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Electrospun gelatin nanofibers as drug carrier: effect of crosslinking on sustained release
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 3 (2016) 3484–3491
www.materialstoday.com/proceedings
International Conference on Advances in Bioprocess Engineering and Technology 2016 (ICABET 2016)
Electrospun gelatin nanofibers as drug carrier: effect of crosslinking on sustained release Anindita Laha, Chandra S. Sharma, Saptarshi Majumdar * *Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Kandi, Sangareddy 502285,Telangana, India
Abstract This study aims to develop a natural biopolymer based nanofabric as an oral drug carrier to deliver a model hydrophobic drug (piperine) in a sustained and controlled manner over prolonged time. For that, we electrospun gelatin nanofabric mesh followed by crosslinking using saturated vapor of glutaraldehyde (GTA) for 8 mins to improve the water resistive properties. Later, we investigated the effect of different crosslinking strategies and found that layer-by-layer crosslinking not only provides an improved structural integrity, thermal and chemical stability of drug but also yields a tight control over sustained drug release up to 8 h.
© 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016. Keywords:Gelatin nanofiber; drug delivery; hydrophobic drug; crosslinking; glutaraldehyde vapor
1. Introduction Controlled drug delivery system delivers pharmaceutical active molecules in a predetermined manner for a defined time interval [1]. The need of using a polymeric based controlled drug delivery system over conventional therapies is to control or monitor the drug concentration in the body within the therapeutic window [1-3]. A diverse range of polymeric drug delivery system has been developed to achieve the different drug release pattern for treatment specific applications [4- 7].
* Corresponding author. Tel.: 91-40-23016087; fax: +91-40-23016032. E-mail address: [email protected]. 2214-7853© 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016.
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Nanofibers have recently been studied as one of the potential polymer drug carrier due to their large surface area and porosity. Electrospinning is one of the most versatile as well as inexpensive way to form continuous fibers with varying morphology from only beads to beaded fibers to fibers in the size range of nano to few microns from natural and synthetic polymers [6, 8]. The potential application of electrospun nanofibers in wound healing, as scaffolds in tissue engineering, affinity membrane, biosensor, drug delivery are explored by different research groups [9-12]. Natural polymers received more attention over synthetic polymers based nanofibers especially in the biomedical area because of their biocompatibility, biodegradability, hydrophilicity and non-toxic effect on physiological system [12, 13]. However, the application of natural polymer in biomedical area is limited due to its poor mechanical and structural stability in aqueous medium. Efforts have been made in terms of polymeric blending, chemical or physical crosslinking to overcome some of these drawbacks. Gelatin is a FDA approved natural polymer which is partially hydrolyzed from collagen. Collagen is the mainly found in the bones, cartilage, tendon and connecting tissues of animals. Due to its biodegradability, non-toxic, biocompatibility and in expensive property, gelatin has been extensively used in food and medical industries [13-15]. However gelatin nanofibers have very poor structural consistency in aqueous conditions. Thus crosslinking of gelatin fibers were investigated to improve the water-resistant and thermo-mechanical properties for biomedical applications [15-18]. In this study, we have crosslinked gelatin nanofiber mesh by exposing it in saturated vapor of GTA for only few minutes. As compared to available literature, we reduced the exposure time substantially from few hours to few minutes to avoid toxicity associate to GTA vapor [18, 19, 20]. In a recent study [21], we found 8 min of saturated GTA vapors exposure sufficient enough to sustain the mesh in aqueous medium for more than 48 h. However the drawback of this direct crosslinking was the excessive shrinkage of the nanofiber mesh. To minimize this shrinkage and achieve more controlled and sustained drug release, we suggest sequential layer-by-layer crosslinking. A comparative study between sequential layer-by-layer crosslinking and crosslinking only after final deposition is made in the present work in terms of morphology, chemical and thermal stability of the mesh. Lastly, we have also investigated the effect of different crosslinking mechanism on in-vitro drug release profiles. Piperine is considered as a model hydrophobic drug in this work. Nomenclature GNF GNF/P GNF/P/C8 GNF/P/LBLC8 GTA GI
gelatin nanofiber mesh piperine loaded gelatin nanofiber mesh piperine loaded gelatin nanofiber mesh with 8min crosslinking piperine loaded gelatin nanofiber mesh with sequential 8min crosslinking glutaraldehyde gastrointestinal tract
2. Experimental Procedures 2.1. Materials Gelatin (Type A, 175 bloom), Piperine (98%), Hydrochloric acid (ACS, 36.5-38.0%), Gluteraldehyde (25% v/v aqueous solution), Acetic acid (glacial, ACS, 99.7+%), Sodium hydroxide pallets (98%), Phosphate buffer saline (pH 7.4) were purchased from Alfa Aesar (A Johnson Matthey Company, India). Deionized water (DI) (Milli Q water 18.1 Ω) was used throughout the experiments. All the chemicals were used as received. 2.2. Fabrication of gelatin fiber To prepare the electrospinning solution, gelatin (type A) was dissolve by 20% (w/v) of the solution weight in acetic acid solution (20% v/v in distilled water) under constant magnetic agitation and at a constant room temperature for 3 h. Piperine was added (2 mg/ml) in the homogeneous gelatin solution to prepare drug loaded
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nanofiber mesh. Gelatin nanofibers mesh (GNF) and drug loaded nanofiber mesh (GNF/P) was fabricated using electrospinning apparatus (E Spin Nanotech Pvt. Ltd, India). The different parameters in this study were: Voltage between tip and collector: 12 kV Solution flow rate: 5 µl/min Needle diameter: 21 gauge Distance between tip and collector: 10 cm Temperature: 25 0C Relative humidity: 30% Metal collector was covered by aluminum foil on which the deposition of 5 ml polymer solution was done to fabricate the mesh. 2.3. Crosslinking mechanism Electrospun GNF/P meshes dissolve in water within few seconds due to its hydrophilicity and high surface area to volume ratio. Therefore, crosslinking was done by exposing the mesh in saturated vapor of GTA (25% v/v aqueous solution) for different time interval. Nanofiber meshes with the aluminum foil were cut into 2 × 2 cm2 and placed inside the closed glass desiccator with 20 ml of GTA solution. GNF/P fabric was then crosslinked using saturated GTA vapor for total 8 min in two different ways: First complete spinning thick fabric and then crosslinking only once in last. Secondly, crosslinking was done in sequential fashion as shown in Fig. 1a. After spinning the first thin layer of GNF/P, it was cross linked for 2 min followed by further deposition of fibers and subsequently crosslinking and so on. 2.4. Degradation and swelling study To check the effect of different crosslinking mechanism on stability of the mesh in-vitro degradation and swelling study was carried out for GNF/P/C8 and GNF/P/LBLC8 in different pH mediums i.e. pH 1.2 and 7.4. Both the cases samples were cut (5 × 5 cm2) and weighed (Mi) and dried samples were placed in 10 ml of PBS (pH 7.4) and 0.1N HCl (pH 1.2) solution at 37 0 C for 50 h. At the fixed time intervals, swelled samples were taken out and weighed (Ms) after blot dried using tissue papers. Similar time interval was maintained and samples were dried in a vacuum oven at room temperature and weighed (Mf). The weight loss (WL%) and swelling degree (SD%) are calculated by using the following equations [20-22] :
Weight loss :
WL(%) (1
Swelling degree: SD(%) (
Mf Mi
) 100
Ms 1) 100 Mi
Where, Mi = Initial sample mass, Mf = Sample mass after an incubation period, Ms = swollen sample mass. 2.5. Morphology of nanofiber mesh The morphology of the nanofiber meshes were examined by table top scanning electron microscopy (SEM) (Phenom world, Model: Pro X). To reduce charging effect, samples were coated with thin gold layer using sputter coater (Excel Instruments, India) for 6 s.
