Chitin dipentanoate as the new technologically usable biomaterial

Materials Science and Engineering C 55 (2015) 50–60

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Chitin dipentanoate as the new technologically usable biomaterial Karolina Skołucka-Szary a,⁎, Aleksandra Ramięga a, Wanda Piaskowska a, Bartosz Janicki b, Magdalena Grala c, Piotr Rieske a, Ewelina Stoczyńska-Fidelus a, Sylwester Piaskowski a a b c

Department of Research and Development, Celther Poland Sp. z o.o., ul. Ostrzykowizna 14A, 05-170 Zakroczym, Poland Silesian University of Technology, Faculty of Chemistry, Department of Physical Chemistry and Technology of Polymers, ul. M. Strzody 9, 44-100 Gliwice, Poland Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

a r t i c l e

i n f o

Article history: Received 27 February 2015 Received in revised form 20 April 2015 Accepted 15 May 2015 Available online 20 May 2015 Keywords: Chitin Esterification Biomaterial Scaffolds

a b s t r a c t In this article, the synthesis of novel biopolymer, chitin dipentanoate (Di-O-Valeryl Chitin, DVCH) has been described. DVCH is a chitin derivative esterified with two valeryl groups at positions 3 and 6 of the Nacetylglucosamine units and it is soluble in common organic solvents like ethanol, methanol, acetone, dichloromethane, 1,2-dichloroethane, N,N-dimethylmethanamide, N,N-dimethylacetamide and ethyl acetate. Highly efficient synthesis (degree of esterification close to 2) of DVCH was achieved by employing a huge excess of valeric anhydride used as both the acylation agent and the reaction medium in the presence of perchloric acid as catalyst. Studies on the DVCH synthesis were aimed at finding optimal conditions (temperature, reaction time) to obtain DVCH with high reaction yield and desirable physicochemical properties. Biological data demonstrate that DVCH is non-cytotoxic in vitro and doesn't exert irritating or allergic effects to animal skin. Thanks to its filmogenic properties, it can be used to manufacture threads, foils, foams and non-woven materials. Moreover, DVCH can be easily processed by salt-leaching method to prepare highly porous structures exhibiting open-cell architecture, that can be further employed in wound dressing therapies and scaffolds for tissue engineering. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Chitin is one of the most widespread natural polysaccharides in nature. Chitinous structures are found in at least 19 animal phyla [1]. It is a major structural constituent of the cell walls of fungi [2,3], the mollusk shells [4], and the exoskeleton of arthropods such as crustaceans [4], insects [5] and arachnids [6]. It is also a characteristic component of yeasts [7], sponges [4,8,9], anthozoans [10] and diatoms [11]. Currently, the main source of chitin production is based on the isolation of chitin from marine invertebrates including crabs, krill, shrimps, lobsters and others (79%), and fungi (21%) [12]. Chitin and its derivatives are applicable in fields such as: food, cosmetics, agriculture, textiles, wastewater treatment, and pharmaceutical industries [2,13,14]. Recent studies have focused on the role of chitin and its derivatives in pharmaceutical and biomedical materials [15,16], especially on their application in wound dressing materials, artificial skin substitute and sutures [17,18] as well as in extreme biomimetics [19]. The main technological limitation of the large scale application of chitin in the pharmaceutical and biomedical industries lies in chitin insolubility in common organic solvents, what significantly hinders its processing. Chitin (α-chitin) resistance to solvents is related to the peculiar characteristic property of inter- and intra-chain hydrogen bonds ⁎ Corresponding author at: Celther Polska Sp. z o.o., Ostrzykowizna 14A, 05-170 Zakroczym, Poland. E-mail address: [email protected] (K. Skołucka-Szary).

http://dx.doi.org/10.1016/j.msec.2015.05.051 0928-4931/© 2015 Elsevier B.V. All rights reserved.

occurring in the polymer chain [20–23]. It has been found that chitin is soluble in aqueous thiourea, alkaline aqueous urea, dimethylformamide/ lithium chloride system, dimethylacetamide/lithium chloride system, ionic liquids [24–26], certain fluorinated solvents such as hexafluoroacetone, hexafluoro-2-propanol, hexafluoroisopropyl alcohol [27,28], methanesulfonic acid [29–31], methanol saturated with calcium chloride dihydrate [32], trichloroacetic acid and dichloroacetic acid systems [28] and others. Many scientific reports focused on improvement of chitin solubility via chemical reaction are available. For example, it has been reported that chitin solubility under neutral or alkali conditions can be obtained via reaction of chitin with cyclic dicarboxylic anhydrides in the presence of aqueous alkanesulfonic acid [33]. In contrast to α-chitin, β-chitin is characterized by weaker intermolecular hydrogen bonding, hence it swells in water (creates a slurry) and is soluble in formic acid [20]. Many solvents used for chitin dissolution are toxic, corrosive, and mutagenic and therefore cannot be used in medicinal applications and, moreover, are inconvenient for industrial production. Consequently, practical use of chitin is based mainly on the use of its amid- and ester-derivatives. The O-acylation with acid anhydrides in the presence of acidic catalyst (methanesulfonic acid or perchloric acid) results in formylated, propionylated or butyrylated chitins, all of which are characterized by good solubility in organic solvents and the lack of solubility in aqueous media [34]. Alternative methods of chitin ester preparation include the synthesis in the presence of fatty acids in trifluoroacetic anhydrides [35]

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

and acylation with butyric acid in the presence of trifluoroacetic anhydride/phosphoric acid system [36]. Chitin esters, mostly dibutyrylchitin, are seen as the prospective biomaterials due to their good solubility in common organic solvents. Their unique properties like biocompatibility, free-radical scavenging, liposolubility and susceptibility to biodegradation and chitin regeneration, allow them to be used in wound dressings or as scaffolds for cartilage and bone repair [37–44]. The aim of this study was to obtain a new biocompatible chitin ester derivative, which could be easily technologically processed (due to its good solubility in organic solvents like ethanol, acetone, DMAC etc.) and would possess appropriate physical, chemical and mechanical properties, giving the opportunity to use it in the field of medical devices, mainly in wound dressing materials. 2. Materials and methods 2.1. Synthesis of chitin dipentanoate (Di-O-Valeryl Chitin) Valeric anhydride (97%, Sigma Aldrich), perchloric acid (70%, Aldrich), acetone (pure, POCH), dimethylacetamide (DMAc, pure, POCH), LiCl (anhydrous, pure, POCH), and NaHCO3 (pure, POCH) were used without further purification. Krill chitin α-type (France Chitine, Orange, France, degree of acylation of 0.98, intrinsic viscosity value of 10.8 dl/g determined in DMAC/5% LiCl at 25 °C and viscosity average molar mass of 197,020 Da) was purified from residual calcium carbonate and other mineral residues. During the purification step, chitin was treated with 2 M HCl for 2 h and then washed with pH 7 distilled water, filtered and dried in a desiccator. Di-O-Valeryl Chitin (DVCH) was prepared by means of a two-step method. In the first step of the synthesis the acylation mixture was prepared. 2.5 ml of perchloric acid was added to valeric anhydride (34 ml) kept in a cooling bath at a temperature ranging from −5 to 0 °C while stirring. In the chitin acylation step, small portions (5 g) of purified chitin were added step by step to freshly prepared acylation mixture kept at a temperature around 0 °C and continuously stirred. The reaction mixture was further homogenized with an anchor-shaped stirrer equipped with flow baffles for approximately 10 min. Subsequently, the synthesis was carried out under conditions (temperature, time) provided in Table 1. Reaction mixtures from all runs were neutralized using aqueous solution of 2.5% NaHCO3, then washed with water with pH 7, filtered and dried. During the neutralization step the reaction mixture was fragmented using the anchor-shaped stirrer equipped with flow baffles. The raw products were dissolved in acetone, filtrated to separate the unreacted chitin, precipitated with distilled water, and dried. Finally, the product was dried in vacuum. Factors such as the yield of reaction, intrinsic viscosity, average molecular weight and molecular weight distribution of obtained products Table 1 The results of DVCH synthesis under various conditions (time, temperature) versus reaction yield and final product intrinsic viscosity. Sample name

