N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate sub-microparticles for oral delivery of insulin—An in vitro evaluation

Colloids and Surfaces B: Biointerfaces 107 (2013) 205–212

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N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate sub-microparticles for oral delivery of insulin—An in vitro evaluation T.A. Sonia, Chandra P. Sharma ∗ Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Trivandrum, Kerala, India

a r t i c l e

i n f o

Article history: Received 22 August 2012 Received in revised form 17 January 2013 Accepted 29 January 2013 Available online 5 February 2013 Keywords: Polydimethylaminoethylmethacrylate Mucoadhesion Insulin Cationic hydrogel Cytotoxicity

a b s t r a c t The present study describes the synthesis and in vitro evaluation of quaternised polydimethylaminoethylmethacrylate for oral delivery of insulin. Quaternisation of the polymer was carried out by conjugating N-hyroxypropyltrimethylammonium chloride to aminoterminated polydimethylaminoethylmethacrylate. Quaternised particles were characterised by particle size, zeta potential measurements, nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), differential scanning calorimetry (DSC) and atomic force microscopy (AFM). In addition, in vitro insulin release experiments, cytotoxic evaluation on L929 & Caco-2 cells, mucoadhesion, enzymatic degradation and tight junction visualisation studies were also performed to evaluate the potential of this matrix for oral delivery of insulin. Results suggest that the quaternised particles exhibited positive zeta potential with a particle size of 513.6 ± 17 nm. Dose-dependent cytotoxic evaluation of quaternised particles on L929 & Caco-2 cells confirmed the nontoxic nature of the matrix. Quaternised particles were more mucoadhesive compared to parent polymer. Adhesive behaviour of mucin with quaternised particles were confirmed by DSC. Moreover these particles exhibited calcium chelating ability and displayed significant inhibitory effect towards trypsin and chymotrypsin. These particles also helped in the opening of tight junctions by disruption of actin filaments and binding to Zona Occludens (ZO-1) proteins. Preliminary studies suggest that the quaternised particles can act as suitable candidates for oral delivery of insulin. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Colloidal carriers (nano/microparticles) based on mucoadhesive polymers are gaining increasing attention nowadays as controlled drug delivery platforms due to their ability to improve the residence time of dosage forms as well as to enhance bioavailability of drugs [1]. Hydrogels (also termed as colloidal gels) are 3 dimensional crosslinked polymeric networks which resemble living tissue and are being extensively used in the development of smart drug delivery systems [2]. Oral insulin delivery for the treatment of diabetes mellitus is still a challenge to the scientific community due to its low bioavailability [3]. Tight junctions present in between intestinal epithelial cells restrict the transport of hydrophilic macromolecular drugs like insulin via paracellular pathway [4]. Carriers for delivery of insulin should be designed in such a way to overcome the barriers of insulin in the gastrointestinal tract and improve the bioavailability. Hydrogels is an interesting category of drug carriers in this scenario as

∗ Corresponding author. Tel.: +91 471 2520214; fax: +91 471 2341814. E-mail addresses: [email protected], [email protected] (C.P. Sharma). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.057

it exhibits excellent chain flexibility and mobility, which makes them suitable for mucosal drug delivery [5]. It can also be tailored to exhibit mucoadhesiveness to facilitate drug targeting, for noninvasive drug administration [6]. Cationic polymers are capable of displacing divalent ions from tight junctions. Moreover, they are expected to improve mucoadhesion [7]. Poly(2-(dimethylamino)ethylmethacrylate) (PD), biocompatible, water soluble polymer finds extensive applications as membranes [8], efficient non-viral gene-delivery vectors [9], antimicrobial agents [10] and biosensors [11].To our knowledge, PD is very least explored for oral drug delivery. Our approach involves the modification of PD hydrogel particles to yield cationic groups on the surface. The presence of cationic charge could enhance the interaction of the particles with the mucus gel layer and may induce an opening of epithelial tight junctions across the intestinal membrane by divalent ion binding. In our previous work, we have reported the synthesis of quaternised PD in which quaternisation was carried out using methyliodide as quaternising agent [12]. Quaternisation of PD in this case resulted in the modification of PD in the main chain itself. Herein, we describe the synthesis of cationic PD hydrogel sub-microparticles for oral insulin delivery using a different quaternising agent, N hydroxypropyltrimethylammonium chloride and