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2.6. Drug-polymer interaction study: To check the functional group of polymer and drug, Fourier Transform Infra-Red spectroscopy (FTIR) (Bruker Corp., Tensor 37) were performed in transmittance mode in 400-4000 cm-1 range with a resolution of 4 cm−1 and 256 scans per samples. Background scan was done at room temperature for all the samples for baseline corrections. 2.7. Thermal stability To check the effect of crosslinking on gelatin nanofiber mesh, Differential Scanning Calorimetry (DSC) was carried out in argon atmosphere using DSC 1 Stare system (Mettler Toledo: Switzerland). It was performed in the range to 35 to 250 0 C with the heating rate of 10 0C/min. 2.8. Drug release mechanism To study the effect of different crosslinking strategies on controlling release of piperine from gelatin nanofiber matrix, in-vitro release study was performed maintaining the physiological conditions. GNF/P/C8 and GNF/P/LBLC8 were cut in 5 × 5 cm2 pieces and placed in 30 ml of release medium with different pH levels (1.2 similar to pH of stomach and 7.4 similar to pH of intestine). The samples were placed in mechanical shaker (Remi RIS-24 plus) for 50 h at 37 0C and 150 RPM, to mimic the physiological conditions. Samples of 3 ml were taken from the release medium at a fixed time intervals and fresh solution was added back to maintain the total volume constant. The amount of piperine present in the medium was analyzed using UV spectrophotometer (Perkin Elmer, Lambda 35) at 342 nm as λmax for piperine. The percentage of cumulative drug release as a function of time was calculated using the following equation: Cumulative amount of release (%) (
Ct ) 100 C
Where, Ct is the amount of piperine released at time t and C∞ refers to total amount of drug loaded in 5 × 5 cm2 sample. All results were performed thrice to confirm reproducibility of the results. 2.9. Statistical analysis The observations are presented as mean ± standard deviation (SD) of three independent experiments to confirm reproducibility of the findings. All the plots were analyzed using Origin Pro 8 software. 3. Results and discussion 3.1. Crosslinking of mesh and assay of water resistivity Gelatin is consisting with both positive and negative amino acids results its polyampholyte nature [19]. Lysine is one of the amino acid with reacts with GTA within very short period of time. Due to the low cost and excellent capability to crosslink collagenous, GTA has been widely used as crosslinking agents, though it has cytotoxicity effects. Thus, the main objective was to reduce the concentration or exposure time of GTA vapor crosslinking to reduce its cytotoxicity [19] as compared to literature [18, 20]. Finally, we were recently able to reduce it to 8 min of exposure in saturated vapor of GTA that was enough to sustain in aqueous medium for more than 48 h [21]. However the crosslinking was not uniform in every layers of the fabric. Thus excessive shrinkage of the mesh was observed. This non-uniformity of the crosslinking leads to less water resistivity as compared to sequential crosslinking. In case of sequential crosslink, the carbonyl group (C=O) of aldehyde can react with amino acid of gelatin in a uniform manner and the compactness of the fabric is better in compared to one time direct final crosslinking. The compactness of the in-between layers may play very important role in terms of water resistivity and further mobility of drug molecules also.
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Fig. 1a showed the schematic diagram of the sequential crosslinking. Here after spinning 2 ml of polymer solution, it was crosslinked for 2 min and followed by further deposition of 1 ml solution and so on. Thus by doing this layer by layer crosslinking, shrinkage in the fabric was reduced with 2 min of exposure. Further deposition of nanofibers as a next layer provided the required support to overcome this shrinkage, if any. Though the total crosslinking time is still 8 min as compared to our previous study [21], layer by layer sequential crosslinking yielded a better uniformity and compactness in the fabric. Fig. 1b and 1c showed the in-vitro degradation of GNF/P/C8 and GNF/P/LBLC8 in pH 1.2 and 7.4. The weight loss (WL) (%) due to hydrolytic degradation in the case of GNF/P/LBLC8 and GNF/P/C8 are 12.3 ± 1.6% and 15.1 ± 1.5% respectively in pH 7.4 after 50 h showed in Fig. 1b. The WL (%) is less in lower pH (1.2) for both the cases (for GNF/P/LBLC8 and GNF/P/C8 7.8 ± 2.8% and 9.5 ± 3.6 % respectively). To understand the reason behind this, swelling study has been done in both the pH (7.4 and 1.2). The swelling degree (SD) (%) of GNF/P/LBLC8 and GNF/P/C8 are 741.1 ± 49.1% and 805.2 ± 36.6% respectively in pH 7.4 after 50 h of deposition in PBS shown in Fig. 1c. GNF/P/LBLC8 showed lesser SD (%) as well as lesser WL (%) in comparison to GNF/P/C8 in both the pH. Interesting with lower pH the SD (%) and WL (%) declined drastically because of the collapse state of protonated amino groups, which is reluctant to swell. Thus, GNF/P/LBLC8 improved water resistivity property than GNF/P/C8. The probable reason behind this phenomenon is the uniformity of the crosslinking through different layers of the gelatin fabric giving better inter-fibrous bonding which creates extra barrier to diffuse water molecules into the fabric. Thus, by reducing the WL (%) and SD (%), it can actually improve the water resistivity of the GNF/P mesh (Fig. 1b and 1c).
