Chitosan: The One and Only? Aminated Cellulose as an Innovative

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Chitosan, the one and only? Aminated cellulose as innovative option for primary amino groups containing polymers Max Jelkmann, Claudia Menzel, Randi Angela Baus, Philipp Ausserhofer, Daniel Baecker, Ronald Gust, and Andreas Bernkop-Schnürch Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01069 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Biomacromolecules

Chitosan, the one and only? Aminated cellulose as innovative option for primary amino groups containing polymers Max Jelkmann1, Claudia Menzel1, Randi A. Baus1, Phillip Ausserhofer1, Daniel Baecker2, Ronald Gust2 and Andreas Bernkop-Schnürch1* 1

Center for Chemistry and Biomedicine,

Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria 2

Center for Chemistry and Biomedicine,

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

Keywords: cellulose derivatives, cationic cellulose, aminocellulose, mucoadhesion, chitosan

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Abstract

The aim of this study was the synthesis and in vitro characterization of aminated cellulose as alternative excipient to chitosan. The aldehyde form of cellulose was generated via oxidative cleavage of vicinal diols by addition of increasing concentrations of sodium periodate. The insertion of primary amines was achieved by reductive amination with ammonia. The degree of substitution

was

calculated

via

primary

amino

group

quantification

using

2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. Mucoadhesiveness was examined adopting the rotating cylinder method and tensile studies using porcine intestinal mucosa. Hydration was evaluated at pH 2-11. Successful formation of aldehydes as well as subsequent introduction of up to 311.61 µmol/g primary amines were proven to correlate with the amount of added periodate. There was a 3- to 14-fold prolongation in the mucosal residence time of the new polymer in comparison to chitosan, as measured by the rotating cylinder method. Although cationic cellulose did not reach the maximum detachment force of chitosan, the total work of adhesion of newly synthesized cellulose derivate was higher than that of chitosan. The higher the degree of amination, the higher was the degree of hydration in neutral and alkaline aqueous media. Compared to chitosan the novel cationic cellulose derivative displays improved mucoadhesive properties as well as sufficient hydration at physiological pH. Therefore, aminated cellulose is a promising alternative to so far used cationic polymers such as chitosan.

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Biomacromolecules

Introduction Chitosan is used in a broad array of industries such as cosmetic, food, water treatment, agricultural, paper, textile and pharmaceutical industries. Within the pharmaceutical arena, it is primarily used as multifunctional excipient. During the last decades, a huge number of drug delivery systems based on this polysaccharide has been published. Chitosan presents several properties that are beneficial for mucosal drug delivery1. Besides release controlling, in situ gelling, transfection improving, permeation enhancing, and efflux pump inhibitory properties, the probably most important feature is the mucoadhesive potential of chitosan facilitating a prolonged time of adhesion on mucosal tissues2. The mucoadhesiveness is based on electrostatic interactions between positively charged amino groups and anionic mucus glycoproteins leading to an intimate contact between the polymer and the mucosal surface. At a pH above 6, however, the mucoadhesive properties are rather weak compared to those of other polymeric excipients as chitosan precipitates. Looking for alternatives in form of positively charged mucoadhesive polymers, only synthetic polymers as for example polyallylamine or polyethylenimine are available that show severe toxicity. Due to this, it is vitally important to investigate new derivatives of biocompatible and non-toxic polymers overcoming these shortcomings. As chitosan and cellulose offers structural similarity, cellulose is highly suitable as a promising alternative after modification to a cationic derivative. Although certain cationic cellulose derivatives such as aminated cellulose obtained via nucleophile displacement have been described in the literature3, none of these excipients has been investigated in the context of mucoadhesion so far. It was therefore the aim of the present study to evaluate the mucoadhesive properties of aminated cellulose and its potential as alternative to chitosan precipitating under most 3 ACS Paragon Plus Environment


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physiological pH conditions. In order to provide high flexibility of polymer chains being likely favorable for the penetration into the mucus and for the hydratability of the polymer leading to high mucoadhesive properties, a novel synthetic pathway resulting in a partial ring opening of glucose units as outlined in Figure 1 was chosen. Aminated cellulose was obtained via oxidative cleavage of 1,2-diols and subsequent reductive amination with ammonia. The resulting aminocellulose was quantitatively examined for its degree of amination. Furthermore, its effects on hydration and zeta potential as well as its mucoadhesive properties and cytotoxicity were investigated.