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the substitution appears as a side chain to the polymer. The derivatisation was confirmed by zeta potential analysis, infrared and nuclear magnetic resonance spectroscopy. Mucoadhesion, in vitro release, tight junction visualisation and permeability studies were carried out to evaluate the efficacy of this hydrogel for oral insulin delivery.

same experimental conditions. FTIR spectra of PDG were recorded in 4000–400 cm−1 region using NICOLET 5700 FTIR spectrophotometer. Thermal properties of the samples were monitored using Q20 DSC Thermal Analyser (TA Instrument USA). Surface morphology of the particles was studied using AFM (JEOL JSPM-5200). 2.5. Swelling studies

2. Methods 2.1. Materials Dimethylaminoethylmethacrylate (DMAEMA), Ethyleneglycoldimethacrylate (EGDMA), N-hydroxypropyltrimethylammoniumchloride (HTC), 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazoliumbromide (MTT), Pluronic F-127, Chymotrypsin, N-Benzoyll-tyrosine p-nitroanilide (BTPNA), N-Benzoyl-l-Arginine Ethyl Ester (BAEE), Fluorescein isothiocyanate (FITC), rhodamine phalloidin, mucin from porcine stomach were obtained from Sigma–Aldrich. Potassium persulfate was obtained from BDH chemicals, Human Insulin, Human Insulin ELISA kit and Calcium kits were purchased from Eli Lilly, Mercodia and Enzyme Technologies Pvt. Ltd., Mumbai, India, respectively. Eudragit L100-55 was a gift from Rohm Pharma, India. Mouse connective tissue fibroblasts, L929 and human epithelial colorectal adenocarcinoma cells, Caco2 cells were obtained from NCCS, Pune. Foetal bovine serum (FBS), Dulbeccos Modified Eagles Medium (DMEM), Hanks Balanced Salt Solution (HBSS) was from Gibco chemicals. 2.2. Synthesis of aminoterminated PD hydrogel nanoparticles Aminoterminated PD hydrogel nanoparticles was prepared by free radical polymerisation in aqueous medium. To 100 ml of water containing a small amount of surfactant (Pluronic F-127), monomer DMAEMA mixed with 800 ␮L cross-linking agent (EGDMA) was added followed by the addition of 0.25 g 2-aminoethane thiol hydrochloride. Potassium persulfate (45 mg) was added after 15 min. The reaction was allowed to stir at 70 ◦ C for 6 h.The resulting white suspension was centrifuged at 10,000 rpm and washed several times with distilled water and dried at vacuum [13]. The parent polymer PD was prepared under the same reaction conditions without the addition of 2-aminoethanethiol hydrochloride. The presence of amino group in the polymer was determined by using Trinitrobenzenesulfonic acid Assay (TNBS Assay) [14]. 2.3. Preparation of N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate (PDG) Quaternization reaction of PD was conducted using Nhydroxypropyltrimethylammonium chloride as the quaternizing agent. Typically, 100 mg of PD was dispersed in alkaline media (40 ml), under stirring at room temperature. Then, 0.5 ml of Nhydroxypropyltrimethylammonium chloride was added dropwise and continuously stirred for 18 h at room temperature. Thereafter the modified polymer (PDG) was recovered by filtration and dried under vaccum. 2.4. Physicochemical characterisation The size and zeta potential of particles was measured using Zetasizer Nano ZS (Malvern Instruments Limited, UK).These measurements were performed in triplicate. 1 H NMR Spectra of PDG were measured in D2 O using 300 MHz spectrometer (Bruker Avance DPX 300) with tetramethylsilane (TMS) as internal standard. As the hydrogel particles are insoluble in deuterated solvents, 1 H NMR spectral analysis was carried out by polymerising DMAEMA without using EGDMA (crosslinking agent) under the

