Magnetic collagen fibers stabilized using functional iron oxide nanoparticles in non-aqueous medium

Abstract

We describe a green and sustainable approach to cross-link collagen fibers in a non-aqueous green medium using oleic acid coated iron oxide nanoparticles. The magnetic nanoparticles coated with oleic acid (OA) were prepared by a simple co-precipitation technique and characterized using X-ray diffraction, UV-Vis-NIR spectroscopy, vibrating sample magnetometer, scanning and transmission electron microscopy. The OA coated nanoparticles, with an average particle size of 5 nm and saturation magnetization of 23.5 emu g⁻¹, were well dispersed in heptane and interacted with the collagen fibers derived from skin trimming wastes. The derived thermo-stable magnetic collagen fibers were further utilized for oil removal applications. We demonstrate that the heptane reaction medium helps in achieving absorption of 8.2 g of used motor oil per g of magnetic collagen fibers with magnetic tracking ability. The approach showcases a simple synthetic protocol for preparing magnetic collagen fibers for possible applications in leather making and environmental protection.

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Introduction

Collagen is the main insoluble fibrous protein in connective tissues.¹ However, fast biodegradation rate and low mechanical strength of the native collagen enforces stabilization for applications such as leather making and biomaterials, either by chemical crosslinking or physical methods.^2,3 Basic chromium(III) sulfate has long been regarded as the most efficient and effective chemical crosslinking agent in leather making.⁴ Leather processing involves a huge quantity of water in different stages of tanning process. After tanning, the water is contaminated with chromium and other chemicals used in leather processing. This wastewater needs to be cleaned at high costs or should be recycled due to the presence of chromium.⁵ Chromium-free tanning and water-less chrome tanning are evolved as some of the green approaches to rectify this problem.^6,7

Cross-linking of collagen using nanoparticles is an inspiring research area. Collagen was cross-linked using tiopronin-modified gold nanoparticles using EDC (1-ethyl-3(3-dimethylaminopropyl)-carbodiimide) coupling.⁸ Tris(hydroxymethyl) phosphine-alanine (THPAL) functionalized gold nanoparticles were used as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure.⁹ In recent years, the design and synthesis of magnetic nanoparticles have gained immense importance due to their broad applicability such as magnetic fluids, catalysis, biomedicine, magnetic resonance imaging, data storage, detection of pesticide and environmental remediation.^10–15 Functionalization of nanoparticles helps to achieve stable particles, prevents uncontrolled growth and modulates the functional property such as conductivity, magnetic moment, etc.¹⁶ It has been shown that magnetite nanoparticles (Fe₃O₄) stabilized with oleic acid (OA) show a reduction in the magnetic moment due to the decreased interaction between the particles in the non-polar solution.¹⁷ We recently interacted citric acid coated super paramagnetic iron oxide nanoparticles (SPIONs) with collagen fibers in aqueous medium for stabilization and oil removal applications.^18,19 It was found that the thermal stability of the stabilized collagen fibers is improved to 86 °C with a saturation magnetization of 0.08 emu g⁻¹ and oil absorption capacity of 2 g g⁻¹.¹⁸ Although these results are sufficient for the intended applications, the use of aqueous medium for the reaction could diminish the collagen stabilization level and oil absorption capacity owing to the poor dispersion of nanoparticles in aqueous medium and possible repulsion of oil on the water containing nanocomposites, respectively. Further the use of water for the stabilization of collagen in leather making is currently viewed adversely due to the generation of wastewater and depletion of precious natural resource.^7,20

Here we propose a novel approach to cross-link collagen fibers in non-aqueous medium employing oleic acid coated iron oxide nanoparticles (IOOANP). Oleic acid (CH₃(CH₂)₇CH=CH(CH₂)₇COOH), a long-chain surfactant, with a cis-double-bond in the middle thereby forming a kink is postulated as an effective stabilizing agent.¹⁶ Heptane, a green solvent, was chosen as the medium, to interact IOOANP with collagen fibers, based on green chemistry principles such as cumulative score, LD₅₀, environment and health hazards and polarity.²¹ The approach is expected to reduce or eliminate the usage of water and generation of wastewater. The developed non-toxic and biocompatible OA functionalized iron oxide nanoparticles were characterized for structure, size and magnetic properties and interacted with collagen fibers derived from trimming waste of leather industry in heptane medium. The stabilized collagen fibers were further characterized for their structure, thermal stability, magnetism and probed for oil removal applications.

