Synthesis and In Vitro Characterization of a Preactivated Thiomer via

Article pubs.acs.org/Biomac

Synthesis and In Vitro Characterization of a Preactivated Thiomer via Polymerization Reaction Laleh Solhi,† Stefan A. Schönbichler,‡ Sarah Dünnhaupt,† Jan Barthelmes,† Heike Friedl,§ Christian W. Huck,‡ and Andreas Bernkop-Schnürch*,† †

Center for Chemistry and Biomedicine, Innrain 80-82, A-6020 Innsbruck, Austria Institut für Analytische Chemie und Radiochemie, Innrain 80-82, A-6020 Innsbruck, Austria § ThioMatrix GmbH, Research Center, Trientlg 65, Innsbruck, Austria ‡

ABSTRACT: The objective of this study was to synthesize 6-(2acryloylamino-ethyldisulfanyl)-nicotinic acid (ACENA) for subsequent copolymerization with acrylic acid (AA) as a new method for synthesis of preactivated thiomers. Copolymerization reactions of ACENA and AA with different molar ratios were performed and the molecular weight (Mw) values of the resulting copolymers were calculated and reported from 3046 to 3271 Da. The disulfide bond content values in the polymer chain were determined from 400 to 544 μmol disulfide bond per gram polymer. The transport enhancement ratio for 0.5% (m/v) solution of poly(acrylic acid) (PAA) was 1.1 using sodium fluorescein (Na-Flu) as model drug, in Ussing-type chambers, whereas it was over 1.9 for 0.5% (m/v) solution of ACENA and AA copolymers. Resazurin cell-viability test showed no significant toxicity for the polymers. Copolymerization of AA and disulfidebond-containing monomers can open new horizons for the preparation of preactivated thiomers taking the better controllability and the huge variety of available monomers and combinations thereof into account.

1. INTRODUCTION The oral administration of drugs is by far the most favored route of application. In many cases, however, the oral bioavailability of drugs is strongly limited by an insufficient uptake from the intestinal mucosa. To overcome this so-called absorption barrier, permeation enhancers are used as auxiliary agents in oral drug delivery systems. Most of these permeation enhancers are small molecules being absorbed much more rapidly than the drug itself.1 Therefore, systemic side effects of these auxiliary agents cannot be excluded. One alternative class of permeation enhancers that has received lots of attention in order to overcome these shortcomings are polymers such as polyacrylates or chitosans. As these polymers will not be absorbed from the gastrointestinal tract, systemic side effects can be precluded and a prolonged permeation enhancing effect provided.1−4 Among this group of polymers, thiomers are of particular interest, as they exhibit comparatively higher permeation enhancing properties along with mucoadhesive, efflux pump, and enzyme−inhibitory effects.5,6 Besides these advantages, however, the synthesis of thiomers is associated with difficulties arising from the chemical nature of thiolmoieties. Synthesis of thiomers goes hand in hand with unintended oxidation reactions leading to the formation of disulfide bonds between the thiol groups of thiomer prior to their reaction with thiol groups on the mucosa. Various efforts have been undertaken to avoid this oxidation such as keeping the reaction in acidic conditions or post reduction of thiomers after synthesis.6 Another approach to solve this problem is the © 2012 American Chemical Society

attachment of mercaptopyridines via disulfide bonds to the polymer chain, resulting in polymers with better oxidation stability. Such preactivated thiomers, however, were so far synthesized by derivatization of well-established polymers such as polyacrylates generally leading to only comparatively low degrees of thiolation.7 To overcome this shortcoming and to open the door to the generation of various further polymers of this class, it was the aim of the present study to develop a synthetic pathway for preactivated thiomers based on polymerization reaction. This reaction might be a promising method to produce preactivated thiomers in a controlled and reproducible manner. Within this study 6-mercaptonicotinic acid (6-MNA) was attached to cysteamine (Cys-Am) via an oxidation reaction between both thiol groups using hydrogen peroxide (H2O2). The purified product was subsequently reacted with acryloyl chloride (ACl) in a two-phase reaction and in the presence of sodium hydroxide (NaOH), resulting in ACENA. Final copolymerization reactions of ACENA with acrylic acid were performed in DMSO using 2,2′-azobis(2-methylpropionitrile) (AIBN) as initiator. These newly synthesized copolymers were investigated in terms of their molecular weight, disulfide bond formation, and cytotoxicity, as well as permeation enhancing effect. Received: May 21, 2012 Revised: August 22, 2012 Published: August 23, 2012 3054

dx.doi.org/10.1021/bm300788d | Biomacromolecules 2012, 13, 3054−3063

Biomacromolecules

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Table 1. Combinations of Comonomers and Initiator in Different Batches name of the product

