S-protected thiolated cyclodextrins as mucoadhesive oligomers for drug delivery

Accepted Manuscript S-protected thiolated cyclodextrins as mucoadhesive oligomers for drug delivery Mulazim Hussain Asim, Ali Moghadam, Muhammad Ijaz, Arshad Mahmood, Roman Xaver Götz, Barbara Matuszczak, Andreas Bernkop-Schnürch PII: DOI: Reference:

S0021-9797(18)30817-8 https://doi.org/10.1016/j.jcis.2018.07.062 YJCIS 23853

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

18 May 2018 14 July 2018 16 July 2018

Please cite this article as: M. Hussain Asim, A. Moghadam, M. Ijaz, A. Mahmood, R. Xaver Götz, B. Matuszczak, A. Bernkop-Schnürch, S-protected thiolated cyclodextrins as mucoadhesive oligomers for drug delivery, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.07.062

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S-protected thiolated cyclodextrins as mucoadhesive oligomers for drug delivery Mulazim Hussain Asim

a, b

, Ali Moghadam

a, e

, Muhammad Ijaz c, Arshad Mahmood d,

Roman Xaver Götz a, Barbara Matuszczak f, Andreas Bernkop-Schnürch a*

a

Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology,

Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria b

Faculty of Pharmacy, Department of Pharmaceutics, University of Sargodha, 40100

Sargodha, Pakistan c d

Department of Pharmacy, COMSATS University, 54000 Lahore, Pakistan Department of Pharmacy, COMSATS Institute of Information Technology, 22060

Abbottabad, Pakistan e f

Institute of Biotechnology, College of Agriculture, Shiraz University, Shiraz, Iran Center for Chemistry and Biomedicine, Department of Pharmaceutical Chemistry,

Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria

*Corresponding author: A. Bernkop-Schnürch, e-mail address: [email protected]; Tel: +43 512 507 58601, Fax: +43 512 507 58699

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ABSTRACT Aim: The purpose of this study was to develop a novel mucoadhesive thiolated and Sprotected gamma cyclodextrin (γ-CD) with an intact ring backbone to assure a prolonged residence time at specific target sites. Method: Thiolated γ-CD was generated through bromine substitution of its hydroxyl groups followed by replacement to thiol groups using thiourea. In the second step, thiol groups were protected by disulfide bond formation with 2-mercaptonicotinic acid (2MNA). Result: Thiolated γ-CD displayed 1385 ± 84 µmol thiol groups per gram of oligomer and the amount of MNA determined in the S-protected oligomer was 1153 ± 41 µmol per gram of oligomer. In-vitro screening of mucoadhesive properties of thiolated and Sprotected γ-CD was done by two methods. Rheological investigation revealed the conjugates non-mucolytic with only a slight increase in viscosity of thiolated and Sprotected γ-CD as compared to unmodified γ-CD, whereas mucoadhesive properties of the new thiolated and S-protected γ-CD performed on freshly excised porcine intestinal mucosa showed 44.4- and 50.9-fold improvement in mucoadhesion, respectively. The new conjugates did not show any cytotoxicity to Caco-2 cells even at a concentration of 1 % (m/v) for 24 hrs. In addition, in-vitro studies of α-amylase degradation of γ-CD, γCD-SH and γ-CD-SS-MNA confirmed that all conjugates are biodegradable. Conclusion: These outcomes predict that these new conjugates of γ-CD might provide a new favorable tool for drug delivery providing a prolonged residence time on mucosal surfaces.

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Keywords: Thiolated gamma cyclodextrin; mucosal drug delivery; mucoadhesion; Sprotected thiomer. 1. Introduction Cyclodextrins (CDs), first discovered in 1891 [1], are cyclic oligosaccharides consisting of six (α-cyclodextrin), seven (β-cyclodextrin), eight (γ-cyclodextrin) or more glucopyranose units linked by α- (1, 4) bonds [2]. CDs bear hydrophobic inner cavities and a hydrophilic outer surfaces [3-5], which are accountable for their ability to encapsulate hydrophilic guest molecules [6]. Due to superior solubilizing and complexing abilities, cyclodextrins are used as complexing agents to enhance the solubility of poorly water-soluble drugs [7]. Furthermore, cyclodextrins are likely considered the smallest drug carriers possessing the ability to interpenetrate within mucus [8]. As they lack mucoadhesive properties, their residence time on mucosal surfaces, however, is comparatively short and in many cases insufficient to guarantee a therapeutic effect of incorporated drugs [9]. In order to overcome this shortcoming, first generation thiolated cyclodextrin conjugates (β-CD-cysteamine) were introduced to the scientific community as novel mucoadhesive delivery system for intra-oral use [10]. This thiolated conjugates formed disulfide linkages with cysteine-rich subdomains of glycoproteins covering mucosal membranes and provided significantly improved mucoadhesive properties [11]. The thiolated conjugates, however, were synthesized under comparatively harsh conditions via oxidative opening of the glucose rings and importantly, significant cytotoxic effects were observed during in-vitro studies on Caco-2 cells due to the cationic nature [9]. These shortcomings were overcome via development of second generation non-ionic 3