Experimental

Materials

Anhydrous ferric chloride (FeCl₃) from Merck Pvt. Ltd., India, ferrous sulphate heptahydrate (FeSO₄·7H₂O) from SD Fine-Chem. Ltd., India, oleic acid from Sigma-Aldrich Ltd., India and ammonia from Ranbaxy Laboratories Ltd., India, were used without further purification. Other chemicals used were of reagent grade. Distilled water was used for all experiments. The hide powder (HP) was prepared from skin trimming waste as detailed elsewhere.¹⁸

Preparation of oleic acid coated iron oxide nanoparticles (IOOANP)

A well-established chemical co-precipitation technique was followed to synthesize magnetic nanoparticles of iron. Aqueous solution of FeCl₃ (8 ml, 16 mM) and FeSO₄·7H₂O (4 ml, 4 mM) were mixed and stirred well using a magnetic stirrer. To this mixture, 25% of NH₄OH was added so as to adjust the pH of the solution to 10. Oleic acid (10 ml) was then added and heated at 95 °C for 5 min. The pH 10 was maintained during the heating process as well. In order to coagulate the oleic acid coated particles, 2 ml dilute HCl solution was added to neutralize the dispersion. The particles were separated from the dispersion by decantation and the top water layer with excess salts was discarded. The particles were washed five times with hot distilled water so as to remove impurities. Finally, water was removed by washing with acetone to yield IOOANP. The same procedure was followed for synthesizing iron oxide nanoparticles (IONP) without oleic acid. This acetone-wet slurry was dispersed in heptane and homogenized. In order to perform X-ray diffraction (XRD) and magnetic measurements, the acetone-wet slurry samples were washed five times with ethanol and then heated to 110 °C for 2 h. The dried samples were collected and characterized.

Interaction of collagen fibers and IOOANP nanoparticles

The hide powder (100 mg) was soaked in heptane (3 ml) for 20 min. Then it was treated with 10% (w/w) IOOANP for 2 h with stirring. A control sample was prepared by treating the hide powder without IOOANP in heptane medium. Subsequently, the control and experimental collagen fiber samples were characterized for thermal stability, morphology and magnetic properties as detailed below.

Characterization

The powder X-ray diffraction patterns of IONP and IOOANP were recorded using a Rigaku Miniflex (II) desktop diffractometer (Ni filtered CuKα radiation with λ = 0.154060 nm) in the 2θ range of 10° to 80° with a step of 0.2°. The obtained XRD patterns were compared with the ICSD reference data for phase identification. The absorbance and reflectance of solid IOOANP were studied using a UV-Vis-NIR spectrophotometer (Cary 5000, Agilent Technologies) against BaSO₄ as a reference. The particle morphology, size and structure of the synthesized nanoparticles and the binding of nanoparticles to collagen fibers were analyzed using a high resolution scanning electron microscopy (HRSEM, FEI Quanta FEG 200). High resolution transmission electron microscopy (HRTEM) of the IOOANP dispersed in heptane was carried out using a JEOL 3010 instrument with a UHR pole piece. The control (treated with heptane alone) and experimental (treated with IOOANP in heptane medium) collagen fiber samples were analyzed for thermal stability using a differential scanning calorimeter (DSC, Model Q200, TA Instruments) by heating the samples from 30 to 120 °C at a heating rate of 2 °C min⁻¹ with N₂ flow of 50 ml min⁻¹. The magnetic measurements of IONP, IOOANP and HP-IOOANP samples were performed with a vibrating sample magnetometer (VSM, Lakeshore VSM 7410) at room temperature.

