 0.003% w/v (to 84% of the baseline value), while the higher concentration of 0.006% w/v was associated with an irreversible effect. It

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Figure 10. The effect of PEGylated MMWCs on the permeability of FD4 across Calu-3 monolayers. mPEG5000-g-MMWC 6.7% D.S. and mPEG1900-g-MMWC 5.4% D.S. were applied to cells at 0.025% w/v in HBSS at pH 7.4, whereas FD4 was applied at 500 µg/mL (both in HBSS/HEPES). Controls 1 and 2 represent control experiments (FD4 applied at 500 µg/mL in HBSS/HEPES) for mPEG5000-g-MMWC 6.7% D.S. and mPEG1900-g-MMWC 5.4% D.S., respectively. Permeability is expressed as apparent permeability coefficient (Papp); results are presented as the mean ( SD (n ) 4).

should be noted that the reversal of TEER occurs for the conjugates applied at the concentrations significantly higher than corresponding unmodified chitosan (0.05 vs 0.003% w/v). This further indicates a lack of irreversible damage that would compromise the monolayer. The tight junction modulation effect of chitosan is believed to be a consequence of its interactions with the tight junction proteins occludin and ZO-1, redistribution of F-actin, destabilization of the plasma membrane,61-63 and its interaction with the protein kinase C pathway.64 Our work clearly shows that chemical conjugation of PEG to MMWC can be achieved in such a way to retain the intrinsic capacity of chitosan to open the tight junctions. However, it is evident from the data that the ionization level of chitosan in its PEGylated form is paramount for its effect on the tight junctions. Although this study demonstrates that conjugation of PEG to chitosan afforded solubility in neutral to slightly alkaline pH media, to facilitate mucosal permeability, the conjugates would have to be applied at a slightly acidic environment. In an in vivo situation, the nasal application of a drug delivery system at pH around 6.0 should not be problematic, considering that the nasal mucosal pH is reported to be approximately 5.5-6.5.65,66 The striking impact of pH on the tight junction opening capacity of the mPEG-g-MMWC conjugates suggests that the effect results from unsubstituted amine groups on the chitosan molecule and their ionization at lower pH, as discussed above. It should be noted from Figure 9 that TEER reversal occurred following application of conjugates to the cell monolayers at the concentration of 0.025% w/v, which is equivalent to applying 0.015 or 0.008% MMWC for mPEG1900-g-MMWC and mPEG5000g-MMWC, respectively. The permanent decrease in TEER associated with MMWC, observed even at a lower concentration of 0.006% w/v, indicates an improved cytotoxicity profile of PEGylated conjugates compared to unmodified MMWC, which is in agreement with the cell viability data. Permeability Studies. Effect of mPEG-g-MMWCs on FD4 Permeability. Effect at pH 7.4. Initially the impact of PEGylated MMWCs (applied at 0.025% w/v) on the permeability of FD4 across confluent Calu-3 monolayers was tested under pH 7.4 conditions (in HBSS/HEPES buffer). Figure 10 depicts that both conjugates, mPEG5000-g-MMWC and mPEG1900-g-MMWC,

Casettari et al.

Figure 11. Effect of mPEG5000-g-MMWC 6.7% D.S. on FD4 permeability across Calu-3 monolayers, applied in pH 6.0 HBSS medium. The conjugate was applied to cells at 0.025% w/v, whereas FD4 was applied at 500 µg/mL (both in HBSS/MES). Permeability is expressed as apparent permeability coefficient (Papp); results are presented as the mean ( SD (n ) 4).