3a 3b 3c 3d 8a 8b 16c 16d 6a 6b 6c 6d

Reaction yield

Reaction time

Intrinsic viscosity

Reaction temperature

[%]

[h]

[dl/g]

[°C]

42.0 52.0 64.4 80.9 59.6 75.8 79.1 83.5 78.1 83.4 84.3 84.8

2 4 6 24 2 4 6 24 2 4 6 24

1.80 1.67 1.59 1.38 1.53 1.38 1.29 0.97 1.12 0.92 0.83 0.53

0

6

21

51

were monitored and measured in the function of reaction conditions. Each synthesis was repeated 5 times. 2.2. Characteristic of DVCH samples 2.2.1. Intrinsic viscosity The intrinsic viscosity [η] of chitin was determined in DMAC/5%LiCl system at 25 °C using the Ubbelohde viscometer. Chitin viscosity average molar mass Mv was calculated using the Mark–Houwink equation [26] [η] = 0.0024 × M0.69 w . The coefficients of intrinsic viscosity [η] of the obtained samples of DVCH were determined in acetone solutions at 25 °C using the Ubbelohde viscometer. 2.2.2. IR spectra IR investigations of samples of purified chitin and obtained products were carried out using an IR-Nexus FT-IR spectrometer (Thermo Nicolet). The IR spectra of chitin samples were recorded by means of the KBr pellet method. IR spectra of DVCH samples were obtained using thin films. Films were casted from 2.0% (w/v) DVCH solutions in acetone (1 ml of polymer solution was poured into a Petri dish of 5.5 cm in diameter and the solvent was evaporated at the ambient temperature). 2.2.3. NMR The 1H and 13C NMR spectra of DVCH samples were recorded using a Brucker Avance III HD 400 Spectrometer. Acetone-D6 (99.9%, Cambridge Isotope Laboratories Inc.) was used as a solvent. 1H NMR was measured by performing 80 scans with a measurement range of 6.4 kHz, while 13C NMR in 8192 scans with a measurement range of 24 kHz. Correlation spectrum HSQC was recorded using standard gradient pulse sequence of the spectrometer, performing 24 scans for each of 256 increments of the t1 time. 2.2.4. The refractive index increment The value of the refractive index (dn/dc) for DVCH was determined using the SEC-3010 dn/dc refractometric detector at a wavelength of λ = 620 nm. For this purpose, six DVCH solutions in DMF were prepared in a graded flask (5 ml) at concentrations ranging from 0.5 g/l to 7.1 g/l. Measurements were conducted at 45 °C. Each measurement was repeated at least three times. Data analysis was performed using the BI-DNDCW software package. For the detector SEC-3010 calibration, a series of polystyrene (Mn = 100 000 g/mol) solutions in DMF, with concentrations ranging from 0.4 g/l to 4.4 g/l, were prepared. 2.2.5. Molecular weights The number average molecular weight (Mn), the weight average molecular weight (Mw) and polydispersity (Mw/Mn) were determined by the gel chromatography method with the measuring system consisting of an Agilent Technologies 1200 isocratic pump, a DAWN HELEOS multi-angle light scattering detector (λ = 658 nm) (Wyatt Technologies), a Δn2010 RI refractive index detector (WGE Dr. Bures) and the PL gel guard, PL gel MIXED-C x2, PSS GRAM 100 Å column system. The measurements were carried out at 45 °C using DMF added with 5 mmol of LiBr as solvent, with the eluent flow set at 1 ml/min. The sample solutions were filtered before being injected into the column using a 0.20 μm SRP 15 filter. The ASTRA 5.3.4.10 (Wyatt Technologies) and PSS WinGPC Unity program packages were used for data analysis. 2.2.6. Elemental analysis Elemental analysis was employed in order to determine the degree of esterification. Analyses of DVCH samples were performed using VARIO EL III Elemental Analyzer (manufactured by Elementar). The desired measured elements (C, N, H) were separated from each other by means of specific adsorption columns (CO2, H2O, N2 adsorption columns) and determined in a thermal conductivity detector (TCD). The

52

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

Fig. 1. Chitin dipentanoate synthesis scheme.

combustion temperature was 115 °C (in the helium atmosphere added with oxygen).

Samples of approximately 11 mg were heated from room temperature up to 600 °C at a rate of 20 °C/min in the nitrogen atmosphere or air.

2.2.7. DSC Thermal analysis of the DVCH samples was conducted using an indium-calibrated TA 2920 DSC apparatus (Thermal Analysis, USA). 5–8 mg specimens were prepared. Thermograms were recorded under nitrogen flow at a heating rate of 10°/min. The glass transition temperature was estimated during heating from 0 °C to 200 °C based on the second heating cycle.

2.2.9. Tensile properties The DVCH polymer films used for determination of mechanical properties were prepared by pouring 7 ml of 4.2% polymer solution in acetone (w/v) into a Petri dish of 10 cm in diameter and evaporating the solvent at ambient temperature. The prepared polymer films were of uniform thickness of 0.4 ± 0.02 mm. The tensile properties of the specimens were probed using the INSTRON 5582 tensile test machine. Rectangular strips with dimensions of 50 × 10 × 0.4 mm cut out from the cast films were uniaxially drawn at room temperature, at a rate of 5 mm/min. The value of each mechanical property was determined as five measurements average.