Swelling characteristics of PDG were carried out separately at pH 1.2 and 6.8 as described elsewhere [15]. Swelling index was calculated as per the following equation: Swelling index =

 (W − W )  s d Wd

× 100%

where Ws is the weight of the swollen particles and Wd is the weight of the dried particles. 2.6. Calcium binding studies The cation binding efficiency of PDG was evaluated with calcium assay kit. Briefly, 5 mg of PDG were dispersed in 1 ml of 1 mM CaCl2 solution and incubated for 1 h followed by centrifugation at 7000 rpm. 200 ␮l of supernatant was used for determining the calcium binding capacity. CaCl2 solution without sample was used as standard. 2.7. Trypsin and chymotrypsin inhibition study Trypsin inhibitory effect of particles was evaluated using BAEE as substrate.1 ml of 0.1% (w/v) particle dispersion in phosphate buffer (pH 7.6) was used for the assay. BAEE in phosphate buffer (pH 7.6), polymer dispersion and 30 U of trypsin solution (in 10 mM HCl) was incubated at 37 ◦ C for 30 min. The enzymatic action was stopped by the addition of 1% trichloroacetic acid solution. Supernatant was analysed by measuring the absorbance for residual trypsin activity at 253 nm using UV/visible spectrometer (Varian Cary 50 Conc.). Control used was without polymer particles and percentage of inhibition was calculated relative to control trypsin [16]. The measurements were carried out thrice. To evaluate the inhibitory properties of PDG towards chymotrypsin, chymotrypsin inhibition assay was performed using the chromogenic substrate, BTPNA. BTPNA, PDG dispersion (0.5%, w/v pH 7.8 Tris–HCl buffer) and 40 U of chymotrypsin solution (in 10 mM HCl) was incubated at 37 ◦ C for 30 min. The reaction was stopped by the addition of 1% trichloroacetic acid solution. Supernatant was analysed by measuring the absorbance at 405 nm spectrophotometrically (Varian Cary 50 Conc.). Control was the same without sample and percentage of inhibition was calculated relative to control chymotrypsin which was taken as 100%. 2.8. In vitro release studies Insulin loading onto PDG was performed by diffusion filling method. 600 ␮l of 400 IU/ml insulin was added to 100 mg PDG and kept for drying at 4 ◦ C. Release studies were carried out as follows. Insulin loaded PDG particles were suspended separately in pH 1.2 and 6.8 buffer. This was then kept in a shaker at 37 ◦ C (50 rpm). At specified intervals of time, 200 ␮l of sample was withdrawn using a micropipette, centrifuged (3000 rpm, 5 min) and the concentration of the supernatant was estimated by Lowry assay [17]. The dissolution medium was replaced with fresh buffer to maintain total volume after each withdrawal. The amount of insulin was calculated from the insulin standard maintained during the assay. Due to the hydrophilic nature of the matrix, insulin loaded particles were coated with Eudragit L 100-55. About 500 ␮l of 5% Eudragit L100-55 in isopropanol was dropped into 50 mg of insulin-loaded

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PDG and dried at 2–4 ◦ C. Encapsulation efficiency of the particles was determined as per the following equation % Encapsulation efficiency =