Fig. 1. (a) Schematic diagram of different crosslinking strategies using saturated vapor of GTA; Assay of water resistivity properties (b) weight loss (WL) (%); (c) swelling degree (SD) (%) of gelatin nanofiber mesh
3.2. Morphology of the nanofiber mesh The morphology of GNF/P, GNF/P/LBLC8 and GNF/P/C8 are presented in Fig. 2a, 2b and 2c respectively. As GNF/P is very sensitive to moisture, while crosslinking with GTA vapor it damages the outer layer of the mesh. The fused fiber morphology is more visible in the case of GNF/P/C8 (Fig. 2c) compared to GNF/P/LBLC8 (Fig. 2b). The probable reason behind this is the 8 min exposure in GTA vapor damages the outer layer of gelatin nanofiber much more in comparison to GNF/P/LBLC8 where the exposure time for last layer is limited to only 2 min. Thus fiber morphology remains intact in case of GNF/P/LBLC8 (Fig. 2b). Though the fiber structure of the outer layer of GNF/P/LBLC8 remains intact even after layer by layer crosslinking, it also gives better water resistivity because of the evenly crosslinked, tightly packed middle layers of the fabric. In contrast, GNF/P/C8 shows the poor crosslinking of in-between layers of the fabric that was also confirmed by higher SD (%) and WL (%) as summarized in Fig. 1b and 1c.
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Fig. 2. SEM images of (a) GNF/P; (b) GNF/P/LBLC8; (c) GNF/P/C8 mesh
3.3. Assay of polymer-drug interaction IR spectra of GNF, GNF/P, GNF/P/C8 and GNF/P/LBLC8 were presented in Fig. 3a. The intensity of absorbent band at 3280.5 cm-1 (N-H stretching vibration) and 1630.5 cm-1 (bending vibration of N-H group) are decreased which indicates the crosslinking in these nanofiber mesh. The shift of peaks towards low wavenumber after 8 min of saturated GTA vapor is also a good indicator of crosslinking, which is more prominently observed in GNF/P/LBLC8. Similar crosslinking effect on gelatin nanofiber mesh is reported by W. Lu et.al [16]. 3.4. Thermal stability of gelatin nanofiber mesh DSC thermograms of GNF/P, GNF/P/C8 and GNF/P/LBLC8 in the range of 35 to 250 0C were carried out to investigate the effect of different crosslinking strategies on thermal stability of nanofibers. Fig. 3b exhibits two endothermic peaks which are characteristic peaks of gelatin [17]. Comparing the DSC curves of GNF/P, GNF/P/C8 and GNF/P/LBLC8, there is a shift in the denaturation temperature towards higher temperature (90.6 to 96.1 to 101.5 0C) is observed. These result extrapolate that sequential layer by layer crosslinking of piperine loaded gelatin mesh (GNF/P/LBLC8) can provide a better thermal stability compared to one time direct crosslinked (GNF/P/C8) fiber mesh. 3.5. Drug release study In order to investigate the effect of different crosslinking strategies on release profile of piperine, in-vitro release study was carried out in the similar pH as per the GI tract. The effect of sequential crosslinking on nanofiber mesh was checked by in-vitro degradation studies, morphology, thermal and chemical stability of the nanofiber matrix. These results lead us to examine the crosslinking effect on drug release profiles. Fig. 3c shows the release profile of piperine from GNF/P/C8 and GNF/P/LBLC8 meshes in different physiological pH (1.2 and 7.4). A time scale of 24 h was chosen for the release study. An initial fast release of drug molecules within 8 h was observed in case of GNF/P/C8. GNF/P/LBLC8 meshes exhibited better control on the fast release of piperine in those initial hours (8 h) of the release study. GNF/P/LBLC8 meshes successfully tailored the initial fast release as well as the overall release of piperine for both the pH. These observations can be explained from previous results of SD (%), WL (%) and morphological images of the mesh. Sequential crosslinking (GNF/P/LBLC8) of the mesh showcased uniform crosslinking through the different layers of the fabric thus showed lesser WL (%) and SD (%) in compared to GNF/P/C8 (Fig. 1b and 1c respectively). GNF/P/LBLC8 showed better chemical and thermal stability in compared to GNF/P/C8 (Fig 3a and 3b respectively).