2.2.8. TGA Thermogravimetric measurements of DVCH samples were performed using a TGA Hi-Res 2950 instrument (Thermal Analysis, USA). Table 2 Synthesis of DVCH under various conditions versus molecular weight, esterification degree and polydispersity of the final product. Sample name

Synthesis conditions Time [h]

Temp. [°C]

Mn [g/mol]

Mw [g/mol]

PD (Mw/Mn)

Elemental analysis %C — 57.71 %H — 7.82 %N — 3.74 %C — 57.60 %H — 7.81 %N — 3.74 %C — 57.11 %H — 7.78 %N — 3.73 %C — 56.64 %H — 7.81 %N — 3.73 %C — 56.74 %H — 7.77 %N — 3.69 %C — 56.92 %H — 7.79 %N — 3.70 %C — 57.06 %H — 7.80 %N — 3.72 %C — 57.36 %H — 7.78 %N — 3.73 %C — 57.34 %H — 7.78 %N — 3.73 %C — 57.82 %H — 7.77 %N — 3.70 %C — 58.01 %H — 7.79 %N — 3.73 %C — 57.98 %H — 7.80 %N — 3.72

3a

2

0

164,700

298,400

1.81

3b

4

0

146,100

270,000

1.85

3c

6

0

145,000

258,800

1.79

3d

24

0

122,900

206,500

1.68

8a

2

6

134,900

212,400

1.58

8b

4

6

122,800

221,200

1.80

16c

6

6

92,600

191,400

2.06

16d

24

6

86,360

136,700

1.58

6a

2

21

88,770

145,000

1.63

6b

4

21

70,730

120,300

1.70

6c

6

21

63,880

105,900

1.66

6d

24

21

38,610

56,610

1.47

2.2.10. Preparation of DVCH scaffolds by salt leaching method The acetone solution of DVCH (3.5% m/v) was poured into a round mold (7 ml of polymer solution per 78.5 cm2 of mold area) previously covered by a porogen layer (NaCl crystals, p.a., POCH, stacking density 1274 g/l, crystal size: a mixture of big crystals with size in the range of 194–733 μm (average crystal size of 452 μm ± 118 μm) and the small ones with size in the range of 4–21 μm (average crystal size of 8.9 μm ± 3.7 μm). The ratio of the amount of polymer dissolved in the solvent to the amount of porogen (m/m) was 1:21. The solvent was evaporated at ambient temperature. Next, the porogen was removed from obtained polymer films by washing them several times with distilled water. The obtained DVCH scaffolds were dried for 24 h at room temperature. The concentration of Cl− in aqueous extracts of dilapidated DVCH scaffolds was checked using the Mohr titration method. 2.2.11. Electron scanning microscopy The structure (pores size) of DVCH scaffolds, size of NaCl crystals used for DVCH scaffold preparation and structure of DVCH polymer films (reference samples) were characterized by means of the scanning electron microscope using the Quanta 3D FEG instrument (FEI) operating in the high vacuum mode and accelerating voltage of 30 kV. The DVCH polymer films used as reference samples were prepared by pouring 7 ml of 3.5% polymer solution in acetone (w/v) into a round mold of 10 cm in diameter and evaporating the solvent at ambient temperature. Prior to examination, the specimens were coated with thin layer of gold by ion-sputtering. 2.2.12. Neutral Red uptake (NRU) assay The NRU procedure was used to assess the cytotoxicity of extracts of di-O-valeryl chitin according to the PN-EN ISO 10993-5 standard using the mouse fibroblasts line BALB/3T3 clone A31 (mouse embryo; ATCC CCL-163) [45]. Di-O-valeryl chitin extract preparation method: A sample of DVCH (η = 1.48 dl/g, Mw = 1.73 × 103) was placed in a 50 ml sterile tube (FALCON), into which the DMEM (Dulbecco's Modified Eagle's Medium, Sigma Aldrich) culture medium with glucose (4.5 g/l) and NaHCO3 (1.5 g/l), supplemented with L-glutamine (4 mM, BioXtra,

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

suitable for cell culture, Sigma Aldrich), penicillin (100 U/ml, BioReagent, suitable for cell culture, Sigma Aldrich), streptomycin (100 μg/ml, BioReagent, suitable for cell culture, Sigma Aldrich), HEPES (25 mM, BioReagent, suitable for cell culture, Sigma Aldrich) and fetal bovine serum (FBS) (5% (v/v), Invitrogen) was added (1 ml medium per 0.2 g of biopolymer including the additional volume of culture medium, which was absorbed by the biopolymer during the incubation) and then shaken in a water bath at 37 °C for 24 h. Subsequently, the test tube was centrifuged (5 min, 1500 g, 25 °C, Beckman GS-6R) and the supernatant was collected into a sterile tube (using a 100 μm nylon filter, BD Biosciences). The extract was then filtered through a 0.22 μm syringe filter (TPP) and a dilution series of the concentrated extract was prepared in the culture medium (once again shaken for 24 h). Positive control preparation procedure: Sodium dodecyl sulfate (SDS, Sigma Aldrich) was dissolved in distilled water (10 mg/ml). The prepared solution was then filtered through a 0.22 μm syringe filter (TPP) and stored at room temperature. Before each experiment, a dilution series of SDS were prepared in the DMEM culture medium containing glucose (4.5 g/l) and NaHCO3 (1.5 g/l), supplemented with L-glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES (25 mM) and fetal bovine serum (10% (v/v)) and shaken in water bath at 37 °C for 24 h. The NRU procedure: The day before the test, the BALB/3T3 fibroblasts, clone A31 in exponential growth phase were placed into 96-well plates (NUNC) in the amount of 3.5 × 103 cells/well and suspended in 100 μl DMEM culture medium containing glucose (4.5 g/l) and NaHCO3 (1.5 g/l), supplemented with L -glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES (25 mM) and FBS (10% (v/v)) and left for incubation overnight. The next day, the medium was removed from the cells. 100 μl of the control medium (DMEM supplemented and shaken as described in the case of the extracted material) was added into each negative control wells (6 repetitions). Aliquots of 100 μl of extract solutions at concentrations from 10% to 100% were added to remaining wells (6 repetitions per each concentration of the extract solution). In the case of positive control,

53

the cells were exposed to a dilution series of SDS in the medium (40–100 μg/ml; 6 repetitions per each concentration of the SDS solution). Then, the 96-well plate was incubated for 24 h at 37 °C in 5% CO2. After this time, the medium was removed from the cells. Subsequently, the cells were washed with PBS, added with 100 μl/well of Neutral Red (NR) in the medium (50 μg/ml, Sigma Aldrich) and further incubated for an additional 3 h. Next, the NR solution was removed, the cells were washed with PBS and added with 150 μl/well of desorbing solution (acetic acid (1%; pure, POCH), ethyl alcohol (50%, pure, POCH), distilled water (49%)) to release the dye from lysosomes. The plate was shaken for 10 min, placed in an ELISA reader (Labsystems Multiskan RC 351, Finland) and the absorbance at 550 nm (with the reference filter at 620 nm) was determined. The results of the viability of cells exposed to different concentrations of the extracts (in relation to control cells) were shown as the mean ± SD from 4 independent experiments. 2.2.13. Skin irritation test In order to verify the DVCH potential irritation effect to the skin, DVCH scaffolds were used. The test was performed in accordance with the PN-EN ISO 10993-10 standard [46] by means of single and repeated applications of DVCH scaffolds on non-corrupted and scarified rabbit (New Zealand White Rabbits (BN)) flanks (the area of 6 cm2). In each test, 3 male rabbits were used. In the case of single application of DVCH scaffolds, the animal observation was carried out 1 h after removing DVCH scaffold from the rabbit skin (4 h of exposure of rabbit skin to DVCH scaffold, occlusive patch was used to hold the scaffold in contact with the skin) and then systematically for 3 consecutive days following the exposure. In the case of repeated application of the DVCH scaffold, the animal skin observation (duration of 5 days, daily application, 4 h of exposure, occlusive patch was used to hold the scaffold in contact with the skin) was performed before and after each application. In both cases, the PBS-wetted gauze was applied adjacent to the place where the DVCH scaffold was used. The experiment was performed

Fig. 2. The 1H NMR spectra of chitin dipentanoate solution in deuterated acetone-d6.

