Influence of palygorskite on the structure and thermal stability of collagen

Applied Clay Science 62-63 (2012) 41–46

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Influence of palygorskite on the structure and thermal stability of collagen Dihan Su a, Chunhua Wang a, Sumei Cai a, Changdao Mu b, Defu Li b, Wei Lin a,⁎ a b

Department of Biomass and Leather Engineering, National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu, Sichuan, China Department of Pharmaceutics and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan, China

a r t i c l e

i n f o

Article history: Received 20 November 2011 Received in revised form 18 April 2012 Accepted 23 April 2012 Available online 17 May 2012 Keywords: Collagen Palygorskite Thermal stability

a b s t r a c t We investigated the influence of palygorskite on the conformation and thermal stability of type I collagen. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR) studies demonstrated that the interaction between collagen and purified palygorskite (PAL) led to the contraction and aggregation of collagen molecules, but did not destroy the triple helix backbone of collagen. Due to the structure of collagen and PAL, the interaction mainly involved hydrogen bonding and electrostatic forces. Atomic force microscopy (AFM) observations clearly displayed the nanorod morphology of PAL, and further revealed the fibril aggregation of collagen in the presence of PAL. Differential scanning calorimetry (DSC) measurements indicated that the PAL-collagen nanocomposites improved the thermal stability in comparison with pure collagen. The present study showed that PAL could modify collagen as a reinforcing agent and preserve the triple helix structure of collagen. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Recently, biopolymer-based nanocomposites or bionanocomposites attracted considerable interests in both academic and industrial fields. They not only displayed the well-known properties of polymer nanocomposites such as improved mechanical properties, higher thermal stability and gas-barrier properties, but also exhibited remarkable advantage in biocompatibility and biodegradability (Darder et al., 2007). The clay mineral-protein hybrids may generate new low-cost materials with functional properties provided by either the biological or inorganic moieties (Chen and Zhang, 2006; Lin et al., 2007; Yuan et al., 2010). Palygorskite (PAL) consists of nano-scaled fibers with exchangeable cations and reactive OH groups on the external and internal surfaces (Wang et al., 2009). Because of its large specific surface area and structural property, PAL is widely used as absorbent (Zheng et al., 2009), catalyst carrier (Frost et al., 2010), rheological modifier, filler (Galan, 1996), and as excipient and active substance in pharmaceutical formulations (Aguzzi et al., 2007). The most abundant type I collagen is the major structural component of connective tissues including skin, bone, tendon. It is also the most investigated protein nowadays due to its commercial and industrial significance as exemplified by the traditional leather industry and current biomedical applications (Covington, 1997; Lee et al., 2001; Riccetto et al., 2006). The hierarchical structure of collagen from the triple helix (~300 nm in length and ~ 1.5 nm in diameter) to microfibrils (~40 nm in diameter), fibrils (100–200 nm in diameter), and further collagen fibers, is well documented (Reich,

2007). However, biocompatible materials based on collagen gel matrix were often limited by poor thermal stability and mechanical strength for their widespread use, since the natural cross-linking and assembly structure of isolated collagens were destroyed during the extraction process (Charulatha and Rajaram, 2003). Designed and developed for artificial biological tissue applications, sepiolite was incorporated into collagen matrices giving rise to hybrid materials with a high degree of organization (Herrera et al., 1995; Olmo et al., 1992). Although clay mineral–collagen nanocomposites can be considered as emerging biomaterials for bone-repair purpose in tissue engineering (Darder et al., 2007), there are limited studies so far. On the other hand, montmorillonite was studied as an environmental friendly tanning agent partially in place of conventional trivalent chrome for leather-making, leading to enhanced hydrothermal stability of hide collagen (Bao and Ma, 2010). Nevertheless, the details of the interaction between the clay mineral particles and proteins are still unclear (Aubin-Tam and Hamad-Schifferli, 2008). The knowledge of the conformational change of collagen induced by clay minerals is considerably insufficient, which is important for understanding the structure of clay mineral-collagen nanocomposites. In the present study, we investigated the influence of purified PAL on the structure of type I collagen molecules. Our aims were to explore the interaction between hierarchical collagen and PAL, and to understand the structure–property relation of bionanocomposites. 2. Experimental 2.1. Materials