Figure 1. Pathway for synthesis of cationic cellulose derivative. Ring structure of polymer is oxidized with sodium periodate and subsequently aminated with ammonia in ethanol as solvent.


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Biomacromolecules

Materials and Methods Materials All chemicals were of analytical grade and obtained from commercial sources. Microcrystalline cellulose (vivapur® MCC 101) was purchased from J. Rettenmaier & Söhne GmbH. Chitosan (high molecular weight, degree of deacetylation > 75%), sodium periodate (NaIO4), ethylene glycol, sodium cyanoborohydride (NaCNBH3), ammonium hydroxide solution (37% NH3), ethanol and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were obtained from Sigma–Aldrich (Vienna, Austria). Regenerated cellulose dialysis tubes (molecular weight cut-off of 3.5 kDa) and Nadir-cellulosehydrate membrane tubings with a molecular mass cut-off of 10-20 kDa were obtained from Carl Roth GmbH & Co (Karlsruhe, Germany). Methods Synthesis of aminated cellulose via reductive amination Oxidative cleavage of cellulose Sodium periodate was used as oxidizing agent to achieve ring opening cleavage of cyclic cellulose. Periodate is known for its very selective and highly efficient cleaving at the C2 and C3 positions of 1,2-diols to an aldehyde form. One gram of cellulose was soaked in 130 mL of demineralized water containing 0.2 M LiCl and 0.2 M MgCl2 to improve solubility by disruption of intermolecular hydrogen bonds thereby promoting the efficiency of sodium periodate4. The effect of sodium periodate content on the level of cleaved diol groups and subsequent amination of the polymer was investigated by addition of increasing quantities (0.2 g, 0.5 g and 0.8 g), dissolved in 20 mL of demineralized water. After 6 h of stirring at 55 °C, pH 7 and light protection, 400 µL of ethylene glycol was added5. To neutralize any remaining excess of NaIO4, the mixture was stirred at room temperature for another hour. Purification of oxidatively cleaved 5 ACS Paragon Plus Environment


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cellulose polymer occurred via filtration using a Büchner funnel and washing with demineralized water. Thereafter, the washed product was dialyzed (Nadir-dialysis tube; molecular weight cutoff of 10-20 kDa; (Roth, Karlsruhe, Germany)) against water for at least seven days, whereby the dialysis medium was changed at least twice daily. To ensure absence of impurities the absorbance of medium was measured at 225 nm. The purified product was freeze dried under reduced pressure (−51 °C, 0.01 mbar, Gamma LSC 1-16, Martin Christ, Osterode, Germany) and stored at 4 °C protected from oxygen until further use. Before the reductive amination, the oxidized polymer was examined via Fehling’s solution to confirm successful ring opening. Amination of the aldehyde groups containing polymer Amination of the oxidatively cleaved polymer was performed via reductive amination. First, 1 g of oxidatively cleaved cellulose was added to 50 mL of ammonium hydroxide solution (37% NH3) and stirred at 80 °C under hermetic and light protected conditions with a reflux cooling to prevent evaporation of ammonia. After three hours of stirring, NaCNBH3 in a weight ratio of 1:1 (NaCNBH3 : polymer) was dissolved in ethanol and added to the reaction mixture. The mixture was stirred at 100 °C for 18 h under hermetic and light protected conditions with a reflux cooling. To purify the aminated cellulose from ammonia, ethanol, and water, the solution was dried in a rotavapor. Then, the product was re-dissolved in water and exhaustively dialyzed against demineralized water for at least three days under light-protection. The medium was changed three times a day and controlled for impurities by absorbance measurement. The purified product was lyophilized under reduced pressure and stored at 4 °C in airtight containers. Characterization of aminated polymer Successful oxidation was examined via Schiff test to quantify the amount of free aldehyde groups after oxidative cleavage of 1,2-diols. Free primary amino groups of the modified polymer 6 ACS Paragon Plus Environment