54

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

Fig. 3. The 13C NMR spectra of chitin dipentanoate solution in deuterated acetone-d6.

on non-corrupted and scarified skin, using the right and left flank of the same rabbit, respectively. In the course of all observations, a special attention was paid to the redness, peeling, swelling and erosion of the rabbit skin being in contact with the DVCH scaffold. 2.2.14. Skin sensitization test In order to verify the potential sensitization skin effect of DVCH, the DVCH scaffolds were used. The Buehler test was carried out in accordance with the PN-EN ISO 10993-10 standard [47] and the OECD Test procedure No. 406: Skin sensitization. For the test, 30 female guinea pigs Dunkin Hartley (10 animals in the control group, 20 animals in the study group) were used. During the induction step, the right flanks

of guinea pigs (the area of 8 cm2) were shaved. In the first group, the DVCH scaffolds were applied (6 h of exposure, occlusive patch was used to hold the scaffold in contact with the skin) and then removed. In the control group, the PBS-wetted gauzes were used instead of the DVCH scaffolds. The procedure described above was repeated also after 6 and 13 days of the test. Twenty-seven days after the beginning of the test, the hair from the left flanks of guinea pigs (both animal groups) was removed and the sensitization (challenge) reaction was performed. On the head-part skin of all animals, the PBS-wetted gauze was applied, whereas on the tail-part skin, the DVCH scaffold was applied instead (6 h of exposure, occlusive patch was used to hold both in contact with the skin). Seven days following the sensitization

C=O

100 90

Amide II

80

Absorbance (%)

a b

C-O valeryl

-CH-CH2-

70

Amide I

-CH3-

60 50 40 30 20

Amide III

10 0 3500

3000

2500

2000

1500

Wavenumber (1/cm) Fig. 4. The IR spectra of krill chitin (a) and example sample of DVCH (b).

1000

500

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

55

reaction, analogical procedure (rechallenge) was repeated using the same animals. The observation of animal skin was carried out 30 and 54 h following the challenge and rechallenge tests. The skin reactions were observed and recorded according to the Magnusson–Kligman Classification [48]. 3. Results and discussion The synthesis revealed chitin dipentanoate (Di-O-Valeryl Chitin, DVCH) as the product, which is a chitin derivative esterified with two valeryl groups at positions 3 and 6 of the N-acetylglucosamine units. Highly efficient synthesis (high degree of esterification) of DVCH was achieved by employing a huge excess of valeric anhydride used as both the acylation agent and the reaction medium and the perchloric acid as the catalyst (Fig. 1). The theoretical consumption of valeric anhydride obligatory for chitin esterification reaction catalyzed with perchloric acid is 2 mol per 1 mol of chitin. However, a part of valeric anhydride (Val2O) undergoes hydrolysis with water present in commercially available HClO4 (~70%). Due to the fact that esterification reaction is carried out under heterogeneous conditions, the amount of reactants arising from the stoichiometry is not sufficient and an excess of acylation mixture is necessary. The mole ratio of valeric anhydride to chitin of 7:1 was chosen, which facilitated adequate mixing of reactants during the reaction and good temperature control. In the course of chitin acylation, the reaction mixture changed its form significantly (depending on reaction conditions, such as time and temperature) from suspension to a form resembling the almost solidified resin. Therefore, the efficient reaction mixture homogenization is extremely important. Poor mixing of the reaction mixture during the acylation step leads to local temperature increases, uneven chitin acylation and results ultimately in formation of products with different esterification degree and higher polydispersity of the final product. Thus, in order to provide effective mixing, an anchor-shaped mixer equipped with flow baffles was used. Uncontrolled temperature rises favor the reduction of DVCH molecular weight as the result of chitin acidic degradation, which takes place in the presence of strong acid simultaneously with the acylation reaction. The chitin acidic degradation occurs via cleavage of the glycosidic bonds (acid hydrolysis), that involves both the protonation of the glycosidic oxygen and the addition of water to produce the reducing sugar end group (SN1 reaction) [49]. The perchloric acid turns out to be excellent catalyst of O-acylation of chitin, as well as of dibutyrylchitin [50]. In all experiments the concentration of HClO4 as the catalyst was 0.5 ml/g of chitin and seems to be sufficient for the products with high degree of esterification to be obtained, regardless of the reaction conditions (Table 2). Additionally, the perchloric acid did not promote the deacetylation of N-acetyl linkage (acidic hydrolysis), what was proved by both the NMR and infrared spectroscopy. Another crucial step in the process of DVCH preparation is the separation of the crude product from the dense reaction mixture. For this purpose, the step of neutralization of valeric acid using 2.5% NaHCO3 aq. with simultaneous grinding of the reaction mixture was performed. Several concentrations of neutralizing agent were tested (the concentration limit of NaHCO3aq. was 10%) and found that none caused any partial deacetylation of NHAc, as confirmed by the 1H NMR, 13C NMR (Figs. 2 and 3) and infrared spectroscopy (Fig. 4). Studies on the synthesis were aimed at finding optimal conditions (temperature, reaction time) for the reaction of chitin esterification with valeric anhydride in the presence of perchloric acid as catalyst. This would allow obtaining high yield of DVCH with desirable Table 3 Theoretical contents of elements in chitin and DVCH.

Chitin dipentanoate (molar mass of a mer 371 g/mol) Chitin (molar mass of a mer 203 g/mol)

C

H

N

58.22% 47.29%

7.82% 6.40%

3.77% 6.89%

Fig. 5. The TGA curves of DVCH a) in nitrogen atmosphere b) in air atmosphere.

physicochemical properties and the degree of esterification close to 2. Numerical values for intrinsic viscosity and DVCH reaction yield under different reaction conditions are presented in Table 1, whereas the number average molecular weight (Mn), weight average molecular weight (Mw), polydispersity index (PD) and results of elemental analysis are summarized in Table 2. The results presented in Tables 1 and 2 indicate that the lower the temperature during the reaction, the higher the intrinsic viscosity and molecular weight of the final products. On the other hand, the longer the reaction time, the higher the reaction yield and the lower the molecular weight (acidic degradation of polymer chain) of DVCH. The highest reaction yields (above 80%) were obtained after 24 h at all temperatures. Molecular weights revealed by gel chromatography (dn/dc = 0.048 ml/g) and intrinsic viscosity of all DVCH samples were significantly lower in relation to the original substrate (chitin, η = 10.8 dl/g determined in DMAC/5% LiCl at 25 °C, viscosity average molar mass of Table 4 The influence of DVCH number average molecular weight on mechanical properties. All samples were prepared by casting from 4.2% acetone solution. No. of sample

Mn [g/mol]

Modulus [MPa]

Strain at yield [mm/mm]

Yield stress [MPa]

Tensile stress at break [MPa]

Tensile strain at break [mm/mm]

6d 6c 6b 6a 3a Wp3

38,610 63,880 70,730 88,770 164,700 184,300

1163 1238 1384 1392 1451 1491

0.02 0.02 0.03 0.03 0.02 0.02

10.1 11.3 18.3 20.6 24.4 25.7

12.4 14.5 24.3 26.7 27.4 29.2

0.03 0.03 0.04 0.04 0.04 0.04

56

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60 30

Stress [MPa]

25

20

15

10

Yield stress [MPa] Tensile stress at break [MPa]

5 4

4,0x10

4

6,0x10

4

8,0x10

10

5

5

1,2x10

5

1,4x10

5

1,6x10

5

1,8x10

DVCH M n[g/mol] Fig. 6. The dependence of tensile yield stress and ultimate stress of DVCH polymer films on DVCH molecular weight Mn.

