N-hydroxysulfosuccinimide esters versus thiomers: a comparative

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N-hydroxysulfosuccinimide esters versus thiomers: a comparative study regarding mucoadhesiveness Christina Leichner, Patrizia Wulz, Randi Angela Baus, Claudia Menzel, Sina Katharina Götzfried, Ronald Gust, and Andreas Bernkop-Schnürch Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01183 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Molecular Pharmaceutics

1

N-hydroxysulfosuccinimide esters versus thiomers: a comparative study regarding

2

mucoadhesiveness

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Christina Leichner1, Patrizia Wulz1, Randi Angela Baus1, Claudia Menzel1, Sina Katharina Götzfried2,

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Ronald Gust2 and Andreas Bernkop-Schnürch1*

6 7

1Center

for Chemistry and Biomedicine,

8

Department of Pharmaceutical Technology,

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Institute of Pharmacy, University of Innsbruck,

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Innrain 80/82, 6020 Innsbruck, Austria

11 12

2Center

for Chemistry and Biomedicine,

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Department of Pharmaceutical Chemistry,

14


15


16 17

*Corresponding Author:

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Center for Chemistry and Biomedicine,

19

Department of Pharmaceutical Technology,

20


21


22

Tel.: +43-512-507 58601

23

Fax: +43-512-507 58699

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e-mail: [email protected]

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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AIM: The objective of the study was to compare polyacrylic acid-N-hydroxysulfosuccinimide

28

reactive esters (PAA-Sulfo-NHS) and polyacrylic acid-cysteine conjugates (PAA-Cys) regarding

29

their mucoadhesiveness.

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METHODS: Polymer conjugates were synthesized in a water free environment and characterized

31

by UV/VIS spectroscopy and FTIR. Water uptake studies were performed and the polymers were

32

further examined for their mucoadhesive properties and cohesiveness using the rotating cylinder

33

method. Tensile force measurements were conducted to define the strength of adhesion to porcine

34

intestinal mucosa. Additionally, polymer-mucus mixtures were assessed for rheological synergism

35

by measuring the increase in dynamic viscosity.

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RESULTS: Both modifications led to a prolonged adhesion time compared to unmodified PAA.

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Fast dissolution of PAA-Sulfo-NHS derivatives was monitored, whereas PAA-Cys tends to

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extensively swell while exhibiting high cohesive properties. Measurements of tensile force

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revealed up to 2.7-fold (PAA-Sulfo-NHS) and 2.3-fold (PAA-Cys) enhancement of the maximum

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detachment force and 7.6-fold (PAA-Sulfo-NHS) and 3.6-fold (PAA-Cys) increase in the total

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work of adhesion. Formation of a gel network between polymer and mucus was confirmed by a

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10.8-fold (PAA-Sulfo-NHS) and 20.8-fold (PAA-Cys) increase in viscosity.

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CONCLUSION: Both types of polymers show high mucoadhesive properties due to the formation

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of covalent bonds with the mucus. As thiolated polymers are capable of forming stabilizing

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disulfide bonds within their polymeric network, they are advantageous over PAA-Sulfo-NHS.

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Key words: Polyacrylic acid, Sulfo-NHS ester, thiomer, mucoadhesion, amino reactive ester

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1. Introduction 2 ACS Paragon Plus Environment

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Availability of the drug at its desired target location is often limited by the short contact time

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between the absorption membrane and the formulation. Especially in the small intestine where

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motility activity is high and transit is fast it is challenging to concentrate the drug at the site of

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action. A promising, and already over years well-established strategy to elongate the residence of

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the delivery system at the mucosal surface comprises the application of mucoadhesive polymers

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(1-5). Thereby, a high local concentration can be achieved together with a reduced diffusion

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distance between mucosal surface and the absorption site connected to a high flux rate (6).

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According to the underlying mechanisms of mucoadhesion, the mucoadhesive can interact with

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mucus substructures, mainly glycoproteins (mucin), via on the one hand rather weak interactions

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like ionic interactions, hydrogen bonds, van-der-Waals forces and hydrophobic interactions, or on

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the other hand by forming strong covalent bonds (7). Thiomers can be regarded as the most

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prominent representatives in the field of mucoadhesion, facilitating strong covalent bonding via

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the formation of disulfide bonds with cysteine-rich mucin domains (8). Furthermore, disulfide

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bonds can also be formed within the thiomer structure resulting in a highly cohesive polymeric

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network (9, 10). One shortcoming of this intra- and inter-molecular cross-linking is the loss of

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available reactive groups towards mucus glycoproteins accompanied by reduced chain flexibility

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and mobility, preventing interpenetration of the mucus gel layer and finally ending up in weakness

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of adhesive forces (11).

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Therefore an alternative covalent binding mucoadhesive polymer type has been introduced in a

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previous study of our research group omitting the above-mentioned susceptibility to self-cross-

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linking. By covalent attachment of N-hydroxysuccinimide (NHS) to the polymeric backbone of

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poly(acrylic acid) (PAA), amino reactive esters were generated with the objective to target amino

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groups of lysine and arginine substructures of mucin glycoproteins (12, 13). Despite their 3 ACS Paragon Plus Environment


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unfavorable rather hydrophobic character these polymer conjugates showed promising results

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regarding mucoadhesive properties (14).

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However, the mucoadhesive properties of such kind of polymeric NHS-esters has not been

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evaluated in direct comparison to thiomers in order to specify the extent of potential pros and cons

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of the two systems. Sulfo-NHS was used in this study instead of NHS by the idea of yielding esters

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with higher hydrophilicity and improved mucoadhesive characteristics (15). It was therefore the

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objective of this study to synthesize PAA derivatives with comparable amount of reactive groups

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and to investigate their mucoadhesiveness via the rotating cylinder method and tensile studies.

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Furthermore, dynamic viscosity measurements of polymer/mucus mixtures were performed to

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allow the assessment of structural changes within the mucus gel network.

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2. Materials and Methods

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2.1. Materials

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Dimethylformamide, acetonitrile and methanol were obtained from VWR chemicals (Radnor, PA,

84

USA).