thiolated cyclodextrins obtained by reductive amination without ring-opening [12]. However, we believe that the full potential of this novel technology has yet by far not been reached. On the one hand, first and second generation thiolated conjugates were developed with non-biodegradable β-CD [13] and on the other hand, free thiol groups on CD backbone are highly susceptible towards oxidation leading to inter- and intramolecular disulfide bond formation prior to interaction with mucus [14]. Therefore, the aim of this study was to synthesize the third generation of thiolated CDs of biodegradable and S-protected nature. As CD, γ-CD was chosen because of being biodegradable, better water-soluble and less toxic [13]. Synthesis was performed by substituting hydroxyl groups for thiol groups on the glucose subunits via bromination followed by reaction with thiourea [15]. S-protection to thiolated conjugate was provided by disulfide bond formation with an aromatic thiol bearing ligand namely 2mercaptonicotinic acid (2-MNA) [16]. The resulting S-protected thiolated γ-CD was evaluated for cytotoxicity, mucoadhesive properties and degradation studies with αamylase. 2. Materials & methods 2.1. Materials Gamma cyclodextrin (γ-CD, 98 %, molecular weight: 1297.12 Da), lithium bromide (LiBr, ≥ 99 %), triphenylphosphine (Ph3P, ≥ 95 %), N,N-dimethylacetamide (DMA, ≥ 99.5 %), N-bromosuccinimide (NBS, ≥ 99 %), thiourea (CS(NH2)2, ≥ 99 %), 2mercaptonicotinic acid (2-MNA, ≥ 98 %), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, ≥ 98 %), Spectra/Por® dialysis tubing MWCO (Molecular weight cut-off) 500-

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1000 Da, Hanks’ balanced salt solution (HBSS), resazurin (7-hydroxy-3H-phenoxazin-3one 10-oxide) sodium salt (dye content ≥ 75%), cysteine (≥ 98 %), sodium borohydride (NaBH4, ≥ 99 %), dimethyl sulfoxide-d6 (DMSO-d6, ≥ 99.9 %), fluorescein diacetate (FDA, ≥ 98 %,), α-amylase (Type II-A, ≥ 1500 units/mg protein), 3,5-dinitrosalicylic acid (DNS, ≥ 98 %), potassium sodium tartrate tetrahydrate (≥ 99 %), sodium sulfite (Na2SO3, ≥99 %), minimum essential eagle medium (MEM), sodium chloride (NaCl), methanol (≥ 95-99 %) and Triton-X 100 were all purchased from Sigma-Aldrich, Vienna, Austria. Cell culture medium was prepared by using MEM powder 9.66 g/L (modified with Earle’s salts, phenol red, 19 amino acids and the non-essential amino acids L-asp, Lasn, L-glu, L-ser, L-ala, L-gly, and L-pro), L-glutamine 2 mM, sodium bicarbonate 2.2 g/L, 1 % penicillin-streptomycin solution (10,000 U/mL) and 10 % fetal bovine serum (FBS). FBS and 100 mM phosphate-buffered saline (PBS) pH 6.8 were purchased from Gibco (Invitrogen, Lofer, Austria). All other materials were of analytical grade and received from commercial sources. 2.2. Synthesis of thiolated γ-CD Primary and secondary hydroxyl groups of gamma cyclodextrin were chosen for substitution with bromine moieties followed by replacement with thiol groups. Thiolated γ-CD was synthesized by replacing the hydroxyl groups of gamma cyclodextrin with bromine and then this bromine was replaced by thiol groups based on a method described previously [17]. Bromination and thiolation as outlined in Fig. 1 were performed under inert conditions (nitrogen gas). γ-CD, NBS, and Ph3P were dried in a