ACENA (g)

ACENA (% molar of total monomers)

AA (μL)

AA (% molar of total monomers)

monomer to solvent (mol L−1)

AIBN (mg)

AIBN (% molar of total monomers)

Cp-1 Cp-2 Cp-3 PAA-4

1.36 0.91 0.45 0

30 20 10 0

769 877 988 1098

70 80 90 100

8 8 8 8

14 14 14 14

0.5 0.5 0.5 0.5

2. EXPERIMENTAL SECTION Materials. 6-MNA, Cys-Am, ACl, L-cysteine hydrochloride anhydrous, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), AIBN, H2O2, NaOH, water HPLC-grade, acetonitrile (ACN) Chromasolv HPLC-grade, trifluoroacetic acid (TFA), ethanol Chromasolv HPLCgrade, sinapinic acid, 3ß-indoleacrylic acid (IAA), DMSO with 0.03% TMS, N-(2-hydroxyethyl) piperazine-N8-(2-ethane-sulfonic acid) (HEPES), resazurin, Triton X-100, phosphate-buffered saline (PBS), and sodium fluorescein (Na-Flu) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All the other solvents were of analytical grade and received from commercial sources. Preparation and Purification of ACENA. The monomer was synthesized using a two-step procedure. In the first step, the intermediate product 6-(2-Amino ethyldisulfanyl) nicotinic acid (AMENA) was prepared. 6-MNA and Cys-Am were attached to each other due to an oxidation reaction of their thiol groups.8 For this, 6-MNA (2 g, 12.89 mM) and Cys-Am (1 g, 12.89 mM) were dissolved in 40 mL DMSO/H2O (80/20, v/v) while stirring. Then H2O2/H2O mixture (3/7, m/m; 1317 μL, 12.89 mM) was added to the solution and stirred at room temperature overnight. The resulting white precipitation was filtered and washed three times with the DMSO/ H2O mixture (100 mL) to remove H2O2, 6-MNA, Cys-Am, and CysAm dimer. The wet cake, containing AMENA and 6-MNA, was redispersed in DMSO and stirred for 30 min, then filtered and washed with DMSO (100 mL) three times. In this way, the two substances were separated and the purified AMENA could be obtained. The second step of the reaction was performed according to the SchottenBaumann reaction.9,10 Briefly, AMENA (2.7 g, 11.65 mM) produced in the first step was dissolved in water (200 mL), using a four-necked 400 mL flask equipped with a stirrer, a thermometer, and two 50 mL dropping funnels. After the mixture was cooled down to 0−5 °C, an acryloyl chloride solution in dichloromethane (11.65 mM) and a 1 M NaOH aqueous solution (23.3 mL, 23.30 mM) were added simultaneously and dropwise under stirring over a total time span of one hour; the temperature was kept at 0−5 °C. When the addition was complete, the reaction mixture was stirred at room temperature for an additional 2 h. Then the aqueous phase was separated from the organic one and its pH was adjusted to 1−2 with a 2 M HCl aqueous solution. A translucent suspension was thus obtained. The white precipitation, which is the pure ACENA was filtered and washed with triflouroacetic acid aqueous solution (pH = 1−2; 100 mL) three times and dried.11 Copolymerisation of ACENA and AA. Different ratios of ACENA and AA were dissolved with AIBN in DMSO (2 mL) according to Table 1. A small amount of 6-MNA (30 mg) was added to all the batches as a chain transfer agent to obtain a monodisperse polymer.12 The reactant solutions were stirred at 80 °C in closed test tubes for 1 h. The resulting viscous reaction mixtures were cooled down and dialyzed against DMSO (4 days) and water (1 day) using a cellulose membrane (Sigma dialysis tubing, benzoylated, avg. flat width 32 mm, molecular mass cutoff 2 kDa) to purify the polymer from small molecules. The dialysis procedure was continued, while the solvent was changed each 12 h and UV-absorbance was measured using a UVmini 1240 spectrophotometer (Shimadzu, Kyoto, Japan) at the wavelengths of 307 nm (corresponding to 6-MNA) and 213 nm (corresponding to AA). On the last day, no UV-absorbance related to 6-MNA or AA was observed in the dialysis solvent. All polymers that were purified by dialysis were then lyophilized by drying frozen polymer solutions at −50 °C and 0.01 mbar (Christ Beta 1−8 K; Osterode am Harz, Germany)13 and stored at 4 °C until further use. The whole synthetic path of the reaction is illustrated in Figure 1.

