You are on page 1of 5

JOURNAL OF OPTOELECTRONICS AND ADVANCED MATERIALS - SYMPOSIA, Vol. 2, No. 1, 2010, p.

140 - 144

Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation and biocompatibility evaluation
S. CAVALU*, F. BANICA, V. SIMONa University of Oradea, Faculty of Medicine and Pharmacy, Oradea 410087 Romania a Babes-Bolyai University, Faculty of Physics & Institute for Interdisciplinary Experimental Research, Cluj-Napoca 400084, Romania
The biocompatibility evaluation of aluminosilicate samples containing iron and dysprosium or yttrium was made with respect to collagen (type I from calf skin) adsorption. The SEM analysis indicates morphological changes on samples surface after incubation in collagen solution. At the same time, the features of ATR-FTIR spectra and the data obtained by deconvolution of the amide I region of adsorbed collagen show qualitative and quantitative diferences compared to the native protein. The secondary structure of collagen is more pronounced modified upon adsorption to yttrium aluminosilicate indicating a lower biocompatibility compared to dysprosium containing sample. Cyclic voltammetry also supports the quantitative investigations by collagen adsorption at the Ag/AgCl electrode surface. The current intensity enhancement and the decrease of the oxidation potential of collagen indicate that collagen adsorption is an irreversible process. (Received April 21, 2009; accepted October 1, 2009) Keywords: Aluminosilicates, SEM, ATR-FTIR, Cyclic voltammetry

1. Introduction Aluminosilicate glasses with iron and yttrium/dysprosium incorporated investigated in this study are of great interest in the treatment of degenerative diseases by hyperthermia and radiotherapy, because they could be used in internal therapy of cancer, both by hyperthermia and local irradiation of the malignant tumours with high energy and short range beta radiation [1, 2]. The ferromagnetic nanoparticles developed in the vitroceramic biomaterial cause heating through hysteresis losses or magnetic relaxation phenomena and can induce the necrosis of the tumours. On the other hand, the yttrium and dysprosium stable isotopes can be activated by neutron irradiation to radioactive isotopes which have convenient properties for cancer radiotherapy [3, 4]. Beside the melt undercooling method used to obtain aluminosilicate systems, the sol-gel synthesis was also tacken into account [5]. The primer condition imposed to materials considered for biomedical applications is biocompatibility dictated by the manner in which their surface interact with blood constituents (erythrocytes, platelets) as well as the proteins [6, 7]. The type and amounts of adsorbed proteins mediate subsequent adhesion, proliferation and differentiation of cells as well as depositing of mineral phases. The behaviour of a protein at an interface is likely to differ considerably from its behaviour in the bulk. Because of the different local environment at the interface, the protein may have the opportunity of adopting a more disordered state exposing its hydrophobic core to the aqueous phase, often called surface denaturation. Denaturation is a process by which hydrogen bonds, hydrophobic interactions and salt linkages are broken and the protein is unfolded. The denaturation of secondary structure involves also changes in ratio among the three common structures: helix, sheets or turns and unordered [8, 10]. FTIR spectroscopy can be used to study protein secondary structure in any state, i.e. aqueous, frozen, dried or even as an insoluble