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desiccator under reduced pressure in the presence of molecular sieves 4 Å. Before experiment LiBr anhydrous was dried at 150 °C in oven. First of all, 1 g of dried γ-CD was dissolved in 100 mL of DMA, the mixture was heated at 90 °C for one hour with constant stirring and then 23 g of LiBr was slowly added. This LiBr-DMA mixture gave homogeneous bromination of γ-CD [17]. The reaction mixture was kept at 90 o C for one hour until LiBr was completely dissolved and then maintained at 70 °C for 18-20 hours until a clear solution was obtained. After cooling the mixture at ice water, 100 mL of 3 % (m/v) NBS and 100 mL of 4 % (m/v) Ph3P (both in DMA solution) were added. The final solution was kept at 70 °C for 3 hrs while stirring. In the following, the brominated γ-CD was precipitated by the addition of 400 mL of acetone (ratio 1:4). The precipitate was filtered with a whatman filter paper, washed three times with acetone, dried in oven at 40 °C, dissolved in water, dialyzed using cellulose membrane (molecular weight cutoff of 500–1000 Da) against water and finally freeze-dried (−51 °C, 0.01 mbar, Gamma LSC 1-16, Martin Christ, Germany). In the following, 1 g of bromo-γ-CD was dissolved in a solution of thiourea (0.5 %; m/v) in 100 mL of DMA and the mixture was stirred at 70 °C for 16 hrs. After cooling the reaction mixture at ice water, 12 mL of 3 M NaOH solution was added slowly to produce thiolated γ-CD. After stirring for 15 min at room temperature, the mixture was neutralized with 18 mL of 3 M H2SO4 solution. The final product was precipitated in acetone as described above, dialyzed using cellulose membrane (molecular weight cutoff of 500–1000 Da) against water, freeze dried and stored at 4 °C until further use.

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2.3. Synthesis of S-protected thiolated γ-CD After thiol groups conjugation to the oligomeric backbone, S-protected thiolated γ-CD was generated by disulfide bond formation between free thiol groups of thiolated γ-CD and 2-mercaptonicotinamide (2-MNA) as shown in Fig. 1. 2-MNA dimer (2, 2′dithiodinicotinic acid) was prepared by oxidation of 2-MNA with hydrogen peroxide under neutral pH conditions to obtain 2-MNA modified oligomers [18]. Briefly, in the first step, 1 g of 2-MNA was dispersed in 12.5 mL of demineralized water by ultrasonication for 30 min. Then pH of the solution was adjusted to 8 using 5.0 M NaOH with constant stirring until a slight yellowish clear solution was obtained. In the following 1.25 mL of hydrogen peroxide (30 %, w/v) was drop-wise added to this solution till a colorless solution (2-MNA dimer; 2, 2′-dithiodinicotinic acid) was obtained. This colorless 2-MNA dimer solution was continuously stirred for 1 hr at room temperature and finally diluted with water to volume of 25 mL. The product was lyophilized and stored at room temperature. The formation of 2-MNA dimer was confirmed by comparing the UV absorption spectra of the monomer and dimer at a wavelength between 200 nm and 400 nm using a UV–VIS spectrophotomer (UVmini1240, Shimadzu Corp., Kyoto, Japan). UV-analyses in case of 2, 2′-DTNA showed a significant peak at 297 nm, whereas the monomer 2-MNA displayed a well-pronounced peak at 307 nm. In the second step, the thiolated cyclodextrin was coupled with aromatic dimeric ligand through disulfide exchange reaction. One gram of thiolated γ-

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CD was hydrated in 100 mL of demineralized water. 2 mL of 2-MNA solution was added drop-wise to thiomer solution buffered to pH 8 using 5 M NaOH. The reaction mixture was incubated overnight at room temperature under constant stirring and then dialyzed for 3 days in the dark using Spectra/Por® dialysis membrane (MWCO: 500-1000 Da) in 5 L of demineralized water. Thereafter, the dialyzed product was freeze-dried at −80 °C under reduce pressure and stored at 4 °C until use. 2.4. Evaluation of the thiol and disulfide content The free thiol groups immobilized on oligomer backbone were measured with Ellman’s reagent [19]. The amount of thiol groups was calculated by using a standard curve of cysteine prepared in exactly the same way as the samples, whereas the disulfide contents were calculated after reduction with NaBH4 [20]. 2.5. Quantification of conjugated 2-mercaptonicotinic acid The degree of S-protected thiol groups was measured photometrically using a method previously described by our research group [18]. Samples of 0.5 mg were dissolved in 500 μL of 0.5 M phosphate buffer solution at pH 8. To liberate 2-MNA, 500 μL of a 2 % (m/v) reduced glutathione solution were added and the mixture was incubated for 1 hr under stirring. The absorbance of free 2-MNA was measured at 354 nm. To ensure that there is no unbound 2-MNA in the sample a control without the addition of GSH was measured at 354 nm. 2.6. Characterization of thiolated and S-protected γ-CD structure IR spectra were recorded on a Bruker ALPHA FT-IR apparatus equipped with a Platinum ATR (attenuated total reflection) module. The 1H NMR spectra were recorded 8