Figure 1. Synthetic path of ACENA and copolymers. Determination of Thiol and Disulfide Bond Content. The amount of thiol moieties on the copolymers was determined photometrically using Ellman’s reagent (DTNB). First, each polymer (0.5 mg) was hydrated in demineralized water (0.25 mL). After addition of 0.5 M phosphate buffer pH 8 (0.25 mL), DTNB 0.03% (m/v, 0.5 mL) was added. The samples were incubated for 2 h at room temperature and the precipitated polymer removed by centrifugation (24000 × g, 5 min). Samples (0.3 mL) were transferred to a 96-well microtitration plate, and the absorbance was immediately measured at 450 nm with a microtitration plate reader (Tecan Austria GmbH, Grödig, Austria). The amount of thiol moieties on the polymer was calculated from a standard curve of L-cysteine hydrochloride in 0.5 M phosphate buffer pH 8 in a concentration of 0.8−25.6 μg mL−1. Disulfide bond content was measured after reduction with NaBH4 and conversion to thiol groups.14 Characterization of ACENA and Polymers. ACENA was characterized using IR, 1H NMR, HPLC-DAD, and MALDI-TOFMS, and polymers were characterized using IR, GPC, and MALDITOF-MS. IR spectra were obtained on a Perkin-Elmer Spectrum 100 instrument (Perkin-Elmer, MA, U.S.A.) equipped with an attenuated-total-reflectance sampling accessory. A total of 20 scans per spectra were taken with a resolution of 1 cm−1 at a temperature of 22 °C. The mass spectra were collected with a Bruker Ultraflex MALDI TOF/TOF (Bruker Daltonics, Bremen, Germany) employing a stainless steel target (MTP 384 target ground steel TF, Bruker Daltonics). For the mass determination of the monomer, sinapinic acid was used as a matrix. A saturated solution of sinapinic acid in water/ ACN (50/50, v/v), containing 1% TFA was prepared and mixed with a 1% (m/v) ethanolic solution of sample (9/1, v/v). Positive ions were detected in reflectron mode. The mass spectra were an average of 200 3055


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Table 2. Yield, 1H NMR Data, and HPLC-DAD Data of the Products for Two Steps of Monomer Synthesis reaction parts

product

yield (%)

step 1

AMENA

90.4

step 2

ACENA

40.3

1

2.65 (CH2-4), 2.95 (CH2-5), 4.78 (H2O), 7.53 (CH-3), 8.24 (CH-2), 8.89 (CH-1), 13.62 (OH) 2.52 (DMSO), 2.87 (CH2-4), 3.39 (CH2-5), 5.60 (CH2-8), 6.117 (CH2-7), 7.94 (CH2-6), 8.27 (CH-3), 8.36 (CH-2), 8.92 (CH-1)