⁎ Corresponding author. Tel.: + 86 28 85460819. E-mail address: [email protected] (W. Lin). 0169-1317/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2012.04.017

Acid soluble collagen was extracted from the fresh adult bovine Achilles tendon in 0.5 M acetic acid with pepsin. The method was

D. Su et al. / Applied Clay Science 62-63 (2012) 41–46

2.2. Sample preparation PAL dispersion was added to the aqueous collagen solutions in acetic acid under magnetic stirring. The dispersion was stirred at room temperature for 3 h, and the pH was adjusted at 4.0 with acetic acid. Subsequently, the dispersions were used for fluorescence measurement or AFM observation. The PAL-collagen samples for FTIR and DSC measurements were formed by casting the mixture on a polytetrafluoroethylene (PTFE) plate and freeze-drying at −20 °C by a vacuum freezing drier (Alpha1-2 LD, Christ, Germany) for 1 day. The final mass ratios of PAL/collagen (m/m related to the mass of dry collagen) were between 0:100 and 8:100. All the samples were freshly prepared just before the measurements.

2.3. Fluorescence measurements The fluorescence emission spectra were recorded on a Hitachi F4010 spectrofluorimeter at wavelengths of 200–800 nm upon excitation by 281 nm at room temperature. The excitation and emission slit widths were both set to 5 nm. The rectangular quartz cells had a path length of 1 cm. The scan rate of 60 nm∙min − 1 and the time constant of 2.0 s were maintained for all the measurements. The blanks corresponding to the matching acetic acid buffer were subtracted to correct the fluorescence background. The L-tyrosine solutions (the same concentration as collagen 0.45 mg∙mL − 1) in the presence of PAL with different concentrations were also examined as reference. The experiments were reproducible within the experimental errors.

2.6. DSC The differential scanning calorimetry (DSC) analyses were conducted on the Netzsch DSC 200 PC between 20 °C and 150 °C with a constant heating rate of 5 °C/min under nitrogen flow. The samples 3 mg were sealed in aluminum pans with an empty aluminum pan as the reference. 3. Results and discussion 3.1. Influence of PAL on the dynamics and conformation of collagen Fluorescence spectroscopy is sensitive to protein dynamics such as the rotational motion of protein side chains, molecular binding, and conformational changes. Intrinsic protein fluorescence from tryptophan (Try), tyrosine (Tyr), and phenylalanine (Phe) groups can be used to monitor changes in the protein microenvironment upon binding to nanoparticles (Mahmoudi et al., 2011). From the interpretation of fluorescence parameters, one can also obtain information about the degree of exposure of the fluorophore to the solvent and the extent of its local mobility (Royer, 2006). Collagen is known to possess 0.5% Trp and 1.3% Phe residues (content in % amino acids), without Try (Reich, 2007). Since the emission of Phe could not be used to study proteins in most cases due to its low quantum yield (Sudhakar et al., 1993), the fluorescence property of Tyr in the PALcollagen dispersion was observed to obtain the information about the collagen dynamic behavior around the Tyr residues. The emission spectra of L-tyrosine solutions with and without PAL upon excitation at 281 nm are shown in Fig. 1a. A broad emission band with the peak at 600 nm was observed for pure L-tyrosine. The addition of PAL did not significantly affect both the position and intensity of the peak. Thus, PAL may not interact with the Tyr directly. In the collagen dispersions (Fig. 1b), the emission maximum shifted to 566 nm revealing a more buried configuration of Tyr residues in

450

0.89% 0.44% 0.22% 0.11% 0

150

0 525

600

675

750

Wavelength / nm

Fourier transform infrared (FTIR) spectra were performed on the Nicolet iS10 FTIR spectrometer using KBr disks at room temperature. All spectra were obtained with a resolution of 4 cm − 1 over the range 400–4000 cm − 1. The spectral plots represent the average of 10 scans.

