surface modification of titanium-based alloys with bioactive molecules using electrochemically fixed...

Surface Modification of Titanium-Based Alloys With BioactiveMolecules Using Electrochemically Fixed Nucleic Acids

J. Michael,1 R. Beutner,2 U. Hempel,3 D. Scharnweber,2 H. Worch,2 B. Schwenzer1

1 Institut fur Biochemie, Technische Universitat Dresden, Bergstr. 66, 01069 Dresden, Germany

2 Max-Bergmann-Zentrum fur Biomaterialien, Institut fur Werkstoffwissenschaft, Technische Universitat Dresden, BudapesterStr. 27, 01069 Dresden, Germany

3 Institut fur Physiologische Chemie, Medizinische Fakultat Carl Gustav Carus, Technische Universitat Dresden,Fiedlerstrasse 42, 01307 Dresden, Germany

Received 1 June 2005; revised 10 November 2005; accepted 27 February 2006Published online 5 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30579

Abstract: A new method of surface modification for titanium (alloys) with bioactive mole-cules was developed with the intention of providing a new basis of implant adaptation forparticular requirements of certain medical indications. Nucleic acid single strands are fixedelectrochemically via their termini (regiospecifically) by growing an oxide layer on Ti6Al7Nbanodically. It could be shown that they are accessible to subsequent hybridization withcomplementary strands at physiological pH. Amount of nucleic acids immobilized and hy-bridized were determined radioanalytically using 32P-labelled nucleic acids. Stable fixationwas attained at and above potentials of 4 VSCE. Up to 4 pmol/cm2 of nucleic acid single strandscould be immobilized and hybridization efficiencies up to 1.0 were reached. Hybridizationefficiency was found to depend on surface density of immobilized oligonucleotides, whilehybridization rates increased when MgCl2 was added. A conjugate consisting of an oligonu-cleotide complementary to the immobilized strand and the hexapeptide GRGDSP with RGDas an integrin recognition site was synthesized. This conjugate was able to bind to integrins onosteoblasts. It was shown that this conjugate binds to the anchor strand fixed on Ti6Al7Nb toan extent comparable with the unconjugated complementary strand. © 2006 Wiley Periodicals,Inc. J Biomed Mater Res Part B: Appl Biomater 80B: 146–155, 2007

Keywords: titanium; DNA electrochemical immobilization; surface hybridization; RGDpeptide

INTRODUCTION

Hitherto progresses in implantology have largely originatedfrom the use of titanium and its alloys. These show goodbiocompatibility based on favorable properties, for examplehigh corrosion resistance in vivo, and toxicological harmless-ness.1–3

Fast and stable integration of metallic implants into thesurrounding tissue (bone, cartilage) may be problematic inthe case of systemic diseases, such as diabetes or osteoporo-sis, and/or other risk factors, such as previous radiation treat-ment or smoking, leading to a need for implant materials withosseoconductive or even osseoinductive surfaces.4–7

This fast and stable integration could be attained by im-mobilization of components of the extracellular matrix(ECM) on the implant surface, as investigated by severalgroups.8–11 A different approach for creating bioactive sur-faces is to immobilize smaller fragments of proteins or arti-ficial peptides containing certain recognition sequences,for example RGD peptides that promote specific cell adhe-sion12–17, which are more stable chemically as well as con-formationally.

Immobilization procedures include covalent attach-ment10,12–15 and adsorption.8,9,11,16 However, immobilizationby adsorption often suffers from weak interactions betweenthe components. Covalent attachment is much more stable,but has certain disadvantages. The main drawback is the needfor additional chemical modification of the surface or thebioactive molecules or both, causing possible changes in thepharmacological and toxicological behavior. Disadvantagesof the methods of surface modification developed so far arethe restriction to one or two components and a commitment

Correspondence to: B. Schwenzer (e-mail: [email protected])

Contract grant sponsor: German Research Foundation (DFG); contract grant num-bers: WO494/14 and SCHW638/3–1

© 2006 Wiley Periodicals, Inc.

146

to certain components and surface properties early in theproduction process.

Based on the above considerations, this contribution isfocused on the development of a method for surface modifi-cation using the specificity of nucleic acid hybridization tomodularize biomaterial surfaces. In a first step, singlestranded nucleic acids (‘anchor strands’) are to be entrappedpartially into a titanium oxide layer during anodic growth(Figure 1).