aggregate, and for this reason it is one of the most used techniques for studying stress induced alterations in protein conformation and for quantifying protein secondary structure. ATR-FTIR can provide important information leading to the development of novel biomaterials as replacements for damaged or diseased natural tissue. The spectral region of amide I (1660 cm-1), amide II (1550 cm-1) and amide III (1300cm-1) are very sensitive to the conformational changes in the secondary structure of proteins. Computational techniques based on the second derivative spectra and deconvolution procedure is used for percentage evaluation of each secondary structure and also the perturbations upon the adsorption to different surfaces [9-12]. Collagen type I is the most abundant protein of the extracellular matrix, a fibrillar triple helical structure that forms gel networks in irregular connective tissue. Collagen is also proline-rich and self assembles into fibrils [13,14]. In the present study, the biocompatibility of aluminosilicate samples incorporating iron and yttrium/dysprosium was evaluated with respect to collagen adsorption. The adsorbed collagen layer on the samples surfaces was investigated by SEM, ATR-FTIR and Cyclic Voltammetry. 2. Materials and methods Reagent grade silicic acid SiOx(OH)4-2x, and nitrates Al(NO3)39H2O, Fe(NO3)3, Y(NO3)3 Dy(NO3)3 were used as starting materials to prepare by sol-gel method [5] 10Dy2O310Fe2O360SiO220Al2O3 (DFSA) and 10Y2O310Fe2O360SiO220Al2O3 (YFSA) samples. The compositions are indicated in mol%. The 110oC dryed sol gels were heat treated at 500C and 1200C. Collagen type I from calf skin (lyophilized) was purchased from Sigma Chemicals. All samples were separatelly incubated for 24 hours at 37 C in 2 mg/mL collagen phosphate buffered solution and, after filtration and drying process, the sample surfaces were analyzed by SEM and ATR FTIR.

Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation ...

141

Scanning Electron Microscopy (SEM) was performed with a JEOL JSM5510 microscope in order to study the morphology of the surfaces, before and after incubation. The FT-IR spectra of the samples before and after incubation were recorded in the region 4000-600 cm-1 by a Bruker EQUINOX 55 spectrometer OPUS software, using an Attenuated Total Reflectance accessory with a scanning speed of 32 cm-1 min-1 and the spectral width 2.0 cm-1. The internal reflection element was a ZnSe ATR plate (50 x 20 x 2 mm) with an aperture angle of 45. A total of 128 scans were accumulated for each spectrum. Spectra were recorded at a nominal resolution of 2 cm-1. The spectra were smoothed with a 9-point SavitskyGolay smooth function to remove the white noise. The second derivative spectral analysis of amide I band was applied to locate positions and assign them to different functional groups. Before starting the fitting procedure, the obtained depths of the minima in the second derivative spectrum and, subsequently, the calculated maximum intensities were corrected for the interference of all neighbouring peaks. All second-derivative spectra, calculated with the derivative function of Opus software, were baselinecorrected, based on the method of Dong and Caughey [10], and area-normalized under the second derivative amide I region, 17001600 cm-1 [15]. Curve fitting was performed by setting the number of component bands found by second-derivative analysis with fixed bandwidth (12 cm-1) and Gaussian profile. The area under each peak was used to calculate the percentage of each component and, finally, to analyze the percentage of secondary structure component [10,15].

Cyclic voltammetric (CV) studies were carried out with a TraceLab 150 system, equipped with a Trace Master interface board, in residual protein solutions. A conventional three-electrode cell was employed incorporating a carbon-paste working electrode (with or without zeolite), a saturated Ag/AgCl reference electrode, and a Pt-wire counter electrode [16]. The supporting electrolyte solutions were 0.05 M phosphate buffer (pH 68) and acetate (pH 5). Voltammetric experiments were carried out in deoxygenated solutions by pure nitrogen. Stock solutions 0.1 M were prepared by dissolving in water the appropriate amount of each compound, usually their potassium salts. Working solutions were prepared by successive dilution of the stock solutions. 3. Results and discussion The as prepared samples and those obtained by 500oC heat treatment are in non-crystalline state, while by the heat treatment applied at 1200oC nanocrystalline structures are achieved. In order to study the morphological details of the samples surfaces, SEM analysis were performed before and after immersion in collagen solution. Fig. 1 clearly illustrates the changes occurred on YFSA and DFSA sample surfaces after the incubation in the solution containing collagen protein. According to the literature [15], once the protein has covered the surface of implants, host cells are no longer able to contact the underlying foreign-body material but only the proteincoated surface. The adsorbed protein layer-rather then the foreign material itself may stimulate or inhibit further biochemical processes.

d e Fig. 1. The morphology of YFSA and DFSA sample surfaces before (a, b) and after incubation (c, d) along with the SEM image of native collagen fibre (e).