on a Varian Gemini 200 spectrometer in D2O with DSS (4, 4-dimethyl-4-silapentane-1sulfonic acid sodium salt) as an internal reference. 2.6.1 Molecular weight determination of CD conjugates by LC-MS Mass spectrometry detection was carried out using Chromaster 5610 MS detector (Hitachi) controlled by a computer running the MSD system manager software (version 2.1). Source conditions were as follows: ionization potential (V): 2000; ion injection (eV): 2.0; counter gas flow (L/min):1.0; AIF temperature (°C):140 and ion source temperature (°C): 80. Sample solutions were prepared by dissolving the γ-CD and γ-CD conjugates in the mobile phase (methanol, water; 85:15, v/v) to reach the concentration of 500 ng/mL. Mass spectral studies were performed in negative electrospray ionization (ESI) mode in the mass range of m/z 600→700 to calculate molecular weight of CD and its conjugates. In order to get clear mass spectrum without any background noise, the conjugates were directly infused using a syringe pump with flow rate of 2 μL min−1 into the mass spectrometer. 2.7. Cytotoxicity studies 2.7.1. Resazurin assay The effect of unmodified, thiolated and S-protected γ-CD on cell viability was evaluated by resazurin assay. For this purpose, Caco-2 cells were purchased from the European Collection of Cell Cultures (ECACC, Health Protection Agency, Porton Down, Salisbury, Wiltshire, United Kingdom). Cells (passage number 25–33) were seeded at a

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density of 2.5 × 104 cells/well in a 24-well plate for 10 days in a final volume of 500 µL of MEM with Earle’s balanced salts supplemented with 2.0 mM L-glutamine, 10 % FBS, and 1 % penicillin-streptomycin at 37 °C in 5 % CO2 environment. The culture medium was refreshed every other day. Resazurin assay was performed as an oxidation-reduction indicator to determine invitro cytotoxicity of thiolated and S-protected γ-CD. When the cells were approximately 80 % confluent (80 % of surface of flask covered by cell monolayer after 8-10 days), they were washed twice with PBS pre-warmed at 37 °C. Test solutions including unmodified γ-CD, thiolated γ-CD and S-protected γ-CD (1 % m/v), positive control prepared in white MEM and negative control (4 % v/v Triton X-100) were added in 500 µL volume in triplicate to the cell culture. Then, the treated cells were incubated at 37 °C in 5 % CO2 environment for 3 and 24 hrs. Afterwards, test solutions were removed and cells were washed twice with prewarmed phosphate buffer saline (PBS). A diluted resazurin solution (2.2 µM) in 500 µL volume was added to each well and cells were incubated for 3 hrs. Supernatant (100 µL) was afterwards transferred to black 96-well plate and fluorescence intensity was measured using microplate reader (M200 spectrometer; Tecan infinite, Grödig, Austria) at a wavelength of 540 nm with background subtraction at 590 nm [21]. Percentage of viable cells was calculated using the following equation:

Experimental values − Negative control Cell Viability (%) = Positive control − Negative control

2.7.2. LDH assay

10

× 100

In parallel to the resazurin assay lactate dehydrogenase (LDH) release assay was also performed. In principle, if test samples damage cell membrane integrity, cytoplasmic LDH is released into culture medium [22]. The amount of LDH leakage is then measured using a commercial test kit (Promega, Madison, WI). In detail, the cells with approximately 80 % confluence were washed with pre-warmed HBSS before being treated with unmodified or modified samples (1 % m/v) as described above. HBSS was used as negative control and Triton-X 100 (4 % m/v) served as positive control. After 3 and 24 hrs of incubation at 37 °C in 5 % CO2 environment, supernatant was collected and LDH assay was performed according to the manufacturer’s instruction. Fluorescence was recorded using microplate reader (M200 spectrometer; Tecan infinite, Grödig, Austria) with an emission wavelength of 590 nm and an excitation wavelength of 560 nm. Percentage of cell toxicity was estimated using the following equation:

Cell toxicity (%) =

Average fluorescence intensity of each sample Average fluorescence intensity of positive control

× 100

2.8. Enzymatic (α-amylase) degradation of γ-CD In-vitro α-amylase degradation of γ-CD was investigated by measuring the formation of reducing sugars (mainly maltose) via a slightly modified method as described previously [13]. 5 mM solutions of unmodified, thiolated and S-protected γ-CD were prepared in 20 mM sodium phosphate buffer (pH 6.9) containing 6.7 mM NaCl at 20 °C. Degradation reaction was started by the addition of α-amylase in a final concentration of 1 unit/mL to 5 mM γ-CD solution. The reaction was kept at 37 °C with continuous