produced a somewhat increased permeability of FD4 compared to the relevant controls. This increase amounted to 1.3-fold and 1.2-fold relative to the controls for mPEG5000-g-MMWC and mPEG1900-g-MMWC, respectively, not reaching statistical significance. At this stage, despite the advantage of reduced toxicity of PEGylated chitosan conjugates, their insignificant permability enhancing potential at pH 7.4 would preclude possible application. That the conjugates did not significantly increase the permeability of FD4 at pH 7.4 was not unexpected considering the small effect on TEER under identical experimental conditions, as discussed above. Effect at pH 6.0. In sharp contrast to the permeability experiment conducted at pH 7.4, the application of mPEG5000g-MMWC 6.7% D.S. at pH 6.0 conditions (0.025% w/v in HBSS/MES) had a remarkable effect on FD4 translocation across the cell monolayers, producing an increase in Papp from 2.5 × 10-8 to 124 × 10-8 cm/s, amounting to an approximately 50-fold permeability enhancement (Figure 11). It must be noted that, in the control experiment, FD4 was applied in an identical medium (HBSS/MES, pH 6.0), confirming that the increase in permeability was not due to exposure of the cells to the slightly acidic pH of the apical medium. It needs to be emphasized that 24 h following the permeability experiment, the reversal of TEER for mPEG5000-g-MMWC reached the baseline value range (95%), indicating that the observed increase in FD4 permeability did not arise from a compromised integrity of the cell monolayers. This substantial effect in permeability was seen despite the fact that, at 6.7% degree of substitution of mPEG5000-gMMWC, the chitosan moiety presents only approximately 31 wt % of the conjugate molecule (Table 2). Effect of mPEG5000-g-LMWC on FD Permeability at pH 6.0. The conjugate based on LMWC, mPEG5000-g-LMWC with 1.6% level of substitution, was applied to the cell monolayers at concentration of 0.003% w/v, at which it did not demonstrate significant toxicity (Figure 8), alongside the two hydrophilic permeability markers, FD4 (4 × 103 g mol-1) and FD40 (40 × 103 g mol-1). The data (Figure 12) illustrates markedly higher permeabilities for both markers, compared to the relevant controls. The extent of permeability enhancement for FD4 reached 37-fold, while the permeability of FD40 increases by a factor of 19, suggesting that the phenomenon is dependent on the molecular weight of the permeant. Again, the measurement of TEER 24 h after the permeability experiment produced values close to the baseline TEER, hence confirming that the permeability enhancement was due to reversible opening of the tight junctions in viable cells.



2863

Figure 12. Effect of mPEG5000-g-LMWC 1.6 D.S.% on FD4 and FD40 permeability across Calu-3 monolayers. The conjugate was applied at 0.003% w/v, whereas FDs were applied at 500 µg/mL. Permeability is expressed as apparent permeability coefficient (Papp); results presented as the mean ( SD (n ) 3).

The trends in permeability achieved with mPEG-g-chitosans at pH 6.0 and 7.4 were, to some extent, expected from the data obtained in TEER studies. Modest changes in TEER produced following the application of PEG-chitosan conjugates at pH 7.4 are reflected in statistically insignificant permeability enhancement, while drastic reductions in TEER associated with application of the PEGylated conjugates at pH 6.0 were reflected in permeability experiments, with a large enhancement of macromolecular permeability of as high as 50-fold. The 19-fold enhancement in permeability of a relatively high Mw macromolecule such as FD40 by mPEG5000-g-LMWC conjugate is significant. Most reported studies which utilized chitosan, or its derivative (TMC), to improve mucosal absorption through tight junction modulation studied peptides with a relatively low molecular mass, including octreotide (1 kDa),67 buserelin (1.3 kDa),68,69 and salmon calcitonin (3.4 kDa).70 The significant improvement of permeability of macromolecules of 40 kDa indicates that the tested conjugate may potentially be useful in facilitating mucosal absorption of proteins in the order of human growth hormone size (22 kDa) or some antibody fragments. Furthermore, it is particularly interesting that permeability enhancement achieved with the synthesized conjugates was superior to that of unmodified chitosan. Table 3 summarizes the extent of permeability enhancement (enhancement ratio) associated with the tested compounds. It shows that the increase in FD4 permeability seen with the conjugates was approximately 3-5 times higher than that obtained with unmodified chitosan. Changes in ZO-1 TJ Protein Distribution with mPEG5000-gMMWC, Applied at Acidic and Neutral pH. In an attempt to further confirm that permeability enhancement seen at pH 7.4 and 6.0 is related to the changes in tight junctions, Figure 13 illustrates the distribution of ZO-1 tight junction protein staining in Calu-3 cell monolayers treated with mPEG5000-g-MMWC at pH 7.4 and 6.0. In cell monolayer treated with the conjugate at

Figure 13. Effect of PEGylated and non-PEGylated MMWC on distribution of ZO-1 tight junction protein: (a) mPEG5000-g-MMWC 6.7% D.S (0.025% w/v, pH 7.4), (b) mPEG5000-g-MMWC 6.7% D.S. (0.025% w/v, pH 6.0), (c) unmodified MMWC (0.003% w/v, pH 6.0), and (d) control (culture medium; scale bar ) 20 µm).