Ci − Cf × 100 Ci

where Ci and Cf are the initial amount of insulin loaded and insulin content in the supernatant solution respectively. ELISA studies were carried out as per standard procedure to determine the biological activity of loaded insulin in PDG. Results were obtained by reading the optical density at 450 nm using micro plate reader (Finstruments Microplate Reader). Circular Dichroism spectra of the released insulin from PDG were recorded on JASCO J-810 spectropolarimeter equipped with a JASCO PTC-423S Peltier type temperature control system. 2.9. Cytotoxicity Cytotoxicity evaluation of the particles was carried out on L929 and Caco-2 cells. Cell lines were seeded into a 24-well plate containing DMEM culture medium with 10% foetal bovine serum. Similarly, Caco-2 cells were seeded into a 24-well plate containing DMEM culture medium with 20% foetal bovine serum. The plates were then incubated for 24 h at 37 ◦ C in a CO2 incubator with a humidified 5% CO2 /95% air atmosphere. Samples dispersed in DMEM culture medium (0.25–1.5 mg/ml) was added to each well. DMEM medium without sample was used as positive control and Triton X-100 was used as negative control. Medium containing particles was removed after incubation for an additional 24 h at 37 ◦ C, and MTT reagent (0.2 mg/ml) was added to each well and incubated for 3 h. MTT was then removed and dimethylsulfoxide was added to dissolve the formazan crystals. The absorbance was read at 570 nm using an automated microplate reader. The final values were plotted as the mean % viability ± standard deviation for four replicates. 2.10. Mucoadhesion studies The animal experiments were done as per the guidelines of Animal Ethics Committee of Sree Chitra Institute for Medical Sciences & Technology. Freshly excised rat intestinal tissue of about 5 cm was taken out, flushed with normal saline to remove the luminal contents and cut open longitudinally. Mucoadhesion testing was conducted in vitro using a texture analyzer (TA.XT plus, Stable Micro Systems, UK) with 0.05 N load cell equipped with mucoadhesive holder as described elsewhere. Maximum detachment force (Fmax ) and work of adhesion (Wad ) is then calculated [18]. Triplicate measurements were made. 2.11. Adsorption studies on mucin PD & PDG hydrogel particles (10 mg), were dispersed in the mucin solutions (2 mg/ml in pH 6.8 phosphate buffer), vortexed and shaken at 37 ◦ C for 2 h. It was then centrifuged at 4000 rpm for 2 min and supernatant was used for the measurement of free mucin content by Lowry assay. The difference between its initial concentration and the concentration found in the dispersion after incubation and centrifugation was used to determine the amount of mucin adsorbed by the PD and PDG. Triplicate determinations were made. The interaction of mucin was further confirmed by DSC (Q 20 DSC thermal analyzer-TA instrument, USA). 2.12. Tight junction visualisation Caco-2 cells (passages 22–28) were grown and maintained in incubator at 37 ◦ C under 5% CO2 and used for experiments 6 days post-seeding. Tight junction visualisation experiments of actin filaments [19] and ZO-1 staining on Caco-2 cells were carried out

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using 1 mg PDG particles in HBSS pretreated cells. After incubation of particles for 2 h in HBSS, the cells were fixed with 300 ␮l of 4% paraformaldehyde for 20 min at room temperature and thereafter the cells were permeabilized with 0.2% Triton X-100 made of 1% (w/v) bovine serum albumin in PBS, for 20 min. The cells were then washed thrice with PBS and blocked with 300 ␮l of 1% BSA for 30 min. Following fixation and permeabilisation, for actin visualisation cells were incubated with 200 ␮l of 0.2 ␮g/ml rhodamine phalloidin solution after removal of bovine serum albumin for 20 min at room temperature. For ZO-1 staining, cells were incubated with 200 ␮l of ZO-1 antibody (0.1 ␮g/ml) overnight at 4 ◦ C Following day, ZO-1 antibody was removed and the cells were treated with 1% Bovine serum albumin for 30 min and washed with PBS. 250 ␮l FITC anti-rabbit IgG was added and the cells were incubated for 1 h at room temperature. Finally cells were washed with PBS, and the images were obtained using fluorescent microscope (Leica DMRB Germany). Fluorescence intensity was measured using Image J software. 2.13. Transepithelial electrical resistance (TEER) measurements Caco-2 cells were seeded into insert of pore size of 0.4 ␮m, 12 mm diameter and 1 cm2 growth. Cell culture medium was added to apical (400 ␮l) and the basal compartment (600 ␮l) and cells were maintained at 37 ◦ C and 5% CO2 . The medium was replaced every 24 h, for 21 days. On the day of the experiments, prior to the beginning of the experiment, the medium was replaced with an equal volume of HBSS and incubated for 1 h. PD and PDG particles (2.5 mg) dispersed in transport medium were added to the apical side of the insert. TEER measurements were performed in order to determine the effect of matrix on opening of tight junctions at different time intervals using a voltmeter with a chopstick electrode (Millicell ERS system). After 2 h, samples were removed to check if the effect of opening tight junctions is reversible. The reduction in the TEER was determined by calculating the change in TEER from initial value. 2.14. Statistical analysis All the results are expressed as mean ± standard deviation. The statistical significance of differences between two groups was analysed using Students’ t-test. Differences were considered to be significant at a level of P < 0.05. 3. Results Synthesis of quaternised Polydimethylaminoethylmethacrylate (PDG) is carried out as shown in Fig. 1. In this reaction, dimethylaminoethylmethacrylate was polymerised in presence of 2-aminoethanethiol hydrochloride to obtain amino terminated PD (PDNH2 ) followed by conjugation of HTC to the amino group of PDNH2 in aqueous alkaline conditions. The presence of amino group in PDNH2 was confirmed by TNBS Assay .The parent polymer, PD showed negligible percentage of amino groups whereas PDNH2 showed 48% of free amino groups. Reaction of N-hydroxypropyltrimethylammonium chloride to amino group of aminoterminated PD takes place via nucleophilic substitution of amino group and cleavage of epoxide ring. The conjugation of N-hydroxypropyltrimethylammonium chloride was confirmed by FTIR and 1 H NMR spectral analysis (Fig. 2(A and B)). IR spectrum shows evidence of the introduction of the quaternary ammonium group on polymer, at 1455 cm−1 (C H bending of trimethylammonium group). Furthermore, the peak at 3400 cm−1 suggests the presence of OH group. The peaks at 0.9, 1.9, 2.7, 3.2,4.1 and 4.6 ppm were attributed to CH3 , CH2 , ( O CH2 CH2 N(CH3 )2 ), ( N(CH3 )3 ), ( CH2 N(CH3 )2 , O CH2 CH2 N(CH3 )3 . 1 H NMR and