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shaking for 4 hrs. Degradation products resulting from this reaction are mainly maltose and to a minor extent glucose. As the degradation products have been investigated in detail previously [23, 24], the reaction was analyzed by quantification of recommended degradation products. Aliquots (250 μL) were withdrawn at predetermined time points and analyzed by reducing sugar method with some changes [13] . Briefly, 250 μL of enzyme-γ-CD solutions were transferred to preheated tubes (95 °C) at predetermined time points in order to denature the enzyme and consequently terminate the reaction. Then 750 μL of a solution consisting of 96 mM 3,5-dinitrosalicylic acid (DNS), 2 M NaOH, 20 % (w/v) potassium sodium tartrate tetrahydrate and 0.05 % (m/v) Na2SO4 was added. The mixture was kept at 95 °C for 15 min before cooling on ice and analyzed using microplate reader (M200 spectrometer; Tecan infinite, Grödig, Austria) at 540 nm. All reactions and analyses were performed in triplicate. 2.9. In-vitro evaluation of mucoadhesive properties Thiolated and S-protected γ-CD, were fluorescent-labelled by incorporation of FDA for the assessment of mucoadhesive properties. Briefly, 20 mg of CDs were dissolved in 20 mL of demineralized water and pH of solutions was adjusted to 6.5 with 100 mM HCl. Then 1 mg of FDA was dissolved in 5 mL of 96 % ethanol and 1 mL of this solution was added to each CD solution. After 24 hrs of stirring at room temperature, the suspensions were filtered in order to eliminate free FDA and freeze dried. For evaluation of mucoadhesive properties, freshly excised porcine intestinal mucosa was collected from a local slaughterhouse. First, porcine small intestine was cleaned and rinsed with 100 mM phosphate buffered saline pH 6.8 [18]. Then, it was cut into

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smaller pieces of 4 × 2 cm and fixed on half cut 50 mL falcon tubes that were placed at an angle of 45º in an incubation chamber at 37 °C and 100 % relative humidity. Afterwards, the mucosa was continuously rinsed with phosphate buffer pH 6.8 for 5 min at a flow rate of 1 mL/min. In the following, 20 mg of FDA-labelled γ-CD samples were separately placed on each mucosa and after 10 min the phosphate buffer flow was restarted. 30 mL of phosphate buffer flowing down the mucosa was collected at following time points: 30, 60, 90, 120, 150 and 180 min. In parallel, reference samples containing 100 % γ-CD were prepared by rinsing the mucus with 30 mL of phosphate buffer and dissolving 20 mg of γ-CD in the collected buffer. To quantitatively hydrolyze FDA to sodium fluorescein, 1 mL of NaOH 5 M was added to 1 mL of each collected samples and the remaining intestine on the falcon tube. Samples were incubated while shaking at 37 °C for 20 min and then centrifuged at 13400 rpm for 5 min at room temperature. Finally, 100 µL of each sample was transferred to the microplate reader (M200 spectrometer; Tecan infinite, Grödig, Austria) and fluorescence intensity was measured at an emission wavelength of 535 nm and exciting wavelength of 485 nm. All experiments were performed in triplicate. 2.10. Viscosity measurement The dynamic viscosity of unmodified, thiolated and S-protected γ-CD was measured in presence of freshly excised porcine intestinal mucus. The mucus was collected and purified as described before [18]. Briefly, porcine mucus was acquired from the small intestine of a freshly slaughtered pig donated by a local slaughter house. To get the mucus, the emptied intestine was cut

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into small pieces and opened lengthwise. Mucus was isolated from the tissue using a scraper. To decontaminate the collected mucus, it was dispersed in NaCl solution (0.1 M) and stirred on ice for one hr. Afterward suspension was centrifuged at 4 ºC and 13,000g for 2 hrs. The supernatant and granular material at the bottom were removed. The purified and homogenized mucus was used immediately. Dynamic oscillatory tests were performed at a frequency of 1 Hz on a plate–plate viscometer (Haake MARS Rheometer, 379-0200; Thermo Electron GmBH, Karlsruhe, Germany). In detail, test samples (0.5 %, w/v) in 200 mM phosphate buffer at pH 7 were mixed with porcine intestinal mucus in a ratio of 1:4 in a petri dish using a spatula. This mixture was incubated at 37 °C without stirring until measurement. The oligomer-mucus mixture was incubated for 0.5, 2, 4 and 6 hrs at 37 °C and then 500 µL was transferred to rheometer plate for measurement. All experiments were repeated three times. 2.11. Statistical analysis The statistical difference among groups was compared by one-way ANOVA and p < 0.05 was considered statistically significant (Graph Pad Prism 5.01). In addition, Student’s t-test was applied with a confidence interval (p < 0.05) for the analysis of two groups. 3. Results and discussion 3.1. Synthesis and characterization of thiolated γ-CD In comparison to the synthetic pathway previously used for CDs [10], thiolated γ-CD was synthesized without ring-opening. Immobilization of thiol groups to γ-CD was carried out via an intermediate brominated derivative. The hydroxyl groups on the 14

