Preparation and drug release properties of ...

Starch/Sta¨rke 2012, 64, 399–407

DOI 10.1002/star.201100158

399

RESEARCH ARTICLE

Preparation and drug release properties of lignin– starch biodegradable films I˙lker Çalgeris, Emrah Çakmakçı, Ays¸e Ogan, M. Vezir Kahraman and Nilhan Kayaman-Apohan Department of Chemistry, Faculty of Science and Letters, Marmara University, Istanbul, Turkey

Starch is one of the most commonly available natural polymers which are obtained from agrosources. It is renewable and abundant in nature. Unfortunately due to its poor mechanical properties and hygroscopic nature, there are some strong limitations to the development of starch-based products. Usually blends of starch are prepared and plasticized with glycerol to improve some of its properties. In this study, lignin was extracted from hazelnut shells and investigated as a potential additive for starch biofilms. The structural characterization of hazelnut lignin was performed by employing UV spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Lignin was blended with corn starch in different ratios to obtain biofilms. Mechanical and thermal properties of the biofilms were enhanced as the lignin amount was increased in the formulations. Water absorption tests were performed at pH 2.0, 4.0, and 6.0. The percent swelling values of the starch/lignin films increased as pH increased. Also, the biofilm exhibiting the best properties was chosen for the drug release studies. Biofilms showed a fast ciprofloxacin (CPF) release within an hour and then the drug release rate decreased. A pH dependent drug release mechanism was also observed according to Koshner–Peppas model. The drug release increased with a decrease in pH.

Received: October 12, 2011 Revised: November 21, 2011 Accepted: November 23, 2011

Keywords: Ciprofloxacin / Drug release / Hazelnut / Lignin / Starch

1

Introduction

Biodegradable plastics have an expanding range of potential applications and have been proposed as a solution for the waste problem [1–3]. They can be produced by biological systems (i.e. microorganisms, plants and animals) or chemically synthesized from biological materials (i.e. sugars, starch, oils and fats) [3, 4]. Starch is one of promising materials for the production of biodegradable plastics and has high potential in food and non-food applications and drug delivery specifically to the colon [5, 6]. It is a renewable, low cost and biodegradable material obtained from a great variety of crops [7]. Since 1970s starch has been incorp-

Correspondence: Professor Ays¸e Ogan, Department of Chemistry, Faculty of Art & Science, Marmara University, 34722 Istanbul, Turkey. E-mail: [email protected] Fax: þ90-216-3478783 Abbreviations: CPF, ciprofloxacin; TPS, thermoplastic starch

ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

orated into polyethylene in order to increase the biodegradability [8]. Usually blends of starch with petrochemicalbased polymers; poly (vinylalcohol) [9] polyethylene, polylactic acid [10], polycaprolactone [11] or gelatin [12] and lignin [13–15] are prepared and plasticized with glycerol or sorbitol to form biofilms with various features [16]. Unfortunately, there are some strong limitations to the development of starch based products due to its poor mechanical properties [17]. The association by blending is a way to overcome the most important weakness of starch as moisture sensitivity and critical aging [11, 15, 18]. Lignin, is one of the most abundant of renewable natural polymers, and a desirable raw material alternative to petroleumderived chemicals, with great possibilities for use as a component in polymeric systems (blends). Different kinds of fractioned or modified lignins (Kraft lignins,) have been investigated in association with thermoplastic starch (TPS). Nut shells contain considerable amount of lignin but they are

Colour online: See the article online to view Fig. 5 in colour.

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˙I. C ¸ algeris et al.

used presently as an energy source or disposed as waste [19]. Alkaline lignin extracted from hazelnut shells may be an attractive material for modification of TPS to improve its mechanical properties [20]. In this study, we aimed to investigate the effect of lignin on the mechanical and thermal properties of glycerol plasticized starch/lignin biofilms. These biofilms were further used in the drug release studies of Ciprofloxacin (CPF) which was utilized as a model drug.