shots. To analyze polymer masses, IAA was used as a matrix. The IAA was prepared as a saturated THF solution and mixed with a 5% (m/v) polymer solution in DMSO in a ratio of 49:1 (v/v). The negative ions were detected in linear mode. Spectra were an average of 400 shots. HPLC-DAD measurements were performed for 25 min with a Shimadzu LC-10AD HPLC-DAD (Shimadzu, Kyoto, Japan) using a Thermo Scientific Bds Hypersil C18 column, 250 × 4 mm, particle size 5 μm (Thermo Fisher Scientific, Waltham, U.S.A.). Solvent A was water with 0.1% TFA and solvent B was ACN. The gradient was from 7 to 70% solvent B in 10 min followed by a washing and equilibration step. Temperature was set to 25 °C. Detection wavelength was 293 nm. The 1H NMR spectra were collected with a Varian Gemini 200 spectrometer (200 MHz), chemical shifts are reported in parts per million, and tetramethylsilane (TMS) was used as an internal standard. 1 H NMR characterization of AMENA was carried out in D2O and for ACENA it was done in deuterated DMSO. The gel permeation chromatography measurements were done in water as solvent using a GPC instrument (Agilent 1100 series, Santa Clara, U.S.A.) and an Aquagel column of 7.5 × 300 mm (ID × L) with a flow rate of 1 mL min−1 at 23 °C. Permeation Studies. Permeation studies were carried out in Ussing-type chambers displaying a volume of 1 mL in the donor and acceptor compartments and a permeation area of 0.64 cm2. To mimic the intestinal fluid, an incubation medium was prepared containing 138 mM NaCl, 1 mM MgSO4, 5 mM KCl, 10 mM glucose, and 2 mM CaCl2 buffered with 10 mM HEPES, pH 6.8. Immediately after sacrificing the rat, 15 cm of the small intestine (duodenum) were excised and mounted in the Ussing chamber. After 15−20 min of preincubation with the medium, the incubation medium of the donor compartment was substituted by 1 mL of the polymers solution (0.5% m/v). Furthermore, each sample contained 0.001% (m/v) Na-Flu as model compound. Samples of 100 μL were withdrawn from the acceptor compartment every 30 min over a time period of 3 h. Samples were immediately replaced by incubation medium (100 μL) equilibrated at 37 °C. The amount of permeated Na-Flu was determined using a Spectra Fluor-fluorimeter (Tecan Austria GmbH, Grö dig, Austria).15 Cumulative corrections were made for the previously removed samples. The apparent permeability coefficients (Papp) for Na-Flu were calculated according to eq 1:

Papp =

Q A×c×t

10.6

avg absorbance value of each triplicate × 100 positive control (3) Statistical Analyses. The results were analyzed and compared using one-way ANOVA and the Tukey’s test at the significance level of 0.05 using SPSS 17 software (IBM, Armonk, U.S.A.). cell viability(%) =

3. RESULTS Characterization of ACENA. The synthetic path of ACENA consists of two steps, first the disulfide bond formation of 6-MNA and Cys-Am giving AMENA, and the second step is the reaction of AMENA and ACl resulting in ACENA. The products of each step were separated, purified and characterized. The yield of each step and 1H NMR and HPLC-DAD data of purified AMENA and ACENA are summarized in Table 2. The total yield of the reaction for synthesis of ACENA was 36.4%. The purity of the products after purification was proved by HPLC-DAD, 1H NMR, and MALDI-TOF-MS. The HPLCDAD chromatograms showed elution times for 6-MNA and 6MNA-dimer of 7.480 and 11.908 min, respectively. The IR and MALDI spectra of ACENA are represented in Figures 2 and 3, respectively. ACENA exhibited strong IR peaks in the range of amide, carboxyl, and carbon−carbon double bond, as shown in Figure 2: 766 cm−1 (C−H bending, alkene), 1100 and 1147 cm−1 (CN stretching, aromatic), 1228 cm−1 (C−O stretching, acid), 1259 cm−1 (C−N stretching, amide), 1368 and 1402 cm−1 (CC stretching, aromatic), 1586 cm−1 (N−H bending, amide), 1616 cm−1 (CC stretching, alkene), 1650 cm−1 (CO stretching, amide), 1699 cm−1 (CO stretching, acid), 2618 cm−1 (O−H stretching, acid), 2817 cm−1 (C−H stretching, alkane), 3060 cm−1 (C−H stretching, aromatic), 3218 cm−1 (C−H stretching, alkene), 3436 cm−1 (N−H stretching, amide).16 The masses found by MALDI-TOF-MS confirm the structure of ACENA: 207.023 (matrix, Mw of sinapinic acid + H−H2O) and 225.067 (matrix, Mw of sinapinic acid + H), 285.138 (Mw of ACENA + H).17 Characterization of Copolymers. The IR spectroscopy data of the Cp-1 and the PAA-4, confirm the expected polymer structures and functionalities. For PAA-4 the peaks were detected at 941 and 997 cm−1 (C−C skeletal vibrations), 1163

(1)

Papp(sample) Papp(control)