197,020 Da) due to the depolymerization of chitin chain during the acylation step in the course of reaction, alike in the case of dibutyrylchitin [50]. The polydispersity index of obtained DVCH varied from 1.47 to 2.06, which suggests a slight distribution of molecular weights in DVCH samples. The polydispersity index of the sample 6a (PD = 1.63; 21 °C, 2 h) is considerably lower than the polydispersity index of dibutyrylchitin obtained under similar reaction conditions (PD = 2.99; 25 °C, 3 h, the ratio of butyric anhydride to chitin of 3.68) [50]. Due to the large differences in the content of each element between chitin and DVCH, the degree of substitution was monitored in all DVCH samples by the elemental analysis (Table 2) and infrared spectroscopy (the example spectra is presented in the Fig. 4). Theoretical contents of elements in chitin [51] and chitin dipentanoate are presented in Table 3.

The results of elemental analysis presented in Table 2 suggest that using the method of synthesis described in the Materials and methods under the reaction conditions presented in Table 1, the DVCH with the degree of esterification close to 2 is obtained. The results of the elemental analysis were confirmed by infrared spectroscopic measurements. All obtained DVCH samples were soluble in many organic solvents including ethanol, methanol, acetone, dichloromethane, 1,2-dichloroethane, N,N-dimethylmethanamide, N,N-dimethylacetamide and ethyl acetate, but insoluble in aqueous media. The properties of solubility of DVCH are similar to those of dibutyrylchitin [50,52]. Thus, the main goal of the study, i.e., obtaining the chitin derivative the solubility of which would be higher compared to that of the chitin itself, has been achieved. The filmogenic and fiber-forming properties of chitin dipentanoate (DVCH can be processed by electrospinning method) have also been observed to be similar as those in the case of dibutyrylchitin [52]. The chemical structure of DVCH was characterized by the 1H and 13C NMR spectroscopy (Figs. 2 and 3 respectively) and infrared spectroscopy (Fig. 4). 1 H NMR (400 MHz, Acetone-d6): δ [ppm] = 0.84–0.95 (m, 6H, – OOC–CH2–CH2–CH2–CH3 valeryl), 1.18–1.46 (m, 4H, –OOC–CH2–CH2– CH2–CH3 valeryl), 1.46–1.71 (m, 4H, –OOC–CH2–CH2–CH2–CH3 valeryl), 1.82 (br. s., 3H, –NH–CO–CH3), 2.16–2.55 (m, 4H, –OOC–CH2–CH2–CH2– CH3 valeryl), 3.64 (br.s. 1H, Glc-H4), 3.75 (br. s., 2 H, Glc-H2,5), 4.16 (br.s. 1H, Glc-H6), 4,46 (br. s., 1H, Glc-H6), 4.67 (br., s., 1H, Glc-H1), 5.09 (br.s., 1H, Glc-H3), 6.98 (br.s., 1H, −NH-CO-CH3). 13 C NMR (101 MHz, Acetone-d6): δ [ppm] = 13.19/13.25 (–OOC– CH2–CH2–CH2–CH3 valeryl), 21.97/22.05 (–OOC–CH2–CH2–CH2–CH3 valeryl), 22.18 (–NH–CO–CH3), 26.85/26.89 (–OOC–CH2–CH2–CH2– CH3 valeryl), 33.49 (–OOC–CH2–CH2–CH2–CH3 valeryl), 54.15 (GlcC2), 62.62 (Glc-C6), 72.81 (Glc-3,4), 75.87 (Glc-C5), 100.82 (Glc-C1), 168.97 (−NH-CO-CH3), 172.24/172.65 (−COO- valeryl). The 1H and 13C NMR spectra (Figs. 2 and 3, respectively) fully confirmed the postulated chemical structure of DVCH. The proton spectrum (Fig. 2) clearly indicates the substitution of the chitin chain with two acyl chains with the defined length (the signals from two methyl groups at the end of the two acyl chains can be seen, with the appropriate

Fig. 7. SEM images of the structure of the DVCH scaffold (sample code: Wp3) a) 150×, b) 750×, c) 2000×, d) cross section 125× and polymer film e) 150×, f) 3500×.

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

Relative cell viability [% of control]

100

80

60

40

20

0 100 %

90%

80%

60%

40%

20%

10%

Fig. 8. Relative viability of the BALB/3T3 mouse fibroblast cells after 24 h of incubation in extraction media with concentrations from 10 to 100% (NRU assay).

values of the integrals of the individual signals). The singlet at 1.82 ppm was assigned to be likely generated by chitin, more precisely by the – CH3 protons of the N-acetyl group. Furthermore, in the 13C NMR spectra, the doubled signals from acyl groups were found, what is consistent with the degree of substitution and different places of such substitution in the N-acetylglucosamine unit. Well defined signals in the range corresponding to the glucosamine unit indicate the high purity of the DVCH and very good repeatability of the monomer. Unambiguous assignment of different signals to corresponding DVCH atoms was confirmed by the analysis of the 2D-HSQC correlation spectrum. The IR spectrum of chitin (curve a in Fig. 4) is characterized by a large band at ca. 3400 cm−1 related to its –OH groups, bands at ca. 2783 cm−1 related to the GlcNHAc units, three bands at 1650 cm−1, 1565 cm−1 and 1412 cm−1 related to amide groups I, II and III, respectively, a band of –CH3 in acetylamide groups at 1376 cm−1; a band of C–O–C group in glucopyranose ring at 1028 cm−1 and the specific band