85

hydroxysulfosuccinimide (Sulfo-NHS), L-cysteine hydrochloride, all buffers and salts were

86

purchased from Sigma-Aldrich (Vienna, Austria).

Poly(acrylic

acid)

450

kDa

(PAA),

N,N'-diisopropylcarbodiimide,

N-

4 ACS Paragon Plus Environment

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2.2. Methods

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2.2.1. Synthesis of polymers

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2.2.1.1.

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Sulfo-NHS was attached to the polymeric backbone of PAA mediated by N,N'-

91

diisopropylcarbodiimide (DIC) as coupling reagent. First, 0.5 g of the polymer was dissolved in

92

dimethlylformamide (DMF) under stirring. Following, 1 mL of DIC and either 150 mg to

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synthesize PAA-Sulfo-NHS 300 or 300 mg Sulfo-NHS in case of PAA-Sulfo-NHS 800 were

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added. After 18 h stirring, the products were purified from any residual amounts of coupling

95

reagent, byproducts and DMF by several washing steps with acetonitrile and dried under vacuum.

96

2.2.1.2.

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PAA-cysteine derivatives were synthesized in the same way as PAA-Sulfo-NHS, except for the

98

addition of L-cysteine HCl instead of Sulfo-NHS. In order to obtain different coupling rates,

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219 mg (PAA-Cys 300) and 544 mg (PAA-Cys 800) of L-cysteine HCl were added to the reaction

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mixture. Reaction time was kept at 18 h. Products were washed with acetonitrile and dried under

101

vacuum. Finally to separate unreacted L-cysteine from the polymers, washing with a mixture of

102

acetonitrile/methanol (10/1 (v/v)) was performed and the products dried. Poly(acrylic acid) only

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treated with the solvents used in the syntheses was prepared as a control polymer for further

104

conducted experiments.

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2.2.2. Characterization of polymers

106

Molecular mass of the newly synthesized PAA derivatives was estimated by static light scattering

107

with a Zetasizer Nano ZSP (Malvern, USA) with a laser wavelength of 633 nm at 25 °C. Polymers

108

were dissolved in a mixture of dimethyl sulfoxide/ 0.5 M NaCl 1:1. The refractive index increment

Synthesis of PAA-Sulfo-NHS

Synthesis of PAA-cysteine



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(dn/dc = 0.147 mL/g) was determined using an Abbe refractometer ATAGO 1T equipped with an

110

ATAGO digital thermometer (ATAGO CO.,LTD., Japan).

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Modification degree of PAA-Sulfo-NHS was determined by the detection of Sulfo-NHS bound to

112

the polymer after alkaline ester hydrolysis (16). Hence, 2 mg of PAA-Sulfo-NHS derivatives were

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dissolved in 1 mL of 0.1 M NaOH followed by absorption measurement at 260 nm (TECAN

114

Infinite M200, Austria GmbH). Liberated Sulfo-NHS in the probes was quantified with respect to

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a calibration curve being based on pure Sulfo-NHS.

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FTIR spectra were taken from PAA, Sulfo-NHS, PAA-Sulfo-NHS 300 and PAA-Sulfo-NHS 800

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using a Perkin Elmer Spectrum 100 ATR-IR spectrometer (Perkin Elmer, Waltham, MA) in an

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arrangement with the software version 6.3.1.0134 (Perkin Elmer). Spectra were recorded with 10

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scans in a wave number range from 4000 to 650 cm-1 and a resolution of 1 cm-1. Measurements

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were performed at 22°C (17).

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NMR spectroscopy was performed on a 400 MHz Bruker Advance equipped with a 1H-13C SEI

122

probe and the spectra were evaluated by TopSpin 3.5 pl 7 software. For the measurements 15 mg

123

of PAA, of Sulfo-NHS and of PAA-Sulfo-NHS were dissolved in D2O. 1H-NMR was recorded for

124

PAA, Sulfo-NHS and PAA-Sulfo-NHS and a 13C-NMR was additionally recorded for PAA-Sulfo-

125

NHS.

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The amount of free thiol groups immobilized at the polymeric backbone of the conjugates was

127

determined photometrically (TECAN Infinite M200, Austria GmbH) using 5,5′-dithiobis (2-

128

nitrobenzoic acid) as described previously (11).

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2.2.3. Assessment of hydrolytic stability

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Stability of the PAA-Sulfo-NHS conjugates was investigated in aqueous media. In brief, test

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polymers were hydrated in either simulated gastric fluid (0.1 M HCl, pH 1.2) or phosphate buffer

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100 mM pH 7.4 in a final concentration of 1 mg/mL. Subsequently, samples were incubated at

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37 °C under shaking at 200 rpm. Aliquots of 120 µL were withdrawn at predefined time points,

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centrifuged at 12,044 x g for 2 min and the supernatant analyzed for Sulfo-NHS content by

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absorption measurement at 260 nm (TECAN Infinite M200, Austria GmbH). In the case of gastric

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fluid pH was raised by the addition of 0.2 M phosphate buffer pH 7.4 in a ratio of 1:1 before

138

analyses as UV detection of Sulfo-NHS is only feasible when the molecule is present in the ionized

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form (16). A Sulfo-NHS calibration curve was subjected to the same procedure.

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2.2.4. Evaluation of toxicity

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2.2.4.1.

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Red blood cell lysis assay was carried out using a protocol previously described with slight

143

modifications (18). First, red blood cell concentrate was diluted 1/10 in phosphate buffered saline

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(PBS). PAA conjugates were added to the cell dilution in final concentrations in the range of 0.05 %

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to 0.5 % (m/v). As positive control resulting in total hemolysis served demineralized water and

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PBS was taken as a negative control with 0 % cell lysis, respectively. Hemolysis was quenched

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after 30 min incubation at 37 °C by putting the samples on ice for 2 min. After that the probes were

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5 min centrifuged at 3000 rpm and the released hemoglobin in the supernatant detected at 540 nm

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(TECAN Infinite M200, Austria GmbH).

Hemolysis assay

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2.2.4.2.

