Cationic starch derivatives as mucoadhesive and soluble excipients in drug delivery

International Journal of Pharmaceutics 570 (2019) 118664

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Cationic starch derivatives as mucoadhesive and soluble excipients in drug delivery Max Jelkmann, Christina Leichner, Claudia Menzel, Verena Kreb, Andreas Bernkop-Schnürch

T

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Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria

A R T I C LE I N FO

A B S T R A C T

Keywords: Starch Amination Cationic starch Polymer Mucoadhesion Drug delivery

The aim of this study was to develop a novel mucoadhesive cationic polymer by introducing primary amino groups to the polymeric backbone of starch. This newly synthesized polymer should exhibit superior properties over chitosan regarding solubility, mucoadhesiveness and cytotoxicity. Increasing amounts of sodium periodate were used to cleave and oxidize vicinal diols under aldehyde formation obtaining three different degrees of modification. In a subsequent step, primary amines were introduced via reductive amination with ammonia. Degree of amination was examined with TNBS-assay and zeta potential measurements. Mucoadhesiveness was investigated by rotating cylinder, tensile studies and rheological measurements. Primary amino groups were successfully attached to the polymer, proven by zeta potential measurements and UV-spectroscopy. Depending on the amount of periodate used in the reaction, coupling rates of up to 514 µmol/g polymer were achieved. All synthesized derivatives showed 100% solubility in a pH range of 1–9. Aminated starch with the highest coupling rate of 514 µmol/g showed a 9.5-fold prolonged retention time on intestinal mucosa and a 2.7-fold higher total work of adhesion on the mucosal tissue compared to chitosan. Furthermore, cytotoxic examinations of all tested polymers showed only a low impact on cell viability after 24 h, whereby starch derivatives possessed even less cell toxic effects than chitosan. Summarizing these results, cationic starch derivatives seem to be promising excipients for mucosal drug delivery with superior properties compared to chitosan, the most examined cationic polymer.

1. Introduction Within the past decades, a massive amount of research on chitosan based drug delivery systems has been published. The reason for this is that chitosan is the only positively charged polysaccharide being monographed in the pharmacopeia because of its beneficial features for mucosal drug delivery, that are all based on its cationic character originating from free amino groups on the polymeric backbone. Besides controlled drug release, in situ gelation, transfection, permeation enhancement, and efflux pump inhibitory properties, the mucoadhesive potential of chitosan might be the most important reason for its great popularity (Bernkop-Schnürch and Dünnhaupt, 2012). The mucoadhesive properties of chitosan are primarily based on electrostatic interactions between positively charged amino groups and anionically charged substructures of mucus glycoproteins such as sialic and sulfonic acid substructures. Due to such mucoadhesive properties, a prolonged retention time on mucosal tissues can be achieved while facilitating improved uptake of drugs (Cohen et al., 2012). In vivo and in vitro

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correlation has been recently shown for buccal application (Baus et al., 2019). Even for gastrointestinal applications, an improved drug bioavailability due to mucoadhesion was shown in humans (Akiyama et al., 1998; Zaghloul et al., 2007). However, compared to other mucoadhesive excipients, chitosan shows only weak mucoadhesive properties (Bernkop-Schnürch and Dünnhaupt, 2012). Moreover, due to its high density of positive charges, chitosan bears the risk of cell membrane disruption by interfering with negative charges of the cellular bilayer (Palma-Guerrero et al., 2009). In search of alternatives to chitosan, one promising approach was the development of aminated cellulose (Jelkmann et al., 2018). Amino groups were introduced to the polymeric backbone via oxidative ring opening followed by reductive amination resulting in improved mucoadhesion of this polysaccharide. However, aminated cellulose has some drawbacks: First, just like chitosan it is hardly soluble in aqueous media, with 80% (m/m) insoluble parts, complicating its use in drug delivery systems. Furthermore, cellulose contains inclusions of β-d-glucans (Liu et al., 2017). Although occurring in different forms inducing different biologic responses, β-d-

Corresponding author. E-mail address: [email protected] (A. Bernkop-Schnürch).

https://doi.org/10.1016/j.ijpharm.2019.118664 Received 24 July 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 Available online 09 September 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Pathway for synthesis of cationic starch derivatives. Initially ring structure was oxidized using sodium periodate and in the following starch was aminated with ammonia.

starch (SS) (Mw = 310–550 kDa) and high molecular weight chitosan (310–375 kDa, > 75% deacetylated) were purchased from Sigma–Aldrich (Vienna, Austria). Regenerated cellulose dialysis tubes (molecular weight cut-off of 3.5 kDa) and Nadir-cellulose hydrate membrane tubing (molecular mass cut-off of 10–20 kDa) were obtained from Carl Roth GmbH & Co (Karlsruhe, Germany). All chemicals were of analytical grade.