pH 7.4, image (a), ZO-1 staining appears throughout the monolayer as continuous “rings” at points of cell-cell contact around the periphery of the cells, similar to control cells incubated with HBSS, image (d). On the contrary, image (b) depicts a considerable loss of staining, indicative of changes in distribution of ZO-1 and tight junction morphology, for the cell monolayer treated with the same mPEG5000-g-MMWC conjugate but at pH 6.0. This loss of staining with mPEG5000-g-MMWC conjugate at pH 6.0 appears more pronounced than that observed with unmodified chitosan (also applied at pH 6.0; c). These observations imply that the TEER and permeability effects seen at pH 6.0 do indeed result from an effect of mPEG5000-g-MMWC at the tight junction level. Conversely, the PEGylated chitosan applied at a pH that did not produce substantial changes in TEER and permeability was also associated with unaltered distribution of ZO-1 protein (observed to be similar to control cell monolayers not subjected to incubation with the copolymer). Overall this work shows that under certain circumstances PEGylation of chitosan can improve physical and biological effects, including solubility and cytotoxicity profile, while preserving the effect on tight junction modulation. In fact, mPEG-conjugated chitosans synthesized in this study exhibited a potent tight junction opening effect, which was actually considerably larger than that of unmodified chitosan. The possible reasons for this phenomenon are unclear, though it may be a consequence of higher equivalent concentrations of chitosans present in the conjugates, the application of which was possible due to reduced untoward effect on cell viability.

Table 3. Permeability Enhancement of Different Permeants by mPEG-g-Chitosan Conjugates and Unmodified Chitosana compound/conditions pH 6.0

pH 7.4 mPEG5000-g-MMWC 6.7% D.S. mPEG1900-g-MMWC 5.4% D.S.

mPEG5000-g-MMWC 6.7% D.S. mPEG5000-g-LMWC 1.6% D.S. mPEG5000-g-LMWC 1.6% D.S. MMWC MMWC a

concentration (% w/v)

permeant

enhancement ratio

0.025 0.025 0.025 0.003 0.003 0.003 0.003

FD4 FD4 FD4 FD4 FD40 FD4 FD10

1.25 1.17 49.6 36.7 18.9 10 10

The experimental conditions, concentration at which the compounds were applied, and solution pH are also shown.

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Also, the present study uses mucus producing, air-liquid interface cultured Calu-3 cell layers where application of unmodified chitosan may result in its pronounced interaction and hence reduced diffusion thorough the mucus layer to reach the cell surface, while the PEGylation may have promoted this process, as it has been discussed in the case of chitosan nanoparticles.71 Finally, it is noteworthy to highlight that the permeabilityenhancing effects of mPEG-g-chitosan conjugates synthesized and tested in this work were found to be superior to those reported for typically used TMC derivatives. For instance, application of TMC to Caco-2 cell monolayers at the physiological pH of 7.4 provided permeability enhancement that was similar to chitosan (later applied at pH 5.6).69 However, other studies found that TMC was not as effective in increasing the absorption rate of [14C]-mannitol8 and [14C]-polyethylene glycol 400072 across Caco-2 monolayers when applied at similar concentrations as chitosan hydrochloride and chitosan glutamate. Furthermore, the higher degree of quarterization of TMC (and, hence, charge density) is a determinant factor for its permeability-enhancing property in neutral environments, but at the same time there is evidence that a higher degree of quarterization in TMC leads to a greater toxicity.73 Furthermore, TMC was demonstrated to possess lower mucoadhesive properties compared to different chitosan salts,74 mucoadhesion being a property important in nasal application. On the other hand, our work shows evidence that conditions can be engineered where, for nasal application in a slightly acidic environment, the conjugation of PEG to chitosan would produce a system that provides both reduced cytotoxicity and improved permeabilityenhancing capacity.

Casettari et al.