Caco-2 cell assay

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The cytotoxic potential of the PAA conjugates was examined on Caco-2 cells employing the

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resazurin assay (19, 20). Briefly Caco-2 cells were seeded on a 24 well plate

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(d = 2.5 × 104 cells/well; 500 µL per well) in minimum essential medium (MEM) supplemented

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with 10 % (v/v) fetal calf serum (FCS) and penicillin/ streptomycin solution (100 units/ 0.1 mg/L)

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and cultured for 1 week at 37 °C in an atmosphere of 5 % CO2 and 95 % relative humidity. During

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the culture period the medium was changed every second day. Afterwards, cells were washed with

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preheated phosphate buffered saline (PBS) and treated with 500 μL of the test solutions. PAA-

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Sulfo-NHS esters were tested in a 0.5 % (m/v) concentration and PAA-Cys derivatives were

160

applied in 0.25 % and 0.5 % (m/v). MEM served as a negative and a 1 % (m/v) solution of Triton

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X-100 as positive control. After an incubation time of 4 h cells were washed with PBS and a 5 %

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(m/v) resazurin solution was added. The cells were incubated for additional 2 h and fluorescence

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of the supernatant was measured at 540 nm excitation wavelength and 590 nm emission

164

wavelength (TECAN Infinite M200, Austria GmbH).

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2.2.5. Preparation of polymer test discs

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Within the executed swelling test and mucoadhesion experiments, polymers were used in the form

167

of compacted discs. For this purpose each polymer was compressed into flat faced discs of 30 mg

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and 5.0 mm diameter using a single punch excentric press (Paul Weber, Remshalden-Grünbach,

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Germany). The applied pressure during disc manufacturing was kept constant at 11 kN for a

170

duration of 30 seconds. Test discs were stored in a dessicator until further use.

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2.2.6. Assessment of swelling behavior

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Water uptake was investigated and as a result of this the swelling behavior of the polymer discs

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evaluated by determining their mass increase gravimetrically as described previously (11).

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Accordingly, discs were fixed with a metal clamp and submerged into preheated phosphate buffer

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100 mM pH 7.4 and incubated at 37 °C. Discs were taken out of the buffer at predetermined time

177

points and weighed.

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2.2.7. Evaluation of mucoadhesive properties

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2.2.7.1.

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The retention time of polymer discs attached to freshly excised porcine intestinal mucosal tissue,

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was evaluated using the rotating cylinder method as described in previous studies of our research

182

group (21). In brief, the mucosa was glued on a stainless steel cylinder using a cyanoacrylate

183

adhesive (Locite, Henkel, Austria) and test discs were attached to the mucosal surface. Afterwards,

184

the cylinder was placed in a dissolution tester (Erweka GmbH, Heusenstamm, Germany) according

185

to the European Pharmacopeia, immersed into 900 mL of a 100 mM phosphate buffer pH 7.4 and

186

rotated at a speed of 100 rpm at 37 °C. Detachment of the polymer discs was observed visually.

187

2.2.7.2.

188

Tensile studies were performed using a texture analyser (TA.XTPlus, Texture Analyser Stable

189

Micro Systems, Winopal Forschungsbedarf GmbH, Elze, Germany) equipped with A/MUC

190

mucoadhesive rig fixture. Briefly, the mucosa was cut into pieces of approximately 2 cm2 and was

191

fixed between two acrylic glass plates and the polymeric test disc was applied to a probe cylinder

192

(P/05, ½” Delrin Cylinder) with a double-sided adhesive tape. Polymer and mucosal surface were

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put in contact by lowering the probe cylinder. Subsequently this contact stage was kept for a 20 min

Rotating cylinder

Tensile force measurement



Page 10 of 39

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incubation time under a constant applied force of 0.01 N. After incubation, the polymer was pulled-

195

up with a speed of 0.1 mm/s until the disc was completely detached from the tissue. The maximum

196

detachment force (MDF) and the total work of adhesion (TWA) were calculated from the recorded

197

measurement data.

198

2.2.8. Mucus collection

199

Fresh porcine intestine fetched from a local abattoir was cut longitudinally and rinsed carefully

200

with physiological saline (NaCl 0.9 % (m/v)) to remove all food remains. Afterwards the mucus

201

was collected by gently scrapping it off from the underlying tissue. The water content of mucus

202

was 78.8 ± 0.2 % (m/m), determined gravimetrically. Rheological measurements using a plate-

203

plate viscometer (RotoViscoTM 158 RT20, Haake GmbH, Karlsruhe, Germany) at a frequency of

204

1 Hz and a sheer stress range of 0.01 Pa–50 Pa yielded a dynamic viscosity of 0.64 ± 0.05 Pas.

205

2.2.9. Rheological investigations

206

Dynamic viscosity measurements were performed with a plate-plate viscometer (RotoViscoTM

207

158 RT20, Haake GmbH, Karlsruhe, Germany) at 37 °C, a frequency of 1 Hz and a sheer stress

208

range of 0.01 Pa–50 Pa. Therefore, test samples were prepared by adding the dry polymer in a final

209

concentration of 1 % (m/m) to a porcine mucus/ buffer (100 mM phosphate buffer, pH 7.4) mixture

210

(5/1). After incubation times of 5 min, 30 min and 120 min, samples were transferred to the plate

211

of the viscometer and the dynamic viscosity was measured.


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2.2.10. Statistical data analysis

213

The software Graph Pad Prism version 5.01 was used for the statistical data analysis. One way

214

ANOVA and Bonferroni t-test were performed with P < 0.05 as the minimal level of significance.

215

3. Results and discussion

216

3.1. Synthesis and characterization of polymers

217

Sulfo-NHS was used in this study in order to yield products with higher solubility than NHS

218

analogues by possessing the same specificity and reactivity (15). As the reactive esters are highly

219

sensitive to hydrolysis, PAA-Sulfo-NHS conjugates were synthesized in a water free environment

220

in order to avoid premature cleavage of the formed ester bond. In virtue of comparability, PAA-

221

Cys was generated under similar conditions (Figure 1). Derivatives with two different coupling

222

rates were obtained by varying the amounts of Sulfo-NHS and L-cysteine in reaction mixtures.