2

Materials and methods

2.1 Materials Starch and glycerin were supplied form Merck-Darmstad. Alkaline lignin was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Finally grounded and dried nut shells were used in this study. Nuts were kindly supplied by a local farmer. Ciprofloxacin hydrochloride was a gift of Atabay Pharmaceutical Company, Turkey.

2.2 Aqueous alkaline lignin extraction Twenty-four grams of grounded nut shell powder was suspended in 1000 mL of 1% w/v aqueous NaOH solution for 5 h around 1008C as described by Parajo et al. [21]. The extract obtained was filtered through filters with 0.45 mm pore diameter and the filtrate was concentrated to 75 mL and stored at 48C until used.

2.3 Measurements 2.3.1 Infrared spectroscopy FT-IR spectrum was recorded on Perkin Elmer Spectrum 100 ATR-FTIR spectrophotometer. The extracted lignin was characterized by comparing its ATR-FTIR spectrum with the spectrum of alkaline lignin obtained from Sigma. Biofilms were dried before Fourier transform infrared (FTIR) analysis. The conditions of analysis were as follows; resolution 2 cm1 and a frequency range of 400–4000 cm1.

2.3.2 Lignin content UV spectroscopy was used to estimate the concentration of aqueous soluble lignin at 215 nm. UV spectrum of alkaline lignin was recorded on UV–Visible Hewlett Packard Spectrophotometer. Alkaline lignin obtained from Sigma Co. was used for the calibration curves. Calibration curves were generated by subjecting increasing amounts of commercial alkaline lignin (0.5–2.5 mg) prepared in 1% aqueous NaOH [22]. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Starch/Sta¨rke 2012, 64, 399–407

2.3.3 Mechanical testing Mechanical properties of the films were determined by standard tensile stress–strain tests in order to measure the modulus (E), ultimate tensile strength tests (s) and elongation at break (e). Stress–strain tests were carried out according to ASTM D638 at room temperature using a universal test machine (Zwick Rolle, 500 N) with a crosshead speed of 10 mm/min. The measurements represent the average of at least five runs.

2.3.4 Thermal analysis Thermogravimetric analyses (TGA) were conducted with Perkin-Elmer Thermo gravimetric analyzer Pyris 1 TGA model. Samples were run from 30 to 5008C with heating rate of 158C/min under air atmosphere. Differentiated scanning calorimetry (DSC) analyses of the films were obtained using a Perkin Elmer DSC Pyris Diamond model device. Samples were run under a nitrogen atmosphere from 30 to 3508C with a heating rate of 108C/min. Nitrogen at a rate of 20 mL/min was used as purge gas. Aluminum pans were filled with 10–20 mg of dry sample and then they were sealed using DSC sample press. An empty pan was used as a reference.

2.3.5 Water absorption test The major drawback of starch-based films is its hydrophilicity. Thus, water absorption measurements at pH 2, 4, and 6 were made. Samples of starch–lignin films were dried, weighed (Wd) and placed in solutions at different pH levels. The swollen samples were taken out at fixed time intervals. After removal of the excess water on the surface via filter paper, the swollen samples were weighed (Ws). The swelling ratios were calculated according to the following equation (1): % Swelling ¼ ½ðWs Wd Þ=Wd 100

(1)

where Ws is the weight of the swollen samples and Wd is the weight of the samples before the swelling test.

2.4 Preparation of starch–lignin polymer films Cornstarch was dried at 1208C for 4 h to eliminate the moisture. Starch–lignin biofilms containing 1.2 wt%, 1.6 wt%, 2 wt%, and 2.4 wt% lignin were prepared. For all these proportions, starch and lignin were added to 20 mL water with constant stirring at 1008C until the mixture became semi-viscous. 0.5 mL of glycerol was added as a plasticizer. Starch–lignin mixture was heated for another 5 min. At the end of 5 min, the mixture was poured onto a Teflon1 plate and left to cure at 308C [23].