The PAL-collagen dispersion (10 μL) was dropped onto a freshly cleaved mica substrate. These samples were dried in a desiccator for 24 h at room temperature. AFM was performed on a Shimadzu SPM-9600 equipped with Si cantilevers in a non-contact (taping) mode. PAL was also characterized by AFM for obtaining height and three-dimensional images. For each sample, the analyses were made at three different points to confirm the consistency of the observed morphology.

PAL / Tyr (g/g)

300

2.4. FTIR spectra

PAL/collagen (g/g) b 0.89% 0.67% 0.44% 0.22% 600 0.11% 0 900

Intensity

2.5. AFM

a

900

Intensity

similar to the procedure previously reported (Mu et al., 2007). The analysis of the extracted collagen by SDS-PAGE (Bio-Rad Powerpac 300, USA) was made in our laboratory to verify its purity and structural integrity (He et al., 2011). The isoelectric point (IEP) of the collagen was at pH 7.4 as measured by potentiometric titration method using the Nano ZS instrument (Malvern Co., UK). Palygorskite was kindly provided by the Jiangsu Huai Yuan Mining Industry Co. Ltd, Jiangsu, China. It was purified by dispersion into sodium hexametaphosphate (0.3 g·L − 1 (NaPO3)6) at 40 °C under intense agitation and ultrasonic vibration (40 kHz, 120 W) for 45 min, followed by centrifugation at 7000 r·min − 1. The resulting upper dispersion, i.e. the purified PAL, was dispersed in acetic acid of pH = 4 as stock solution. The zeta potential of the PAL particles in the acetic acid was about −30 mV measured by the Nano ZS instrument (Malvern Co., UK), showing its negative surface charge. All chemical reagents used were of analytical grade.

Intensity

42

600

C collagen= 0.45 mg/mL

300 0

1

2

3

4

C PAL / (10-3 mg/mL)

300

0 540

560

580

600

Wavelength / nm Fig. 1. Fluorescence emission spectra of L-tyrosine (a) and collagen (b) at different contents of PAL upon excitation at 281 nm in pH 4 acetic acid solutions.

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collagen (Royer, 2006). The fluorescence intensity increased with the content of PAL, indicating that the interaction of PAL with collagen induced conformational changes, which in turn affected the local environment of the Tyr residues in the polypeptide chains and its fluorescence. Generally, aromatic residues like Tyr are often fully or partially buried in the hydrophobic environment of protein interiors. When the protein unfolds or is destabilized by the presence of nanoparticles, they become exposed to the hydrophilic surroundings exhibiting a red-shifted emission (Aubin-Tam and Hamad-Schifferli, 2008; Royer, 2006). Here, the PAL-collagen dispersions did not show a red-shift or blue-shift of the emission maximum at 566 nm, indicating that collagen triple-helixes did not unfold to random coils by binding to PAL. Thus, the increased fluorescence intensity could result from mutually interacting Tyr residues in close proximity, induced by the collagen adopted on the surface of PAL nanoparticles (Mente, 2006; Shang et al., 2007). The reason is the formation of the hydrogen bonds between the collagen chains and the Si―OH groups of PAL, and the electrostatic interaction of collagen with the negative surface charges of PAL. It is known that aspartic and glutamic acid side

chain carboxyls have pKa values 3.8 and 4.2 respectively, thus the carboxyl groups of collagen are partially ionized at pH 4 (Covington, 1997). Therefore, the apparent charge of collagen remains positive

a PAL T (%)

642 1655

Fig. 3. Schematic illustration showing the interaction of PAL and collagen.