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Fig. 1. Preparation of N-hydroxypropyltrimethylammonium polydimethylaminoethyl methacrylate.

FTIR spectra confirmed the successful incorporation of quaternised moiety to PD. DSC studies also confirmed the cationisation of PD (Fig. 3(A and B)). The endothermic peak at 75.8 ◦ C of PD (Fig. 3(A) was shifted to 67 ◦ C in PDG (Fig. 3(B)). An endotherm at about 218 ◦ C was also observed for PDG. The shift in endothermic peaks confirmed the derivatisation of the polymer. The particle size of PDG was found to be 513.6 ± 17.45 nm with a polydispersity index of 0.439 and particle size of insulin loaded PDG was found to be 646.5 ± 8.25 nm with a polydispersity index of 0.441. AFM image shown in Fig. 4(A) confirms the morphology and size of PDG. Zeta potential of PDG was found to be 35.1 ± 1.21 mV whereas that of unmodified polymer was 21.8 ± 2.31 mV. The swelling profile of PDG at pH 1.2 and 6.8 is depicted in Fig. 4(B). PDG particles exhibited high percentage of swelling at acidic pH whereas the degree of swelling was lower at intestinal pH. P-value as determined by Students’ t-test was found to be statistically significant. The maximum detachment force, Fmax and work of adhesion, Wad of PDG measured by using texture analyser are depicted in Fig. 5. Fmax and Wad was higher for PDG compared to parent polymer, PD. P-value was found to be less than 0.05 and hence the result is statistically significant. The adsorption studies of mucin with PD

& PDG are shown in Supplementary Fig. 1. Adsorption studies on mucin showed that a significant amount of mucin was adsorbed onto the particle which was further confirmed by DSC. Mucin thermogram showed endotherms at 98 ◦ C and 221 ◦ C (Fig. 3(C)). In DSC thermograms of mucin complexed with PDG (Fig. 3(D)), the endothermic peak at 67 ◦ C of PDG was shifted to 81 ◦ C in mucin complexed with PDG. An endotherm at about 240 ◦ C for mucin complexed PDG, was also observed. The shift in endothermic peaks confirmed the interaction of mucin with the polymer. This evidenced that some amount of mucin had been adsorbed onto PDG through electrostatic interaction. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.01.057. Fig. 6 shows the in vitro release profile of Eudragit coated insulin loaded PDG at gastric and intestinal pH. PDG exhibited encapsulation efficiency of 85.5 ± 3.24%. Due to the swelling nature of the particles at pH 1.2, the particles were coated with Eudragit L 10055. About 1.5% of insulin was released at gastric conditions whereas about 30% of insulin was released at intestinal pH at first hour. The results are statistically significant as the P-value was found to be less than 0.05. ELISA and Circular dichroism studies proved that the insulin released from PDG was capable of retaining its biological