Starch/Sta¨rke 2012, 64, 399–407

2.5 Preparation of drug loaded biofilms For the preparation of the drug loaded biofilms, 2 wt% lignin containing biofilms were prepared as described in Section 2.4. Dried biofilms were then placed in vials containing 15 mL of saturated aqueous solution of Ciprofloxacin (172 mg/L) for 72 h at 208C. The amount of Ciprofloxacin incorporated in the biofilms was calculated as the difference between the initial and final concentrations in the solution, from absorbance measurements at 277 nm. Then the biofilms were dried in an oven at 408C [24].

2.6 Release studies Release studies were performed as described by Wang et al. [24] The drug loaded films were suspended in glass vessels containing 20 mL of buffer solutions at different pHs (pH 1.0, 3.6, 5.0, and 7.4) and incubated on a shaking water bath at 378C. The buffer solutions used in this study are: pH 1.0 (0.1 M HCl solution, acts as simulated gastric liquid), pH 3.6 and pH 5.0 (10 mM solution acetate buffer), and pH 7.4 (10 mM NaH2PO4–Na2HPO4-buffered solution, acts as simulated intestinal liquid). At appropriate time intervals 3 mL of the solutions were withdrawn and the amount of ciprofloxacin hydrochloride released from the drug loaded films were evaluated by UV spectrophotometry at 277 nm. Then an equal volume of the same dissolution medium was added back to maintain a constant volume. All the experiments were done in triplicate.

3

Results and discussion

3.1 FTIR and UV investigation In this study, lignin was extracted from a renewable and a cheap source; hazel nut. The extracted lignin was charac-

401 terized by ATR-FTIR analysis and UV spectrophotometric measurements. When the ATR-FTIR spectra of extracted lignin and alkaline lignin obtained from Sigma Co. were compared; almost identical spectra were obtained. In both spectra the broad peaks between 3600 and 3200 cm1 are due to the alcoholic and phenolic –OH absorptions. The broad peaks at around 1600 and 1400 cm1 are attributed to the aromatic structures (Fig. 1). The amount of the extracted lignin was determined by measuring the UV absorbance at 205–215 nm. The calibration curve was prepared by using 1 wt% NaOH solutions of the standard lignin in the concentration range of 0.5–2.5 mg/100 mL. The amount of lignin was found as 2.4 g/100 mL. Then the extracted lignin was used to prepare starch– lignin biofilms. Glycerol and sorbitol were used as plasticizers. The films prepared with sorbitol were not evaluated because they were too brittle to be tested. Starch–lignin biofilms containing 1.2 wt%, 1.6 wt%, 2 wt%, and 2.4 wt% lignin were prepared (Table 1). Samples were designated as TPSLX where X stands for the weight fraction of lignin. For example, TPSL2.4 means that the weight fraction of lignin is 2.4%. The lignin containing films were brown in color and were found to darken as the lignin amount was increased. When the lignin concentration was above 2.4 wt%, it was found that the films did not dry up and formed tacky surfaces. Blends that contain alkaline wood lignin up to 30 wt% were reported previously [23]. However, properties of the resulting films are highly dependent on the origin of the lignin and the extraction method. In our study lignin was extracted from hazelnut shells, so its properties may be different from those obtained from wood and other sources. The ATR–FTIR spectra of the biofilms can be seen in Fig. 2. In these spectra, peaks between 3600 and 3200 cm1 are attributed to the OH groups of the starch, glycerol, and lignin. The absorption bands at 2900 cm1 region are assigned to the C–H stretching vibrations in

Figure 1. ATR-FTIR spectra of (a) standard alkaline lignin (solid line) and (b) extracted lignin (dashed line). ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Starch/Sta¨rke 2012, 64, 399–407

Table 1. The recipe for the preparation of formulations

Sample

Starch (g)

Lignin (g)

Glycerol (mL)

Water (mL)

TPSL0 TPSL1.2 TPSL1.6 TPSL2.0 TPSL2.4

1 1 1 1 1

0.000 0.012 0.016 0.020 0.024

0.5 0.5 0.5 0.5 0.5

20 20 20 20 20

glycerol and starch. The peaks at 1076 and 990 cm1 are characteristics of anhydroglucose ring O–C stretching of starch. The peak occurred at 1645 cm1 is due to the presence of bound water in starch [25].