Conjugates may be synthesized from complementarystrands and various bioactive molecules and hybridized to thesurface modified with anchor strands. This work deals withthe basic scientific principles of such a modular system thatmay be used for designing tailored biological implant sur-faces. The objects of investigation have been the dependenceof immobilization stability of the nucleic acid single strands(anchor strands) on electrochemical conditions (e.g., polar-ization potential) and on oligonucleotide concentration, thedependence of anchor strand surface density on immobiliza-tion conditions (potential, oligonucleotide concentration), andthe dependence of hybridization efficiency on anchor strandsurface density. Further investigations included the influenceof Mg2� on hybridization efficiency and rate.

Based on the abovementioned investigations, coupling ofa nucleic acid single strand with an RGD-containing moietywas performed to obtain a conjugate with preserved RGDactivity, proven by testing receptor–ligand interaction be-tween the RGD–oligonucleotide–conjugate and osteoblasts.These experiments were accompanied by investigations ofthe hybridization ability of the conjugate to electrochemicallyentrapped anchor strands.

MATERIALS AND METHODS

Preparation of Titanium Alloy Samples

Samples of Ti6Al7Nb with a diameter of 16 mm and a heightof 2 mm were turned from bar stocks and ground down to a

grain size of P600 (26 �m). Subsequently, sample etchingwas carried out for 2 min in a mixture of 0.4M hydrofluoricacid and 1M nitric acid at room temperature (RT).

Nucleic Acids

All nucleic acids used were single stranded oligodeoxyribo-nucleotides (ODN). Synthesis and modifications (phosphor-ylation, aminohexylation) were performed by ThermoElectron Corp. (Ulm, Germany). Radioactive labelling (5�-phosphorylation using �-32P-dATP and 3�-extension with�-32P-dCTP) was carried out by Hartmann Analytic GmbH(Braunschweig, Germany). Purification of the ODNs wasperformed by the suppliers using RP-HPLC.

The anchor strand (AS) was a 5�-phosphorylated 60merhaving following sequence: 5�-CCA AAC CCG TCA ATCAAG TCT ACA CTG TTC CCA AAC CCG TCA ATCAAG TCT ACA CTG TTC-3�

The complementary (CS) and noncomplementary (NS)strands were 31mers that were not phosphorylated, that is,they carried hydroxyl groups on both termini.

Sequence of CS: 5�-CAG TGT AGA CTT GAT TGACGG GTT TGG GAA C-3�. Sequence of NS: 5�-TTC CCAAAC CCG TCA ATC AAG TCT ACA CTG C-3�

Conjugation of CS With GRGDSP

A conjugate of CS with the hexapeptide GRGDSP (carryingRGD as a recognition sequence for integrins) was synthe-sized. Synthesis followed a slightly modified routine for thecoupling of alkaline phosphatase to ODN18 using disuccin-imidyl suberate (DSS) (Sigma) to link 5�-aminohexylated CSand GRGDSP (Bachem, Switzerland).

The intermediate (activated ODN) of ODN and DSS wasobtained in a linkage reaction by mixing ODN solved in 1MNaHCO3 (pH � 8.5–9.0) and DSS solved in mole sieve-driedDMSO for 2 min. Purification was immediately done using aSephadex™-G-25 (Pharmacia, Sweden) column (300 mm �A7 mm) in a refrigerated chamber with sodium acetate

Figure 1. Principle of the immobilization system.

Journal of Biomedical Materials Research Part B: Applied BiomaterialsDOI 10.1002/jbmb

147IMMOBILIZATION OF ODN STRANDS ON TITANIUM ALLOY SURFACES

(1 mM; pH 4) as eluent. The united intermediate fractionswere concentrated in a Centricon-3™ Concentrator at 4°Cand 7500g.

Instantly, equimolar amounts of activated ODN andGRGDSP in reaction buffer (3M NaCl, 100 mM NaHCO3,1 mM MgCl2, 0.05% NaN3, pH � 8.2–8.3) were mixed andkept in the dark at room temperature for 20 h. The formedconjugate was concentrated and rebuffered (0.5M Tris–HCl,pH 7.5) in a Centricon-3™ Concentrator at 4°C and 7500g.The overall yield of the synthesis was 84%.

Fractions of the conjugate were used for radiolabelling by3�-extension with �-32P-dCTP, for the determination of theconcentration via UV absorbance, for hybridization experi-ments, and for further concentration and rebuffering withPBS at pH 7.4 as needed in the cell adhesion experiment.