223

Efficiency of the ester bond formation can be considered as higher compared to amide bond

224

formation of PAA-Cys, as lower molar ratios of ligand to polymer were sufficient to obtain a

225

comparable coupling rate (Table 1).

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Figure 1: Reaction scheme of carbodiimide mediated syntheses. PAA-Sulfo-NHS (A) was built via ester bond formation between PAA and Sulfo-NHS. PAA-Cys (B) was synthesized via covalent attachment of L-cysteine to the polymer resulting in an amide bond.

231 232 233 234 235

Table 1: Amounts of attached reactive groups at the polymeric backbone of polyacrylic acid dependent on the used weighed portions in the syntheses. Data are illustrated in form of mean values of at least three measurements including standard deviation. Polymer

PAASulfo-NHS 300 PAASulfo-NHS 800 PAA-Cys 300 PAA-Cys 800

Synthesis amounts [mol] PAA LSulfocysteine NHS 1.0 0.1

-Sulfo-NHS [µmol/g] Mean SD

-SH [µmol/g] Mean

SD

363.0

76.8

-

-

1.0

-

0.2

885.5

89.0

-

-

1.0

0.2

-

-

-

326.3

73.4

1.0

0.5

-

-

-

815.3

18.2

236 12 ACS Paragon Plus Environment

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237

Static light scattering experiments revealed an average molecular mass of 412 ± 43.0 kDa for PAA-

238

Sulfo-NHS 300 and 490 ± 14.8 kDa for PAA-Sulfo-NHS 800. In case of the PAA control a

239

molecular mass of 353 ± 14.9 kDa was detected.

240

Upon alkaline hydrolysis of PAA-Sulfo-NHS, Sulfo-NHS could be quantified at 260 nm and the

241

attached amount of ligand was calculated. Free thiol groups immobilized on the backbone of PAA

242

were determinated by Ellman´s assay.

243

Successful formation of the PAA-Sulfo-NHS conjugate was additionally to UV detection

244

confirmed by FTIR spectroscopy (spectra are presented in the supporting information in Figure S1-

245

S3). Polyacrylic acid displayed the typical C-H stretching vibrations at about 2941 cm-1. The

246

carboxyl group was characterized by a wide OH stretch from 3176 to 2583 cm-1, which

247

superimpose the C-H stretching bands. The C=O stretch of carboxylic acid appeared at 1699 cm- 1,

248

those of C-O exist at 1232 cm-1. In the spectrum of Sulfo-NHS characteristic bands were at

249

3149 cm-1 (O-H), 3005 cm-1 to 2934 cm-1 (C-H), 1702 cm-1 (C=O). The PAA-Sulfo-NHS

250

spectrum showed a reduction of the broad OH band, being an indicator for esterification of COOH

251

groups with Sulfo-NHS. Esterification led to characteristic carbonyl bands at 1734 cm-1. Another

252

indication for the formation is the disappearing of the characteristic carbonylfinger band of the acid

253

group at 1700 cm-1. Furthermore, amide I bands were detectable at 1614 (22). Rising of a new peak

254

at 3338 cm-1 could not be associated to characteristic group signals according to literature.

255

The formation of PAA-Sulfo-NHS was furthermore characterized via NMR- spectroscopy

256

(supporting information Figure S4-S7). The 1H-NMR spectra of PAA-Sulfo-NHS in D2O shows

257

the typical peaks at 1.60 – 2.0 ppm for the CH2-group and at 2.44 ppm for the methine-group of

258

PAA and the geminale peaks corresponding to the CH2 group at 3.06 and 3.27 ppm as well the dd

259

at 4.35 ppm of N-hydroxysulfosuccinimide. The small shift of 0.20 ppm of the CH2- and the 13 ACS Paragon Plus Environment


Page 14 of 39

260

methine-group in corresponding to the NMR spectra of PAA (see supporting information) into the

261

downfield evidenced the esterification of PAA and Sulfo-NHS.

262

3.2. Assessment of hydrolytic stability

263

Although the polymers were mainly used in dry form within the experiments focusing on adhesive

264

properties, the reactivity in hydrated form is important. As the stability of reactive thiol groups on

265

polymers has already been described in literature (23), stability of PAA-Cys derivatives was not

266

further evaluated. NHS ester stability is reported in literature with half-lives ranging between

267

1 hour at pH 8 and 25 °C (23) and 10 min at 8.6 at 4 °C (24) with higher hydrolytic stability at

268

pH 5 and 6 (25). Along pH and temperature, hydrolysis of hydrophilic esters is strongly dependent

269

on the hydration behavior of the polymer backbone. Ester hydrolysis was therefore investigated in

270

more detail. Visual observations confirmed complete hydration of the polymers within less than

271

30 min. Consequently, ester hydrolysis happened fast under both applied pH conditions with a half-

272

life of around 17.5 min (PAA-Sulfo-NHS 300) and 8 min (PAA-Sulfo-NHS 800) at pH 1.2 and 7-8

273

min for both modifications at pH 7.4 (Figure 2). The total amount of Sulfo-NHS having been

274

hydrolytically cleaved at pH 1.2 and 7.4 correlated to the amount of covalently attached Sulfo-

275

NHS as listed in Table 1.


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276 277 278 279

Figure 2: Time dependent ester hydrolysis in 0.1 M HCl (A) and 100 mM phosphate buffer pH 7.4 (B). Illustrated values are means of at least three experiments ± standard deviation. PAA-SulfoNHS 300 (■) and PAA-Sulfo-NHS 800 (▲).