ATR-FTIR spectra of both 500C and 1200C heat treated samples, before and after incubation in collagen solution, are presented in Fig. 2. The dominant bands around 1087 cm-1 are assigned to the stretching vibration

of Si-O-Si and Al-O-Al bonds, while the Al-O stretching vibrations of tetrahedral AlO4 groups are related with the bands at around 789 cm-1. Other weak absorption bands at around 912 cm-1 are present in the spectra of the samples

142

S. Cavalu, F. Banica, V. Simon

treated at 1200C, also attributed to the silica lattice [17]. The intensity of these bands is significantly reduced upon incubation. One can observe that collagen is preferentially adsorbed to the samples treated at 500C, emphasized by the characteristic amide I at 1624/1635 cm-1 and imide II at 1429/1418 cm-1. As a reference, the FTIR spectrum of native collagen is shown in Fig. 3, pointing out the features characteristic of amide I, II and III which are the most intense vibrational modes. The present study is focused on the amide I behavior, which is due primarily to the stretching vibrations of the peptide carbonyl group. As shown in Fig. 2 (b, d), the amide bands of adsorbed collagen are shifted towards lower wavenumber upon adsorption (compared with the amide bands of the native protein). According to the literature, the intensity of amide I band of collagen decreases markedly upon denaturation, and after deconvolution, four prominent components are present both in the native or denaturated protein spectrum [13,18]. That the relative intensities of these four peaks vary with the extent of collagen-fold or triple helix content speaks to the point that they are clearly conformationally dependent. Specific components within the fine structure of amide I adsorbed collagen is correlated with different states of hydrogen bonding associated with the local conformations of the alpha chain peptide backbones. This heterogeneity can arise either from intrinsic basicity differences in the strengths of the hydrogen bonds associated with the carbonyls [19].

Deconvolution of amide I band of native collagen and adsorbed to our aluminosilicate samples with iron and yttrium/dysprosium is shown in Fig. 4 a,b,c and the assignment of the components in Table 1 was made on the basis of the previous reported studies, along with the quantitative analysis.
0.250 0.225 0.200 0.175

Absorbance (a.u.)

1546 Amide II

1640 Amide I

0.150 0.125 0.100 0.075 0.050 0.025 0.000

1800

1600

1400

1228 Amide III 1200

1000
-1

800

600

W avenum ber cm

Fig. 3. ATR FTIR spectrum of the lyophilized collagen type I, used to prepare the protein solution.

Table 1. Assignment and relative area of amide I components of native collagen, or respectively adsorbed to 500oC heat treated aluminosilicate samples containing iron and yttrium/dysprosium. Collagen amide I native collagen adsorbed to YFSA adsorbed to DFSA
0.22 0.20

(cm-1) 1640 1635 1624

helix A (%) 44.6 34.0 40.2

(cm-1) 1653 1640 1641

helix A (%) 23.5 44.0 25.5

(cm-1) 1666 1663 1657

helix A (%) 23.1 12.0 23.5

(cm-1) 1710 1673 1670

turns A (%) 8.8 10 10.8

(a)

000020

(b)

Absorbance (a.u.)

0.18 0.16 0.14 0.12 0.10 0.08

000010

000005

000000

000005

Absorbance (a.u.)

000015

1580

1600

1620

1640

1660

1680
-1

1700

1720

1740

1600

1610

1620

1630

1640

1650

1660
-1

1670

1680

1690

Wavenumber cm

Wavenumber cm

00018 00016 00014 00012 00010 00008 00006 00004 00002 00000 00002 1610 1620 1630 1640 1650
-1

(c)

Absorbance (a.u.)

1660

1670

1680

Wavenumber cm

Fig. 4. Deconvoluted amide I absorption band of native collagen (a) and adsorbed collagen to YFSA (b) and DFSA (c) samples.

Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation ...