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It is interesting to note that, though the total crosslinking time is same (8 min) for both GNF/P/C8 and GNF/P/LBLC8, GNF/P/LBLC8 showed a sustained release of piperine for 24 h. The probable reason behind this observation might be the uniformity of crosslinking and tightly packed structure of intermediate layers of the mesh. It formed strong inter-fibrous bonding which created extra barrier to diffuse drug molecules in the release medium. Thus GNF/P/LBLC8 exhibited a better stability of the vehicle and also played major role in achieving the sustained release over a period of time. On the other hand, the hierarchy of crosslinking degree among the intermediate layers of the fabric leads fast release of drug molecules in the initial hours. Fig. 3c illustrates the drug release profiles in different pH. The cumulative release (%) for piperine after depositing the GNF/P/LBLC8 and GNF/P/C8 meshes in pH 7.4 for 24 h are 55.2 ± 3.7 % and 65.4 ± 4.6% respectively. The initial fast releases of piperine within 8 h are successfully tailored by approx. 20% in the case of GNF/P/LBLC8 (40.6 ± 1%) compared to GNF/P/C8 (59.7 ± 2.9%). Thus sequential crosslinking not only toned down the initial fast release, it controlled the overall release profiles. Typically, the vehicle has to pass through the harsh condition (pH 1.2) of stomach within 4-5 h followed by the absorption site i.e small intestine (pH 7.4). We have studied the release profiles in pH 1.2 for 24 h for both the meshes. It is observed that the cumulative release (%) for GNF/P/LBLC8 and GNF/P/C8 meshes in pH 1.2 after 24 h are 46.3 ± 7.4% and 59.4 ± 3.5% respectively which is substantially decreased in case of GNF/P/LBLC8 and GNF/P/C8 meshes after 4h (28.9 ± 3.6% and 43.3 ± 1.9% respectively). The cumulative release of piperine is less in lower pH for both the samples. This might be caused because of the ionization of amino group present in gelatin in a low pH medium, which discourages formation of H-bonds with water molecules resulting less swelling of the mesh. Thus in lower pH, the protonated amino groups collapse and doesn’t swell much. Due to less swelling of the mesh, drug molecules don’t get sufficient osmotic pressure to travel from the mesh to release medium. Releasing less drug in lower pH (pH 1.2: similar to stomach) is good as the absorbance site of the drug is intestine. It is observed that, compared to GNF/P/C8, GNF/P/LBLC8 mesh doesn’t swell much in lower pH resulting less WL (%) after more than 48 h. Thus GNF/P/LBLC8 mesh shows better control on piperine release compared to GNF/P/C8. These results provide a better understanding to the crosslinking of electrospun gelatin nanofibers and thus to facilitate their use as potential drug delivery system with controlled release.
Fig. 3. (a) FT-IR of crosslinked and noncrosslinked GNF; (b) DCS thermogram of crosslinked and noncrosslinked GNF; (c) In-vitro cumulative release of piperine from different crosslinked samples.
4. Conclusion In conclusion, we successfully fabricated drug loaded gelatin nanofiber mesh by electrospinning and also crosslinked by GTA vapor with two different strategies. Interestingly, only 8 min crosslinking yielded desired stability of the mesh in aqueous medium for more than 48 h. However non-uniformity and shrinkage problems are addressed by sequential layer by layer crosslinking (2+2+2+2 min) of GNF/P mesh, keeping total crosslinking time
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constant (8 min). The crosslinking was even in different layers of the mesh and formed strong inter-fibrous bonding which created extra barrier to diffuse drug molecules in the release medium. Drug release profiles clearly suggest that this sequential crosslinking of piperine loaded gelatin nanofabric improved the mesh further in terms of water resistivity, thermal and chemical stability and therefore can be used as a potential drug carrier with sustained release over an extended period of time interval. Acknowledgements We acknowledge Indian Institute of Technology Hyderabad for providing necessary research infrastructure to carry out this work. References [1] K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M. Shakesheff, Chem. Rev. 99 (1999) 3181-3198. [2] R. S. Langer, NA Peppas, Biomaterials 2 (1981) 201-214. [3] R. S. Langer, NA Peppas, AIChE J. 49 (2003) 2990-3006. [4] A. Laha, U. Bhutani, K. Mitra and S. Majumdar, Mater. Manuf.Processes, 2015, DOI: 10.1080/10426914.2015.1070422. [5] U. Bhutani, A.Laha, K. Mitra and S.Majumdar, Mater Lett, DOI:10.1016/j.matlet.2015.10.114 [6] X. Hu , S. Liu , G. Zhou, Y. Huang, Z. Xie, X. Jing, J. Controlled Release 185 (2014) 12–21. [7] G. Fundueanu, M. Constantin, P. Ascenzi, Biomaterials 29 (2008) 2767–2775. [8] A. Furmhals, US patent 1,975,504, 1934. [9] T.J. Sill, H.A. von Recum, Biomaterials 29 (2008) 1989–2006. [10] P. Zahedi, I. Rezaeian, S.-O. Ranaei-Siadat, S.-H. Jafari, P. Supaphol, Polym. Adv. Technol. 21 (2010) 77–95. [11] T. Kowalczyk, A. Nowicka, D. Elbaum, T.A. Kowalewski, Biomacromolecules 9 (2008) 2087–2090. [12] J. Zeng, X. Xu, X. Chen, Q. Liang, X. Bian, L. Yang, X. Jing, J. Controlled Release 92 (2003) 227–231. [13] H. Nie, J. Li, A. He, S. Xu, Q. Jiang, C. C. Han, Biomacromolecules 11 (2010) 2190-2194. [14] S.Young, M. Wong, Y. Tabata, A.G. Mikos, J. Controlled Release 109 (2005) 256– 274. [15] C-H. Huang, C-Y. Chi, Y-S. Chen, K-Y. Chen, P-L. Chen, C-H. Yao, Mater Sci Eng C Mater Biol Appl 32 (2012) 2476–2483. [16] W. Lu,M. Ma,H. Xu,B. Zhang,X. Cao,Y.Guo, MaterialsLetters 140 (2015)1–4. [17] Y. Su, X. Mo, J.Controlled Release 152 (2011) e230–e232. [18] Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Polymer 47 (2006) 2911–2917. [19] S. Farris, J. Song, Q. Huang, J. Agric. Food Chem. 58 (2010) 998–1003. [20] D.M. Correia, J. Padrão, L.R. Rodrigues, F. Dourado, S. Lanceros-Méndez , V. Sencadas, Polym Test 32 (2013) 995–1000. [21] A. Laha, S. Yadav, S. Majumdar, C.S. Sharma, Biochem. Eng. J. DOI: 10.1016/j.bej.2015.11.001. [22] D.C. Aduba Jr., J. A. Hammer, Q. Yuan, W. A. Yeudall, G. L. Bowlin, H. Yang, Acta Biomater 9 (2013) 6576–6584.