280

3.3. Evaluation of toxicity

281

As Sulfo-NHS ester structures are frequently used for the labeling of cell surface proteins, PAA-

282

Sulfo-NHS polymers might show a damaging effect to the cell membrane (15, 26). In vitro

283

erythrocyte lysis can be considered as a valid test to predict membrane damaging effects of the test

284

compounds in vivo. As cell membrane rupture correlates with cytotoxicity in general, the

285

experimental set-up can also be used to assess the polymer safety (18). Hemolysis was calculated

286

as a percentage of the 100 % lysis value (Figure 3). According to the ASTMF756-00(2000)

287

standard, materials with hemolysis rates greater than 5 % are regarded as hemolytic. Except of 15 ACS Paragon Plus Environment


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PAA-Sulfo-NHS 800 at a concentration of 0.5 % (m/v) all other tested polymer concentrations did

289

not possess any hemolytic potential. When subtracting the negative control from the sample values,

290

hemolysis deriving from the pure polymer was 6.3 ± 0.4 %. One explanation for this finding might

291

be the afore mentioned possible covalent binding of PAA via amide bond formation to cell surface

292

proteins, ending up in membrane interference and cell damage. Sulfo-NHS alone did not induce

293

any noticeable erythrocyte lysis in a concentration corresponding to the amount maximal included

294

in a 0.5 % test solution of PAA-Sulfo-NHS 800. PAA-Cys conjugates induced hemolysis in the

295

range of 0.4 % for PAA-Cys 300 and PAA-Cys 800 in 0.5 % (m/v) concentration, and 0.1 %

296

hemolysis for PAA-Cys 300 in 0.05 % (m/v). Additionally cell permeation and systemic uptake of

297

PAA-Sulfo-NHS and pure Sulfo-NHS seem to be unlikely due to the negative charge of the

298

sulfonate group (27). Summarizing, harmful effects of both modified polymer types on the cell

299

membrane can be widely excluded. Nevertheless, cytotoxicity was studied on Caco-2 cells (Figure

300

4). Sulfo-NHS esters did not evoke any toxic effect, whereas the thiolated polymers displayed a

301

concentration dependent effect on cells. These findings can be explained by a primarily physical

302

effect caused by a layer of completely swollen polymer settled on the surface of the cell monolayer,

303

hindering the access of nutrients and oxygen. In vivo the polymer will likely not get into contact

304

with the mucosal membrane in such high concentration because of limited diffusion across the

305

mucus layer. Moreover PAA-Cys derivatives can be considered as generally safe as their

306

innocuousness could even be proven within a clinical study applied on the sensitive mucosal

307

surface of the eye (28).


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308 309 310 311

Figure 3: Percentage of hemolysis caused by the controls, Sulfo-NHS and different concentrations of the PAA-Sulfo-NHS and PAA-Cys conjugates. Values represent data of at least three replications plus standard deviation.



Page 18 of 39

312 313 314 315 316

Figure 4: Percentage of Caco-2 cell viability caused by the controls, PAA-Sulfo-NHS derivatives in 0.5 % (m/v) (A) and PAA-Cys conjugates (B) in 0.25 % (m/v) (light gray) and 0.5 % (m/v) (dark gray). Cells were incubated for 4 h. Values represent data of at least three replications plus standard deviation.

317

3.4. Assessment of swelling behavior

318

A sufficient amount of swelling is necessary to properly hydrate the polymer and expose available

319

adhesive sites for bond formation. Therefore, the capacity of water uptake was investigated by

320

monitoring the percentage of mass increase over time (Figure 5). PAA-Cys discs exhibited a high

321

water uptake capacity and moreover polymer dissolution in comparison to the PAA control was

322

retarded. During the first 45 min, PAA-Cys conjugates possessed similar water uptake, compared 18 ACS Paragon Plus Environment

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323

to PAA. Maximum swelling was reached after 180 min with around 400 % of the initial disc weight

324

for PAA-Cys 300 and PAA-Cys 800 completed swelling after 420 min by 960 % mass increase.

325

This observation can be explained by the formation of intra polymeric disulfide bridges,

326

contributing to a highly cohesive structure with a high water uptake capacity. In contrast, the

327

behavior of PAA-Sulfo-NHS was strikingly different, as discs were slightly swelling. PAA-Sulfo-

328

NHS 300 was about 3.1-fold and PAA-Sulfo-NHS 800 around 2.3-fold less swelling than the PAA

329

control, and the polymers rather start dissolving after 30 to 45 min. This fast dissolution can be

330

explained by the hydrophilicity of the sulfonate group, favoring additional to the carboxylic groups

331

on the PAA backbone, strong hydrogen bonding with water molecules followed by fast

332

disentanglement of the single polymer chains.

333



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334

335 336 337 338 339

Figure 5: Time dependent water uptake of polymer discs in 100 mM phosphate buffer pH 7.4. Illustrated values are means of at least three experiments ± standard deviation. A: PAA control (●), PAA-Sulfo-NHS 300 (■) and PAA-Sulfo-NHS 800 (▲). B: PAA control (●), PAA-Cys 300 (■) and PAA-Cys 800 (▲).


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340

3.5. Evaluation of mucoadhesive properties

341

3.5.1. Rotating cylinder

342

Within this study adhesive as well as cohesive properties of polymer discs can be evaluated. Along

343

with the direct interactions of the polymer and mucus layer, the test material´s cohesiveness

344

furthermore prolongs the retention time on the mucosa. Due to the modification, adhesion time of

345

PAA-Sulfo-NHS 300 was 2.5-fold increased and a higher modification led to a 2.9-fold increase

346

for PAA-Sulfo-NHS 800 respectively (Figure 6). Having a closer look at the mechanism of

347

covalent bond formation, the dependence on the environmental pH has to be taken into

348

consideration in order to explain the findings. Amide bond formation between Sulfo-NHS esters

349

and primary amino groups has its pH optimum in a range of 7 to 9. Within a previous study,

350

reactivity towards glycine and hence adhesion to mucus was already established under

351

physiological pH for PAA-NHS esters (14). Accordingly, it can be assumed that PAA-Sulfo-NHS

352

will likely react in the same way under the formation of a powerful mucoadhesive junction. Thiol

353

groups tend to continuous oxidation at pH values above 5. Within the buffer pH 7.4 intra- and inter

354

polymeric disulfide bridges can easily be formed additionally to fast covalent binding to the

355

mucosa. As opposed to the Sulfo-NHS conjugates, a highly cohesive polymer network was built

356

and the adhesion time was on account of this additively extended by 5.3-fold (PAA-Cys 300) and

357

7.9-fold (PAA-Cys 800) compared to unmodified PAA. Observations were in good agreement with

358

the results of the water uptake study, as a fast dissolution of PAA-Sulfo-NHS and excessive

359

swelling of PAA-Cys could be confirmed.