Fig. 2. (A) IR and (B) NMR spectra of N-hydroxypropyltrimethylammonium polydimethyl aminoethylmethacrylate.

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Fig. 3. DSC thermograms of (A) polydimethylaminoethylmethacrylate, (B) N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate and (C) mucin and (D) mucin complexed with N-hydroxypropyltrimethylammonium polydimethyl aminoethylmethacrylate.

Fig. 4. (A) AFM of N-hydroxypropyltrimethylammonium polydimethylaminoethyl methacrylate and (B) swelling profile of N-hydroxypropyltrimethylammonium polydimethyl aminoethylmethacrylate at pH 1.2 and 6.8. Indicated values are the mean of three measurements (±SD) (n = 3).

Fig. 5. Force (A) and work of adhesion (B) of N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate. Each bar is the mean of three measurements (±SD) (n = 3).

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Fig. 6. In vitro release profile of N-hydroxypropyltrimethylammonium polydimethyl aminoethylmethacrylate at pH 1.2 and 6.8. Each data point is the mean of three measurements (±SD) (n = 3).

activity (92%) and conformation of insulin (Supplementary Fig. 2). There was no significant change in the peaks of ␣ and ␤ helix. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.01.057. The nontoxic nature of PDG has been established through MTT assay on L929 cells. Both the modified and unmodified particles showed 80–90% cell viability at a concentration of 0.25–1.5 mg/well. MTT Assay of different concentration of PDG on Caco-2 cells (0.25–1.5 mg/ml) displayed 85–90% cell viability

(Fig. 7), which further confirmed the nontoxic nature of PDG (P < 0.05). The amount of calcium that binds to the particles was determined from the free concentration of calcium before and after binding. PDG exhibited a calcium binding capacity of 18%. The ability of the particles to protect insulin from trypsin and chymotrypsin degradation was determined using BTPNA and BAPNA as substrate. PDG exhibited a higher chymotrypsin and trypsin inhibitory effect compared to PD (Supplementary Fig. 3) at neutral pH (P < 0.05). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.01.057. The calcium binding efficacy of PDG helps in the opening of tight junctions as visualised from fluorescent micrographs in Supplementary file Fig. 4 and Fig. 8. The control cells showed continuous nondisrupted F-actin rings, which is required for maintenance of tight junction integrity whereas PDG showed a total disruption of F- actin filaments (Supplementary file Fig. 4). On ZO-1 visualisation (Fig. 8), there was a decrease in fluorescence intensity of PDG treated cells compared to control cells. The decrease in fluorescence intensity was quantitatively evaluated using Image J software. As evident from Supplementary file Fig. 6 mean grey value was found to be low for PDG treated cells compared to control cells. From this data we can conclude that PDG binds to ZO1 proteins. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.01.057. Opening of tight junctions was further confirmed by TEER measurements on Caco-2 cells. It was observed that the TEER values remained constant in the control during the experiment and PDG were able to reduce the TEER values (P < 0.05) by >50%

Fig. 7. % Cell viability of different concentration of N-hydroxypropyltrimethylammonium polydimethylaminoethylmethacrylate on (a) L929 and (b) Caco-2 cells. Each value represents the mean of three measurements (±SD) (n = 3).

Fig. 8. Fluorescent micrographs of ZO-1 visualisation of control cells and Caco-2-cells complexed with N-hydroxypropyltrimethylammonium polydimethylaminoethyl methacrylate (PDG).