ScienceDirect Materials Today: Proceedings 3 (2016) 3484–3491
www.materialstoday.com/proceedings
International Conference on Advances in Bioprocess Engineering and Technology 2016 (ICABET 2016)
Electrospun gelatin nanofibers as drug carrier: effect of crosslinking on sustained release Anindita Laha, Chandra S. Sharma, Saptarshi Majumdar * *Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Kandi, Sangareddy 502285,Telangana, India
Abstract This study aims to develop a natural biopolymer based nanofabric as an oral drug carrier to deliver a model hydrophobic drug (piperine) in a sustained and controlled manner over prolonged time. For that, we electrospun gelatin nanofabric mesh followed by crosslinking using saturated vapor of glutaraldehyde (GTA) for 8 mins to improve the water resistive properties. Later, we investigated the effect of different crosslinking strategies and found that layer-by-layer crosslinking not only provides an improved structural integrity, thermal and chemical stability of drug but also yields a tight control over sustained drug release up to 8 h.
© 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016. Keywords:Gelatin nanofiber; drug delivery; hydrophobic drug; crosslinking; glutaraldehyde vapor
1. Introduction Controlled drug delivery system delivers pharmaceutical active molecules in a predetermined manner for a defined time interval [1]. The need of using a polymeric based controlled drug delivery system over conventional therapies is to control or monitor the drug concentration in the body within the therapeutic window [1-3]. A diverse range of polymeric drug delivery system has been developed to achieve the different drug release pattern for treatment specific applications [4- 7].
* Corresponding author. Tel.: 91-40-23016087; fax: +91-40-23016032. E-mail address: [email protected]. 2214-7853© 2015 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering and Technology 2016.
Laha et al./ Materials Today: Proceedings 3 (2016) 3484–3491
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Nanofibers have recently been studied as one of the potential polymer drug carrier due to their large surface area and porosity. Electrospinning is one of the most versatile as well as inexpensive way to form continuous fibers with varying morphology from only beads to beaded fibers to fibers in the size range of nano to few microns from natural and synthetic polymers [6, 8]. The potential application of electrospun nanofibers in wound healing, as scaffolds in tissue engineering, affinity membrane, biosensor, drug delivery are explored by different research groups [9-12]. Natural polymers received more attention over synthetic polymers based nanofibers especially in the biomedical area because of their biocompatibility, biodegradability, hydrophilicity and non-toxic effect on physiological system [12, 13]. However, the application of natural polymer in biomedical area is limited due to its poor mechanical and structural stability in aqueous medium. Efforts have been made in terms of polymeric blending, chemical or physical crosslinking to overcome some of these drawbacks. Gelatin is a FDA approved natural polymer which is partially hydrolyzed from collagen. Collagen is the mainly found in the bones, cartilage, tendon and connecting tissues of animals. Due to its biodegradability, non-toxic, biocompatibility and in expensive property, gelatin has been extensively used in food and medical industries [13-15]. However gelatin nanofibers have very poor structural consistency in aqueous conditions. Thus crosslinking of gelatin fibers were investigated to improve the water-resistant and thermo-mechanical properties for biomedical applications [15-18]. In this study, we have crosslinked gelatin nanofiber mesh by exposing it in saturated vapor of GTA for only few minutes. As compared to available literature, we reduced the exposure time substantially from few hours to few minutes to avoid toxicity associate to GTA vapor [18, 19, 20]. In a recent study [21], we found 8 min of saturated GTA vapors exposure sufficient enough to sustain the mesh in aqueous medium for more than 48 h. However the drawback of this direct crosslinking was the excessive shrinkage of the nanofiber mesh. To minimize this shrinkage and achieve more controlled and sustained drug release, we suggest sequential layer-by-layer crosslinking. A comparative study between sequential layer-by-layer crosslinking and crosslinking only after final deposition is made in the present work in terms of morphology, chemical and thermal stability of the mesh. Lastly, we have also investigated the effect of different crosslinking mechanism on in-vitro drug release profiles. Piperine is considered as a model hydrophobic drug in this work. Nomenclature GNF GNF/P GNF/P/C8 GNF/P/LBLC8 GTA GI
gelatin nanofiber mesh piperine loaded gelatin nanofiber mesh piperine loaded gelatin nanofiber mesh with 8min crosslinking piperine loaded gelatin nanofiber mesh with sequential 8min crosslinking glutaraldehyde gastrointestinal tract
2. Experimental Procedures 2.1. Materials Gelatin (Type A, 175 bloom), Piperine (98%), Hydrochloric acid (ACS, 36.5-38.0%), Gluteraldehyde (25% v/v aqueous solution), Acetic acid (glacial, ACS, 99.7+%), Sodium hydroxide pallets (98%), Phosphate buffer saline (pH 7.4) were purchased from Alfa Aesar (A Johnson Matthey Company, India). Deionized water (DI) (Milli Q water 18.1 Ω) was used throughout the experiments. All the chemicals were used as received. 2.2. Fabrication of gelatin fiber To prepare the electrospinning solution, gelatin (type A) was dissolve by 20% (w/v) of the solution weight in acetic acid solution (20% v/v in distilled water) under constant magnetic agitation and at a constant room temperature for 3 h. Piperine was added (2 mg/ml) in the homogeneous gelatin solution to prepare drug loaded
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nanofiber mesh. Gelatin nanofibers mesh (GNF) and drug loaded nanofiber mesh (GNF/P) was fabricated using electrospinning apparatus (E Spin Nanotech Pvt. Ltd, India). The different parameters in this study were: Voltage between tip and collector: 12 kV Solution flow rate: 5 µl/min Needle diameter: 21 gauge Distance between tip and collector: 10 cm Temperature: 25 0C Relative humidity: 30% Metal collector was covered by aluminum foil on which the deposition of 5 ml polymer solution was done to fabricate the mesh. 2.3. Crosslinking mechanism Electrospun GNF/P meshes dissolve in water within few seconds due to its hydrophilicity and high surface area to volume ratio. Therefore, crosslinking was done by exposing the mesh in saturated vapor of GTA (25% v/v aqueous solution) for different time interval. Nanofiber meshes with the aluminum foil were cut into 2 × 2 cm2 and placed inside the closed glass desiccator with 20 ml of GTA solution. GNF/P fabric was then crosslinked using saturated GTA vapor for total 8 min in two different ways: First complete spinning thick fabric and then crosslinking only once in last. Secondly, crosslinking was done in sequential fashion as shown in Fig. 1a. After spinning the first thin layer of GNF/P, it was cross linked for 2 min followed by further deposition of fibers and subsequently crosslinking and so on. 2.4. Degradation and swelling study To check the effect of different crosslinking mechanism on stability of the mesh in-vitro degradation and swelling study was carried out for GNF/P/C8 and GNF/P/LBLC8 in different pH mediums i.e. pH 1.2 and 7.4. Both the cases samples were cut (5 × 5 cm2) and weighed (Mi) and dried samples were placed in 10 ml of PBS (pH 7.4) and 0.1N HCl (pH 1.2) solution at 37 0 C for 50 h. At the fixed time intervals, swelled samples were taken out and weighed (Ms) after blot dried using tissue papers. Similar time interval was maintained and samples were dried in a vacuum oven at room temperature and weighed (Mf). The weight loss (WL%) and swelling degree (SD%) are calculated by using the following equations [20-22] :
Weight loss :
WL(%) (1
Swelling degree: SD(%) (
Mf Mi
) 100
Ms 1) 100 Mi
Where, Mi = Initial sample mass, Mf = Sample mass after an incubation period, Ms = swollen sample mass. 2.5. Morphology of nanofiber mesh The morphology of the nanofiber meshes were examined by table top scanning electron microscopy (SEM) (Phenom world, Model: Pro X). To reduce charging effect, samples were coated with thin gold layer using sputter coater (Excel Instruments, India) for 6 s.