“MMWC”, 1H NMR calculation of the degree of substitution of the mPEG-g-chitosan conjugates, and Calu-3 cell monolayer characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18)

Conclusion Different graft copolymers of methoxy-polyethylenglycol (1.9 and 5.0 × 103 g mol-1) and chitosans (Mn 20 and 53 × 103 g mol-1) with varying degrees of substitution (from 1.5 to 6.7%) were synthesized in this work. PEGylation of two chitosans featuring low (LMWC 20-70 × 103 g mol-1) and medium molecular weights (MMWC 50-150 × 103 g mol-1) increased their solubility in aqueous solutions in the 2.0-9.6 pH range. There were considerable improvements in cytotoxicity of chitosans with PEGylation, with toxicity of the conjugates found to be dependent on the degree of PEGylation. TEER and permeability experiments showed that the synthesized copolymers exhibited strong tight junction opening effects, though a slightly acidic pH was required, suggesting the necessity for chitosan to be in its protonated form for an effect on epithelial tight junctions, as suggested previously by other authors.56 Remarkably, the permeability experiments found that mPEGg-chitosan conjugates produced a notably larger permeability enhancing effect relative to unmodified chitosan. Our results therefore suggest that mPEG-g-chitosan conjugates may find applications as absorption promoters in macromolecular therapeutics formulations designed for nasal administration. Acknowledgment. The authors thank Mr. Mahmoud E. Soliman, Drug Delivery and Tissue Engineering Division, School of Pharmacy, Nottingham, for assistance with synthetic procedures and Prof. Steve Harding’s group, School of Biosciences, University of Nottingham, for the SEC-MALLS measurements. Supporting Information Available. FT-IR spectra of mPEG1900-g-MMWC (D.S.: 5.4%) and Protasan Cl 113

(19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38)

Tauzin, B. Pharm. Biotechnol. Report 2008. Illum, L. Eur. J. Pharm. Sci. 2000, 11, 1–18. Stolnik, S.; Shakesheff, K. Biotechnol. Lett. 2009, 31, 1–11. Illum, L. Pharm. Res. 1998, 15, 1326–1331. Dyer, A. M.; Hinchcliffe, M.; Watts, P.; Castile, J.; Jabbal-Gill, I.; Nankervis, R.; Smith, A.; Illum, L. Pharm. Res. 2002, 19, 998–1008. Artursson, P.; Lindmark, T.; Davis, S. S.; Illum, L. Pharm. Res. 1994, 11, 1358–1361. Sieval, A. B.; Thanou, M.; Kotze´, A. F.; Verhoef, J. C.; Brussee, J.; Junginger, H. E. Carbohydr. Polym. 1998, 36, 157–165. Van der Merwe, S. M.; Verhoef, J. C.; Verheijden, J. H. M.; Kotze´, A. F.; Junginger, H. E. Eur. J. Pharm. Biopharm. 2004, 58, 225–235. Le Dung, P.; Milas, M.; Rinaudo, M.; Desbrieres, J. Carbohydr. Polym. 1994, 24, 209–214. Kast, C. E.; Bernkop-Schnu¨rch, A. Biomaterials 2001, 22, 2345–2352. Muzzarelli, R. A. A.; Tanfani, F.; Emanuelli, M.; Mariotti, S. Carbohydr. Res. 1982, 107, 199–214. Liu, X. F.; Guan, Y. L.; Yang, D. Z.; Li, Z.; Yao, K. D. J. Appl. Polym. Sci. 2001, 79, 1324–1335. Rinaudo, M.; Dung, P. L.; Gey, C.; Milas, M. Int. J. Biol. Macromol. 1992, 14, 122–128. Harris, J. M.; Evelyn, C. S.; Martha, G. C.; Paley, M. S.; Manssur, Y.; James, M. V. A.; Donald, E. B. J. Polym. Sci., Polym. Chem. Ed. 1984, 22, 341–352. Saito, H.; Wu, X.; Harris, J. M.; Hoffman, A. S. Macromol. Rapid Commun. 1997, 18, 547–550. Ohya, Y.; Cai, R.; Nishizawa, H.; Hara, K.; Ouchi, T. STP Pharm. Sci. 2000, 10, 77–78. Jeong, Y.-I.; Kim, D.-G.; Jang, M.-K.; Nah, J.-W. Carbohydr. Res. 2008, 343, 282–289. Mao, S.; Shuai, X.; Unger, F.; Wittmar, M.; Xie, X.; Kissel, T. Biomaterials 2005, 26, 6343–6356. Jintapattanakit, A.; Junyaprasert, V. B.; Kissel, T. J. Pharm. Sci. 2009, 98, 4818–4830. Sugimoto, M.; Morimoto, M.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1998, 36, 49–59. Hu, F.-Q.; Meng, P.; Dai, Y.-Q.; Du, Y.-Z.; You, J.; Wei, X.-H.; Yuan, H. Eur. J. Pharm. Biopharm. 2008, 70, 749–757. Wu, J.; Wang, X.; Keum, J. K.; Zhou, H.; Gelfer, M.; Avila-Orta, C. A.; Pan, H.; Chen, W.; Chiao, S. M.; Hsiao, B. S.; Chu, B. J. Biomed. Mater. Res., Part A 2007, 80A, 800–812. Bentley, M. D.; Roberts, M. J.; Harris, J. M. J. Pharm. Sci. 1998, 87, 1446–1449. Zhang, X.; Zhang, H.; Wu, Z.; Wang, Z.; Niu, H.; Li, C. Eur. J. Pharm. Biopharm. 2008, 68, 526–34. McCloskey, B. D.; Ju, H.; Freeman, B. D. Ind. Eng. Chem. Res. 2009, 49, 366–373. Lebouc, F.; Dez, I.; Desbrie`res, J.; Picton, L.; Madec, P.-J. Polymer 2005, 46, 639–651. Bae, K. H.; Moon, C. W.; Lee, Y.; Park, T. G. Pharm. Res. 2008, 26, 93–100. Amiji, M. M. Carbohydr. Polym. 1997, 32, 193–199. Ouchi, T.; Nishizawa, H.; Ohya, Y. Polymer 1998, 39, 5171–5175. Yoksan, R.; Matsusaki, M.; Akashi, M.; Chirachanchai, S. Colloid Polym. Sci. 2004, 282, 337–342. Fangkangwanwong, J.; Mitsuru, A.; Toshiyuki, K.; Suwabun, C. Biopolymers 2006, 82, 580–586. Morpurgo, M.; Bayer, E. A.; Wilchek, M. J. Biochem. Biophys. Meth. 1999, 38, 17–28. Jang, M.-K.; Nah, J.-W. Bull. Korean Chem. Soc. 2003, 24, 1303– 1307. Jiang, X.; Dai, H.; Leong, K. W.; Goh, S.; Mao, H.; Yang, Y. J. Gene Med. 2006, 8, 477–487. Witschi, C.; Mrsny, R. J. Pharm. Res. 1999, 16, 382–390. Florea, B. I.; Meaney, C.; Junginger, H. E.; Borchard, G. AAPS Pharm. Sci. 2002, 4, E12. Li, L.; Mathias, N. R.; Heran, C. L.; Moench, P.; Wall, D. A.; Smith, R. L. J. Pharm. Sci. 2006, 95, 326–335. Yang, T.; Hussain, A.; Paulson, J.; Abbruscato, T. J.; Ahsan, F. Pharm. Res. 2004, 21, 1127–1136.