glucans are generally considered pro-inflammatory causing strong immune responses (Gefroh et al., 2013; Liu et al., 2017). It was therefore the aim of the study to develop a biodegradable, mucoadhesive excipient representing an alternative for chitosan with improved mucoadhesive properties and at the same time without the shortcomings of aminated cellulose. On purpose, starch was chosen as polysaccharide backbone and amino groups were introduced by ring opening and reductive amination as outlined in Fig. 1. This resulted in a novel polymeric excipient since all former studies on aminated starch introduced cationic groups like secondary or mainly tertiary amines for other applications like paper industry (Wang and Xie, 2010) or wastewater treatment (Pal et al., 2005). In regards to pharmaceutical use, cationic starches were widely investigated as potential agent in gene delivery (Engelberth et al., 2015; Engelberth et al., 2017; Nagasaki et al., 2004; Rekha and Sharma, 2009; Thiele et al., 2017; Yamada et al., 2014) and its potential in controlled drug release (Azagury et al., 2014; Mulhbacher et al., 2001). However, cationic starch was so far scarcely evaluated as mucoadhesive excipient. Merely, the commercially available dextran derivative DEAE bearing a tertiary amine was investigated for its potential use as mucoadhesive excipient (Miyazaki et al., 2003). In the present study, a new synthesis pathway to generate an aminated starch containing primary amino groups was realized and the novel polymer was investigated as a mucoadhesive excipient and alternative to chitosan.

2.2. Methods 2.2.1. Synthesis of cationic starch derivatives by reductive amination 2.2.1.1. Oxidative cleavage of polysaccharide ring structure. In order to identify the most suitable kind of starch, starches of different origin were tested, that are all listed in Table 1. NaIO4 was used to accomplish ring-opening of polysaccharide structure by oxidative cleavage. In detail, 2 g of starch was dispersed in 200 ml of demineralized water and 20 ml of a NaIO4 solution were added. To achieve different extent of oxidation of starch increasing concentrations (94 mM, 234 mM and 374 mM) of NaIO4 were applied. Periodate concentrations were kept equal to those applied in recently published research modifying cellulose for the same purpose to facilitate comparisons between the modified derivatives (Jelkmann et al., 2018). The mixtures were stirred for 2 h at room temperature under controlled pH 7 and light protection. To stop the oxidative reaction 500 µl of ethylene glycol were admixed and stirred for one further hour at room temperature to neutralize any remaining surplus of NaIO4 (Sirvio et al., 2011). To purify the polymer the product was dialyzed in regenerated cellulose dialysis tubes (molecular weight cut-off of 3.5 kDa) against water. Dialysis was performed with frequently, of at least two times per day, changing of dialysis medium until no signal at 225 nm via UV–Vis analysis was detectable. The purified product was freeze dried under reduced pressure (−51° C, 0.01 mbar, Gamma LSC 1–16, Martin Christ, Germany) and stored at 4 °C under hermetic conditions till further use. Prior further use for synthesis, Fehling’s solution was utilized as a qualitative test to confirm successful ring-opening of the polysaccharide. In detail, equal volumes of a 0.28 M copper(II) sulfate

2. Materials and methods 2.1. Materials Corn (CS) (amylose content = 25–29%; gelation temperature = 75 °C), potato (PoS) (amylose content = 20–22%; gelation temperature = 65 °C) and pea (PeaS) (amylose content = 35%; gelation temperature = 69 °C) starch were kindly gifted by Roquette (Lestrem, France). Sodium periodate (NaIO4), ethylene glycol, sodium cyanoborohydride (NaCNBH3), ammonium hydroxide solution (32% NH3), ethanol, 2,4,6-trinitrobenzenesulfonic acid (TNBS), soluble 2

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– 43.38 59.67 51.77

0 0.06 0.13 0.21

solution and a solution containing 1.2 M potassium sodium tartrate and 2.5 M sodium hydroxide were added to 30 mg of the polymer and subsequently heated to 100 °C. A brown precipitate indicated the formation of aldehydes and confirmed the ring opening. Quantification of free aldehyde groups was performed adopting Schiff test to all products. In brief, samples were dissolved in water and mixed with three parts of Schiff reagent to a final polymer concentration of 0.125% (m/v). After one hour of incubation at 40 °C samples were measured as duplicate at 570 nm using a Tecan infinite M200 spectrophotometer (Grödig, Austria) and aldehyde content was computed using a formaldehyde calibration curve. 2.2.1.2. Amination of the polymer. Initially, 1 g of oxidatively cleaved polymer was added to 25 ml of ammonium hydroxide solution (32% NH3) and stirred at 50° C under light protected and hermetic conditions with a reflux cooling to retain evaporating ammonia for three hours. Next, NaCNBH3 in a weight ratio of 1:1 (NaCNBH3:polymer) was dissolved in 50 ml of ethanol, added to the reaction mixture and stirred at 50 °C while keeping the same reaction conditions. After three days of reaction, the resulting aminated starch was purified. Therefore, the remains of ammonia and ethanol of reaction mixture were vaporized with a rotavapor until dryness and the dry product was resolved in 50 ml of demineralized water. To remove remaining impurities the polymer was exhaustively dialyzed against demineralized water for at least seven days under light-protection. Medium was changed three times a day until UV–Vis spectral analysis (190 nm to 800 nm) did not exhibit remaining impurities in dialysis medium anymore. The purified product was lyophilized under reduced pressure and stored at 4 °C in air-tight containers. 2.2.2. Characterization of aminated polymer Free primary amino groups of modified polymer were photometrically quantified by 2,4,6-trinitrobenzenesulfonic acid (TNBS-test) (Snyder and Sobocinski, 1975). In detail, the polymer was dissolved in a 0.5% (m/v) NaCl solution to a final concentration of 0.2% (m/v). Thereafter, TNBS was dissolved in 8% NaHCO3 (m/v) to a final concentration of 0.1% (m/v) and added in a weight ratio of 1:1. The mixture was incubated at 37 °C for 90 min, subsequently centrifuged and measured as duplicate at 450 nm using a Tecan infinite M200 spectrophotometer (Grödig, Austria). Calculation of primary amines was conducted using a calibration curve of concentrations ranging from 0.013 µM to 0.413 µM. Within this work, the extent of modification is described by the degree of substitution (D.S.) displaying the average number of cationic substituents (NH2) per anhydroglucose unit. Taking into account that a D.S. equal 1 states one cationic group per anhydroglucose unit on average, the theoretically highest possible D.S. is 2 since the ringopening cleavage offers two aldehydes per monomer for further amination. The D.S. is calculated using the equation below:

0 4.25 10.6 17

D. S. =

starch starch starch starch soluble soluble soluble soluble SS ASS1 ASS2 ASS3

162∙W 100∙14 − (W ∙16)

162: molar mass of anhydroglucose; W: percentage (w/w) of Nitrogen; 14: molar mass of Nitrogen; 16: molar mass of NH2 (cationic substituent group). pH dependent solubility of unmodified and modified starch in comparison to chitosan was investigated as previously described (Hauptstein et al., 2013). All polymers were set up as 0.5% (m/v) mixtures of polymer in buffer solutions of different pH values. A 0.2 M citric acid – sodium citrate buffer system was used for examinations of pH from 3 till 6 and a 0.1 M Trizma buffer was used to investigate in neutral to slightly basic pH range of 7–9. Additionally, chitosan, native and aminated starch were dissolved in water 0.5% (m/v) and pH was adjusted to 3 using 5 M HCl. Afterwards, 0.1 M NaOH was added slowly to the test solutions to increase the pH in order to determine the pH value of beginning precipitation.

0.43 ± 0.12

– 71 ± 7 51 ± 6 34 ± 10

not detectable 344.67 ± 121.73 529.99 ± 63.15 996.93 ± 108.37

not detectable 149.50 ± 17.50 316.26 ± 14.60 516.14 ± 100.18

0 0.05 0.12 0.20 – 40.15 65.94 59.83 0 4.25 10.6 17 starch starch starch starch Corn Corn Corn Corn CS ACS1 ACS2 ACS3

3.16 ± 0.936

– 81 ± 3 75 ± 5 57 ± 6

not detectable 299.96 ± 48.98 448.25 ± 46.32 858.31 ± 138.42

not detectable 120.45 ± 5.04 295.57 ± 58.95 513.50 ± 69.15

0 0.04 0.09 0.22 – 42.71 53.81 56.33 0 4.25 10.6 17 starch starch starch starch Pea Pea Pea Pea PeaS APeaS1 APeaS2 APeaS3

5.95 ± 1.94

– 77 ± 8 64 ± 4 43 ± 14

not detectable 220.04 ± 31.84 440.85 ± 51.83 990.97 ± 177.82

not detectable 93.98 ± 15.00 237.23 ± 51.35 558.17 ± 75.49

0 0.06 0.11 0.19 – 47.71 36.48 46.26 not detectable 147.71 ± 8.27 275.95 ± 40.06 475.50 ± 121.20 0 4.25 10.6 17 starch starch starch starch Potato Potato Potato Potato PoS APoS1 APoS2 ApoS3

7.1 ± 1.92

– 56 ± 7 41 ± 9 29 ± 11

not detectable 309.63 ± 42.58 756.47 ± 111.56 1027.93 ± 115.78

% of implemented aldehydes Amount of amino groups [µmol/g] Amount of aldehyde groups [µmol/g] Amount of periodate [mM/g] Yield of syntheses [%] MW [106] Original polymer Polymer

Table 1 Characterization of modified starch. Values indicate means ± standard deviation of at least three experiments.

Degree of substitution

M. Jelkmann, et al.

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thickness and a diameter of 5.0 mm.

2.2.3. Determination of molecular weights Estimation of the molecular weights was performed using an established static light scattering method (Zhong et al., 2006). In detail, samples were stirred in DMSO containing 50 mM LiBr until solution got clear. A minimum of five different concentrations in a range between 0.001 and 0.02 mg/ml were analyzed using a Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK). The required sample concentration range and the molecular weight as the result of subsequent measurements was calculated by the zetasizer software adopting a (dn/ dc) refractive index increment of 0.066 and a solvent refractive index of 1.479.

