SIMONA CAVALU_Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation and biocompatibility evaluation

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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. SIMON

a

University of Oradea, Faculty of Medicine and Pharmacy, Oradea 410087 Romaniaa Babes-Bolyai University, Faculty of Physics & Institute for Interdisciplinary Experimental Research, Cluj-Napoca400084, Romania

The biocompatibility evaluation of aluminosilicate samples containing iron and dysprosium or yttrium was made with respectto 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 deconvolutionof the amide I region of adsorbed collagen show qualitative and quantitative diferences compared to the native protein. Thesecondary 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 quantitativeinvestigations by collagen adsorption at the Ag/AgCl electrode surface. The current intensity enhancement and thedecrease 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 andyttrium/dysprosium incorporated investigated in this studyare of great interest in the treatment of degenerativediseases by hyperthermia and radiotherapy, because theycould be used in internal therapy of cancer, both byhyperthermia and local irradiation of the malignanttumours with high energy and short range beta radiation[1, 2]. The ferromagnetic nanoparticles developed in thevitroceramic biomaterial cause heating through hysteresislosses or magnetic relaxation phenomena and can inducethe necrosis of the tumours. On the other hand, the yttriumand dysprosium stable isotopes can be activated byneutron irradiation to radioactive isotopes which haveconvenient properties for cancer radiotherapy [3, 4].Beside the melt undercooling method used to obtainaluminosilicate systems, the sol-gel synthesis was alsotacken into account [5].

The primer condition imposed to materials consideredfor biomedical applications is biocompatibility dictated bythe manner in which their surface interact with bloodconstituents (erythrocytes, platelets) as well as the proteins[6, 7]. The type and amounts of adsorbed proteins mediatesubsequent 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 thedifferent local environment at the interface, the proteinmay have the opportunity of adopting a more disorderedstate exposing its hydrophobic core to the aqueous phase,often called surface denaturation. Denaturation is a process by which hydrogen bonds, hydrophobic interactions andsalt linkages are broken and the protein is unfolded. The

denaturation of secondary structure involves also changesin ratio among the three common structures: α helix, β sheets or turns and unordered [8, 10]. FTIR spectroscopycan be used to study protein secondary structure in anystate, i.e. aqueous, frozen, dried or even as an insoluble

aggregate, and for this reason it is one of the most usedtechniques for studying stress induced alterations in protein conformation and for quantifying proteinsecondary structure. ATR-FTIR can provide importantinformation leading to the development of novel

biomaterials as replacements for damaged or diseasednatural tissue. The spectral region of amide I (1660 cm

-1),

amide II (1550 cm-1

) and amide III (1300cm-1

) are verysensitive to the conformational changes in the secondarystructure of proteins. Computational techniques based onthe second derivative spectra and deconvolution procedureis used for percentage evaluation of each secondarystructure and also the perturbations upon the adsorption todifferent surfaces [9-12]. Collagen type I is the mostabundant 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 andyttrium/dysprosium was evaluated with respect to collagenadsorption. The adsorbed collagen layer on the samplessurfaces was investigated by SEM, ATR-FTIR and CyclicVoltammetry.

2. Materials and methods

Reagent grade silicic acid SiOx(OH)4-2x, and nitratesAl(NO3)3·9H2O, Fe(NO3)3, Y(NO3)3 Dy(NO3)3 were usedas starting materials to prepare by sol-gel method [5]10Dy2O3·10Fe2O3·60SiO2·20Al2O3 (DFSA) and10Y2O3·10Fe2O3·60SiO2·20Al2O3 (YFSA) samples. Thecompositions are indicated in mol%. The 110

oC dryed sol

gels were heat treated at 500°C and 1200°C. Collagen typeI from calf skin (lyophilized) was purchased from SigmaChemicals. All samples were separatelly incubated for 24hours at 37 °C in 2 mg/mL collagen phosphate bufferedsolution and, after filtration and drying process, the samplesurfaces 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 performedwith a JEOL JSM5510 microscope in order to study themorphology 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, usingan Attenuated Total Reflectance accessory with a scanningspeed of 32 cm-1 min-1 and the spectral width 2.0 cm-1. Theinternal reflection element was a ZnSe ATR plate (50 x 20x 2 mm) with an aperture angle of 45°. A total of 128scans were accumulated for each spectrum. Spectra wererecorded at a nominal resolution of 2 cm-1. The spectrawere smoothed with a 9-point Savitsky–Golay smoothfunction to remove the white noise. The second derivativespectral analysis of amide I band was applied to locate positions and assign them to different functional groups.Before starting the fitting procedure, the obtained depthsof 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 thederivative function of Opus software, were baseline-corrected, based on the method of Dong and Caughey[10], and area-normalized under the second derivativeamide I region, 1700–1600 cm

-1[15]. Curve fitting was

performed by setting the number of component bandsfound 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 componentand, finally, to analyze the percentage of secondarystructure component [10,15].