carbohydrate backbone were initially substituted with bromine followed by its replacement with thiol groups. Hydroxyl groups at terminal site of polysaccharide chain are likely favored for this type of substitution, as primary hydroxyl groups display a higher reactivity to nucleophilic substitution reactions than secondary hydroxyl groups [25, 26]. From stability point of view the intermediary step was important as bromination of carbohydrates is a temperature dependent reaction and higher temperatures result in degradation of carbohydrates with an insignificant degree of substitution [27].The thermal degradation pattern of cyclodextrins is similar to that of cellulose and temperature of decomposition strongly depend on the degree of substitution [28]. Bromination of carbohydrates was mostly proceeded at 90 °C but recoveries of the products were much lower at this temperature as cellulose was degraded [27]. In order to achieve a higher degree of bromination and at the same time to avoid degradation of CD, the reaction was initially proceeded at 90 °C for just 1 hr and afterwards continued at 70 °C for 18-20 hrs [12]. Furthermore, bromine conjugation was found dependent on the concentration of NBS and Ph3P during the reaction. Thiolated CDs show an almost two times higher degree of substitution (DS) when a twice as high concentration of Ph3P and NBS is applied in the reaction. This effect might be explained by the higher degree of bromination on the backbone of the oligomer [12]. However, an increasing concentration of thiourea in the later step did not affect the thiolation efficiency identifying the degree of bromination to be controlling the degree of thiolation [15]. Thiolation of CDs in LiBr-DMA mixture generates homogeneous bromination of CDs, as shown by our previous research work [12]. An efficiently regioselective bromination was achieved using NBS/Ph3P in LiBr15

DMA mixture. Additionally, LiBr-DMA mixture is useful for chemical modification [29] and also produces high degrees of uniform substitution [30]. In bromination reaction, yield was 95.4 % with 1.9 degree of substitution. Thiolated γ-CD appeared as odorless, white, fibrous structured oligomer that was soluble at physiological pH in aqueous media. Thiolation reaction yield was almost 90 %. The amount of immobilized thiol groups and disulfide bonds formed during the reaction were quantified by Ellman’s reagent as shown in Table 1. Results showed that oxidation of thiol groups to disulfides could not be completely excluded during the preparation process. The successful immobilization of thiol groups to γ-CD was further confirmed via FT-IR spectroscopy. FT-IR spectra of γ-CD-SH describe significant differences from unmodified γ-CD, as shown in supplementary Fig S1. The strong peak around 1140 cm1

and moderate peak at 613 cm-1 are interpreted as CS stretching vibrations in case of γ-

CD-SH [31]. 3.2. Synthesis and characterization of S-protected thiolated γ-CD Thiolated CDs (CD-SH) exhibit –SH groups that are less stable and can be easily oxidized at pH ≥ 5 unless sealed under inert conditions [14]. This too early oxidation of thiol groups before getting into contact with the mucus layer might deteriorate the interactions between thiolated CDs and mucus layer. Consequently, free –SH groups cannot participate in thiol/disulfide exchange reactions and this deteriorating factor reduces the efficiency of CDs-SH, mainly in body regions where pH is raised. To overcome this shortcoming, thiolated γ-CDs were S-protected to enhance stability of thiol groups and to subsequently improve their mucoadhesive properties [32].

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The formation of disulfide bonds between thiol groups of thiolated γ-CD and the aromatic ligand 2-mercaptonicotinic acid (2-MNA) was achieved as previously described by our research group [16]. The suitable molar ratio of thiol groups on the thiomer and aromatic ligand was found to be 1:2. The lyophilized γ-CD-SS-MNA was a water soluble, white and odorless powder of fibrous structure. Reaction yield of S-protected thiolated CD was almost 90 %. The degree of S-protection of thiol groups was quantified spectrophotometrically using a method previously described by our research group [18].

Chemical characterization of S-protected thiolated γ-CD showed high

amount of MNA conjugated to CD are shown in table 1. Comparison the 1H NMR spectrum of γ-CD-SS-MNA with those of the unmodified γCD and γ-CD-SH indicates that in γ-CD-SS-MNA there are additional signals in the aromatic area between 7.6 and 8.6 ppm, which are characteristic for the protons of the pyridine moiety of the 2-thionicotinic acid subunit (Supplementary Fig. S2). 3.2.1 Molecular weight determination of CD conjugates by LC-MS Liquid chromatography-mass spectrometry under optimized conditions with negative ESI mode provided a highly selective method for determination of molecular weight of γCD and its derivatives. Direct infusion through a syringe pump was used to achieve most suitable mass spectrometry conditions. The solutions were filtered through a 0.45 μm membrane filter prior to transferring into ESI-MS. All samples were operated in negative polarity mode. Methanol and water (85:15 v/v) was identified as most appropriate eluent. The doubly charged state of ions at m/z 647.0, 686.1 and 686.3 represented γ-CD, γ-CD-SH and γ-CD-SS-MNA, respectively. The spectrum of γ-CD