1195

3418

3614

3550

1087 1036

4000

3000

2000

470 983 1000

Wavenumber / cm-1

b

A B

C

T (%)

D

3085 3330 4000

3000

2000

1000

Wavenumber / cm-1

c

T (%)

A B C

1658

1553 1500

1240 1034

D 983

1000

470 500

Wavenumber / cm-1 Fig. 2. FTIR spectra of PAL (a), collagen and PAL-collagen (b and c) at different contents of PAL (A: 0%, B: 4%, C: 6%, D: 8%).

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Fig. 4. AFM images of the purified palygorskite (PAL).

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at this pH (b IEP). Other types of forces such as coordination to Al 3+ ions in PAL and van der Waals interaction might also contribute (Shang et al., 2007). The increase of the overall fluorescence intensity was PALconcentration dependent (inset in Fig. 1a). At lower PAL contents (≤1 × 10 − 3 mg∙mL − 1) or lower PAL-collagen mass ratios, the interaction of PAL and collagen caused a sudden increase of the intensity of emission, which was likely due to the abrupt contraction of the collagen peptide chains. With increasing PAL content, this effect became less pronounced. Further increasing the PAL content (>1% collagen), the opacity of the solutions increased (data not show), indicating partial aggregation of collagen. Therefore, it can be speculated that the added PAL induces the contraction of collagen chains and further the aggregation of the molecules. Fig. 2a shows the typical FTIR spectrum of PAL. The characteristic bands are very similarly reported (Augsburger et al., 1998; Frost et al., 1998). The peak at 3614 cm − 1 is assigned to the stretching mode of the hydroxyl groups which are coordinated to the octahedral magnesium (aluminum) and the tetrahedral silicon. The bands around 3550 cm − 1 and 3418 cm − 1 correspond to the bending vibration of coordinated water and physically adsorbed water (zeolitic water). The asymmetrical bending vibration of the coordinated and adsorbed water is reflected by the band at 1650 cm − 1. Typically, the stretching vibrations of the Si―O―Si bridge and the O―SiO3 terminal group appear at 1200–600 cm − 1. Namely, the bands at 1195 cm − 1 and 642 cm − 1 correspond to the antisymmetric and symmetric modes of the Si―O―Si bridge, which is characteristic of PAL (Mendelovici and Portillo, 1976). The strong bands at 1087, 1036

and 983 cm − 1 are assigned to the antisymmetric mode of the terminal group O―SiO3. The region between 600 and 410 cm − 1 in the spectrum is complex, with contributions from Si―O―Si and O―SiO3 bending vibrations and lattice modes. The FTIR spectra of pure collagen and PAL-collagen are compared in Fig. 2b. The intact fibrillar collagen has a special triple helix conformation which is characterized by the amide bands in IR spectra (Doyle et al., 1975; He et al., 2011). The bands at 3350 cm − 1 and 3087 cm − 1, normally denoted as amide A and B bands, are mainly associated with the N―H stretching vibrations. The amide I band at 1658 cm − 1 is dominantly attributed to the stretching vibrations of peptide C O groups. The amide II absorbance at 1553 cm − 1 arises from the N―H bending vibrations coupled to C―N stretching vibrations. The Amide III band centered at 1240 cm − 1 is assigned to the C―N stretching and N―H bending vibrations from amide linkages, as well as wagging vibrations of CH2 groups in the glycine backbone and proline side chains (Madhan et al., 2005). As revealed before, the denaturation of collagen reduced the intensity for all these major amide bands and shifted the amide I band toward lower wavenumbers (Li et al., 2009) since the amide I vibrational band was the most sensitive to the collagen triple-helix backbone conformation (Payne and Veis, 1988). The positions of the four amide bands did not change with increasing PAL content (Fig. 2b, c), indicating that the collagen triple helix in the composites may not be destroyed by the PAL addition (He et al., 2011). Nevertheless, the intensity of the amide A, I, II and III bands increased, and the amide A band was broadened. This suggested that strong non-covalent interactions between collagen and PAL may be involved (He et al., 2011).