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2.6. Drug-polymer interaction study: To check the functional group of polymer and drug, Fourier Transform Infra-Red spectroscopy (FTIR) (Bruker Corp., Tensor 37) were performed in transmittance mode in 400-4000 cm-1 range with a resolution of 4 cm−1 and 256 scans per samples. Background scan was done at room temperature for all the samples for baseline corrections. 2.7. Thermal stability To check the effect of crosslinking on gelatin nanofiber mesh, Differential Scanning Calorimetry (DSC) was carried out in argon atmosphere using DSC 1 Stare system (Mettler Toledo: Switzerland). It was performed in the range to 35 to 250 0 C with the heating rate of 10 0C/min. 2.8. Drug release mechanism To study the effect of different crosslinking strategies on controlling release of piperine from gelatin nanofiber matrix, in-vitro release study was performed maintaining the physiological conditions. GNF/P/C8 and GNF/P/LBLC8 were cut in 5 × 5 cm2 pieces and placed in 30 ml of release medium with different pH levels (1.2 similar to pH of stomach and 7.4 similar to pH of intestine). The samples were placed in mechanical shaker (Remi RIS-24 plus) for 50 h at 37 0C and 150 RPM, to mimic the physiological conditions. Samples of 3 ml were taken from the release medium at a fixed time intervals and fresh solution was added back to maintain the total volume constant. The amount of piperine present in the medium was analyzed using UV spectrophotometer (Perkin Elmer, Lambda 35) at 342 nm as λmax for piperine. The percentage of cumulative drug release as a function of time was calculated using the following equation: Cumulative amount of release (%) (
Ct ) 100 C
Where, Ct is the amount of piperine released at time t and C∞ refers to total amount of drug loaded in 5 × 5 cm2 sample. All results were performed thrice to confirm reproducibility of the results. 2.9. Statistical analysis The observations are presented as mean ± standard deviation (SD) of three independent experiments to confirm reproducibility of the findings. All the plots were analyzed using Origin Pro 8 software. 3. Results and discussion 3.1. Crosslinking of mesh and assay of water resistivity Gelatin is consisting with both positive and negative amino acids results its polyampholyte nature [19]. Lysine is one of the amino acid with reacts with GTA within very short period of time. Due to the low cost and excellent capability to crosslink collagenous, GTA has been widely used as crosslinking agents, though it has cytotoxicity effects. Thus, the main objective was to reduce the concentration or exposure time of GTA vapor crosslinking to reduce its cytotoxicity [19] as compared to literature [18, 20]. Finally, we were recently able to reduce it to 8 min of exposure in saturated vapor of GTA that was enough to sustain in aqueous medium for more than 48 h [21]. However the crosslinking was not uniform in every layers of the fabric. Thus excessive shrinkage of the mesh was observed. This non-uniformity of the crosslinking leads to less water resistivity as compared to sequential crosslinking. In case of sequential crosslink, the carbonyl group (C=O) of aldehyde can react with amino acid of gelatin in a uniform manner and the compactness of the fabric is better in compared to one time direct final crosslinking. The compactness of the in-between layers may play very important role in terms of water resistivity and further mobility of drug molecules also.
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Fig. 1a showed the schematic diagram of the sequential crosslinking. Here after spinning 2 ml of polymer solution, it was crosslinked for 2 min and followed by further deposition of 1 ml solution and so on. Thus by doing this layer by layer crosslinking, shrinkage in the fabric was reduced with 2 min of exposure. Further deposition of nanofibers as a next layer provided the required support to overcome this shrinkage, if any. Though the total crosslinking time is still 8 min as compared to our previous study [21], layer by layer sequential crosslinking yielded a better uniformity and compactness in the fabric. Fig. 1b and 1c showed the in-vitro degradation of GNF/P/C8 and GNF/P/LBLC8 in pH 1.2 and 7.4. The weight loss (WL) (%) due to hydrolytic degradation in the case of GNF/P/LBLC8 and GNF/P/C8 are 12.3 ± 1.6% and 15.1 ± 1.5% respectively in pH 7.4 after 50 h showed in Fig. 1b. The WL (%) is less in lower pH (1.2) for both the cases (for GNF/P/LBLC8 and GNF/P/C8 7.8 ± 2.8% and 9.5 ± 3.6 % respectively). To understand the reason behind this, swelling study has been done in both the pH (7.4 and 1.2). The swelling degree (SD) (%) of GNF/P/LBLC8 and GNF/P/C8 are 741.1 ± 49.1% and 805.2 ± 36.6% respectively in pH 7.4 after 50 h of deposition in PBS shown in Fig. 1c. GNF/P/LBLC8 showed lesser SD (%) as well as lesser WL (%) in comparison to GNF/P/C8 in both the pH. Interesting with lower pH the SD (%) and WL (%) declined drastically because of the collapse state of protonated amino groups, which is reluctant to swell. Thus, GNF/P/LBLC8 improved water resistivity property than GNF/P/C8. The probable reason behind this phenomenon is the uniformity of the crosslinking through different layers of the gelatin fabric giving better inter-fibrous bonding which creates extra barrier to diffuse water molecules into the fabric. Thus, by reducing the WL (%) and SD (%), it can actually improve the water resistivity of the GNF/P mesh (Fig. 1b and 1c).