Immobilization Procedure

Immobilization was carried out in a specially designed con-ical cell made from acrylic glass. A circular sample area of1.8 cm2 was exposed to the electrolyte solution. A platinumwire acted as a counter electrode. Potential was measuredversus a saturated calomel electrode, using an agarose-filledplastic hose as a salt bridge (Figure 2).

Filling the cell with 3 mL of electrolyte solution (40 mMacetate buffer; pH 4.0; defined amount of ODN) was fol-lowed by 15 min adsorption time allowing the anchor strandto adsorb to the sample surface. Subsequently, anodic polar-ization was carried out. For electrochemical treatment a po-tentiostat/galvanostat system Voltalab 4.0 combined with ahigh voltage booster HVB100 (Radiometer Analytical,Copenhagen) was used.

In the case of adsorption experiments, treatment was thesame but polarization was omitted.

After anodization the sample was washed inside the cellthree times for 2 min with 3 mL of 40 mM acetate buffer (pH4.0). Samples were air-dried in a hood before quantification.

Desorption Procedure

Samples underwent a desorption procedure to remove merelyadsorbed but not entrapped anchor strand molecules and to

test immobilization stability. The desorption procedure con-sisted of two steps of 15 min and one step of 30 min. Threemilliliters of fresh 50 mM Tris–HCl (pH 7.5) were used foreach step. Samples were then dried and quantified. A fourthdesorption step lasted for 24 h in 3 mL of 50 mM Tris–HCl(pH 7.5) in order to desorb nonentrapped molecules to alarger extent, and was concluded by drying and quantifica-tion.

Hybridization Procedure

After desorption, samples were placed into 12-well plates(NUNC, Denmark) and 100 �L of a Tris–HCl-buffered (50mM, pH 7.5) solution of CS or NS (c � 400 nmol/l),respectively, were placed on the sample surfaces. At leastfour out of the 12 wells were left empty and filled with waterto maintain a high degree of humidity, thus minimizingevaporation from the surfaces. This first hybridization stepwas terminated after 30 min by rinsing the samples withTris–HCl buffer (50 mM, pH 7.5). Drying and quantificationwas followed by a second hybridization step, similar to thefirst except for the presence of an additional 10 mM MgCl2 inthe hybridization solution.

Quantification of the Amount of Substance onSample Surfaces

For quantification, radiolabelling of the nucleic acids with 32Pwas used. In all experiments distinct amounts of radiolabelledODNs were mixed with the corresponding nonlabelled ones.Percentage of labeled molecules in the mixture was in therange of 0.01 to 1%.

Count rates of samples were determined in a shieldedchamber using a passivated implanted planar silicon detector(PIPS)-based spectrometry system (Canberra/Ortec™). Theenergy of emitted electrons (Emax � 1.7 MeV) is high enoughto permeate the TiO2 layer (maximum 15 nm) on top of thesample and the air between sample and detector withoutsignificant loss of signal.

Counting efficiency (quotient count rate by true radioac-tivity) was 28%. Amounts of substance bound were calcu-lated from count rates, taking into consideration countingefficiency, labelling percentage and decay.

Cell Adhesion Experiments

Rat calvarial osteoblasts were labelled for 24 h with [3H]Thy-midin and incubated with 100 �M cycloheximide (CHX) for3 h, followed by trypsinization. Three fractions of the cellswere separately incubated in 100 �M CHX (serum-free me-dium) for 20 min, whereof two contained 80 �mol/LGRGDSP and GRGDSP-CS, respectively, aiming at a satu-ration of the cell receptors (i.e. blocking them for adhesionsurfaces). Adhesion on polystyrene cell-culture plates wasperformed for 1 h by seeding 25,000 cells/cm2 in 100 �MCHX (serum-free medium).

RESULTS

The experimental data are presented as bar graphs in thefollowing sections. In all of these diagrams the bars indicate

Figure 2. Design of the electrochemical cell.


148 MICHAEL ET AL.

the mean and the error bars refer to the standard deviation ofthe data.

Immobilization

In Figure 3 surface densities of AS are shown after immobi-lization from an electrolyte containing 40 nmol/L ODN. Themere adsorption procedure is compared to potentiostatic (ps)or galvanostatic (gs) polarization, where different potentialsup to 15 VSCE were used. Surface density of AS ranges from0.8 to 1.3 pmol/cm2 and is nearly independent of the appliedpotential and the polarization mode used. The same indepen-dence of the immobilization potential after immobilization isobserved for higher concentrations of ODN in the electrolyteas shown in Figure 4(a). As clearly to be seen in Figure 4(b),desorption at pH 7.5 leads to a distinct decline of surfacedensity by �90% in the case of merely adsorbed AS, whilst30–40% of the electrochemically immobilized AS are re-tained. Retention increases with higher potentials of 8 and15 VSCE [Figure 4(b)].