3.2 Mechanical properties The effect of lignin content on mechanical properties of the films was examined by stress–strain measurements. The results are listed in Table 2. The mechanical properties of the lignin-free films were not determined because suitable films could not be prepared. And the films produced from only starch and glycerol were too brittle to be mechanically tested. From the results it can be seen that the modulus of the biofilms increase and the elongation at break values decrease with increasing the lignin content. These results show that a strong interaction occurs between the lignin and starch. Thus it can be said that the presence of lignin greatly improves the mechanical strength of the biofilms. Although the concentration of lignin was very low, due to the rigid structure of lignin, starch–lignin biofilms with bet-

ter mechanical properties were prepared. On the other hand, a dramatic decrease in the modulus was observed for the films containing the highest amount of lignin. This decrease may arise from the phase separation between the hydrophobic lignin and the hydrophilic starch moieties. At low lignin contents the phase separation can be reduced by the strong interactions between the functional groups of lignin and starch. Glycerol may contriubute to the miscibility of the phases. But at lignin concentration; 2.4 wt%, phase separation cannot be tolerated and so the strength of the material decreases. Also glycerol concentration may not be enough to tolerate the incompatibility between the two phases. Phase separation phenomena in starch/polymer blends were observed in many studies [26–28]. In these studies, the mechanical and thermal properties of the starch/polymer blends were found to be closely related with the phase separation phenomena. The decrease in the mechanical properties of the films was also attributed to the agglomeration of lignin [23]. In our case the decrease in the modulus of the films was attributed to the reduction of the crystalline phase when the amount of lignin was 2.4 wt%. The evidence for the loss of crystallinity can be seen in the DSC spectra of the films. From the results displayed in Table 2, it can be seen that while the tensile modulus of the TPSL2.4 films was decreased, the tensile strength of the films was doubled. The increase in tensile strength may be attributed to the re-formation of crystalline domains by the orientation of the lignin molecules during stretching of the films while being tested. Thus it can be said that the use of high amounts of lignin for the preparation of starch–lignin biofilms distorts the crystalline phase of lignin and results in a more amorphous structure.

Figure 2. ATR-FTIR spectra of the biofilms containing (a) 0 wt% lignin, (b) 1.2 wt% lignin, (c) 1.6 wt% lignin, (d) 2.0 wt% lignin, and (e) 2.4 wt% lignin. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Table 2. Tensile properties of the biofilms

Sample

Tensile modulus (N/mm2)

TPSL1.2 TPSL1.6 TPSL2.0 TPSL2.4

282 589 670 171

4.1 6.8 8.3 3.6

Tensile strength (N) 28 31 33 60

1.9 2.6 3.0 3.8

Table 3. Thermal properties of the hybrid films at air atmosphere Elongation at break (%) 47 35 21 97

2.4 3.7 1.7 5.6

Sample

T10 (8C)a)

Max. weight loss (8C)

Char yield (%)

TPSL0 TPSL1.2 TPSL1.6 TPSL2.0 TPSL2.4

248 220 227 228 227

318 267 281 287 295

11 24 25 27 28

3.3 Thermal behaviors

a) T10 is the temperature at 10% weight loss.