Toxicity and Permeability Enhancement of Chitosan (39) Seki, T.; Kanbayashi, H.; Chono, S.; Tabata, Y.; Morimoto, K. Int. J. Pharm. 2007, 338, 213–218. (40) Teijeiro-Osorio, D.; Remunan-Lopez, C.; Alonso, M. J. Eur. J. Pharm. Biopharm. 2009, 71, 257–263. (41) Teijeiro-Osorio, D.; Remunan-Lopez, C.; Alonso, M. J. Biomacromolecules 2009, 10, 243–249. (42) Liu, L.; Xu, X.; Guo, S.; Han, W. Carbohydr. Polym. 2009, 75, 401– 407. (43) Prego, C.; Torres, D.; Fernandez-Megia, E.; Novoa-Carballal, R.; Quinõa´, E.; Alonso, M. J. J. Controlled Release 2006, 111, 299–308. (44) Aktas, Y.; Yemisci, M.; Andrieux, K.; Gursoy, R. N.; Alonso, M. J.; Fernandez-Megia, E.; Novoa-Carballal, R.; Quinoa, E.; Riguera, R.; Sargon, M. F.; Celik, H. H.; Demir, A. S.; Hincal, A. A.; Dalkara, T.; Capan, Y. l.; Couvreur, P. Bioconjugate Chem. 2005, 16, 1503–1511. (45) Beri, R. G.; Walker, J.; Reese, E. T.; Rollings, J. E. Carbohydr. Res. 1993, 238, 11–26. (46) Illum, L. Eur. J. Pharm. Sci. 2000, 11, 1–18. (47) Stolnik, S.; Shakesheff, K. Biotechnol. Lett. 2009, 31, 1–11. (48) Braunova´, A.; Pechar, M.; Ulbrich, K. Collect. Czech. Chem. Commun. 2004, 69, 1643–1656. (49) Kulbokaite, R.; Ciuta, G.; Netopilik, M.; Makuska, R. React. Funct. Polym. 2009, 69 (10), 771–778. (50) Gorochovceva, N.; Makuska, R. Eur. Polym. J. 2004, 40, 685–691. (51) Deng, L.; Qi, H.; Yao, C.; Feng, M.; Dong, A. J. Biomater. Sci., Polym. Ed. 2007, 18, 1575–1589. (52) Hu, Y.; Jiang, H.; Xu, C.; Wang, Y.; Zhu, K. Carbohydr. Polym. 2005, 61, 472–479. (53) Chan, P.; Kurisawa, M.; Chung, J. E.; Yang, Y.-Y. Biomaterials 2007, 28, 540–549. (54) Du, J.; Hsieh, Y.-L. Cellulose 2007, 14, 543–552. (55) Kudsiova, L.; Lawrence, M. J. J. Pharm. Sci. 2008, 97, 3998–4010. (56) Schipper, N. G. M.; Vårum, K. M.; Artursson, P. Pharm. Res. 1996, 13, 1686–1692. (57) Carrenõ-Go´mez, B.; Duncan, R. Int. J. Pharm. 1997, 148, 231–240. (58) Mao, S.; Germershaus, O.; Fischer, D.; Linn, T.; Schnepf, R.; Kissel, T. Pharm. Res. 2005, 22, 2058–2068.