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and its derivatives showed base peak ions at mass-to-charge ratio (m/z) in range of 1.3 kDa as shown in supplementary Fig S3-S5. 3.3. Cytotoxicity The cell viability of both thiolated and S-protected γ-CD on Caco-2 cells was assessed by utilization of two methods: resazurin and LDH assays. Cells were treated with γ-CD-SH and γ-CD-SS-MNA at higher concentrations (1 %) as compare to 0.5 % concentration of cationic oligomers (β-CD) that were found toxic [12]. Results showed over 94 % cell viability for the concentration tested after 3 and 24 hrs of incubation (Fig. 2). The thiolated and S-protected γ-CD did not show significant toxicity (p < 0.05). The newly synthesized thiolated and S-protected γ-CD can be considered as relatively safe for in-vivo applications. Results of LDH assay are shown in Fig. 3. Again both thiolated and S-protected γ-CD showed no significant difference in comparison to the negative control after 3 and 24 hrs. These results are in good accordance with the resazurin results. 3.4. Enzymatic (α-amylase) degradation of γ-CD The degradation products of α-amylase and γ-CD reaction are reducing sugars mainly maltose. It has already been demonstrated that the rate-determining step in the degradation of γ-CD is the ring opening [28]. Hence, it can be expected that once the γCD ring is opened, the degradation reaction will lead to formation of primarily maltose and, to a much less extent, glucose. Full degradation of γ-CD will create 20 mM reducing products due to the formation of 4 mM maltose per mM γ-CD [13]. α-Amylase hydrolyzes γ-CD by a multiple attack mechanism [33-36]. According to this mechanism,

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an encounter between cyclodextrin and α-amylase results in a ring-opening reaction to produce smaller oligosaccharides with subsequent cleavage of the initial product. Results showed the same degradation kinetic for γ-CD, thiolated and S-protected γCD (Fig. 4). The results of the reactions are to a high extent compatible with previous results, which showed about 72 % degradation of γ-CD after 4 hrs at 37 °C using enzyme concentrations of 50 nM [23]. As quantitative hydrolyses of γ-cyclodextrin by αamylase is independent of the cyclodextrin concentration [23], higher concentration of enzyme used in this reaction resulted in degradation of γ-CD, thiolated and S-protected γ-CD just in less time. 3.5. Viscosity measurement There is a correlation between the increase in viscosity of the mucus and mucoadhesive properties during interaction with a mucoadhesive oligomer [37]. Thiolated and S-protected CD were examined for their mucoadhesive potential. Thiomers, with free thiol moieties, form covalent bonds through oxidative coupling of thiol moieties and/or by thiol-disulfide exchange reaction of the thiol groups present in oligomer and cysteine-rich subdomains of glycoproteins in the mucus layer [38]. Thiolated and S-protected γ-CD were found non-mucolytic in nature. Rheological investigations of γ-CD, thiolated and S-protected γ-CD /mucus mixtures were carried out on modified γ-CD and unmodified CD as control. In detail, the viscosity of unmodified CD/mucus mixture does not change significantly from the viscosity of mucus/buffer mixture, used as a control.

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Thiomers interact with mucin glycoproteins in many different ways with increase in viscosity of thiomer/mucin blends, as they interact with cysteine-rich subdomains found in mucin leading to the formation of new disulfide bonds [39]. CDs described in this study, however, exhibit only one thiol group and not many. Consequently, they could act like mucolytic agent such as N-acetyl-cysteine by breaking disulfide bonds via thiol/disulfide exchange reactions within the mucus. Unmodified CDs were not expected to be mucolytic. Thiolated γ-CD demonstrates a 1.5- fold higher mucus viscosity, whereas S-protected γCD prompted 1.6- fold higher mucus viscosity as compared to mucus-buffer mixture (Fig. 5). This minor difference in viscosity between thiolated and S-protected thiomers might be explained by free hydroxyl groups interacting with mucosal surface. Moreover, mucoadhesion can be influenced by several physical and chemical parameters for example electrostatic forces. S-protected CDs interact with mucin glycoproteins in many different ways. S-protected thiomers contain hydroxyl groups in their structure which can easily undergo a nucleophilic attack on the carboxyl group of aspartic acid and glutamic acid substructures of mucus [39]. Moreover, hydroxyl groups are able to form hydrogen bonds with other functional groups, therefore, interaction between the oligomer and gastric mucus is possible [18]. Results of thiolation showed that a ~1.8 hydroxyl groups per γ-CD molecule were thiolated whereas other OH- groups were found free. 3.6. Evaluation of mucoadhesive properties on freshly excised porcine intestinal mucosa