Fig. 5. AFM images of pure collagen and PAL-collagen at the mass ratio of 100:8 on mica. Collagen concentration: 3 × 10− 2 mg∙mL− 1 (a and b), and 3 mg∙mL− 1 (a′ and b′).

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Especially, the band intensity at 1034 and 983 cm − 1 (Fig. 2c) from the terminal group O―SiO3 of PAL did not increase in parallel to the band at 1658 cm − 1 from the bending vibration of water (corresponding to 1655 cm − 1 in Fig. 2a). Several mechanisms may be involved in the interaction of PAL with collagen, such as electrostatic and van de Waals forces, hydrogen bonding, and ligand exchange (Aguzzi et al., 2007; Mahmoudi et al., 2011). As discussed above, at pH 4, the overall surface charge of PAL was negative and the collagen molecules had a positive charge below their isoelectric point (pH 7.4) which obviously strengthened the electrostatic interaction. Many carbonyl, carboxyl, side chain hydroxyl, amino and amide groups of collagen provided interacting sites for hydrogen bonds with PAL. Therefore, it was conceivable that the interaction of collagen and PAL led to the contraction and aggregation of collagen molecules without destroying the backbone structure ( Fig. 3). 3.2. AFM morphology Fig. 4 shows the PAL had a unique straight rod-like structure with 20–70 nm in diameter and several hundreds nanometers to one micrometer in length. The PAL nanorods were well dispersed in acidic dispersion showing no obvious impurity. It also indicated that the described purification improved the dispersion of PAL and the dispersion stability. Fig. 5 presents the morphological changes of collagen induced by PAL. The pure type I collagen (Fig. 5a) in diluted acetic acid solution (3 × 10 − 2 mg∙mL − 1) exhibited the typical fibrillar structure on the mica substrate as previously reported (He et al., 2011; Maeda, 1999). In the presence of PAL (Fig. 5b for 8% PAL), entangled bundles of fibrils were observed, indicating the aggregation induced by PAL. In contrast, when the collagen concentration increased to 3 mg∙mL − 1 (Fig. 5a′), individual fibrils could not be clearly observed and a feltlike structure of randomly packed molecule was formed, as observed by Mertig et al. (1997). The PAL nanorods were randomly dispersed in the collagen matrix and coated with aggregated collagen molecules showing a roughened and banded surface (Fig. 5b′). The AFM images directly revealed the influence of PAL on the fibrillar structure of collagen and showed the occurrence of fibrils aggregation. Thus PAL can be used as an effective cross-linking agent in the preparation of collagen-based nanocomposites. 3.3. Thermal stability The endothermic peak in Fig. 6 is related to the conformational transition of collagen from the triple helix to the random coil upon heating (Pietrucha, 2005). And the temperature of thermal denaturation (Td) strongly depends on the water content in collagen and

endothermal

Heat flow / (mW/mg)

0

-3

A B

-6

C 40

80

120

D 160

T / °C Fig. 6. DSC curves of PAL-collagen at different PAL contents (A: 0%, B: 4%, C: 6%, D: 8%). Td : 89.0 °C, 105.4 °C, 117.4 °C, and 122.8 °C.