2.2.7. Examination of in vitro mucoadhesion via the rotating cylinder Mucoadhesion was evaluated in vitro via the rotating cylinder method according to a method described previously (Bernkop-Schnürch and Steininger, 2000) in a modified procedure. Retention time of polymers on freshly excised porcine mucosa provide insight into mucoadhesive properties of the tested polymers. Besides test discs of native, aminated starch and chitosan were attached to mucosal tissue that was previously fixed on a stainless steel cylinder (diameter, 4.4 cm; height, 5.1 cm; apparatus four-cylinder, USP XXIII). A dissolution test apparatus (confirming to the USP) filled up with 100 mM phosphate buffer pH 6.8 at 37° C and a circumferential speed of 50 rpm was used to constitute the rotating cylinder. The duration until detachments of test discs was documented.

2.2.4. Determination of zeta potential To appraise the impact of induced primary amino groups on the cationic character of the synthesized polymer all polymers were examined regarding their surface zeta potential. Next to chitosan, all samples of native and aminated starch were measured at room temperature in a concentration of 1 mg/ml. Halogen free buffer solutions were used to exclude the high influence of salt concentration, notably of halogen containing salts. Buffer solutions in the pH range between 3 and 7 were prepared using a citric acid – phosphate buffer system. Laser Doppler Micro-electrophoresis was used to determine surface zeta potential via Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK) in conjunction with disposable folded capillary cells (DTS1070). Mean zeta potential was computed as mean electrophoretic mobility via Smoluchowski’s equation of at least three samples per polymer.

2.2.8. Tensile studies The maximum detachment force (MDF) and the total work adhesion (TWA) was assessed to survey mucoadhesive properties and was estimated by trapezoidal rule. A previously established protocol was followed (Dünnhaupt et al., 2012). For this purpose, freshly excised native porcine intestinal mucosa was fixed on the base of a heavy beaker and placed on a balance positioned on a height-adjustable laboratory stand. The beaker was subsequently and carefully filled with phosphate buffer (100 mM; pH 6.8; 37° C) to avoid flush down of any mucus. The test discs of unmodified and modified starch as well as chitosan were fixed on a stainless steel flat cylinder (8 mm in diameter, 0.3 g in weight) with a cyanoacrylate adhesive. The cylinder was hanged on a string over a laboratory rack and the fixed test disc was installed above the mucosal tissue. The height-adjustable laboratory stand with the balance was elevated to a light touch of test discs and mucosa. After incubation time about 15 min the balance was continuously lowered with a constant velocity of 1.0 mm/s. A balance-linked personal computer collected one data point each second (Sarta Collect software; Sartorius AG, Austria) enabling to compute MDF and TWA.

2.2.5. Rheological investigations Oscillatory shear experiments of polymers were accomplished on a plate-plate combination viscometer under temperature control (Haake MARS Rheometer, 379-0200, Thermo Electron GmbH, Karlsruhe, Germany; Rotor: PP35 Ti, d = 35 mm). To determine the linear viscoelastic range preliminary strain sweep evaluations have been conducted at a constant frequency of 1 Hz and defined gap of 0.5 mm. On basis of this linear range, oscillatory measurements were executed at a shear rate of 0.01–50 Pa at 37 °C. Prior to rheological examinations, mucus was collected from freshly obtained porcine small intestine and purified following an established method (Wilcox et al., 2015). In detail, intestine was sliced and mucus was carefully scrapped of the mucosa and subsequently mixed with sodium chloride solution (0.1 M) in a ratio of 5 ml per gram mucus. Mixture was stirred for one hour prior to centrifugation for 2 h at 10.400 g. The supernatant was wasted as well as the coarse impurities located at the bottom. These purification steps were once repeated. During the whole purification procedure, temperature was kept at 4 °C. Polymers were dissolved in phosphate buffer (0.1 M, pH 6.8) in a concentration of 1% (m/v) and incubated for 30 min at 37° C prior to their usage. Application of the rheological synergism method was used to investigate mucoadhesive properties of the native, oxidized and aminated polymer. In detail, polymer solutions were mixed with freshly extracted and purified porcine intestinal mucus in a volume ratio of 1:1. The rheological measurements of mucus/polymer mixtures were performed immediately after sample preparation and after a pre-incubation of samples for 15 min at 37 °C. Each analysis was performed in triplicate and the mean increase on viscosity was calculated. To normalize the initial viscosity and to exclude any polymeric effects, viscosities of mucus, polymer solutions and the initial polymer/mucus mixtures were evaluated and offset against the final viscosity. This allows comparison of viscosity increase without imperfections due to initial polymer/mucus viscosities.