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of the initial value (Supplementary file Fig. 5). The decrease in TEER value can be attributed to the opening of the tight junctions via the calcium ion binding. On removal of samples after 2 h, and substitution of medium with fresh DMEM, the monolayers started to recover slowly and a slight increase in resistance towards the initial values was observed which implied that the PDG did not cause permanent damage to the tight junctions. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.01.057.

4. Discussion In this investigation, polydimethylaminoethylmethacrylate was successfully cationised with N-hydroxypropyltrimethylammonium chloride to improve the positive charge density and hence the mucoadhesive properties of the hydrogel. Mucoadhesive properties of polymers are not only expected to prolong the residence time of the dosage forms at the site of administration but may also enhance drug permeability through the epithelium by opening the tight junctions between the cells [20]. IR and NMR spectra (Fig. 2(A and B)) confirmed the successful quaternisation of PD to form strong cationic network. Quaternisation was also confirmed by zeta potential value. PDG exhibits high positive charge density at physiological pH. Zeta potential is an important property of drug carrier. The zeta potential values gives an indication of the potential stability of the system [21]. Aggregation behaviour of the particles is reported to be likely to diminish in a suspension having large positive or negative zeta potential value. It was beneficial for the colloidal stability in the aqueous solution. The size of the PDG particles was found to be in the submicron ranges, which was further confirmed by AFM (Fig. 4(A)). Mucus layer present in intestinal epithelial cells is composed of hydrated glycoproteins, which consists of anionic sialic residues [22]. The high positive density of PDG helps them to interact with anionic sialic acid residues of mucus. Moreover the flexible nature of hydrogel helps them to interpenetrate across the mucus network, which is one of the important prerequisites of mucoadhesive behaviour [23]. PDG exhibited high swelling behaviour at acidic pH and comparatively low degree of swelling (Fig. 4(B)) at intestinal pH. This behaviour may be explained by the overshooting effect of PD as described by Li et al. [24]. On contact of polymer chains to the mucus, swelling enables mechanical entangling of polymer chains and subsequent formation of hydrogen bonds and/or electrostatic interactions between the polymer and components of the mucosa. Polymer chain flexibility is required for diffusion of chains and their entanglement with mucin [25]. As evident from the adsorption studies of mucin on particle surface (Supplementary file Fig. 1), 73% of mucin was adhered in the case of PDG. This can be attributed to the formation of hydrogen bond between the hydroxyl group present in PDG as well as electrostatic interaction between positive charges of NH3 + with the negatively charged sialic acid residues of the mucus. Mucoadhesive nature of particles was further confirmed by texture analyser measurements which is based on measuring the force/work required to detach the polymers from the mucosal layer. As texture analysis more closely simulates the in vivo situation, measurement of detachment force will be more useful [26]. For this analysis, the mucoadhesion force of polymers on the rat intestinal tissue was measured, and force and work of adhesion was evaluated. The maximum detachment force for PD and PDG was calculated from the force versus distance curve. Fmax and Wad were higher for PDG (Fig. 5) when compared to quaternised PD reported earlier. The increased mucoadhesivity of the PDG particles may be due to the formation of hydrogen bond and electrostatic interaction.