Results and discussion

Characterization of IONP and IOOANP

XRD patterns of as-synthesized IONP and IOOANP are shown in Fig. 1a. It is seen that the magnetite (Fe₃O₄) co-existed with hematite (α-Fe₂O₃) in both IONP and IOOANP. The major diffraction peaks in IONP are indexed to hematite (α-Fe₂O₃), which matches with the Joint Committee on Powder Diffraction Standards (JCPDS) database (File no. 33-0664). The peaks at 2θ = 24.2, 32.8, 35.8, 40.4, 49.6, 54.2, 57.4, 62.9 and 64.1 correspond to the (012), (104), (110), (113), (024), (116), (018), (214) and (300) planes of the rhombohedral phase. The remaining diffraction peaks in IONP are indexed to magnetite (Fe₃O₄) corresponding to inverse spinel crystal structure matching with the reported value (JCPDS 19-0629). The peaks at 30.6, 35.8, 43.7 and 58.54 correspond to (220), (311), (400) and (511) planes of Fe₃O₄.²² The X-ray diffraction pattern of IOOANP shows that the strongly magnetic Fe₃O₄ phases are dominating in comparison to weakly magnetic α-Fe₂O₃. The particle size of IONP and IOOANP calculated based on the width of the strongest diffraction line (110) and (311) using the Debye–Scherrer formula is 12 and 5 nm, respectively. In order to verify the presence of both species, we carried out UV-Vis-NIR spectroscopy (Fig. S1†) for the IOOANP sample and the results suggest the strong presence of broad inter-valence charge transfer (IVCT) absorption band in the IR region around 0.6 eV (∼2066 nm) corresponding to magnetite (Fe₃O₄).²³ However, the characteristic d–d electron pair transition (EPT) band at 2.3 eV (540 nm) corresponding to hematite is not seen in our spectra.^24,25 This may be due to very low amount of hematite species present in IOOANP in comparison to magnetite. This is later verified using magnetization studies.

Fig. 1 (a) XRD patterns of IONP and IOOANP. HRSEM images of (b) IONP and (c) IOOANP dispersed in heptane.

HRSEM images of both IONP and IOOANP show that the particles are spherical and agglomerated (Fig. 1b and c). In the absence of surface coating in IONP (Fig. 1b), the attractive force between the nanoparticles increases due to the increase in the large surface area to volume ratio and the nanoparticles are agglomerated in order to reduce the surface energy.^16,26 The results also indicate that the IOOANP form clusters with large aggregates in spite of particles being coated by OA (Fig. 1c).²⁷ This may be due to the OA coating is not sufficiently thick to keep the particles from aggregating. The morphology of OA-coated IOOANP, dispersed in heptane, was further examined using high resolution transmission electron microscopy, as shown in Fig. 2. It is evident that the particles are nearly spherical in shape with an average particle size of 5 ± 1 nm. This is in agreement with the size calculated from the XRD data using Debye–Scherrer formula. The measured lattice spacing from the individual IOOANP particle was found to be 0.25 nm (Fig. 2b), which reflects the (311) lattice plane of inverse spinel Fe₃O₄.²⁸

Fig. 2 HRTEM images of IOOANP dispersed in heptane at (a) lower and (b) higher magnification.

Fig. 3 displays room temperature magnetization curves (M − H) of IONP and IOOANP nanoparticles probed using a vibrating sample magnetometer. Both IONP and IOOANP display a ferromagnetic like hysteresis curve. Further, they exhibit a perfect saturation magnetization even at small magnetic fields of <1 T with reasonably high magnetic moment. The saturation magnetization of IOOANP is observed to be 23.5 emu g⁻¹, which is slightly higher than that of the uncoated IONP (∼14.8 emu g⁻¹). This may be due to the OA coating is not being sufficiently thick leading to magnetic interactions between the particles. As also corroborated, both IONP and IOOANP show minimal coercivity of 7.5 and 1.9 G, respectively (Fig. S2†) suggesting that the particles are weakly ferromagnetic rather than a typical superparamagnetic. The coercivity in these two samples may be due to the magnetic interactions between the particles since the particles have not been isolated in a matrix or fluid.²⁹ In the case of IOOANP, although the nanoparticles are coated with OA for protection, the coating may be thin leading to less magnetic interaction between the particles and thereby reduced coercivity in comparison to IONP. We also observe here that the M − H loop was shifted to negative direction while showing the coercivity in the magnified plots (Fig. S2†). This may be due to the exchange bias in the samples since they consist of both hematite and magnetite. Hematite is known to exhibit canted antiferromagnetic property above the Morin transition at 260 K and below its Néel temperature at 956 K.³⁰ Therefore, these observations suggest that the nanoparticles synthesized in this study consist of antiferromagnetic hematite and ferromagnetic magnetite at room temperature, as also seen in the XRD analysis.