2.2.9. In vitro cytotoxicity studies Human colon cancer Caco-2 cells were used to investigate cytotoxical characteristics of native and aminated starch, chitosan and polyethylenimine. Cells were cultured in 48-well plates with 0.25 ml of minimum essential medium (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 until a confluence of 80% with periodic media change. Resazurin assay is detecting active metabolism of cells and was method of choice for cytotoxicity studies. Operating principle of this assay is based on detection of the fluorescent (red) resorufin that arise out of reduction of the non-fluorescent (blue) resazurin correlating to the number of viable cells (Vega-Avila and Pugsley, 2011). Cells were washed twice with preheated (37° C) phosphate buffer saline (PBS) prior to addition of 0.5% polymeric solutions in MEM, negative control (MEM) and positive control (1% (v/v) Triton® X-100 in MEM) in multiple to the cell culture in 0.25 ml volume. After 2, 4 and 24 h of incubation period at 37 °C, 5% CO2 in air and 95% relative humidity cell medium was removed. Cells were washed twice with PBS. 250 µl of a resazurin solution was added per well and incubated for further 2 h at 37 °C, 5% CO2 in air and 95% relative humidity. Afterwards each well was measured as duplicate with a Tecan infinite M200 spectrophotometer (Grödig, Austria) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Percentages of viable cells were calculated in compliance with a negative control (100% viability) and a positive control (0% viability) (Iqbal et al., 2012).

2.2.6. Preparation of test discs To compress unmodified and modified polymer as well as chitosan a single punch excentric press (Paul Weber, Remshalden-Grünbach, Germany) was used. All test discs consisted of 30 mg compacted by at a constant compaction pressure of 11 kN. Test discs displayed 2.0 mm

2.2.10. Statistical data analysis Data were expressed as the mean ± SD and analyzed using one way ANOVA and a Bonferroni post-test with p < 0.05. All statistical tests 4

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were performed using Prism 5.01 (GraphPad). 3. Results and discussion 3.1. Synthesis and characterization of cationic starch derivatives Initially, molecular weights of starches from different origins were analyzed and respective values are depicted in Table 1. Cationic starch derivatives from different starch sources were generated in a two-step synthesis. First, the ring structure of the polysaccharide backbone was opened by oxidative cleavage. NaIO4 was therefore chosen as most suitable oxidizing agent due to its high selectivity, forming aldehydes by cleaving on the C2 and C3 position of 1,2-diols. Successful ring opening and aldehyde formation of all starch derivatives was confirmed by Fehling’s solution, facilitating differentiation between aldehydes from ketones. Here, Fehling’s solution presents only a qualitative test providing results very quickly. In the second step, reductive amination was utilized in order to obtain novel cationic polymers. The reaction with ammonium hydroxide solution leads to formation of an imine intermediate that is in the following reduced by NaCNBH3 (Peter et al., 2005). All obtained starch derivatives turned out as brownish colored and odorless lyophilisates of fibrous structure. The amount of aldehydes after ring opening was quantified adopting Schiff test and the amount of primary amines on the polymeric backbone was evaluated utilizing TNBS forming highly chromogenic yellow/orange colored derivatives with primary amines. Results of all synthesized starch derivatives obtained by executing three independent syntheses are listed in Table 1. Depending on the amount of NaIO4 added to the reaction mixture, different substitution degrees were obtained. Comparison between starches of different sources exhibited almost similar degrees of substitutions on same extent of oxidative cleavage. Considering yields of synthesis, a dependence of applied sodium periodate was evaluated Supporting data of previous research (Jelkmann et al., 2018; Wei et al., 2016). Future studies may enable further optimization of the synthesis processes. Possible yield improving actions might be the usage of low periodate concentrations in combination with longer reaction times (Veelaert et al., 1994) and performing precipitation of the polymer in alcohol instead of using dialysis for purification. All starch derivatives showed 100% solubility within seconds over the total measured pH range. In contrast, chitosan showed rapid hydration only at pH 1–2. At pH 3–5 it hydrated within 2–3 h and at pH 5–6 only by stirring overnight. At pH values above 6, reflecting physiological conditions, it was not soluble at all validating literature (Jimtaisong and Saewan, 2014). Concerning solubility of the aminated cellulose of previous research (Jelkmann et al., 2018), the full hydration of aminated starch derivatives accomplish a fivefold improvement for the highest modification up to a 15-fold increase regarding the lowest modification degree. This enhanced solubility enables an easier handling of cationic polymer in aqueous media of full pH range whereas chitosan is restricted to acidic solvents.

Fig. 2. Zeta-potential of unmodified ○, cationic corn starch ACS1 ●, ACS2 ▴ and ACS3 ■ and chitosan ◊ are displayed as means ± standard deviation of at least three experiments. Investigation was performed in a pH range between three and seven.