Fig. 1. (a) Schematic diagram of different crosslinking strategies using saturated vapor of GTA; Assay of water resistivity properties (b) weight loss (WL) (%); (c) swelling degree (SD) (%) of gelatin nanofiber mesh
3.2. Morphology of the nanofiber mesh The morphology of GNF/P, GNF/P/LBLC8 and GNF/P/C8 are presented in Fig. 2a, 2b and 2c respectively. As GNF/P is very sensitive to moisture, while crosslinking with GTA vapor it damages the outer layer of the mesh. The fused fiber morphology is more visible in the case of GNF/P/C8 (Fig. 2c) compared to GNF/P/LBLC8 (Fig. 2b). The probable reason behind this is the 8 min exposure in GTA vapor damages the outer layer of gelatin nanofiber much more in comparison to GNF/P/LBLC8 where the exposure time for last layer is limited to only 2 min. Thus fiber morphology remains intact in case of GNF/P/LBLC8 (Fig. 2b). Though the fiber structure of the outer layer of GNF/P/LBLC8 remains intact even after layer by layer crosslinking, it also gives better water resistivity because of the evenly crosslinked, tightly packed middle layers of the fabric. In contrast, GNF/P/C8 shows the poor crosslinking of in-between layers of the fabric that was also confirmed by higher SD (%) and WL (%) as summarized in Fig. 1b and 1c.
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Fig. 2. SEM images of (a) GNF/P; (b) GNF/P/LBLC8; (c) GNF/P/C8 mesh
3.3. Assay of polymer-drug interaction IR spectra of GNF, GNF/P, GNF/P/C8 and GNF/P/LBLC8 were presented in Fig. 3a. The intensity of absorbent band at 3280.5 cm-1 (N-H stretching vibration) and 1630.5 cm-1 (bending vibration of N-H group) are decreased which indicates the crosslinking in these nanofiber mesh. The shift of peaks towards low wavenumber after 8 min of saturated GTA vapor is also a good indicator of crosslinking, which is more prominently observed in GNF/P/LBLC8. Similar crosslinking effect on gelatin nanofiber mesh is reported by W. Lu et.al [16]. 3.4. Thermal stability of gelatin nanofiber mesh DSC thermograms of GNF/P, GNF/P/C8 and GNF/P/LBLC8 in the range of 35 to 250 0C were carried out to investigate the effect of different crosslinking strategies on thermal stability of nanofibers. Fig. 3b exhibits two endothermic peaks which are characteristic peaks of gelatin [17]. Comparing the DSC curves of GNF/P, GNF/P/C8 and GNF/P/LBLC8, there is a shift in the denaturation temperature towards higher temperature (90.6 to 96.1 to 101.5 0C) is observed. These result extrapolate that sequential layer by layer crosslinking of piperine loaded gelatin mesh (GNF/P/LBLC8) can provide a better thermal stability compared to one time direct crosslinked (GNF/P/C8) fiber mesh. 3.5. Drug release study In order to investigate the effect of different crosslinking strategies on release profile of piperine, in-vitro release study was carried out in the similar pH as per the GI tract. The effect of sequential crosslinking on nanofiber mesh was checked by in-vitro degradation studies, morphology, thermal and chemical stability of the nanofiber matrix. These results lead us to examine the crosslinking effect on drug release profiles. Fig. 3c shows the release profile of piperine from GNF/P/C8 and GNF/P/LBLC8 meshes in different physiological pH (1.2 and 7.4). A time scale of 24 h was chosen for the release study. An initial fast release of drug molecules within 8 h was observed in case of GNF/P/C8. GNF/P/LBLC8 meshes exhibited better control on the fast release of piperine in those initial hours (8 h) of the release study. GNF/P/LBLC8 meshes successfully tailored the initial fast release as well as the overall release of piperine for both the pH. These observations can be explained from previous results of SD (%), WL (%) and morphological images of the mesh. Sequential crosslinking (GNF/P/LBLC8) of the mesh showcased uniform crosslinking through the different layers of the fabric thus showed lesser WL (%) and SD (%) in compared to GNF/P/C8 (Fig. 1b and 1c respectively). GNF/P/LBLC8 showed better chemical and thermal stability in compared to GNF/P/C8 (Fig 3a and 3b respectively).