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the degree of cross-linking between the chains (Tsereteli et al., 1997). Here, Td increased with increasing PAL content as expected, indicating the improved thermal stability of collagen. This effect was due to PAL as cross-linking agent as discussed above (Mu et al., 2007). The increased thermal stability may also contain a contribution from the nanorods acting as a heat barrier in the collagen matrix (Li and Wang, 2005; Zhu et al., 2001). The increase of Td was favorable for maintaining the triple helix structure of collagen which was connected with the mechanical property and the biocompatibility of collagen-based materials (Mu et al., 2010). Therefore, the mechanical strength of the collagen was expected to increase by the formation of nanocomposites with PAL (Lebaron et al., 1999). 4. Conclusions The addition of PAL to collagen led to contraction and aggregation of collagen molecules but did not destruct the triple-helix backbone structure. The interaction between collagen and PAL involved hydrogen bonds, electrostatic forces, as well as coordination binding and van de Waals attraction. In comparison with pure collagen, PALcollagen nanocomposites showed enhanced thermal stability with increasing PAL content. The results are of significance for developing new applications of palygorskite and fabrication of novel collagenbased functional materials. Acknowledgements The financial support of National Natural Science Foundation (NNSF) of China (21074074), Science and Technology Planning Project of Sichuan Province (2010HH0004) and Ph.D. Programs Foundation of Ministry of Education of China (20090181110061) is gratefully acknowledged. References Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Applied Clay Science 36, 22–36. Aubin-Tam, M.E., Hamad-Schifferli, K., 2008. Structure and function of nanoparticle– protein conjugates. Biomedical Materials 3, 034001. Augsburger, M.S., Strasser, E., Perinoa, E., Mercaderb, R.C., Pedregosa, J.C., 1998. FTIR and Mossbauer investigation of a substituted palygorskite: silicate with a channel structure. Journal of Physics and Chemistry of Solids 59, 175–180. Bao, Y., Ma, J.Z., 2010. The interaction between collagen and aldehyde-acid copolymer/ MMT nano-composite. Journal of the Society of Leather Technologists and Chemists 2, 53–58. Charulatha, V., Rajaram, A., 2003. Influence of different crosslinking treatments on the physical properties of collagen membranes. Biomaterials 24, 759–767. Chen, P., Zhang, L., 2006. Interaction and properties of highly exfoliated soy protein/ montmorillonite nanocomposites. Biomacromolecules 7, 1700–1706. Covington, A.D., 1997. Modern tanning chemistry. Chemical Society Reviews 26, 111–126. Darder, M., Aranda, P., Ruiz-Hitzky, E., 2007. Bionanocomposites: a new concept of ecological, bioinspired, and functional hybrid materials. Advanced Materials 19, 1309–1319. Doyle, B.B., Bendit, E.G., Blout, E.R., 1975. Infrared spectroscopy of collagen and collagen-like polypeptides. Biopolymers 14, 937–957. Frost, R.L., Cash, G.A., Kloprogge, J.T., 1998. Rocky Mountain leather, sepiolite and palygorskite—an infrared emission spectroscopic study. Vibrational Spectroscopy 16, 173–184. Frost, R.L., Xi, Y.F., He, H.P., 2010. Synthesis, characterization of palygorskite supported zero-valent iron and its application for methylene blue adsorption. Journal of Colloid and Interface Science 341, 153–161. Galan, E., 1996. Properties and applications of palygorskite-sepiolite clays. Clay Minerals 31, 443–453. He, L.R., Mu, C.D., Shi, J.B., Zhang, Q., Shi, B., Lin, W., 2011. Modification of collagen with a natural cross-linker, procyanidin. International Journal of Biological Macromolecules 48, 354–359. Herrera, J.I., Olmo, N., Turnay, J., Sicilia, A., Bascones, A., Gavilanes, J.G., Lizarbe, M.A., 1995. Implantation of sepiolite–collagen complexes in surgically created rat calvaria defects. Biomaterials 16, 625–631. Lebaron, P.C., Wang, Z., Pinnavaia, T.J., 1999. Polymer-layered silicate nanocomposites: an overview. Applied Clay Science 15, 11–29. Lee, C.H., Singla, A., Lee, Y., 2001. Biomedical applications of collagen. International Journal of Pharmaceutics 221, 1–22. Li, A., Wang, A.Q., 2005. Synthesis and properties of clay-based superabsorbent composite. European Polymer Journal 41, 1630–1637.

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