8.9

resazurin measurements at day 14 after plating. The medium was replaced by 0.5% solution of polymers in colorless MEM (0.5 mL) after washing the cells with PBS (500 μL) two times. Experiments were performed in triplicate. The plate was incubated at 37 °C, 5% CO2, 95% relative humidity for 24 h. The standard assay setup included the following: blank (culture medium alone), positive control (culture medium with the cells), and negative control (5%, v/v, Triton-X 100 in MEM). For the assay, a resazurin solution in colorless MEM (1/19, v/v) was thawed. After washing the cells with PBS solution two times, 500 μL of the thawed resazurin solution were transferred into each well of the plate and incubated for 24 h at 37 °C. The absorbance of the supernatant was measured at a wavelength of 540 nm with background subtraction at 590 nm after 3 and 24 h, with a spectrophotometer (iEMS, Labsystems, Finland). The cell viability percent was calculated using eq 3:

where Papp is the apparent permeability coefficient (cm s−1), Q is the total amount permeated within the incubation time (mg), A is the diffusion area of the Ussing chamber (cm2), c is the initial concentration of the marker in the donor compartment (mg cm−3), and t is the total time of the experiment (s). Transport enhancement ratios (R) were calculated from Papp values according to the eq 2: R=

HPLC-DAD elution time of the product (min)

H NMR of the product (ppm)

(2)

Cytotoxic Effects. This test was performed on Caco-2 cells and the medium was composed of 80% minimum essential medium (MEM), 20% FCS, and 1% penicillin−streptomycin liquid. The cells were plated in 24-well plates at a density of 1 × 105 cells/well in a final volume of 0.5 mL cell culture medium, mentioned above, and incubated at 37 °C in an atmosphere of 5% CO2, 95% relative humidity. The medium was changed 48 h after plating and subsequently every second day. Caco-2 cell cultures were used for 3056


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Figure 2. IR spectrum of ACENA.

and 1236 cm−1 (C−O stretching, acid), 1316 cm−1 (O−H inplane bending, acid), 1405 cm−1 (C−H bending, CH2), 1705 cm−1 (CO stretching, acid), 2923 cm−1 (C−H stretching, CH2), 3013 cm−1 (C−H stretching, CH).18 The peak data for Cp-1 includes 955 and 982 cm−1 (C−C skeletal vibrations), 1163 and 1236 cm−1 (C−O stretching, acid), 1316 cm−1 (O−H in plane bending, acid), 1405 cm−1 (C−H bending, CH2), 1705

cm−1 (CO stretching, acid), 2923 cm−1 (C−H stretching, CH2), and 3013 cm−1 (C−H stretching, CH), which are similar to PAA-4, and the new peaks at 818 and 848 cm−1 (C−H out of plane bending, aromatic), 1068 and 1025 cm−1 (C−H in plane bending, aromatic), 1140 and 1186 cm−1 (CN stretching, aromatic), 1260 cm−1 (C−N stretching, amide), 1366 and 1452 cm−1 (CC stretching, aromatic), 1563 cm−1 3057


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Figure 3. MALDI-TOF-MS spectrum of ACENA.

(N−H bending, amide), 1623 cm−1 (CO stretching, amide), 1657 cm−1 (CO stretching, acid, ACENA), 2615 cm−1 (O− H stretching, acid, ACENA), 3064 cm−1 (C−H stretching, aromatic), and 3243 cm−1 (N−H stretching, amide).19 Cp-1 displayed 109.7 ± 39.8 μmol thiol group and 543.5 ± 82.3 μmol disulfide bond per gram polymer, while thiol group moieties for Cp-2 and Cp-3 were 96.5 ± 31.5 and 85.1 ± 16.6 μmol g−1. The disulfide bond contents for Cp-2 and Cp-3 were 483.2 ± 75.3 and 399.9 ± 69.2 μmol g−1, respectively. Results showed that thiol groups are formed in a constant ratio with disulfide groups during the polymerization process. The weight percentage of ACENA in polymer was easily calculated from the disulfide bond contents. These amounts were calculated: 15.3% for Cp-1, 13.6% for Cp-2, and 11.3% for Cp-3. The MALDI-TOF-MS spectra of the synthesized polymers are displayed in Figure 4. According to MALDI-TOF-MS spectra, the Mp (molecular weight of the highest peak of the spectrum), Mn, Mw, and polydispersity index (PDI) were 2880 Da, 3044 Da, 3271 Da, 1.07 for Cp-1; 2611 Da, 2837 Da, 3046 Da, 1.07 for Cp-2; 2613 Da, 2983 Da, 3203 Da, 1.07 for Cp-3;