143

Curve fits to the amide I native collagen reveals four Gaussian components at 1640, 1653, 1666 and 1710 cm-1 representing helix-related hydrogen-bounded set of carbonyls. According to the literature, the highest frequency carbonyl absorption peak represents the weakest H-bonded system [18]. Beside the characteristic frequencies of helix conformation, the peak located in the higher region, at 1710 cm-1, represent the formation of an antiparallel -sheet structure (or turns). Both the intensity and the location of the characteristic peaks are modified upon adsorption. As a general behaviour, one can
0.14 0.12

observe a shift toward lower frequencies, a decrease in helix content and concomitant increase of turn percentage upon adsorption, as a consequence of denaturation. Comparing the quantitative results in table 1, we can remark that the sample ASY10Fe10 appear to be more susceptible to conformational changes due to the adsorption process, since spectral alteration reflected on the components percentage is more obvious as compared with the native protein. In terms of biocompatibility, we suggest that dysprosium/iron aluminosilicate present a beter behavior with respect to collagen adsorption.
0.12 0.10

1087

Y FS A beforeincubation

1087 1624

Y FSA after incubation 1428

Absorbance (a.u.)

Absorbance (a.u.)

0.10 0.08

0.06 0.04 0.02 0.00

500 C

798

0.04 0.02 0.00

500 C

789 912

-0.02 -0.04 -0.06

1200 C

-0.02 -0.04

1200 C

500

1000

1500

2000
-1

2500

3000

-0.08

500

789 912
1000

0.06

789

0.08

1500

2000
-1

2500

3000

W avenum ber cm

W avenum ber cm

a
0.16 0.14 0.12 0.10

b
DFSA before incubation
0.18 0.16 0.14 0.12 0.10 0 1

1087

1087

DSFA after incubation

Absorbance (a.u.)

798

0.08 0.06 0.04 0.02 0.00

797

0.06 0.04 0.02 0.00

855

784

-0.02 -0.04 -0.06 -0.08 -0.10

1200C

-0.02 -0.04 -0.06

1200 C

500

790

500C

1000

1418

500 C

Absorbance (a.u.)

1500

1635
2000
-1

0.08

2500

3000

500

1000

1500

2000
-1

2500

3000

Wavenumber cm

Wavenumber cm

Fig. 5. ATR FTIR spectra of the samples YFSA and DFSA heat treated at 500C and 1200C, recorded before and after incubation in collagen solution.

Cyclic voltammetry measurements were also carried out in residual collagen solutions using a carbon paste electrode modified with zeolite after an original method [20]. The goal was to study the effect of zeolite/carbon paste electrode concentration on the accumulation of

collagen. Cyclic volatmograms at different collagen concentrations were registered with modified carbon paste electrode (Fig. 5) exhibiting a strong anodic peak at +0.25 V vs. Ag/AgCl electrode whose intensity varies directly proportional to the collagen concentration of solution.

144

S. Cavalu, F. Banica, V. Simon

2.5x10

-5

References
YFSA/500

DFSA/500

YFSA/1200

DFSA/1200 0.0 0.2 0.4 0.6 0.8 1.0

E (V)

Fig. 6. The cyclic voltammograms for different residual collagen solutions using modified carbon paste electrodes at pH 7 and scan rate 100 mV/s.