Page 22 of 39

360 361 362 363 364

Figure 6: Mucoadhesive properties of PAA, PAA-Sulfo-NHS 300, PAA-Sulfo-NHS 800, PAACys 300 and PAA-Cys 800. The time of adhesion on freshly excised porcine intestinal mucosa was determined via the rotating cylinder method in 100 mM phosphate buffer pH 7.4. The graph shows the means of at least three experiments ± standard deviation (***P < 0.001).

365

3.5.2.Tensile force measurement

366

Tensile studies rank among the most established and used mucoadhesive test systems. In general,

367

the higher the mucoadhesion of polymer, the higher are the resulting maximum detachment force

368

and total work of adhesion. Both types of PAA derivatives exhibited higher adhesive forces,

369

leading to increased MDF and TWA values compared to the control (Figure 7). The maximum

370

detachment force reflects the strength of the covalent bond formed between polymer and mucus,

371

whereas the total work of adhesion represents the total energy needed to completely detach the

372

polymer from the mucosa. The improvements in case of MDF were 2.7-fold (PAA-Sulfo-

373

NHS 300), 2.3-fold (PAA-Sulfo-NHS 800 and PAA-Cys 300) and 2.0-fold (PAA-Cys 800),

374

respectively. Upon modification stated enhancements in TWA were 7.6-fold (PAA-Sulfo-

375

NHS 300), 6.9-fold (Sulfo-NHS 800), 3.0-fold (PAA-Cys 300) and 3.6-fold (PAA-Cys 800)

376

compared to the unmodified PAA control. Interestingly, the recorded MDF and TWA were higher 22 ACS Paragon Plus Environment

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377

for the Sulfo-NHS derivatives, especially when comparing the higher modified polymers. Taking

378

the results of the water uptake study and rotating cylinder into consideration, mentioned distinct

379

differences might be explained by different performances regarding cohesiveness and dissolution.

380

Fast swelling ability of thiomers promotes the formation of a highly stable and cohesive cross-

381

linked polymeric network. Accompanied by high cross-linking within the PAA-Cys structure,

382

possible shortcomings could be a decreased amount of free thiol groups needed for covalent

383

binding to mucins on the one hand, resulting in lower MDF, and on the other hand overhydrating

384

of the polymer could provide a slippery mucilage, and thus consequential reduce TWA (6, 7). The

385

high dissolution rate of the PAA-Sulfo-NHS derivatives favors disentanglement of the polymeric

386

network. In the space of a previous study, interpenetration of loose polymeric chains into the mucin

387

network was pointed out as one of the key features influencing mucoadhesion (29). Based on the

388

higher molecular mobility and flexibility of the polymer, non-cross-linked polymers like PAA-

389

Sulfo-NHS, should be able to interpenetrate the mucus gel layer more deeply giving rise to

390

enhanced adhesion forces (30). The existing pH gradient of mucus from the luminal side (pH 5.5)

391

to the apical side (pH 7.4), combined with the pH optima of the covalent bond formation is

392

supporting that theory. As a result, it can be suspected that Sulfo-NHS esters are diffusing the

393

mucus mesh more deeply before they react and thiomers stay rather at the surface of the mucus

394

layer. In contrast to the amide bond, disulfide bonds are reversible under physiological conditions

395

as they can be reduced by glutathione within the mucus. Consequently disulfide bonds might be

396

less stable resulting in lower adhesion forces.

397

In comparison to the PAA-NHS ester of the previous work, with comparable amount of reactive

398

groups (data not shown), stronger adhesive forces were measured upon modification with Sulfo-

399

NHS resulting in an additional 1.4-fold improvement of MDF and a 2.6-fold enhancement of TWA

400

(14). 23 ACS Paragon Plus Environment


Page 24 of 39

401 402 403 404 405

Figure 7: The graphs display the mucoadhesive properties of PAA, PAA-Sulfo-NHS 300, PAASulfo-NHS 800, PAA-Cys 300 and PAA-Cys 800 determined via tensile studies. Figure part A shows the maximum detachment force (MDF) and B the total work of adhesion (TWA). Values are means ± standard deviation of at least three experiments (***P < 0.001).

406

3.6. Rheological investigations

407

Rheological measurements can provide information about the structure of the polymer-mucin

408

network. Increasing viscosity of mucus/polymer mixtures in comparison to the control can be

409

explained by the interactions between the mucus gel network and the polymer molecules. Sufficient 24 ACS Paragon Plus Environment

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410

depth of polymer chain interpenetration with mucins is a crucial requirement for physical

411

entanglement, conformational changes and secondary chemical interactions (6). Main occurring

412

chemical interactions are non-covalent ones like ionic interactions, hydrogen bonding, van-der-

413

Waals forces and covalent bonds as disulfide or amide bonds. Thiomers are able to build a strong

414

three-dimensional network by the formation of disulfide bridges with cysteine-rich subdomains of

415

the mucus glycoproteins (9). According to the theory PAA-Sulfo-NHS esters should also be able

416

to covalently bind to arginine or lysine mucin substructures via amide bond formation. Upon

417

formation of adhesive joints, gel strengthening can occur together with increased resistance to

418

elastic deformation (7).

419

Thus after 2 h incubation time the viscosity of polymer/mucus mixtures increased significantly,

420

except for the PAA control (Figure 8). Beside weak hydrogen bonds, PAA is not forming a covalent

421

network with the mucus and adhesive interactions can only occur to a minor extent. Depending on

422

the degree of modification the viscosity of the samples increased, pointing out the building of gel-

423

like structures. Dynamic viscosity increased up to 2.6-fold (PAA-Sulfo-NHS 300), 10.8-fold

424

(PAA-Sulfo-NHS 800), 10.3-fold (PAA-Cys 300) and 20.7-fold (PAA-Cys 800), respectively.