amination. This confirms the successful introduction of positive charges to the polymeric backbone due to amination. Chitosan, however, showed a substantially higher positive zeta potential than all starch derivatives. These results are in good agreement with the amount of amino groups on the polymeric backbone of all tested polymers. With a theoretical free amine number of around 4000 µmol per gram polymer, chitosan bears a 8-fold higher amount of positive charges compared to ACS3. Furthermore, the zeta potential is known to influence the mucoadhesiveness of a polymer due to ionic interactions of the polymer with highly negatively charged mucin particles (Bogataj et al., 2003; Takeuchi et al., 2005). 3.3. Mucoadhesion studies Different starch sources were tested regarding their potential as mucoadhesive cationic chitosan-alternatives. Results from preliminary mucoadhesion studies revealed superiority for corn starch over all other tested starch types (see Supplementary data). Pea starch showed lower mucoadhesive potential than corn starch, but higher mucoadhesive strength than potato starch. Soluble starch revealed in tensile force evaluation the highest adhesive strength of all four starch-sources on mucosal tissue but very poor results in rotating cylinder studies due to rapid disintegration. Furthermore, corn starch evinced highest transformation of aldehydes to amines in combination with highest yields within starches of different sources. Thus, corn starch, also referred to as maize starch being one of the most prominent starches used in the field of pharmaceutical technology, present the most promising characteristics for mucoadhesive dosage forms. Accordingly, data shown and discussed in the following all refer to aminated corn starch with three different degrees of substitution. Data were compared to chitosan as the most prominent cationic polymer.

3.2. Zeta potential measurements Successful reductive amination of polysaccharides should result in a pH-dependent zeta potential change due to introduction of positive charges to the polymeric backbone. Since chitosan is not soluble at pH values above 6, measurements were only performed up to this value. As illustrated in Fig. 2, CS, ACS1, ACS2 and ACS3 showed a slightly negative zeta potential over a pH range of 5 to 6. This might be due to a low number of carbonyl groups on oxidized starch molecule fragments. At pH 7, zeta potentials of all starch solutions considerably dropped, probably due to excess of free hydroxide ions in the solution. Highest differences were observed at pH 3, where almost all amino groups are protonated and positively charged. At this pH, significant differences (p < 0.05) between all starch derivatives could be observed, with increasingly positive zeta potentials depending on the degree of

3.3.1. Rheological investigations Mucoadhesive potential of polymers can be evaluated by measuring the dynamic viscosity of polymer/mucus mixtures (Madsen et al., 1998; Mortazavi and Smart, 1994). Mucoadhesive interactions between polymers and mucus glycoproteins cause the formation of a network resulting in an increased viscosity compared to the sum of viscosity values of the single components. This mechanism called rheological synergism occurs for all kind of mucoadhesive interactions, namely hydrophobic interactions, van der Waals forces, hydrogen bonds, 5

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dimensional structure of the polymer as well as its swelling ability on the mucosa. Since chitosan is insoluble in the neutral environment of the mucosa, only amino groups on the surface of the test discs can interact with the mucosa (Makhlof et al., 2008). In contrast, aminated starch derivatives swell in the mucus gel layer increasing the number of interacting groups by penetrating the mucus layer to some extent. This explains also the considerably lower TWA of CS compared to the MDF, since TWA calculation contains the distance between the starting point and the point of detachment. Due to swelling and penetration into the mucus layer, this distance could be elongated. Despite major advantages with respect to penetration and mucosal wetting of polymer, mucoadhesive properties can be diminished due to the dissolution of polymeric disc by longer exposure to gastrointestinal fluid triggered by the fast hydration. This was observed in tensile studies as well. The endpoint of chitosan examination was set by detachment of the test disc from the mucosal surface whereas analysis of cationic starches was terminated by disintegration of test discs. This might also be taken into account regarding possible added drugs. Enhanced spreading of the polymer will increase the area of contact between mucosa and drug leading to better bioavailability but might also lead to a faster release of drugs.

Fig. 3. Rheological measurements. Viscosities of 1% solutions of polymers were examined after mixing with porcine intestinal mucus in a ratio of 1:1 and an incubation time of 15 min. Indicated values were corrected by subtracting initial viscosity values. Imaged are means ± standard deviation from at least three measurements. Plotted stars declare level of significances in comparison to native corn starch.

3.3.3. Rotating cylinder A third mucoadhesion study was used in order to investigate mucoadhesive potential closer to in vivo conditions. While rheological experiments and tensile force evaluations mainly focus on the mechanism of polymer/mucus interactions and the adhesive strength on the mucosa, rotating cylinder studies assess the actual prolongation of retention time, probably being the most important parameter to evaluate mucoadhesive dosage forms. Furthermore, the rotating cylinder method is highly reproducible and takes into account several parameters like shear forces, swelling and cohesiveness. As can be seen in Fig. 5, amination considerably increased the time of adhesion with a 10.5-fold longer retention time of ACS3 compared to CS. The noticeable shorter retention time of ACS1 shows the importance of high substitution degrees. In contrast, the mucoadhesive potential of covalently mucus binding polymers is less dependent on their coupling degree. Although chitosan has a high density of positive charges, it attached to the mucosal tissue for only 31 min, being in the same range as unmodified starch and ACS1. In contrast, a significant difference between chitosan and ACS2 (p < 0.01) and ACS3 (p < 0.001) were determined. As mentioned above, this might be due to its poor swelling capacity preventing amino groups under the disc’s surface from interacting with the mucosa. As mucoadhesive properties are strengthened with increasing molecular weight, the molecular weights of derivatives were analyzed to investigate their impact on mucoadhesive properties of derivatives. All derivatives exhibited a decrease in molecular weight with increasing degree of modification being in accordance with previous research of aminated cellulose (Jelkmann et al., 2018) and research about oxidized starch (Zuo et al., 2017). More in detail, ACS1 exhibited a molecular weight of 2.8 ± 0.318∙106 synonymous a loss of molecular weight about 11 ± 10% whereas ACS showed a loss of 43 ± 10% and ACS3 of about 50 ± 23%. With respect to all investigations of mucoadhesiveness, there nevertheless was a clear improvement depending on the degree of modifications further emphasizing the gained mucoadhesive forces due to cationic modification.