and 2490 Da, 2801 Da, 3046 Da, 1.09 for PAA-4, respectively. For the Cp-1 and PAA-4, GPC measurements were performed in addition to MALDI-TOF mass-spectrometry to compare the results and ensure the reliability of MALDI-TOF-MS results. The GPC chromatogram of these two samples included a high and narrow peak, which is shown in Figure 5. Mp, Mn, Mw, and PDI values, calculated from GPC chromatograms, were 2624 Da, 2657 Da, 2916 Da, 1.1 for Cp-1 and 2425 Da, 2525 Da, 3020 Da, 1.2 for PAA-4, respectively. The MALDI-TOF-MS results are in good agreement with values obtained from GPC. Evaluation of Permeation Enhancement Studies. Permeation enhancers are added to increase the bioavailability of noninvasive administered drugs, which show just a limited absorption based on their charge and molecular mass.20 Thiolated polymers are known to exhibit a strong permeation enhancing effect for the paracellular uptake of drugs. To evaluate this effect of the synthesized copolymers, permeation studies were performed in Ussing chambers. The influence of 0.5% of polymers for the cumulative transport of Na-Flu, widely used as marker for the paracellular pathway, was plotted as 3058


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Figure 4. MALDI-TOF-MS spectra of (a) Cp-1, (b) Cp-2, (c) Cp-3, and (d) PAA-4, measured in negative linear mode with IAA as matrix using a stainless steel target.

cumulative transport over a time period of 180 min.15,21 The transport enhancement ratio (R) was calculated for all the polymers and determined to be 2.5 for Cp-1, 2.1 for Cp-2, 1.9 for Cp-3, and 1.1 for PAA-4. Percentage of permeated Na-Flu is shown in Figure 6 and demonstrated an increased transport of Na-Flu across the intestinal mucosa for all copolymers. Comparison of the permeation enhancing properties of PAA4 and Cp-1, for instance, revealed a stronger effect of the latter one (different from PAA-4, p < 0.05). Furthermore, the influence on the permeation enhancing effect of copolymers with different amounts of ACENA in the backbone of the

polymer was studied. The results confirmed that the synthesized preactivated thiomer (Cp-1) was able to improve the permeated Na-Flu level and the permeation enhancement ratio. Nevertheless, the increase of the ACENA-content in range of the synthesized copolymers did not statistically increase the amount of Na-Flu permeated through the membrane within 3 h of incubation (no significant difference among Cp-1, Cp-2, and Cp-3 (p < 0.05)). Evaluation of Cytotoxicity by Resazurin Assay. Cell viability, determined by resazurin test, was performed after 3 and 24 h exposure to the polymer solutions. The cell viability 3059


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Figure 5. GPC chromatogram of CP-1 (a) and PAA-4 (b).

data are shown in Figure 7. As it is demonstrated in the figure, the cell viability in all cases was more than 80%, confirming the nontoxicity of all samples during 3 and 24 h of exposure. The viability of PAA-4 after 3 h is significantly higher than the viability of Cp-1 and Cp-2 after 24 h (p < 0.05), which can be due to the higher moiety of ACENA in Cp-1 and Cp-2 and the longer time of exposure.

4. DISCUSSIONS The first step of monomer-synthesis included a mild oxidation of thiol groups to a disulfide bond. It was important not to use more than a stoichiometric amount of oxidizer (H2O2) or to avoid too strong oxidizing agents, otherwise the oxidation of thiol groups would continue further to sulfenic or sulfonic acids.22 The second step was categorized under the reactions between a primary amine and acyl chloride, known as 3060


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Figure 6. Permeation enhancing effect of copolymers (0.5%, m/v) in comparison to Na-Flu and PAA homopolymer. Indicated values are means of three experiments ± SD. The permeation enhancement of Cp-1 is different from that of PAA-4 and Na-Flu (p < 0.05).

Figure 7. Resazurin test results after 3 and 24 h (Cell viability mean ± SD). The viability of PAA-4 after 3 h is significantly higher than the viability of Cp-1 and Cp-2 after 24 h (p < 0.05).