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In vitro release studies of insulin-loaded PDG carried out at gastric and intestinal pH showed a burst release (Fig. 6) due to the fast swelling nature of these particles. For oral insulin delivery systems, minimal release in gastric pH is appropriate as it increases the bioavailability of the loaded insulin compared to that of a matrix which does not display this property [15]. Therefore PDG were coated with Eudragit L-100. Eudragit coated insulin loaded PDG showed a slow release at gastric pH and a burst release at intestinal pH. ELISA results suggest that PDG were capable of preserving biological activity of encapsulated insulin. Moreover there was no significant difference in the secondary structure of insulin released from insulin loaded PDG and native insulin as observed in Supplementary file Fig. 2. In our study, cytotoxicity was studied to monitor the dose dependent activity of PDG particles. Remarkably, no significant cytotoxicity of PDG was observed at a concentration range of 0.25–1.5 mg/well on L929 cells and Caco-2 cells (Fig. 7(A and B). The results were found to be statistically significant (P < 0.05). Similar results were obtained by Keely et al. [27] on cytotoxic evaluation of quaternised PD. Thus, it could be concluded that the PDGs are suitable for oral insulin delivery. The chelating ability of PDG with divalent metal ions like calcium is expected to inhibit protease degradation. The deprivation of divalent cations like calcium indirectly inhibits the proteolytic enzyme like trypsin and chymotrypsin (Supplementary file Fig. 3). This property of PDG could be useful in reducing the enzymatic degradation of insulin on oral administration. Chelating calcium from tight junction can disrupt the structure of tight junctions [28]. PDG interacts with the epithelial tight junctions inducing a redistribution of actin filaments (Supplementary file Fig. 4) and binding to the tight junction protein, ZO-1.The decrease in fluorescence intensity of PDG treated cells may be due to the loss of ZO-1 from sites of cell–cell contact [19]. This ultimately leads to the opening of tight junctions across the intestinal epithelium as visualised from fluorescent micrographs in Fig. 8. Literature suggests the role of some non-specific interactions such as Van der Waals and electrostatic interaction of polymer with enzymes for inhibition process [29]. For effective oral administration of protein drugs like insulin, the drugs should be absorbed in the small intestine. Insulin is readily degraded by the high acidic conditions in the stomach. Free insulin cannot passively diffuse across epithelial cells due to its high molecular weight (5800 Da) and low lipophilicity [30]. The tight junction restricts the diffusion of hydrophilic molecules. Hence its ability of insulin to pass through tight junctions is very limited. It is well documented in literature that when encapsulated as part of a polymeric carrier, these carriers may be able to facilitate both paracellular and transcellular transport of proteins [31,32]. PDG being mucoadhesive is expected to reduce the proteolytic attack. Fluorescent microscopy confirmed the interaction of PDG with F-actin which leads to the opening of the tight junctions via paracellular pathway. This data is in agreement with previous reports, which shows that cationic polymers such as chitosan/or its derivatives can induce structural separation in F-actin distribution of the tight junctions in Caco-2-cells [17,33]. This is well supported by the reduction in TEER values. TEER is a measure of integrity of tight junctions and will exhibit reduction in values if the tight junctions are opened [34]. The high mucoadhesive nature of PDG did not allow complete removal of the particles without damaging the cells and this might be the reason for the gradual increase in resistance. Moreover, incubation with PDG on Caco-2 cells for 24 h did not alter the viability of the monolayers. This data is in agreement with previous reports by Kotze et al. which states that complete removal of the trimethylated chitosan without damaging the cells was found to be difficult due to high viscosity and adhesivity of the polymer. Here also, the author obtained only a gradual increase in resistance and did not obtain 100% recovery [35,36]. Thus, PDG having high

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positive charge modulate tight junction opening by binding to the Zona-occludens proteins and actin filament dislocation. Therefore, the development of PDG particles could help in developing specific bioadhesive interactions with the intestinal mucosa. 5. Conclusion Mucoadhesive, nontoxic, cationic hydrogel particles PDG, based on synthetic polymer PD was successfully synthesised by conjugating HTC to amino terminated PD and characterised by various physicochemical and biological characterisation. PDG having high positive charge displayed calcium binding property, which helped in the loosening of epithelial tight junctions by actin filament dislocation and binding to ZO-1 proteins. Therefore the present study highlighted the importance of chemical modification on synthetic polymer for enhancing mucoadhesion and thereby making it suitable carrier for mucosal delivery of proteins. Acknowledgements Authors thank Dr. K. Radhakrishnan, Director and Dr. G.S. Bhuvaneswar, Head, BMT Wing, SCTIMST, Thiruvananthapuram, for providing facilities. The authors also thank CSIR for Senior Research Fellowship. This study was supported by the Department of Science and Technology, Government of India through the project ‘Facility for nano/microparticle based biomaterials – advanced drug delivery systems’ #8013, under the Drugs & Pharmaceuticals Research Programme. References [1] [2] [3] [4]

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