Cyclic voltammetric (CV) studies were carried outwith a TraceLab 150 system, equipped with a TraceMaster interface board, in residual protein solutions. Aconventional three-electrode cell was employedincorporating a carbon-paste working electrode (with or without zeolite), a saturated Ag/AgCl reference electrode,and a Pt-wire counter electrode [16]. The supportingelectrolyte solutions were 0.05 M phosphate buffer (pH 6-8) and acetate (pH ≤5). Voltammetric experiments werecarried out in deoxygenated solutions by pure nitrogen.Stock solutions 0.1 M were prepared by dissolving inwater the appropriate amount of each compound, usuallytheir potassium salts. Working solutions were prepared bysuccessive dilution of the stock solutions.

3. Results and discussion

The as prepared samples and those obtained by 500oCheat treatment are in non-crystalline state, while by theheat treatment applied at 1200

oC nanocrystalline structures

are achieved.In order to study the morphological details of the

samples surfaces, SEM analysis were performed beforeand after immersion in collagen solution. Fig. 1 clearlyillustrates the changes occurred on YFSA and DFSAsample surfaces after the incubation in the solutioncontaining 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 underlyingforeign-body material but only the protein–coated surface.The adsorbed protein layer-rather then the foreign materialitself may stimulate or inhibit further biochemical processes.

a b c

d e

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

ATR-FTIR spectra of both 500°C and 1200°C heattreated samples, before and after incubation in collagensolution, are presented in Fig. 2. The dominant bandsaround 1087 cm

-1are assigned to the stretching vibration

of Si-O-Si and Al-O-Al bonds, while the Al-O stretchingvibrations 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

isfocu

hydrogen bonds associated with the carbonyls [19].

reported studies, along with theq

Fig. gen

type I, used to prepare the protein solution.

Table 1. Assignment and relati dsorbed to 500oC heat treated uminosilicate sample ng iron and yttrium/dysprosium.

helix helix helix turns

treated at 1200°C, also attributed to the silica lattice [17].The intensity of these bands is significantly reduced uponincubation. One can observe that collagen is preferentiallyadsorbed to the samples treated at 500°C, emphasized bythe characteristic amide I at 1624/1635 cm-1 and imide IIat 1429/1418 cm

-1. As a reference, the FTIR spectrum of

native collagen is shown in Fig. 3, pointing out thefeatures characteristic of amide I, II and III which are themost intense vibrational modes. The present study

sed on the amide I behavior, which is due primarily tothe 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 thenative protein). According to the literature, the intensity of amide I band of collagen decreases markedly upondenaturation, and after deconvolution, four prominentcomponents 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 clearlyconformationally dependent. Specific components withinthe fine structure of amide I adsorbed collagen iscorrelated with different states of hydrogen bondingassociated with the local conformations of the alpha chain peptide backbones. This heterogeneity can arise either from intrinsic basicity differences in the strengths of the

Deconvolution of amide I band of native collagen andadsorbed to our aluminosilicate samples with iron andyttrium/dysprosium is shown in Fig. 4 a,b,c and theassignment of the components in Table 1 was made on the basis of the previous

ua e analysis.ntitativ

1800 1600 1400 1200 1000 800 600

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

1 2 2 8

A m i d e I I I

Wavenumber cm-1

A b s o r b a n c e

( a . u . )

1 6 4 0

A m i d e I

1 5 4 6

A m i d e I I

3. ATR FTIR spectrum of the lyophilized colla

ve area of amide I components of native collagen, or respectively aal s containi

α α αCollagenamide I ν ν ν ν (cm-1) A (%) (cm-1) A (%) (cm-1) A (%) (cm-1) A (%)

native

collagen

1640 44.6 1653 23.5 1666 23.1 1710 8.8

ad o 1635 34.0 1640 44.0 1663 12.0 1673 10sorbed tYFSA

ad oDFSA

1624 40.2 1641 25.5 1657 23.5 1670 10.8sorbed t

1580 1600 1620 1640 1660 1680 1700 1720 1740

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Wavenumber cm-1

(a)

A b s o r b a n c e

( a . u . )

1600 1610 1620 1630 1640 1650 1660 1670 1680 1690000005

000000

000005

000010

000015

000020 (b)

Wavenumber cm-1

A b s o r b a n c e

( a . u . )

1610 1620 1630 1640 1650 1660 1670 16800002

0000

0002

0004

0006

0008

0010

0012

0014

0016

0018

(c)

Wavenumber cm-1

A b s o r b a n c e

( a . u . )

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

mod ed upon adsorption. As a general behaviour, one can

sent a

beter behavior with respect to collagen adsorption.