Schotten−Baumann reactions. In these reactions, as shown in Figure 1, an amide was formed together with a proton and a chloride ion. The addition of a base was required to absorb the acidic proton or the reaction will not proceed. In this reaction, a two-phase solvent system was used, consisting of water and an organic solvent. The base within the water phase neutralized

the acid generated during the reaction, while the acyl chloride was added gradually in organic phase.11 The purification of the products in each step was carried out by using solubility differences and eventually resulted in products of high purity. Purity was proven by HPLC-DAD, 1H NMR, and MALDITOF-MS. The 1H NMR data, IR spectroscopic data, and 3061


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assumed that glutathione in reduced form (GSH) is able to inhibit PTP activity via disulfide bond formation with its active site cysteine, leading to an enhanced opening of the tight junctions.26 However, this inhibitory effect of GSH is limited by being rapidly oxidized to GSSG. Thiomers seem to prevent the oxidation of GSH, and being attached on the mucosa as a result of their high mucoadhesive properties can shift the balance between GSSG and GSH on the membrane to the side of GSH, which leads to increased tight junction opening and subsequent permeation enhancement.1 Explanation for the mechanism of preactivated thiomers regarding permeation enhancement is still nebulous and only partially understood. Nevertheless, one reason for this enhanced permeation might be based on the more reactive thiol groups in the form of disulfide bonds available for interaction with the mucosa, which leads rapidly to a high GSH concentration and the opening of tight junctions. While thiomers mainly react with other thiol groups through oxidative coupling over a long period, preactivated thiomers interact subsequently by thiol disulfide exchange reaction under release of the pyridyl substructure.27 The alamar blue used in the cytotoxicity assay is nontoxic to the cells and reduced by the cells into resorufin (the pink fluorophor). This reduction is directly proportional to the number of viable cells.28 The reason for the higher toxicity of Cp-1 and Cp-2 might be due to the protonation of amine groups of ACENA on the chain.29 The higher the content of ACENA in the copolymer, the more positive charges are located on the chains. Cytotxicity of polymers is directly related to their surface charge density. Because PAA-4 lacks ACENA, it showed the lowest cytotoxicity.30 In future investigations ATRP, RAFT, or other free-radical polymerization techniques could be performed to increase the molecular weight and to improve permeation enhancing properties of the copolymers. Due to the controllability and variety of the polymerization reactions, synthesizing copolymers in the way described within this study can open new horizons for the preparation of various further preactivated thiomers.

masses obtained from MALDI-TOF-MS measurements were in full agreement with the proposed structures. Comparing IR spectra data of Cp-1 and PAA-4, additional peaks in the region from 1450 to 1660 cm−1 in the Cp-1 spectrum occurred, which were associated with the presence of aromatic and amide structures. In both IR spectra of PAA-4 and Cp-1 there was no peak at 1616 cm−1, which corresponds to bending CC alkene, while it could be easily found in the IR spectrum of ACENA in Figure 2. Considering that all the polymers were purified from small molecules, these data proved the presence of ACENA in the Cp-1 and the loss of CC alkene in both PAA-4 and Cp-1 as a consequence of the polymerization process. It was assumed that high steric hindrance is the reason for the failed homopolymerization of ACENA. Due to the appearance of a single narrow peak in GPC, the nearly Gaussian-distributed peaks in the MALDI-TOF-MS spectra and the appearance of specific absorbances of ACENA in the IR spectra of the polymers, it can be concluded that the synthesized polymers are copolymers of ACENA and AA. The weight percentage of ACENA in the polymer chains was not as high as the amount that was injected in each batch to the reactor, and according to the disulfide bond content test results, it increased while the higher amount of ACENA was present in the reaction mixture. Precise interpretation of the comonomer composition in copolymer chains depended on the reactivity ratios (rr) of ACENA and AA. As rr of ACENA was not reported in literature, further research on this topic could open new aspects of this subject.23 The presence of low amounts of thiol groups in the copolymer chains corresponded to the detachment of the 6MNA unit from the polymer backbone during the polymerization reaction due to the high temperature of reaction mixture. The trace amount of free 6-MNA that was used in the polymerization step as the chain transfer agent can also be a cause of disulfide bond cleavage according to the thiol-disulfide exchange mechanism.24 These effects could be confirmed by the appearance of a yellow color during the polymerization reaction. As the trace amount of 6-MNA, used as chain transfer agent, was responsible for a very pale yellow color, but the thiol group formation during the reaction results in a more yellowish reaction mixture compared to the beginning of the polymerization. The decomposition of ACENA in the polymer chain and the cleavage of disulfide bonds resulted in formation of thiol groups on some sites of the polymer. These thiol groups can be responsible for the improved permeation of Na-Flu caused by Cp-1 and the higher permeation enhancement ratio of copolymers in comparison with PAA-4.1 The MALDI-TOFMS spectra and GPC results demonstrated a narrow PDI (

Synthesis and In Vitro Characterization of a Preactivated Thiomer via

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