57

of the β (1 → 4) glycoside bridge at approx. 895 cm−1 [53]. The crystalline structure of the type α chitin contains the amide I split peaks at 1650 cm−1 and 1620 cm−1. The first one (1650 cm−1) is attributed to the hydrogen bonding of the C_O group of chitin to the –NH group of an adjacent chitin chain, and the second one with an –OH group on the same chitin chain (1620 cm−1) [54]. In the IR spectrum of DVCH (curve b in Fig. 4) a band of reduced absorption can be observed at approx. 3400 cm−1 that is related to the absence of hydroxyl groups occurring in chitin. However, new absorption band characteristic of esters of aliphatic acids appeared around 1740 cm−1, 1450 cm−1 and 1250 cm−1. Absorption bands appearing at 2900 cm−1, 790 cm−1 and 740 cm−1 are strong and specific for the –CH2 and –CH3 aliphatic groups, the content of which is much higher in DVCH compared to chitin. Three band characteristics of the amide group of chitin are present in the spectrum of DVCH although their positions are slightly changed compared to the spectrum of chitin. Both the first and the third band corresponding to the amide groups are shifted towards higher wave numbers while the second one is shifted slightly in the opposite direction (towards the lower wave numbers). The occurrence of all three amide bands in DVCH spectrum may confirm that applied conditions of DVCH synthesis did not cause any changes in the degree of deacetylation in GlcNAc units. The band at 1620 cm−1 is not observable in the DVCH spectrum which suggests the high degree of substitution of hydroxyl groups by valeryl ones in the chitin units. The band of β (1 → 4) glycoside bridge is also apparent in the DVCH spectrum at ca. 940 cm−1 and is shifted towards the higher wave numbers in relation to the initial chitin spectrum. The IR spectra of all samples studied are similar, which confirms the homogeneity of the obtained product. Thermal and thermo-oxidative stability of DVCH was studied by thermogravimetric analysis (TGA) in an inert (N2) and oxidative (air) atmosphere, respectively. The TGA weight loss curves obtained in nitrogen and air are presented in Fig. 5a and b, respectively. When compared to inert conditions (in N2 atmosphere), heating DVCH up in an oxidative atmosphere (air) leads to faster degradation, which is due to its peroxidation. This manifests in a noticeable decrease

Fig. 9. Micrographs of the BALB/3 T3 cells after 24 h of incubation with a) control medium b) 100% extraction medium c) 60% extraction medium d) 10% extraction medium, (Olympus IX170 Microscope, 100×).

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

Relative cell viability [% of control]

58

100

Col 3

80

60

40

20

0 100

90

80

70

60

50

40

SDS concentration (µg /ml) Fig. 10. The relative viability of the BALB/3T3 mouse fibroblast cells after 24 h of incubation with SDS in the function of the SDS concentration (NRU assay).

of the temperature of degradation. Td (taken as the temperature of the maximum weight loss rate) of DVCH in the air, which is lower by approximately 10 °C comparing to the degradation temperature in the N2 atmosphere. For DVCH in nitrogen atmosphere, T90 and T50 (10 and 50 wt.% of the weight loss, respectively) are 306.1 °C and 340.8 °C respectively, which are both similar to values obtained for dibutyrylchitin, in the case of which the T90 and T50 values 324.9 °C and 358.1 °C were found, respectively [55]. The DSC heating thermogram obtained for chitin dipentanoate shows the existence of an endothermic peak in the vicinity of 75 °C, a value which is similar to that reported for dibutyrylchitin [56]. The glass temperature (Tg) was found to be 150 °C. To eliminate the thermal history of the polymer, thermal properties were determined based on the second heating cycle.

Due to the good solubility of DVCH in organic solvents such as acetone or ethanol, the preparation of polymer thin films by casting or porous structures by means of the salt-leaching method (described in Materials and methods) appeared quite easy. The tensile properties of DVCH in the form of thin films were tested in relation to the molecular weight of DVCH. The results are presented in Table 4 and Fig. 6. All DVCH samples tested demonstrated a semi-ductile behavior and fractured soon after passing the yield point. The data presented in Table 4 and Fig. 6 show that the tensile properties of DVCH films depend on the molecular weight. The modulus, stress at yield, stress at break, as well as strain at break, increase continuously with increasing molecular weight of DVCH. As can been seen in Fig. 6, the increasing trend of both the yield stress and the stress at break is very similar. Moreover, an increase of modulus with molecular weight results in higher mechanical strength of DVCH films. The elongation at break, although increasing slightly with molecular weight, remains at low level, not exceeding 4%. Consequently, DVCH samples with higher molecular weight behave like a rigid plastic, which can bear relative large stress, but cannot withstand much elongation before fracturing. The porous structures, potentially useful as scaffolds, were attempted to be produced with the salt leaching method. The structure of the DVCH scaffolds obtained in this way consists of a uniform network of interconnected channels, as illustrated by SEM micrographs presented in Fig. 7. This structure is characterized by a high degree of open-porosity with pores of various sizes. Two populations of pores were found: the big ones with the size in the range of 150–780 μm (average pore size of 435 μm ± 168 μm) and the small ones with the size in the range of 4–22 μm (average pore size of 7.7 μm ± 3.3 μm). The absence of salt crystals (used in preparation and then washed out) in the final structure of the DVCH scaffold was confirmed by Mohr titration method. We believe, that due to their unique structure, the DVCH scaffolds can be prospectively used in research related to cellular response associated with the surface characteristics and the concept of cell niche, as well as in standard biocompatibility tests.

Fig. 11. Micrographs of the BALB/3T3 cells after 24 h of incubation with a) control medium b) SDS solution (100 μg/ml) c) SDS solution (80 μg/ml) d) SDS solution (60 μg/ml), (Olympus IX170 Microscope, 100×).

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

A standardized NRU procedure was used to investigate the in vitro cytotoxicity of the novel Di-O-Valeryl Chitin biopolymer. Relative viability of the BALB/3 T3 clone A31 of mouse fibroblasts in the presence of extracts in the concentration range of 10–100% is presented in Fig. 8. Micrographs in Fig. 9 show the cell densities of control cells in comparison to cells exposed to extracts of biopolymer following 24 h of exposure. In either case, the fibroblasts demonstrated a normal proliferation. Extracts did not affect the BALB/3 T3 cells' morphology (the cells' size and shape were the same as in the case of fibroblasts cultured in the control medium). The cell monolayer formation was observed for all extracts. In the case of all extract concentrations, the average viability of BALB/3T3 cells was above 80% of that in the case of the negative control, suggesting that DVCH had no cytotoxicity effect on BALB/3T3 fibroblasts. The effect of increasing concentrations of sodium dodecyl sulfate (the positive control) on BALB/3T3 fibroblasts viability is presented in Fig. 10. The IC50 (95% CI) for SDS is 87.3 μg/ml (86.0–88.7), the micrographs of the BALB/3T3 cells following the 24-hours incubation with SDS are presented in Fig. 11 In order to verify the DVCH potential irritation and sensitization effect to the skin (in vivo tests, in accordance with PN-EN ISO 10993-10: 2011 Biological evaluation of medical devices — Part 10: Testing for irritation and skin sensitization), the DVCH scaffold made by means of the leaching method was used. It has been found, that during the exposure of the male rabbit (BN) skin to DVCH scaffolds (Skin Irritation Test) as well as at the time following such exposure, the appearance and behavior of the animal did not differ from normality. There were no skin reactions including redness, peeling, swelling and erosion appearing after both the single and repeated applications of DVCH scaffolds on the rabbit skin. This result is indicative of no irritation effect of DVCH scaffolds to the skin. The same applies also to the sensitization test (the Buehler Test), in the case of which the appearance, body behavior and the body weight of female Dankin Hartley pigs did not differ from the normal condition throughout the observation period. In both, control an treated group, the challenge and rechallenge tests revealed no skin reactions.