425

In case of thiomers, the recorded rheological synergism is not only attributed to polymer-mucin

426

interactions, rather the occurrence of intramolecular oxidation under physiological conditions

427

additionally enhances in situ gelling, explaining the higher values in total dynamic viscosity

428

compared to those of the PAA-Sulfo-NHS samples. Unlike thiomers, PAA-Sulfo-NHS esters are

429

not building intramolecular cross-links and the determined extent of increase in viscosity is only

430

caused by direct interactions between polymer and mucin via physical entanglement together with

431

amide bond formation.



Page 26 of 39

432

Attributed to the replacement of NHS by the more hydrophilic Sulfo-NHS, a further 2.3-fold

433

increase in viscosity could be achieved after 2 h incubation. This finding might be explained by

434

virtue of additional non-covalent bindings like ionic interactions between the charged sulfonate

435

group and mucin substructures, or hydrogen bonds (14).

436 437 438 439 440 441 442

Figure 8: Comparison of the dynamic viscosity of the PAA conjugates with intestinal porcine mucus after different time of incubation. Polymers were incubated in a final concentration of 1 % (m/m) in mucus/buffer (100 mM phosphate buffer pH 7.4) mixtures (5/1) at 37 °C. White bars represent the PAA control, light grey bars show the lower modification and dark grey stands for the high modification degree. Figure part A displays results for PAA-Sulfo-NHS and part B illustrates the data of the PAA-Cys derivatives.

443


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444

4. Conclusion

445

Within this study a new type of mucoadhesive covalently binding polymers was compared to

446

thiomers. Both modifications resulted in improved adhesive forces on porcine intestinal mucosa

447

compared to the unmodified polyacrylic acid and furthermore rheological synergism could be

448

confirmed. As a result of the replacement of NHS by Sulfo-NHS, more hydrophilic conjugates

449

could be obtained with the advantage of increased tensile forces and advanced rheological

450

synergism. The major shortcoming induced by the highly hydrophilic character of PAA-Sulfo-

451

NHS derivatives is their high dissolution rate, what makes it difficult to act as a proper drug

452

delivery matrix as strong mucoadhesive joints alone will not do the job. In contrast, thiomers are

453

able to build a stable, cross-linked, highly cohesive intramolecular polymeric network beside

454

covalent disulfide bond formation to mucins upon oxidation under physiological conditions.

455

Hence, a diffusion controlled release of an incorporated drug is possible. In conclusion, thiomers

456

seem to be more advantageous, as disulfide bonds can be regarded as the most important bridging

457

structure in mucus and copying nature seems to be effective in order to generate powerful drug

458

delivery excipients.

459

Associated Content

460

Supporting Information

461

IR and 1H-NMR spectra of educts and PAA-Sulfo-NHS polymer.

462

Sulfo-NHS.

13C-NMR

spectrum of PAA-

463



Page 28 of 39

464

Acknowledgments

465

The Austrian Research Promotion Agency FFG [West Austrian BioNMR 858017] is kindly

466

acknowledged.

467

Reference

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1. Caramella C, Bonferoni MC, Rossi S, Ferrari F. Rheological and tensile tests for the assessment of polymer-mucin interactions1994. 213-7 p. 2. Smart JD, Kellaway IW, Worthington HE. An in-vitro investigation of mucosa-adhesive materials for use in controlled drug delivery. The Journal of pharmacy and pharmacology. 1984;36(5):295-9. 3. Grabovac V, Guggi D, Bernkop-Schnürch A. Comparison of the mucoadhesive properties of various polymers. Advanced drug delivery reviews. 2005;57(11):1713-23. 4. Albarkah YA, Green RJ, Khutoryanskiy VV. Probing the Mucoadhesive Interactions Between Porcine Gastric Mucin and Some Water-Soluble Polymers. Macromolecular bioscience. 2015;15(11):1546-53. 5. Martins ALL, de Oliveira AC, do Nascimento CMOL, Silva LAD, Gaeti MPN, Lima EM, et al. Mucoadhesive Properties of Thiolated Pectin-Based Pellets Prepared by Extrusion-Spheronization Technique. Journal of pharmaceutical sciences. 2017;106(5):1363-70. 6. Madsen F, Eberth K, Smart JD. A rheological examination of the mucoadhesive/mucus interaction: the effect of mucoadhesive type and concentration. Journal of controlled release : official journal of the Controlled Release Society. 1998;50(1-3):167-78. 7. Smart JD. The basics and underlying mechanisms of mucoadhesion. Advanced drug delivery reviews. 2005;57(11):1556-68. 8. Shen J, Wang Y, Ping Q, Xiao Y, Huang X. Mucoadhesive effect of thiolated PEG stearate and its modified NLC for ocular drug delivery. Journal of Controlled Release. 2009;137(3):217-23. 9. Bernkop-Schnürch A. Thiomers: a new generation of mucoadhesive polymers. Advanced drug delivery reviews. 2005;57(11):1569-82. 10. Gök MK, Demir K, Cevher E, Özsoy Y, Cirit Ü, Bacınoğlu S, et al. The effects of the thiolation with thioglycolic acid and l-cysteine on the mucoadhesion properties of the starch-graft-poly(acrylic acid). Carbohydrate polymers. 2017;163:129-36. 28 ACS Paragon Plus Environment