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It is interesting to note that, though the total crosslinking time is same (8 min) for both GNF/P/C8 and GNF/P/LBLC8, GNF/P/LBLC8 showed a sustained release of piperine for 24 h. The probable reason behind this observation might be the uniformity of crosslinking and tightly packed structure of intermediate layers of the mesh. It formed strong inter-fibrous bonding which created extra barrier to diffuse drug molecules in the release medium. Thus GNF/P/LBLC8 exhibited a better stability of the vehicle and also played major role in achieving the sustained release over a period of time. On the other hand, the hierarchy of crosslinking degree among the intermediate layers of the fabric leads fast release of drug molecules in the initial hours. Fig. 3c illustrates the drug release profiles in different pH. The cumulative release (%) for piperine after depositing the GNF/P/LBLC8 and GNF/P/C8 meshes in pH 7.4 for 24 h are 55.2 ± 3.7 % and 65.4 ± 4.6% respectively. The initial fast releases of piperine within 8 h are successfully tailored by approx. 20% in the case of GNF/P/LBLC8 (40.6 ± 1%) compared to GNF/P/C8 (59.7 ± 2.9%). Thus sequential crosslinking not only toned down the initial fast release, it controlled the overall release profiles. Typically, the vehicle has to pass through the harsh condition (pH 1.2) of stomach within 4-5 h followed by the absorption site i.e small intestine (pH 7.4). We have studied the release profiles in pH 1.2 for 24 h for both the meshes. It is observed that the cumulative release (%) for GNF/P/LBLC8 and GNF/P/C8 meshes in pH 1.2 after 24 h are 46.3 ± 7.4% and 59.4 ± 3.5% respectively which is substantially decreased in case of GNF/P/LBLC8 and GNF/P/C8 meshes after 4h (28.9 ± 3.6% and 43.3 ± 1.9% respectively). The cumulative release of piperine is less in lower pH for both the samples. This might be caused because of the ionization of amino group present in gelatin in a low pH medium, which discourages formation of H-bonds with water molecules resulting less swelling of the mesh. Thus in lower pH, the protonated amino groups collapse and doesn’t swell much. Due to less swelling of the mesh, drug molecules don’t get sufficient osmotic pressure to travel from the mesh to release medium. Releasing less drug in lower pH (pH 1.2: similar to stomach) is good as the absorbance site of the drug is intestine. It is observed that, compared to GNF/P/C8, GNF/P/LBLC8 mesh doesn’t swell much in lower pH resulting less WL (%) after more than 48 h. Thus GNF/P/LBLC8 mesh shows better control on piperine release compared to GNF/P/C8. These results provide a better understanding to the crosslinking of electrospun gelatin nanofibers and thus to facilitate their use as potential drug delivery system with controlled release.
Fig. 3. (a) FT-IR of crosslinked and noncrosslinked GNF; (b) DCS thermogram of crosslinked and noncrosslinked GNF; (c) In-vitro cumulative release of piperine from different crosslinked samples.
4. Conclusion In conclusion, we successfully fabricated drug loaded gelatin nanofiber mesh by electrospinning and also crosslinked by GTA vapor with two different strategies. Interestingly, only 8 min crosslinking yielded desired stability of the mesh in aqueous medium for more than 48 h. However non-uniformity and shrinkage problems are addressed by sequential layer by layer crosslinking (2+2+2+2 min) of GNF/P mesh, keeping total crosslinking time
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constant (8 min). The crosslinking was even in different layers of the mesh and formed strong inter-fibrous bonding which created extra barrier to diffuse drug molecules in the release medium. Drug release profiles clearly suggest that this sequential crosslinking of piperine loaded gelatin nanofabric improved the mesh further in terms of water resistivity, thermal and chemical stability and therefore can be used as a potential drug carrier with sustained release over an extended period of time interval. Acknowledgements We acknowledge Indian Institute of Technology Hyderabad for providing necessary research infrastructure to carry out this work. References [1] K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M. Shakesheff, Chem. Rev. 99 (1999) 3181-3198. [2] R. S. Langer, NA Peppas, Biomaterials 2 (1981) 201-214. [3] R. S. Langer, NA Peppas, AIChE J. 49 (2003) 2990-3006. [4] A. Laha, U. Bhutani, K. Mitra and S. Majumdar, Mater. Manuf.Processes, 2015, DOI: 10.1080/10426914.2015.1070422. [5] U. Bhutani, A.Laha, K. Mitra and S.Majumdar, Mater Lett, DOI:10.1016/j.matlet.2015.10.114 [6] X. Hu , S. Liu , G. Zhou, Y. Huang, Z. Xie, X. Jing, J. Controlled Release 185 (2014) 12–21. [7] G. Fundueanu, M. Constantin, P. Ascenzi, Biomaterials 29 (2008) 2767–2775. [8] A. Furmhals, US patent 1,975,504, 1934. [9] T.J. Sill, H.A. von Recum, Biomaterials 29 (2008) 1989–2006. [10] P. Zahedi, I. Rezaeian, S.-O. Ranaei-Siadat, S.-H. Jafari, P. Supaphol, Polym. Adv. Technol. 21 (2010) 77–95. [11] T. Kowalczyk, A. Nowicka, D. Elbaum, T.A. Kowalewski, Biomacromolecules 9 (2008) 2087–2090. [12] J. Zeng, X. Xu, X. Chen, Q. Liang, X. Bian, L. Yang, X. Jing, J. Controlled Release 92 (2003) 227–231. [13] H. Nie, J. Li, A. He, S. Xu, Q. Jiang, C. C. Han, Biomacromolecules 11 (2010) 2190-2194. [14] S.Young, M. Wong, Y. Tabata, A.G. Mikos, J. Controlled Release 109 (2005) 256– 274. [15] C-H. Huang, C-Y. Chi, Y-S. Chen, K-Y. Chen, P-L. Chen, C-H. Yao, Mater Sci Eng C Mater Biol Appl 32 (2012) 2476–2483. [16] W. Lu,M. Ma,H. Xu,B. Zhang,X. Cao,Y.Guo, MaterialsLetters 140 (2015)1–4. [17] Y. Su, X. Mo, J.Controlled Release 152 (2011) e230–e232. [18] Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Polymer 47 (2006) 2911–2917. [19] S. Farris, J. Song, Q. Huang, J. Agric. Food Chem. 58 (2010) 998–1003. [20] D.M. Correia, J. Padrão, L.R. Rodrigues, F. Dourado, S. Lanceros-Méndez , V. Sencadas, Polym Test 32 (2013) 995–1000. [21] A. Laha, S. Yadav, S. Majumdar, C.S. Sharma, Biochem. Eng. J. DOI: 10.1016/j.bej.2015.11.001. [22] D.C. Aduba Jr., J. A. Hammer, Q. Yuan, W. A. Yeudall, G. L. Bowlin, H. Yang, Acta Biomater 9 (2013) 6576–6584.