covalent bonds and, as in the present case electrostatic interactions. Rheological investigations were performed with CS, ACS1, ACS2 and ACS3 as well as the intermediate product of ACS3, the aldehyde containing corn starch (aldCS) of maximum executed ring opening. One benefit of aminated starch vs chitosan is its solubility in physiological media above pH 6. In contrast, chitosan is not forming a gel like network in gastrointestinal environment and could not be investigated for rheological synergism due to poor solubility in mucus and also in phosphate buffer. Results from rheological measurements are illustrated in Fig. 3 and show higher viscosity for aminated starch derivatives vs native starch. ACS1 caused a 19-fold, ACS2 a 31-fold and ACS3 a 50-fold higher dynamic viscosity compared to CS indicating the formation of a gel-like network based on ionic complex formation between polymeric amino groups and carboxyl groups of mucus glycoproteins. This network is gaining density and structure strength with increasing number of amino groups leading to a positive correlation between substitution degree and dynamic viscosity. Results of the aldehyde containing form of corn starch did not affect the viscosity in a significant level emphasizing the effect of induced amino groups on rheological synergism.

3.3.2. Tensile force evaluation Tensile studies were performed in order to investigate the actual adhesive strength between starch conjugates and the mucosa. Therefore, the force that has to be applied to detach the test disc from the tissue was measured (Dünnhaupt et al., 2012). The maximum detachment force (MDF) as well as the total work of adhesion (TWA) for chitosan, CS, ACS1, ACS2 and ACS3 are illustrated in Fig. 4. In contrast to rheological experiments, solubility in neutral environment is not necessary to assess adhesive strength on the mucosal tissue; hence, chitosan could be evaluated despite its insolubility in phosphate buffer pH 6.8. Depending on the degree of substitution, enhanced adhesive strength could be observed. TWA and MDF were 7.1-fold and 4.3-fold higher for ACS3 compared to CS. Furthermore, ACS2 and ACS3 showed higher mucoadhesion than chitosan achieved by a significant (p < 0.01 and p < 0.001) increase of TWA whereas no significant differences of MDF between all aminated starches and chitosan were detected. This result is quite surprising regarding the amount of amino groups in both molecules, being higher in chitosan with around 4000 µmol per gram polymer than in ACS3 with 514 µmol per gram. Consequently, not only the electrostatic binding strength between the functional groups and the total number of these groups have to be considered, but also additional parameters such as the three-

3.4. Cell viability studies Cytotoxicity investigations were performed since almost all cationic charged polymers are known for their cell membrane disrupting effect and their cytotoxic potential (Dutta et al., 2008; Seow et al., 2013). Especially synthetic polymers with free amino groups, like for example polyethyleneimine (PEI) or polyallylamine show a noticeable negative effect on the integrity of cell membranes. Chitosan, on the other hand is 6

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Fig. 4. Dynamic tensile studies. Diagram displays results of chitosan, unmodified (CS) and modified polymer (ACS1, ACS2 and ACS3) on freshly excised mucosa. Total work adhesion (TWA) is represented in Figure A and the maximum detachment force (MDF) is illustrated in Figure B. Figure C displays an exemplary force vs displacement graphs indicating a typical curve progression. Indicated values are means ± standard deviation of at least three experiments. Significant levels of results were depicted in comparison with the native polymer (black stars) and compared to chitosan (grey stars).

Fig. 6. Cytotoxicity investigations. Effects of cationic and native starch, chitosan and polyethyleninime (PEI) on cell viabilities are pictured in histogram for CaCo-2 cell monolayer for exposure time of 2 h, 4 h and 24 h. Portrayed are means ± SD from at least three experiments. Fig. 5. Rotating cylinder. Mucoadhesive properties of native and cationic starch derivative as well as chitosan were examined on freshly excised porcine intestinal mucosa. The graph shows means of at least three experiments ± standard deviation. Significant levels of results were indicated in comparison to the native polymer by black stars and to chitosan via grey stars.