2865

(59) Schipper, N. G. M.; Olsson, S.; Hoogstraate, J. A.; deBoer, A. G.; Vårum, K. M.; Artursson, P. Pharm. Res. 1997, 14, 923–929. (60) Kotze´, A. F.; Lueβen, H. L.; de Boer, A. G.; Verhoef, J. C.; Junginger, H. E. Eur. J. Pharm. Sci. 1999, 7, 145–151. (61) Dodane, V.; Amin Khan, M.; Merwin, J. R. Int. J. Pharm. 1999, 182, 21–32. (62) Fang, N.; Chan, V.; Mao, H.-Q.; Leong, K. W. Biomacromolecules 2001, 2, 1161–1168. (63) Thanou, M.; Verhoef, J. C.; Junginger, H. E. AdV. Drug DeliVery ReV. 2001, 50, S91–S101. (64) Smith, J. M.; Dornish, M.; Wood, E. J. Biomaterials 2005, 26, 3269– 3276. (65) Fabricant, N. D. Arch. Otolaryngol. 1941, 34, 150–163. (66) England, R. J. A.; Homer, J. J.; Knight, L. C.; Ell, S. R. Clin. Otolaryngol. Allied Sci. 1999, 24, 67–68. (67) Florea, B. I.; Thanou, M.; Junginger, H. E.; Borchard, G. J. Controlled Release 2006, 110, 353–361. (68) Lueβen, H. L.; de Leeuw, B. J.; Langemeyër, M. W. E.; de Boer, A. G.; Verhoef, J. C.; Junginger, H. E. Pharm. Res. 1996, 13, 1668– 1672. (69) Kotze´, A. R.; Lueβen, H. L.; de Leeuw, B. J.; de Boer, B. G.; Verhoef, J. C.; Junginger, H. E. Pharm. Res. 1997, 14, 1197–1202. (70) Sinswat, P.; Tengamnuay, P. Int. J. Pharm. 2003, 257, 15–22. (71) Bravo-Osuna, I.; Vauthier, C.; Chacun, H.; Ponchel, G. Eur. J. Pharm. Biopharm. 2008, 69, 436–44. (72) Kotze, A. F.; Lueszen, H. L.; de Leeuw, B. J.; de Boer, G.; Coos Verhoef, J.; Junginger, H. E. J. Controlled Release 1998, 51, 35–46. (73) Hagenaars, N.; Verheul, R. J.; Mooren, I.; de Jong, P. H. J. L. F.; Mastrobattista, E.; Glansbeek, H. L.; Heldens, J. G. M.; van den Bosch, H.; Hennink, W. E.; Jiskoot, W. J. Controlled Release 2009, 140, 126–133. (74) Snyman, D.; Hamman, J. H.; Kotze, A. F. Drug DeV. Ind. Pharm. 2003, 29, 61–69.

BM100522C

Effect of PEGylation on the Toxicity and Permeability Enhancement of

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