We can observe that the current related to both heat treated samples at 1200C presents a higher intensity compared to those treated at 500C, suggesting the preferential collagen adsorption to the last one. The current enhancement was remarkable, and additionally, a significant decrease in the oxidation potential of collagen can be distinguished (more than 100 mV) when the electrode is modified with zeolite. This behavior, which was observed at different concentrations of collagen and at several scan rates potential, clearly demonstrates that the zeolite mediate the electrocatalytically properties of collagen [20,21]. No cathodic peak was observed in the reverse scan, indicating that the adsorption of collagen at zeolite modified electrode is an irreversible process. 4. Conclusions Iron and yttriun/disprosium aluminosilicate systems prepared by sol-gel route and heat treated at 500C and 1200C were characterized using SEM, ATR FTIR spectroscopy and cyclic voltammetry. The biocompatibility of the samples was evaluated with respect to collagen adsorption. Qualitative and quantitative analysis of amide I features by deconvolution and curve fitting reveals that the samples containing with iron and disprosium present a beter behavior with respect to collagen adsorption. SEM images reveal different degree of collagen adsorption toward the dysprosium/yttrium samples. Cyclic voltammetry carried out in residual collagen solutions indicates preferential collagen adsorption onto the samples heat treated at 500C as an irreversible process, that is in agreement with the ATRFTIR results. Acknowledgements The study was supported by the scientific research project CEEX 100/2006-MATNANTECH of the Romanian Excellence Research Program.

[1] U. O. Hfeli, W. K. Roberts, G. J. Pauer, S. K. Kraeft, R. M. Macklis, Applied Radiation and Isotopes 54, 869 (2001). [2] W. S. Roberto, M. M. Pereira, T. P. R. Campos, Artificial Organs 27(5), 420 (2003). [3] D. Cacaina, R. Viitala, M. Jokinen, H.Ylnen, M. Hupa, S. Simon, Key Engineering Materials, Bioceramics 17(284-286), 411 (2005). [4] V. Simon, D. Eniu, A. Takcs, K. Magyari, M. Neumann, S. Simon, J. Non-Cryst. Solids 351(30-32), 2365 (2005). [5] V. Simon, D. Eniu, A. Gritco, S. Simon, J. Optoelectr. Adv. Mater. 9(11), 3368 (2007). [6] W. Aken, Steen Dawis ed., Kluwer academic publishers, Norwell, Massachusetts, USA, 1989. [7] K. Vijayanand, D. K. Pattanayak, T. R. Rama Mohan, R. Banerjee, Trends Biomater. Artif. Organs 18(2), 73 (2005). [8] A. Dong, J. D. Meyer, J. L Brown, M. C. Manning, J. F. Carpenter, Arch. Biochem. Biophys 383, 148 (2000). [9] M. Van de Weert, P. I. Haris, W. E. Hennink, D. J. A. Crommelin, Analytical Biochemistry 297, 160 (2001). [10] A. Dong, W. S. Caughey, Methods Enzymol. 232, 139 (1994). [11] G. Damian, S. Cavalu, Asian Chem. Letters 9(1-2), 3 (2005). [12] S. Cavalu, V. Simon, J. Optoelectron. Adv. Mater. 9(11), 3297 (2007). [13] L.J. Juszczak, J. Biol. Chem. 279(9), 7395 (2004). [14] S. Leikin, V. A. Parsegian, W. H. Yang, G. E. Walrafen, Proc. Nat. Acad.Sci. USA 94, 11312 (1997). [15] S. Tunc, M. F. Maintz, G. Steiner, L. Vasquez, M. T. Pham, R. Salzer, Colloids Surf. B 42, 219 (2005). [16] Y. Xie, H. Liu, N. Hu, Bioelectrochemistry 70(2), 311 (2007). [17] A. Gritco, M. Moldovan, R. Grecu, V. Simon, J. Optoelectron. Adv. Mater. 7(6), 2845 (2005). [18] K. J. Payne, A. Veis, Biopolymers 27, 1749 (1988). [19] J. D. Whittle, N. A. Bullett, R. D. Short, C. W. I. Douglas, A. P. Hollander, J. Davies, J. Mater. Chem. 2, 2726 (2002). [20] M. Boji, L. Roman, R. Sndulescu, R. Oprean, Analiza i controlul medicamentelor 2, ed. Intelcredo, 557 (2003). [21] S. S. Khaloo, M. K. Amini, S. Tangestaninejad, S. Shahrokhian, R. Kia, J. Iranian Chem. Soc. 1(2), 128 (2004). ______________________ * Corresponding author: scavalu@rdslink.ro

I (A)