Fig. 3 Room temperature M − H curve of as-synthesized (a) IONP and (b) IOOANP probed using a vibrating sample magnetometer. Insets show that the respective samples are trapped on the side wall of the glass vial when placed near the permanent magnets (∼1000 Oe).

Interaction of collagen fibers with IOOANP in heptane medium and its applications

Water is one of the poor solvent for nanoparticles leading to lower extent of interaction and stability.³¹ Here, we employed IOOANP to stabilize collagen fibers in heptane medium. DSC is a most convenient method for measuring the denaturation (shrinkage) temperature of collagen or thermal stability of collagen. Shrinkage temperature or hydrothermal stability is the temperature at which noticeable shrinkage occurs irreversibly when collagen matrix is gradually heated in aqueous medium. Fig. 4 shows the DSC traces of control (in heptane alone) and experimental (treated with IOOANP in heptane medium) collagen fiber samples. The shrinkage temperature of pristine collagen fibers in heptane medium is 78.1 °C. It is interesting to note that this value is much higher than that generally observed in water medium, which is around 65 °C.^18,27 This means that heptane itself provides some stabilization to collagen molecules compared to water. This may be mainly due to the low dielectric constant of heptane as well as dehydration of collagen fibers. Sufficient decrease in medium dielectric constant may lead to progressive strengthening of internal hydrogen bonding network in the collagen molecules thereby overcoming the destabilizing tendency.^32,33 As can be seen, the shrinkage temperature of the collagen fibers treated with IOOANP is elevated to 90.1 °C. This may be due to the electrostatic interaction between carboxylic groups of oleic acid coated on the nanoparticles and side chain amino functional groups of collagen molecules. Since the size of IOOANP nanoparticles is around 5 nm, it can only interact with the collagen molecules on the boundary of penta-fibril assembly and it may not enter the sub-nano to nanometer pores present in the microfibrils. The thermal stability obtained in this study is slightly higher than that was achieved using citric acid coated iron oxide nanoparticles.¹⁸ This improvement may be attributed to the use of heptane medium instead of water. HRSEM images of collagen fibers treated with IOOANP are shown in Fig. 5. The images clearly show the binding of IOOANP with the collagen fibers. Higher magnification image (Fig. 5b) depicts the presence of IOOANP particles on the surface of the collagen fibers probably through electrostatic interactions. The extent of diffusion and cross-linking of IOOANP particles is largely influenced by the size of the individual nanoparticles and their aggregated state. While the single nanoparticles may diffuse and interact up to fibrillar assembly, the aggregated bulky particles tend to interact largely at the fiber and fiber bundle level.

Fig. 4 DSC traces of control (in heptane alone) and experimental (treated with IOOANP in heptane medium) collagen fiber samples.

Fig. 5 HRSEM images of collagen fibers treated with IOOANP at (a) lower and (b) higher magnification.