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The mucoadhesive properties of oligomers are subject to interactions between functional groups of the mucus gel layer and the mucoadhesive oligomer [40, 41]. Most important interactions responsible for mucoadhesive properties are ionic interactions and chain entanglements, but CDs lack such interactions. Thiolation is therefore perhaps the only way to enhance mucoadhesive properties of CDs. Non-covalent bonds also play a significant role in mucus-oligomer interaction, so anionic thiolated oligomers showed less pronounced adhesion time on mucosal surface due to lack of ionic interaction [42]. In contrast to cationic thiolated oligomers, anionic oligomers must overcome the electrostatic repulsion to attach to mucus layer [43]. Mucoadhesion study of thiolated and S-protected γ-CD on porcine intestinal mucosa confirmed that this type of thiomer shows strongly improved mucoadhesive properties. In detail, γ-CD-SH and γ-CD-SS-MNA exhibited 44.4- and 50.9- fold higher mucoadhesion in comparison to unmodified γ-CD, respectively. As shown in Fig. 6, more than 44 % of thiolated and more than 51 % S-protected γCD remained on the intestinal mucosa after 3 hrs. The unmodified γ-CD washed off the mucosa in a considerable short time period. These data demonstrate that γ-CD-SH and γ-CD-SS-MNA are able to adhere and remain for longer period of time on the intestinal mucosa. 4. Conclusion In this study novel thiolated S-protected γ-CDs with intact ring structure were developed. These novel conjugates were biodegradable, non-mucolytic and showed 60-fold improvement in mucoadhesion as compared to unmodified oligomer. Conjugates, were

21

furthermore non-toxic as compared to cationic ring-opened thiolated oligomers (α and βCD) [10, 44]. The ligands had no toxic effect on cells even in high concentration (1 %), while the first generation thiolated CDs showed a time and concentration dependent toxicity [12]. These favorable properties will likely to be useful for these novel Sprotected γ-CDs for localized drug delivery on mucosal surfaces. Acknowledgements This work was supported by the Higher Education Commission (HEC), Pakistan and the Austrian Agency for International Cooperation in Education and Research (OeAD), Austria.

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Fig. 1. Presumptive chemical substructure of γ-CD thiolation and S-protection: Bromination of γ-CD with NBS and Ph3P in LiBr-DMA and substitution of thiol groups using thiourea. 2-MNA dimer is covalently attached to thiolated γ-CD via disulfide bond formation.

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Fig. 2. Histogram shows cell viability of S-protected thiolated gamma cyclodextrin (γCD-SS-MNA) compared to thiolated gamma cyclodextrin (γ-CD-SH) and unmodified gamma cyclodextrin (γ-CD) after 3 and 24 hours. Viability assays were performed on Caco-2 cells using resazurin. As negative control MEM without phenol red was used and the positive control was Triton™ X-100 (4 % m/v). Indicated values represent an average of at least three experiments (±SD).

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Fig. 3. LDH test performed on Caco-2 cell line with γ-CD (1 %), γ-CD-SH (1 %) and γ-CD-SS-MNA (1 %) for 3 and 24 hrs. The results are expressed as % cytotoxicity of

cells (means ±SD; n = 3).

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Fig. 4. Degradation of γ-CD (●), γ-CD-SH (■) and γ-CD-SS-MNA (▲) by α-amylase in sodium phosphate buffer (pH 6.9) containing 6.7 mM NaCl at 20 °C. The results are given as means ± standard deviation (n=3).

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Fig. 5. Dynamic viscosity (Pas) measurement of γ-CD, γ-CD-SH & γ-CD-SSMNA with mucus at a frequency of 1 Hz on a plate-plate viscometer with test samples (0.5 %, w/v) in 200 mM phosphate buffer at pH 7 mixed with porcine intestinal mucus in a ratio of 1:4 at 37 °C. Results are expressed as means ±SD (n = 3). * differs from unmodified CD, p-value < 0.05.

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Fig. 6. Time course of percentage of remaining unmodified (●), thiolated (■) and Sprotected (▲) γ-CD on porcine intestinal mucosa continuously rinsed with 100 mM phosphate buffer pH 6.8 at 37 °C and 100 % relative humidity. Indicated values are means ± SD of three experiments. (**P <0.01, ***P <0.001)

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Table 1. Amount of thiols (-SH), disulfide groups (-S-S-) and MNA on γ-CD-SH and γ-CD-SS-MNA. Indicated values are means ± standard deviation of three experiments.

Conjugates γ-CD-SH γ-CD-SS-MNA

-SH (µmol/gram) 1385±84 20±3

-S-S- (µmol/gram) 180±48 1124±74

Graphical abstract

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MNA (µmol/gram) ---1153±41