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11. Marschütz MK, Bernkop-Schnürch A. Thiolated polymers: selfcrosslinking properties of thiolated 450 kDa poly(acrylic acid) and their influence on mucoadhesion. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2002;15(4):387-94. 12. Yan JX, Packer NH. Amino Acid Analysis of Mucins. In: Corfield AP, editor. Glycoprotein Methods and Protocols: The Mucins. Totowa, NJ: Humana Press; 2000. p. 113-9. 13. Nanda JS, Lorsch JR. Labeling a protein with fluorophores using NHS ester derivitization. Methods in enzymology. 2014;536:87-94. 14. Menzel C. How to avoid syringes by mucosal drug delivery : addressing the mucus gel layer [Dissertation]: University of Innsbruck; 2018. 15. Staros JV. N-hydroxysulfosuccinimide active esters: bis(Nhydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982;21(17):3950-5. 16. Miron T, Wilchek M. A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. Analytical biochemistry. 1982;126(2):433-5. 17. Schonbichler SA, Bittner LK, Pallua JD, Popp M, Abel G, Bonn GK, et al. Simultaneous quantification of verbenalin and verbascoside in Verbena officinalis by ATR-IR and NIR spectroscopy. Journal of pharmaceutical and biomedical analysis. 2013;84:97-102. 18. Mahmoud DB, Shukr MH, Bendas ER. In vitro and in vivo evaluation of self-nanoemulsifying drug delivery systems of cilostazol for oral and parenteral administration. International journal of pharmaceutics. 2014;476(1-2):60-9. 19. Jennings P, Koppelstaetter C, Aydin S, Abberger T, Wolf AM, Mayer G, et al. Cyclosporine A induces senescence in renal tubular epithelial cells. Am J Physiol Renal Physiol. 2007;293(3):F831-8. 20. O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421-6. 21. Bernkop-Schnürch A, Steininger S. Synthesis and characterisation of mucoadhesive thiolated polymers. International journal of pharmaceutics. 2000;194(2):239-47. 22. Martínez-Mancera FDea. Derivatzation and spectroscopic characterization of a biopolymer based on L-lysine with D-biotin analogs: copoly(L-lysine)-graft-(E-N-[X-D-biotinyl]-L-lysine). Química Nova. 2015;39(1):44-8. 29 ACS Paragon Plus Environment


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23. Staros JV, Wright RW, Swingle DM. Enhancement by Nhydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Analytical biochemistry. 1986;156(1):220-2. 24. Cuatrecasas P, Parikh I. Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry. 1972;11(12):2291-9. 25. Mattson G, Conklin E, Desai S, Nielander G, Savage MD, Morgensen S. A practical approach to crosslinking. Molecular Biology Reports. 1993;17(3):167-83. 26. Elia G. Biotinylation reagents for the study of cell surface proteins. PROTEOMICS. 2008;8(19):4012-24. 27. Staros JV. N-hydroxysulfosuccinimide active esters: bis(Nhydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982;21(17):3950-5. 28. Hornof M, Weyenberg W, Ludwig A, Bernkop-Schnürch A. Mucoadhesive ocular insert based on thiolated poly(acrylic acid): development and in vivo evaluation in humans. Journal of Controlled Release. 2003;89(3):419-28. 29. Imam ME, Hornof M, Valenta C, Reznicek G, Bernkop-Schnürch A. Evidence for the interpenetration of mucoadhesive polymers into the mucous gel layer. Stp Pharma Sciences. 2003;13(3):171-6. 30. Duchene D, Ponchel G. Principle and investigation of the bioadhesion mechanism of solid dosage forms. Biomaterials. 1992;13(10):709-14.

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88x66mm (300 x 300 DPI)



Figure 1: Reaction scheme of carbodiimide mediated syntheses. PAA-Sulfo-NHS (A) was built via ester bond formation between PAA and Sulfo-NHS. PAA-Cys (B) was synthesized via covalent attachment of L-cysteine to the polymer resulting in an amide bond. 177x126mm (300 x 300 DPI)


Page 32 of 39

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Figure 2: Time dependent ester hydrolysis in 0.1 M HCl (A) and 100 mM phosphate buffer pH 7.4 (B). Illustrated values are means of at least three experiments ± standard deviation. PAA-Sulfo-NHS 300 (■) and PAA-Sulfo-NHS 800 (▲). 82x118mm (300 x 300 DPI)



Figure 3: Percentage of hemolysis caused by the controls, Sulfo-NHS and different concentrations of the PAA-Sulfo-NHS and PAA-Cys conjugates. Values represent data of at least three replications plus standard deviation. 104x100mm (300 x 300 DPI)


Page 34 of 39

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Figure 4: Percentage of Caco-2 cell viability caused by the controls, PAA-Sulfo-NHS derivatives in 0.5 % (m/v) (A) and PAA-Cys conjugates (B) in 0.25 % (m/v) (light gray) and 0.5 % (m/v) (dark gray). Cells were incubated for 4 h. Values represent data of at least three replications plus standard deviation. 82x133mm (300 x 300 DPI)



Figure 5: Time dependent water uptake of polymer discs in 100 mM phosphate buffer pH 7.4. Illustrated values are means of at least three experiments ± standard deviation. A: PAA control (●), PAA-Sulfo-NHS 300 (■) and PAA-Sulfo-NHS 800 (▲). B: PAA control (●), PAA-Cys 300 (■) and PAA-Cys 800 (▲). 82x133mm (300 x 300 DPI)


Page 36 of 39

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Figure 6: Mucoadhesive properties of PAA, PAA-Sulfo-NHS 300, PAA-Sulfo-NHS 800, PAA-Cys 300 and PAACys 800. The time of adhesion on freshly excised porcine intestinal mucosa was determined via the rotating cylinder method in 100 mM phosphate buffer pH 7.4. The graph shows the means of at least three experiments ± standard deviation (***P < 0.001). 82x78mm (300 x 300 DPI)



Figure 7: The graphs display the mucoadhesive properties of PAA, PAA-Sulfo-NHS 300, PAA-Sulfo-NHS 800, PAA-Cys 300 and PAA-Cys 800 determined via tensile studies. Figure part A shows the maximum detachment force (MDF) and B the total work of adhesion (TWA). Values are means ± standard deviation of at least three experiments (***P < 0.001). 82x152mm (300 x 300 DPI)


Page 38 of 39

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Figure 8: Comparison of the dynamic viscosity of the PAA conjugates with intestinal porcine mucus after different time of incubation. Polymers were incubated in a final concentration of 1 % (m/m) in mucus/buffer (100 mM phosphate buffer pH 7.4) mixtures (5/1) at 37 °C. White bars represent the PAA control, light grey bars show the lower modification and dark grey stands for the high modification degree. Figure part A displays results for PAA-Sulfo-NHS and part B illustrates the data of the PAA-Cys derivatives. 82x123mm (300 x 300 DPI)


N-hydroxysulfosuccinimide esters versus thiomers: a comparative

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