The magnetic property of the collagen fibers treated with IOOANP was analyzed using a VSM at room temperature (Fig. 6a). It is seen that the IOOANP treated collagen fibers exhibit a weak ferromagnetic like behavior with saturation magnetization value of 0.38 emu g⁻¹ with minimal coercivity of 1.4 G (Fig. S3†). It is well known that the pristine collagen fibers are absolute diamagnetic material.^34,35 This clearly indicates that the collagen fibers are transformed into magnetic upon treatment with IOOANP as also seen in the inset of Fig. 6a, where it is seen that the magnetic collagen fibers are trapped on the top of the glass vial against the gravitational force under magnetic field (∼1000 Oe). Indeed, magnetization obtained in this study is markedly improved in comparison to our earlier report primarily due to the reaction in heptane medium.¹⁸ The weak magnetic ability of the treated collagen fibers may be due to the low offer of IOOANP and low-density distribution of IOOANP over the large surface area of collagen fibers. Although these magnetic collagen fibers show a small ferromagnetic property, it could be potentially used for applications such as oil removal, magnetic actuation and tracking. To demonstrate the selective removal of oil with tracking to the desired places in the oil contaminated water bodies, the magnetic collagen fibers were added to the used motor oil–water mixture in a Petri dish and allowed to absorb oil. Permanent magnets (field ∼1000 Oe) were used to track the magnetic collagen fibers such that it can absorb oil more effectively from all over the surface of used motor oil–water mixture in the Petri dish (Movie S1†). We have also calculated the maximum oil absorbing capacity of the magnetic collagen fibers and it is found to be 8.2 g g⁻¹ for the used motor oil (Fig. 6b). This was achieved spontaneously within few minutes. This value is much higher than our earlier report on the use of analogous magnetic collagen fibers prepared in aqueous medium.¹⁸ The higher absorption achieved in this study is primarily due to the use of heptane as the reaction medium, which resulted in fibers with less inter-fiber cohesion after drying leading to more oil absorption. Evaporation of water leads to fiber compaction and inter-fiber cohesion in the aqueous medium, probably due to charge interactions, mimicking conventional air drying. On the other hand, solvents assist in fibril separation after solvent evaporation leading to a great internal surface.³⁶ Nevertheless, it may be possible to further improve the oil absorption capacity by employing porous magnetic collagen sponge like structures instead of fibers, prepared through freeze drying technique, which can be explored in the future.

Fig. 6 (a) Room temperature M − H curve of collagen fibers stabilized using IOOANP probed using a vibrating sample magnetometer. Inset shows that the magnetic collagen fibers are trapped on the top of the glass vial against the gravitational force under permanent magnets (∼1000 Oe); (b) oil absorption capacity of the magnetic collagen fibers as a function of time based on used motor oil.

Conclusions

Here, we have prepared magnetic collagen fibers using oleic acid coated iron oxide nanoparticles in hexane medium. XRD and UV-Vis-NIR spectroscopic analysis reveal that the nanoparticles comprise both α-Fe₂O₃ and Fe₃O₄ and the presence of later is dominated in the oleic acid coated iron oxide nanoparticles. Electron microscopic analyses show that the IOOANP have average particle size of 5 nm and evidences the presence of Fe₃O₄ planes. Magnetic measurements provide sufficient data to support the fact that the particles are weakly ferromagnetic rather than superparamagnetic. We show that IOOANP can stabilize collagen fibers effectively so as to elevate the shrinkage temperature up to 90 °C when reacted in heptane medium. The derived magnetic collagen fibers were fruitfully used to remove used motor oil from water with magnetic tracking ability. The results suggest possible potential applications of stable magnetic collagen fibers in leather making, biomedical and environmental protection industries.

Acknowledgements

Financial support from CSIR under XII^th plan project “Research Initiatives for Waterless Tanning” (RIWT-CSC0202) is greatly acknowledged. We also thank SAIF and Prof. T. Pradeep, Dept. of Chemistry, IIT Madras for providing the VSM, HRSEM and HRTEM facility. CSIR-CLRI Communication no. 1120.