viability was significantly (p < 0.05) affected by chitosan. In contrast, CS, ACS1, ACS2 and ACS3 all showed a less cell toxic effect compared to chitosan, most likely due to the lower degree of amino groups as pictured out in Fig. 6. It is assumed that the molecular weight of chitosan possess an influence on its cytotoxicity (Bretado Aragón et al., 2018; Wiegand et al., 2010). Considering this factor, the even higher molecular weight of starch derivatives cause no impairing of the cell viability. Therefore, non-toxic and high molecular weight cationic polymers can be achieved by the present synthesis. Regarding the ISO 10993–5 (tests for in vitro cytotoxicity), aminated starch with a viability above 90% over 24 h is in a well acceptable range, however, chitosan with a viability slightly above 70% is just fitting the criteria (Standardization, 2009). Since aminated starch derivatives show

used in various dosage forms as one of very few less toxic positively charged polymers. Like all other aminated polymers, chitosan also interferes with the negatively charged cell membrane. However, due to its biodegradability and the ability to reversibly open tight junctions, it is more biocompatible than other positively charged polymers (Croisier and Jérôme, 2013). Despite these toxicity lowering factors, chitosan still shows cytotoxic potential (Huang et al., 2004). This could also be observed in the present study: After an incubation time of 24 h, cell 7

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increased mucoadhesive potential and coincidently lower cell toxic effects compared to chitosan, they seem to be superior excipients for mucosal drug delivery.

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4. Conclusion Within this study, aminated starch as a novel cationic excipient for mucosal drug delivery has been established. As pioneer, starch has been aminated by ring opening and reductive amination with ammonia. Showing superior mucoadhesive properties and less cytotoxic effects than chitosan as well as higher solubility than aminated cellulose, aminated starch could be a proper candidate to replace chitosan in formulations used so far in mucosal drug delivery. Targeting of other mucosal surfaces such as vagina or rectum is also imaginable and cationic charges enable furthermore the preparation of particulate systems. Future trends could include the introduction of covalently mucus binding groups, as for example thiol bearing ligands, to the polymeric backbone of aminated starch to even further increase its mucoadhesive potential. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.118664. References Akiyama, Y., Nagahara, N., Nara, E., Kitano, M., Iwasa, S., Yamamoto, I., Azuma, J., Ogawa, Y., 1998. Evaluation of oral mucoadhesive microspheres in man on the basis of the pharmacokinetics of furosemide and riboflavin, compounds with limited gastrointestinal absorption sites. J. Pharm. Pharmacol. 50, 159–166. Azagury, A., Amar-Lewis, E., Mann, E., Goldbart, R., Traitel, T., Jelinek, R., Hallak, M., Kost, J., 2014. A novel approach for noninvasive drug delivery and sensing through the amniotic sac. J. Control. Release 183, 105–113. Baus, R.A., Haug, M.F., Leichner, C., Jelkmann, M., Bernkop-Schnurch, A., 2019. In vitroin vivo correlation of mucoadhesion studies on buccal mucosa. Mol Pharm 16, 2719–2727. Bernkop-Schnürch, A., Dünnhaupt, S., 2012. Chitosan-based drug delivery systems. Eur. J. Pharm. Biopharm. 81, 463–469. Bernkop-Schnürch, A., Steininger, S., 2000. Synthesis and characterisation of mucoadhesive thiolated polymers. Int J Pharm 194, 239–247. Bogataj, M., Vovk, T., Kerec, M., Dimnik, A., Grabnar, I., Mrhar, A., 2003. The correlation between zeta potential and mucoadhesion strength on pig vesical mucosa. Biol. Pharm. Bull. 26, 743–746. Bretado Aragón, L., Jiménez Mejía, R., López-Meza, J., Loeza-Lara, P., 2018. Composites of silver-chitosan nanoparticles: a potential source for new antimicrobial therapies. Cohen, S., Coue, G., Beno, D., Korenstein, R., Engbersen, J.F., 2012. Bioreducible poly (amidoamine)s as carriers for intracellular protein delivery to intestinal cells. Biomaterials 33, 614–623. Croisier, F., Jérôme, C., 2013. Chitosan-based biomaterials for tissue engineering. Eur. Polym. J. 49, 780–792. Dünnhaupt, S., Barthelmes, J., Rahmat, D., Leithner, K., Thurner, C.C., Friedl, H., Bernkop-Schnürch, A., 2012. S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties. Mol. Pharm. 9, 1331–1341. Dutta, T., Garg, M., Jain, N.K., 2008. Poly(propyleneimine) dendrimer and dendrosome mediated genetic immunization against hepatitis B. Vaccine 26, 3389–3394. Engelberth, S.A., Hempel, N., Bergkvist, M., 2015. Chemically modified dendritic starch: a novel nanomaterial for siRNA delivery. Bioconjug. Chem. 26, 1766–1774. Engelberth, S.A., Hempel, N., Bergkvist, M., 2017. Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery. J. Photochem. Photobiol., B 168, 185–192. Gefroh, E., Hewig, A., Vedantham, G., McClure, M., Krivosheyeva, A., Lajmi, A., Lu, Y., 2013. Multipronged approach to managing beta-glucan contaminants in the downstream process: control of raw materials and filtration with charge-modified nylon 6,6 membrane filters. Biotechnol. Prog. 29, 672–680. Hauptstein, S., Bonengel, S., Griessinger, J., Bernkop-Schnürch, A., 2013. Synthesis and characterization of pH tolerant and mucoadhesive (thiol–polyethylene glycol)

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