4. Conclusions Di-O-Valeryl Chitin, an ester chitin derivative is a technologically friendly biopolymer. Good DVCH solubility in several organic solvents (ethanol, DMAC, DMSO, acetone) results from the presence of two valeryl groups at C-3 and C-6 positions of its molecule. The procedure of the preparation of Di-O-Valeryl chitin using the perchloric acid as an esterification catalyst employed in this study has numerous advantages including simplicity of the reaction, high reaction yield and high degree of product purity. The use of strong acids, such as the mixture of valeric anhydride and 70% perchloric acid in the esterification process, facilitated the significant depolymerization of chitin. By choosing appropriate conditions it was possible to prepare DVCH with a sufficiently high molecular weight. Esterification of chitin under conditions presented in this article (i.e., the reagent ratio, the synthesis temperature and time) promoted the DVCH synthesis with high reaction yield and substitution degree as well as polydispersity index close to 2. DVCH also preserves the filmogenic properties of chitin, so it can easily be used to manufacture threads, foils, foams and scaffolds as well as non-woven materials. Biological data indicate that DVCH is non-cytotoxic to fibroblasts and does not exert irritating or allergic effects to animal skin. Moreover, DVCH appears to ensure better handling and mechanical resistance than chitin. Since DVCH is soluble in organic solvents but insoluble in water, it can easily be processed by the saltleaching method to prepare large-size porous structures exhibiting open-cell architecture with pores of various sizes easy to obtain. These structures can further be employed in wound dressing therapies and scaffolds for tissue engineering.

59

5. Competing interest Patent Pending: Rieske P., Stoczynska-Fidelus E., Piaskowski S., Skołucka-Szary K., Piaskowska W., and Ramięga A. have submitted a patent application on this technology (P-409240). The authors declare that they have no competing interests. Acknowledgment This study was sponsored by the Polish Agency for Enterprise Development, Grant No. POIG.01.04.00-10-020/10. A special gratitude to professor Zbigniew Bartczak for all advices relating to this work. References [1] P. Wilmer, Invertebrate Relationships: Patterns in Animal Evolution, Cambridge University Press, 1990. [2] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (7) (2006) 603–632. [3] M. Kaya, I. Akata, T. Baran, A. Mentes, Physicochemical properties of chitin and chitosan produced from medical fungus (Fomitopsis pinicola), Food Biophys. 10 (2) (2014) 162–168. [4] H. Ehrlich, Chitin and collagen as universal and alternative templates in biomineralization, Int. Geol. Rev. 52 (7–8) (2010) 661–699. [5] H. Merzendorfer, L. Zimoch, Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases, J. Exp. Biol. 206 (24) (2003) 4393–4412. [6] M. Kaya, O. Seyyar, T. Baran, S. Erdoğan, M. Kar, A physicochemical characterization of the fully acetylated chitin structure isolated from two spider species, Int. J. Biol. Macromol. 65 (2014) 553–558. [7] P.A. Roelofsen, I. Hoette, Chitin in the cell wall of yeasts, Antonie Van Leeuwenhoek 17 (1) (1951) 297–313. [8] H. Ehrlich, M. Maldonado, K. Spindler, C. Eckert, T. Hanke, R. Born, C. Goebel, P. Simon, S. Heinemann, H. Worch, First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (Demospongia: Porifera), J. Exp. Zool. Part B Mol. Dev. Evol. 308 (4) (2007) 347–356. [9] H. Ehrlich, J. Keith Rigby, J.P. Botting, M.V. Tsurkan, C. Werner, P. Schwille, Z. Petrášek, A. Pisera, P. Simon, V.N. Sivkov, D.V. Vyalikh, S.L. Molodtsov, D. Kurek, M. Kammer, S. Hunoldt, R. Born, D. Stawski, A. Steinhof, V.V. Bazhenov, T. Geisler, Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta, Sci. Rep. 3 (3497) (2013). [10] B.A. Juárez-de la Rosa, P. Quintana, P.L. Ardisson, J.M. Yáñez-Limón, J.J. Alvarado-Gil, Effects of thermal treatments on the structure of two black coral species chitinous exoskeleton, J. Mater. Sci. 47 (2) (2012) 990–998. [11] E. Brunner, P. Richthammer, H. Ehrlich, S. Paasch, P. Simon, S. Ueberlein, K.H. van Pée, Chitin-based organic networks — an integral part of cell wall biosilica from the diatom Thalassiosira pseudonana, Angew. Chem. Int. Ed. 48 (51) (2009) 9724–9727. [12] R.A.A. Muzzarelli, Chitin, Pergamon, New York, 1977. [13] P.R. Austin, C.J. Brine, J.E. Castle, J.P. Zikakis, Chitin: new facets of research, Science 212 (4496) (1981) 749–753. [14] L. Illum, Chitosan and its use as a pharmaceutical excipient, Pharm. Res. 15 (9) (1998) 1326–1331. [15] H. Ehrlich, Biomimetic potential of chitin-based composite biomaterials of poriferan origin, in: A.J. Ruys (Ed.), Biomimetic Biomaterials: Structure and Applications, Woodhead Publishing 2013, pp. 47–67. [16] A. Anitha, S. Sowmya, P.T.S. Kumar, S. Deepthi, K.P. Chennazhi, H. Ehrlich, M. Tsurkan, R. Jayakumar, Chitin and chitosan in selected biomedical applications, Prog. Polym. Sci. 39 (9) (2014) 1644–1667. [17] R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (3) (2011) 322–327. [18] E. Khor, L.Y. Lim, Implantable applications of chitin and chitosan, Biomaterials 24 (23) (2003) 2339–2449. [19] M. Wysokowski, I. Petrenko, A.L. Stelling, D. Stawski, T. Jesionowski, H. Ehrlich, Poriferan chitin as a versatile template for extreme biomimetics, Polymers 7 (2) (2015) 235–265. [20] K. Kurita, Controlled functionalization of the polysaccharide chitin, Prog. Polym. Sci. 26 (9) (1921–1971) 2001. [21] A. Almond, J.K. Sheenan, Predicting the molecular shape of polysaccharides from dynamic interactions with water, Glycobiology 13 (4) (2003) 255–264. [22] M. Vincendon, NMR conformational analysis of chitin in lithium chloride solution, in: R.A.A. Muzzarelli, C. Jeuniaux, G. Gooday (Eds.), Chitin in Nature and Technology, Proceedings of the Third International Conference on Chitin and Chitosan, Plenum Press, Italy, NY 1985, pp. 343–345. [23] G.A. Vikhoreva, I.N. Gorbacheva, L.S. Gal’braikh, Synthesis and properties of water — soluble derivatives of chitin: a review, Fibre Chem. 31 (4) (1999) 274–278. [24] X. Hu, Y. Du, Y. Tang, Q. Wang, T. Feng, J. Yang, et al., Solubility and property of chitin in NaOH/urea aqueous solution, Carbohydr. Polym. 70 (4) (2007) 451–458. [25] B. Chen, K. Sun, K. Zhang, Rheological properties of chitin/lithium chloride, N,N-dimethyl acetamide solutions, Carbohydr. Polym. 58 (1) (2004) 65–69.