The thermal properties of the starch–lignin biofilms were characterized by TGA in air atmosphere. Figure 3 shows the TGA thermograms of the biofilms and the results are given in Table 3. As it is seen that the samples exhibited a 10% weight loss at around 220–2508C and rapid losses were obtained over the temperature range 260–3208C. Thermogravimetric analyses showed that with the increase in lignin content, the initial and the final weight loss temperatures shifted to lower values. The decrease in the thermal stability of lignin containing biofilms can be attributed to the impurities in the lignin extract. The char yields at 5008C were also collected. The higher the lignin content in the films, the more char residue was observed. These high char yields result from the thermally stable aromatic structures in the lignin backbone. Lignins depending on the origin and the method of extraction, can exhibit high thermal stabilities, high char formations

[29, 30]. Since lignin produces high char yield, it may be used as a flame retardant. The DSC spectra of the TPS/ lignin blends are shown in Fig. 4. From the DSC spectra of the biofilms, we could not be able to detect glass transition temperatures (Tg). Reported glass transition temperatures for starch-based materials are inconsistent due to the complexity of thermal behavior of starches used and different measurement conditions [31]. Also it was reported that the glass transition temperatures of the starch containing materials vary with the amount of the plasticizer and water [32]. In our study the glass transition temperatures may be below 308C due to the high amount of plasticizer and water in the composition. On the other hand we were able to detect endothermic peaks at high temperatures. The melting temperature of TPSL0 was found as 1838C. When lignin was added, two new peaks appeared at around

Figure 3. TGA curves of the biofilms with different lignin content at air atmosphere. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Figure 4. DSC thermograms of the biofilms (a) TPSL0, (b) TPSL1.2, (c) TPSL1.6, (d) TPSL2.0, and (e) TPSL2.4.

1508C and 2808C except for TPSL2.4, in which only a large endotherm was observed at 1808C. These endothermic peaks were attributed to the glass transition and the melting temperature of lignin, respectively [33]. The large endothermic peak in the spectrum of the TPSL2.4 is due to the deformation of the crystalline phase of lignin. In this case the absence of the peak corresponding to the melting temperature of lignin indicates that lignin transforms into an amorphous phase from its semi-crystalline structure. Thus, as discussed in Section 3.2, the modulus of TPSL2.4 decreases.

3.4 Swelling measurements The water absorption and swelling are important factors that determine the rates of drug release. Water absorption tests were performed at three different pHs. The plots of percent swelling ratios of the biofilms with different lignin contents versus time can be seen in Fig. 5a–c. When these plots are examined, it is clearly seen that the water absorption of the biofilms increase with increasing the lignin content. This can be related with increasing phase separation between the two phases as the lignin domains increase. When the amount of lignin is increased in the biofilms, the system becomes more incompatible in water and starch prefers to bind water molecules rather than lignin. Moreover we must point out that the percent swelling values increase with an increase in pH. However, a slight decrease was observed when pH was changed from 2 to 4. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

When the lignin content was highest (TPSL2.4), the swelling percentage did not change even pH was shifted from 4 to 6.

3.5 Drug release properties TPSL2.0 was chosen for drug release studies since its mechanical properties were enhanced compared to other formulations. Ciprofloxacin was used as a model drug. It was found that the drug release was accelerated with decrease in pH (Fig. 6). This result agrees with swelling studies. At higher pHs the swelling ratio of the biofilms increases and thus both the amount of absorbed water and drug increases. At all pHs, linear steep phases of the drug loaded biofilms indicate fast release of the drug. In order to investigate the drug release kinetics, several mathematical models have been developed [34]. In this study, drug release mechanism of starch/lignin biofilms was evaluated using Korsmeyer–Peppas model which is suitable for the analysis of polymeric systems [35]. In this model, the mechanism of drug release was analyzed according to the following equation (2): Mt =M1 ¼ kt n

(2)

where Mt/M1 is fraction of drug released at time t, k is the rate constant and n is the release exponent which is used to characterize different release mechanisms. To determine the exponent of n, first 60% drug release data (Mt/M1