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 weakestH-bonded system [18]. Beside the characteristicfrequencies of α helix conformation, the peak located inthe higher region, at 1710 cm-1, represent the formation of an antiparallel β-sheet structure (or turns). Both theintensity and the location of the characteristic peaks are

observe a shift toward lower frequencies, a decrease in α

helix content and concomitant increase of turn percentageupon adsorption, as a consequence of denaturation.

Comparing the quantitative results in table 1, we can

remark that the sample ASY10Fe10 appear to be moresusceptible to conformational changes due to theadsorption process, since spectral alteration reflected onthe components percentage is more obvious as comparedwith the native protein. In terms of biocompatibility, we

suggest that dysprosium/iron aluminosilicate preifi

500 1000 1500 2000 2500 3000-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

YFSA

after incubation

1200oC

7 8 9

500

o

C

Wavenumber cm-1

A b s o r b a n c e ( a . u . )

1 4 2 8 1 6 2 4

1087

7 8 9

9 1 2

a b

500 1000 1500 2000 2500 3000-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Wavenumber cm-1

A b s o r b a n c e

( a . u . )

1200°C

500°C

DSFA

after incubation1087

1 4 1 8

1 6 3 5

7 9 0

7 9 8

c d

SA and DFSA heat treated at 500

Fig. 5. ATR FTIR spectra of the samples YF °C and 1200°C, recorded before and after incubation in collagen solution.

.25V vs. Ag/AgCl electrode whose intensity varies directly proportional to the collagen concentration of solution.

Cyclic voltammetry measurements were also carried

out in residual collagen solutions using a carbon pasteelectrode 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 collagenconcentrations were registered with modified carbon paste

electrode (Fig. 5) exhibiting a strong anodic peak at +0

500 1000 000 2500 30001500 2

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

1087

500oC

1200oC

YFSA

before incubation

A b s o r b a n c e ( a . u . )

7 9 8

7 8 9

9 1 2

Wavenumber cm-1

500 1000 000 2500 3000

-0.06

1500 2

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

500oC

1200oC

DFSA

before incubation

Wavenum

A b s o r b a n c e

( a . u . )

7 8 4

8 5 5

7 9 7

1087

ber cm-1



144 S. Cavalu, F. Banica, V. Simon

0.0 0.2 0.4 0.6 0.8 1.0

DFSA/1200

YFSA/1200

DFSA/500

YFSA/500

2 . 5 x 1 0 - 5

I ( A )

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 heattreated samples at 1200°C presents a higher intensitycompared to those treated at 500°C, suggesting the preferential collagen adsorption to the last one. Thecurrent enhancement was remarkable, and additionally, asignificant decrease in the oxidation potential of collagencan be distinguished (more than 100 mV) when theelectrode is modified with zeolite. This behavior, whichwas observed at different concentrations of collagen and atseveral scan rates potential, clearly demonstrates that the

zeolite mediate the electrocatalytically properties of collagen [20,21]. No cathodic peak was observed in thereverse scan, indicating that the adsorption of collagen atzeolite modified electrode is an irreversible process.

4. Conclusions

Iron and yttriun/disprosium aluminosilicate systems prepared by sol-gel route and heat treated at 500°C and1200°C were characterized using SEM, ATR FTIR spectroscopy and cyclic voltammetry. The biocompatibility of the samples was evaluated with respectto collagen adsorption. Qualitative and quantitativeanalysis of amide I features by deconvolution and curvefitting reveals that the samples containing with iron anddisprosium present a beter behavior with respect tocollagen adsorption. SEM images reveal different degreeof collagen adsorption toward the dysprosium/yttriumsamples. Cyclic voltammetry carried out in residualcollagen solutions indicates preferential collagenadsorption onto the samples heat treated at 500°C as anirreversible process, that is in agreement with the ATR-FTIR results.

Acknowledgements

The study was supported by the scientific research

project CEEX 100/2006-MATNANTECH of theRomanian Excellence Research Program.

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______________________ *Corresponding author: [email protected]

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SIMONA CAVALU_Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation and biocompatibility evaluation