60

K. Skołucka-Szary et al. / Materials Science and Engineering C 55 (2015) 50–60

[26] M. Terbojevich, C. Carraro, A. Cosani, Solution studies of the chitin-lithium chlorideN,N-dimethylacetamide system, Carbohydr. Res. 180 (1) (1988) 73–86. [27] F.A. Rutherford, P.R. Austin, in: R.A.A. Muzzarelli, E.R. Pariser (Eds.),Proceedings of the First International Conference on Chitin/Chitosan, MIT Sea Grant Report MITSG 78-7 1978, pp. 182–192. [28] C.K.S. Pillai, W. Paul, C.P. Sharma, Chitin and chitosan polymers: chemistry, solubility and fiber formation, Prog. Polym. Sci. 34 (7) (2009) 641–678. [29] H. Tamura, T. Hamaguchi, S. Tokura, Destruction of rigid crystalline structure to prepare chitin solution, in: J. Boucher, K. Jamieson, A. Retnakaran (Eds.), Advances in Chitin Science, Proceedings of the Ninth International Conference on Chitin and Chitosan, European Chitin Society, Montreal 2004, pp. 94–97. [30] R.C. Capozza, Spinning and shaping poly-(N-acetyl-D-glucosamine), US Patent 3,988,411, 1976. [31] R.C. Capozza, Solution of poly-(N-acetyl-D-glucosamine), US Patent 3,989,535, 1976. [32] S. Tokura, N. Nishi, in: M.B. Zakaria, W.M.W. Muda, P. Abdullah (Eds.), Chitin and Chitosan, Universiti Kebangsaan, Malaysia 1994, p. 67. [33] H. Moeller, A. Rathjens, R. Wachter, Chitin und Chitosanderivate, Verfahren und Verwendung, DE 19826953 C1, 1999. [34] K. Kaifu, N. Nishi, T. Komai, S. Tokura, O. Somorin, Studies on chitin. V. formylation, propionylation and butyrylation of chitin, Polym. J. 13 (1981) 241–245. [35] B.Y. Yang, Q. Ding, R. Montgomery, Preparation and physical properties of chitin fatty esters, Carbohydr. Res. 344 (3) (2009) 336–342. [36] L.R. Bhatt, B.M. Kim, K. Hyuan, K.H. Kang, C. Lu, K.Y. Chai, Preparation of chitin butyrate by using phosphoryl mixed anhydride system, Carbohydr. Res. 346 (5) (2011) 691–694. [37] R.A.A. Muzzarelli, M. Guerrieri, G. Goteri, C. Muzzarelli, T. Armeni, R. Ghiselli, M. Cornelissen, The biocompatibility of dibutyryl chitin in the context of wound dressings, Biomaterials 26 (29) (2005) 5844–5854. [38] S. Piełka, D. Paluch, J. Staniszewska-Kuś, B. Żywicka, L. Solski, L. Szosland, A. Czarny, E. Zaczyńska, Wound healing acceleration by a textile dressing containing dibutyrylchitin and chitin, Fibres Text. East. Eur. 2 (41) (2003) 79–84. [39] A. Błasińska, J. Drobnik, Effects of nonwoven mats of Di-O-butyrylchitin and related polymers on the process of wound healing, Biomacromolecules 9 (3) (2008) 776–782. [40] G. Schoukens, P. Kiekens, I. Krucińska, New bioactive textile dressing materials from dibutyrylchitin, Int. J. Cloth. Sci. Technol. 21 (2/3) (2009) 93–101. [41] L. Szosland, Synthesis of highly substituted butyryl chitin in the presence of perchloric acid, J. Bioact. Compat. Polym. 11 (1) (1996) 61–71. [42] E. Castagnino, M. Francesca Ottaviani, M. Cangiotti, M. Morelli, L. Casettari, R.A.A. Muzzarelli, Radical scavenging activity of 5-methylpyrrolidinone chitosan and dibutyryl chitin, Carbohydr. Polym. 74 (3) (2008) 640–647.

[43] A. Błasińska, I. Krucińska, M. Chrzanowski, Dibutyrylchitin nonwoven biomaterials manufactured using electrospinning method, Fibres Text. East. Eur. 4 (48) (2004) 51–55. [44] L. Szosland, I. Krucińska, R. Cisło, D. Paluch, J. Staniszewska- Kus, L. Solski, Synthesis of dibutyrylchitin and preparation of new textiles made from dibutyrylchitin and chitin for medical applications, Fibres Text. East. Eur. 9 (2001) 54–57. [45] PN-EN ISO 10993–5, Biological evaluation of medical devices — part 5: tests for in vitro cytotoxicity, 2009. [46] PN-EN ISO 10993–10:2011 Biological evaluation of medical devices - Part 10: Testing for irritation and skin sensitization - OECD Test Procedure No. 404: Acute Dermal Irritation/Corrosion, 2002; B.5 Procedure: "Acute toxicity - irritant/corrosive to the skin". [47] Biological evaluation of medical devices - Part 10: Testing for irritation and skin sensitization - B.6 Procedure "sensitization" (Acts. Office. Commission Regulation EC No 440/2008 of 30 May 2008). [48] E. Schlede, R. Eppler, Testing for skin sensitization according to the notification procedure for new chemicals: the Magnusson and Kligman test, Contact Dermatitis 32 (1) (1995) 1–4. [49] J.T. Edward, Stability of glycosides to acid hydrolysis — a conformational analysis, Chem. Ind. 36 (1955) 1102–1104. [50] L. Szosland, J. Szumielewicz, Effect of reagents and catalyst concentration on the process of chitin esterification and o the reaction product, Fibres Text. East. Eur. 9 (2003) 49–60. [51] J. Szumielewicz, L. Szosland, Determination of chitin esterification degree, Progr. Chem. Appl. Chitin Deriv. IX (2003) 41–48. [52] I. Krucińska, A. Komisarczyk, M. Chrzanowski, D. Paluch, Producing wound dressing materials from chitin derivatives by forming nonwovens directly from polymer solution, Fibres Text. East. Eur. 15 (5/6) (2007) 64–66. [53] A. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan and chitin produced from silkworm crysalides, Carbohydr. Polym. 64 (1) (2006) 98–103. [54] J. Jin, P. Hassanzadeh, G. Perotto, W. Sun, M.A. Brenckle, D. Kaplan, F.G. Omenetto, M. Rolandi, A biomimetic composite from solution self-assembly of chitin nanofibers in a silk fibroin matrix, Adv. Mater. 25 (32) (2013). [55] P. Sobecki, B. Pabin-Szafko, New homogenous blends of dibutyrylchitin and aliphatic polyesters, Progr. Chem. Appl. Chitin Deriv. XV (2010). [56] A. Wszyszczyński, Xia Qu, L. Szosland, E. Adamczak, L.A. Linden, J.F. Rabek, Blends of poly(ethylene oxide) with chitosane acetate salt and dibutyrylchitin: structure and morphology, Polym. Bull. 34 (4) (1995) 493–500.