Notes and references

1. G. N. Ramachandran and G. Kartha, Nature, 1954, 174, 269–270.
2. A. Anumary, P. Thanikaivelan, M. Ashokkumar, R. Kumar, P. K. Sehgal and B. Chandrasekaran, Soft Mater., 2013, 11, 181–194.
3. M. Ragothaman, T. Palanisamy and C. Kalirajan, Carbohydr. Polym., 2014, 114, 399–406.
4. P. Thanikaivelan, V. Geetha, J. R. Rao, K. J. Sreeram and B. U. Nair, J. Soc. Leather Technol. Chem., 2000, 84, 82–87.
5. P. Thanikaivelan, J. R. Rao, B. U. Nair and T. Ramasami, Crit. Rev. Environ. Sci. Technol., 2005, 35, 37–79.
6. V. Sundar, J. Raghava Rao and C. Muralidharan, J. Cleaner Prod., 2002, 10, 69–74.
7. S. Selvaraj, A. Rathinam, R. R. Jonnalagadda and T. Palanisamy, J. Cleaner Prod., 2015, 87, 567–572.
8. L. Castaneda, J. Valle, N. Yang, S. Pluskat and K. Slowinska, Biomacromolecules, 2008, 9, 3383–3388.
9. J. S. Graham, Y. Miron and M. Grandbois, J. Mol. Recognit., 2011, 24, 477–482.
10. Q. A. Pankhurst, J. Connolly, S. Jones and J. Dobson, J. Phys. D: Appl. Phys., 2003, 36, R167.
11. M. Lewin, N. Carlesso, C.-H. Tung, X.-W. Tang, D. Cory, D. T. Scadden and R. Weissleder, Nat. Biotechnol., 2000, 18, 410–414.
12. T. Hyeon, Chem. Commun., 2003, 927–934.
13. C. Sun, J. S. Lee and M. Zhang, Adv. Drug Delivery Rev., 2008, 60, 1252–1265.
14. G. Reiss and A. Hutten, Nat. Mater., 2005, 4, 725–726.
15. A. H. Lu, E. e. L. Salabas and F. Schüth, Angew. Chem., Int. Ed., 2007, 46, 1222–1244.
16. A. K. Gupta and M. Gupta, Biomaterials, 2005, 26, 3995–4021.
17. L. Zhang, R. He and H.-C. Gu, Appl. Surf. Sci., 2006, 253, 2611–2617.
18. P. Thanikaivelan, N. T. Narayanan, B. K. Pradhan and P. M. Ajayan, Sci. Rep., 2012, 2, 230.
19. P. Thanikaivelan, T. Narayanan, B. Gupta, A. Reddy and P. Ajayan, J. Nanosci. Nanotechnol., 2015, 15, 4504–4509.
20. R. Bagatin, J. J. Klemeš, A. P. Reverberi and D. Huisingh, J. Cleaner Prod., 2014, 77, 1–9.
21. C. Capello, U. Fischer and K. Hungerbühler, Green Chem., 2007, 9, 927–934.
22. S.-J. Liu, C.-H. Huang, C.-K. Huang and W.-S. Hwang, Chem. Commun., 2009, 4809–4811.
23. J. Tang, M. Myers, K. A. Bosnick and L. E. Brus, J. Phys. Chem. B, 2003, 107, 7501–7506.
24. T. Grygar, J. Dědeček, P. Kruiver, M. Dekkers, P. Bezdička and O. Schneeweiss, Catena, 2003, 53, 115–132.
25. N. Li, S. Jayaraman, S. Y. Tee, P. S. Kumar, C. J. J. Lee, S. L. Liew, D. Chi, T. A. Hor, S. Ramakrishna and H.-K. Luo, J. Mater. Chem. A, 2014, 2, 19290–19297.
26. D. Kim, Y. Zhang, W. Voit, K. Rao and M. Muhammed, J. Magn. Magn. Mater., 2001, 225, 30–36.
27. W. Yu, H. Xie, L. Chen and Y. Li, Colloids Surf., A, 2010, 355, 109–113.
28. X. Teng and H. Yang, J. Mater. Chem., 2004, 14, 774–779.
29. M. P. Morales, S. Veintemillas-Verdaguer, M. I. Montero, C. J. Serna, A. Roig, L. Casas, B. Martínez and F. Sandiumenge, Chem. Mater., 1999, 11, 3058–3064.
30. M. F. Hansen, C. B. Koch and S. Mørup, Phys. Rev. B: Condens. Matter Mater. Phys., 2000, 62, 1124–1135.
31. Y. Xu, Y. Qin, S. Palchoudhury and Y. Bao, Langmuir, 2011, 27, 8990–8997.
32. R. Usha and T. Ramasami, J. Therm. Anal. Calorim., 2008, 93, 541–545.
33. A. E. Russell, Biochem. J., 1973, 131, 335–342.
34. M. Ashokkumar, N. T. Narayanan, A. L. M. Reddy, B. K. Gupta, B. Chandrasekaran, S. Talapatra, P. M. Ajayan and P. Thanikaivelan, Green Chem., 2012, 14, 1689–1695.
35. M. Ashokkumar, K. M. Sumukh, R. Murali, N. T. Narayanan, P. M. Ajayan and P. Thanikaivelan, Carbon, 2012, 50, 5574–5582.
36. K. J. Bienkiewicz, Physical chemistry of leather making, Krieger Publishing Co. Inc., 1983.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16181e

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