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were quantified photometrically with 2,4,6-trinitrobenzenesulfonic acid (TNBS-test)6. Molecular weights of native cellulose (NC), AC1, AC2 and AC3 were determined via viscosity measurements according to McCormick et al.7. Native cellulose and aminated cellulose were dissolved in N,N-dimethylacetamide containing 9% (w/w) lithium chloride by heating to 150 °C and subsequent slowly cooling down to room temperature. Fourier transform infrared (FT-IR) spectroscopy analysis The effective amination of cellulose was monitored by infrared spectroscopy employing an Alpha FT-IR Spectrometer (Bruker, Billerica, USA) in arrangement with its associated software Opus Version 7.0 (Bruker). Therefore, the intermediate products obtained by different degrees of oxidation of cellulose as well as the final products (AC1/AC2/AC3) were assessed. The FT-IR spectra of the compounds were recorded with 32 scans in a wavenumber range covering 4000 cm-1 to 400 cm-1 and exerting a resolution of 1 cm-1. The measurements of the samples were conducted at 22 °C. Determination of zeta potential Zeta potential differences of all polymers were investigated to examine the impact of induced cationic groups. To analyze the influence of temperature on zeta potential, samples were measured at room temperature and at 37 °C. Furthermore, the stability of the zeta potential was determined after 72 h of incubation under continuous shaking of samples. Concerning the impact of salt concentration, especially halogen containing salts, two halogen free buffer systems were used. Solutions of pH 3 and pH 4 were prepared as 0.1 M citric acid -Na2HPO4 buffers and in the pH range between 5 and 8 as phosphate buffer solutions as combination of potassium dihydrogen phosphate and sodium phosphate dibasic dihydrate using Sørensen method. There was no necessity of pH adjustment with HCl for solutions of all pH values. Laser Doppler Micro7 ACS Paragon Plus Environment


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electrophoresis was applied to determine the zeta potential via Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK) in conjunction with disposable folded capillary cells (DTS1070). Zeta potential was calculated as mean electrophoretic mobility via Smoluchowski’s equation of at least three samples per polymer. Hydration studies Hydratability was determined by filtration as described previously8, 9. In detail, 0.1% (m/v) samples of chitosan, native cellulose and aminated cellulose were prepared in a citric acid buffer solution (0.1 M) pH 2, 0.1 M phosphate buffer pH 6.8 and a buffer solution pH 11 (0.1 M glycine, 0.12 M NaCl, 0.1 M NaOH). After shaking at 900 rpm for 30 min, samples were filtrated to retain non-hydrated polymer. The amount of non-hydrated polymer was determined gravimetrically, after drying the supernatant at 50 °C until constant weight. An additional investigation was performed to confirm data of this filtration method by means of UV/VIS spectroscopy10. More precisely, samples of chitosan, native and modified cellulose were prepared in the same way as for the filtration method. Immediately after preparation, samples of 1 mg/mL were analyzed with repeated measurements until there were no further changes of AUC due to precipitation and sedimentation of insufficiently hydrated polymer. Wavelength range was set between 200 nm and 500 nm on a UV-Vis spectrophotometer UVmini-1240 (Shimadzu. Kyōto, Japan) and recorded with UV Probe 2.33 software. The percentage of hydrated polymer was calculated setting the terminal AUC in relation to the origin AUC. Preparation of test discs A single punch excentric press (Paul Weber, Remshalden-Grünbach, Germany) was used to compress chitosan, native and modified cellulose into 30 mg test discs at a constant compaction pressure of 11 kN. Test discs displayed 2.0 mm thickness and a diameter of 5.0 mm. 8 ACS Paragon Plus Environment

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Examination of in vitro mucoadhesion via the rotating cylinder In vitro mucoadhesion was investigated by a modified method of the rotating cylinder according to a method described previously11, whereby the retention time on porcine intestinal mucosa was determined for chitosan as well as native and modified cellulose. After fixation of the mucosa on a stainless-steel cylinder (diameter, 4.4 cm; height, 5.1 cm; apparatus fourcylinder, USP XXIII) test discs of chitosan, native and aminated cellulose were applied. The rotating cylinder was immersed into 900 mL of 0.1 M phosphate buffer, pH 6.8 at 37 °C and agitated at a circumferential speed of 100 rpm using a dissolution apparatus DT 700 (Erweka GmbH, Heusenstamm, Germany). Time point of detachment of test discs was recorded visually. Tensile studies Determination of the total work of adhesion (TWA) and the maximum detachment force (MDF) was carried out to investigate mucoadhesive properties, according to a previously established method12. In more detail, freshly excised native porcine intestinal mucosa was glued on the bottom of a beaker that was subsequently carefully filled with phosphate buffer (0.1 M; pH 6.8; 37 °C) and placed on a balance. Test discs of chitosan, native and aminated cellulose were fixed on a stainless steel flat cylinder (8 mm in diameter, 0.3 g in weight) by means of a cyanoacrylate adhesive and hung on a string over a laboratory rack. The fixed tablet was placed over the mucosa, the height-adjustable laboratory stand with the balance was lifted until contact of mucosa and tablet was provided. After 20 min incubation, the platform with the balance was continuously lowered with a velocity of 1.0 mm/s. One data point each second was recorded by a balance-linked personal computer (Sarta Collect software; Sartorius AG, Austria) to calculate MDF and TWA.



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Biodegradability Enzymatic biodegradability was studied by tartrate/3,5-dinitrosalicylic acid assay. In detail, the color reagent solution was freshly prepared by slowly adding 8 mL of sodium potassium tartrate solution (5.3 M in NaOH, 60 °C) to 12 mL of water (60 °C) and subsequent slow addition of 20 mL of 3,5-dinitrosalicylic acid solution (96 mM, 60 °C). Modified polymers and native cellulose as positive control were dissolved in a final concentration of 1% (m/v). Enzymatic degradation was induced with cellulase, from Aspergillus Niger, in a final concentration of 5 units/mL. One mL of sample (polymer and positive control) was mixed with 1 mL of enzyme for exactly 30 min at 37 °C. Samples without enzyme served as negative control. After incubation, the negative controls were complemented with the lacking amount of enzyme solution and color reagent was added to all samples in a final volume ratio of 1:2. The samples were immediately heated to 100 °C for exactly 15 min. Afterwards, samples were kept on ice for 3 min. Then, 9 mL of water was added and the samples were measured photometrically at 540 nm. In vitro cytotoxicity studies Cell viability was evaluated utilizing two different cell lines, namely human embryonic kidney cells 293 (HEK293) and human colon cancer Caco-2 cells. Cell lines were cultured in 24-well plates with 0.5 mL of MEM (pH 7.4) containing phenol red, Earle’s balanced salts supplemented with 10% fetal bovine serum, 2.0 mM L-glutamine, and 1% penicillin-streptomycin at 37 °C in a 5% CO2 environment with periodic media change until a confluence of 80% was reached. Chosen method for cytotoxicity studies was a resazurin assay that detects active metabolism of cells. It is based on reduction of the non-fluorescent (blue) resazurin to fluorescent (red) resorufin whereby 10 ACS Paragon Plus Environment

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the extent of this reaction is correlating to the number of viable cells13. Preheated (37 °C) phosphate buffered saline (PBS) was used to wash cells twice before either 500 µL of 0.5% (m/v) polymeric solutions in MEM, negative control (MEM) and positive control (1% (v/v) Triton® X-100 in MEM) were added. Cell medium was removed after 24 h of incubation at 37 °C, 5% CO2 in air and 95% relative humidity. Cells were washed twice with PBS. 250 µL of a resazurin solution was added per well and cells were incubated for further 2 h at 37 °C, 5% CO2 in air and 95% relative humidity. Afterwards each well was measured in duplicate with a Tecan infinite M200 spectrophotometer (Grödig, Austria) at a wavelength of 540 nm with background subtraction at 590 nm. Percentages of viable cells were calculated in comparison to a negative control (100% viability) and a positive control (0% viability), respectively14. Statistical data analysis Data were expressed as the mean ± standard deviation (SD) and analyzed using one way ANOVA and a Bonferroni post-test with p

Chitosan: The One and Only? Aminated Cellulose as an Innovative

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