A strong dependence of surface density on ODN concen-tration in the electrolyte was observed for the applied range asshown in Figure 5. In a concentration range between 40 and400 nmol/L, surface densities of AS after desorption increasefrom 0.7 to 4.0 pmol/cm2. However, at a concentration of800 nmol/L saturation seems to be reached.

Hybridization

The influence of immobilization potential on the binding ofCS and NS, respectively, is considered in Figure 6. Surfacedensities of CS are highest (1.5 pmol/cm2) for immobilizationat 4 and 8 VSCE [Figure 6(a)]. For samples with merelyadsorbed anchor strands approximately 50% less CS wasdetected compared with 4 VSCE or 8 VSCE, which can betraced back to low quantities of AS remaining after desorp-tion. The lowest surface densities of CS (�0.5 pmol/cm2)were detected on samples with AS immobilized at 14.5 VSCE.Surface densities of NS are comparably low (�0.5 pmol/cm2)

for all immobilization conditions. The binding ratios (BR) ofCS and NS, defined as the quotient of surface density of CSand NS, respectively, by surface density of AS, are presentedin Figure 6(b). With BRCS � 1.1 the value is highest forsamples with solely adsorbed AS and decreases to 0.2 for animmobilization potential of 14.5 VSCE. Potentials of 4 VSCE

and 8 VSCE lead to BR values of approximately 0.4.In all hybridization experiments shown in Figure 6, dis-

tinctly higher amounts of CS and NS could be bound duringthe second hybridization step in the presence of 10 mmol/LMgCl2 compared to the first step without MgCl2.

Figure 3. Surface densities of anchor strand (AS) at various immobi-lization conditions (40 nM AS in 40 mM acetate buffer, pH � 4.0;potentiostatic polarization (ps) 4–14.5 VSCE, galvanostatic polariza-tion (gs) 15 VSCE) compared to adsorption.

Figure 4. Surface density of anchor strand (AS) for different immo-bilization potentials (400 nM AS in 40 mM acetate buffer, pH � 4.0).a: absolute values; b: normalized diagram (AS density after immobi-lization � 1).

Figure 5. Surface density of anchor strand (AS) for different ASconcentrations in the electrolyte (40 mM acetate buffer, pH � 4.0, ps8 VSCE).



Results of hybridization experiments in dependence ondifferent surface densities of AS are shown in Figure 7. Inthis diagram immobilization conditions were disregarded andeach data point represents an individual sample.

Below a surface density of 0.8 pmol/cm2 of immobilizedanchor strand, BR may reach values above 1.0 with a maxi-mum of 3.5. However, for CS the observed values of BR arealways higher than for NS. With rising quantity of fixedanchor strand BR values of CS and NS decrease to 0.36 and0.04, respectively.

Results of a hybridization experiment with a conjugate ofCS and a cell adhesion peptide (GRGDSP) as a model systemfor biologically active molecules are compared to the valuesof nonconjugated CS in Figure 8. For this experiment, im-mobilization of the anchor strand was performed in an elec-trolyte containing 400 nmol/L AS at 8 VSCE. After bothhybridization steps BR for this conjugate reached 0.4, whichis exactly the same as for unconjugated CS. However, theconjugate appears to bind mainly during the second hybrid-ization step in the presence of MgCl2, while the bindingduring the first step was clearly decreased compared to CSalone.

Cell Adhesion Experiment

Biological activity of the GRGDSP–CS–conjugate was testedwith an inhibition test of cell adhesion.

In Figure 9, numbers of adherent cells (expressed as mea-sured 3H-activity) on polystyrene cell-culture plates areshown after incubation with the GRGDSP–CS–conjugate,with unconjugated GRGDSP (positive control) and withoutblocking substance (negative control). For both GRGDSP

Figure 6. Influence of immobilization potential on binding of comple-mentary strand (CS) and noncomplementary strand (NS) Immobiliza-tion: 400 nM AS in 40 mM acetate buffer, pH � 4.0; Hybridization: 400nM CS or NS in 50 mM Tris–HCl, pH � 7.5). a: surface density; b:binding ratio.

Figure 7. Dependence of binding ratios of complementary strand(CS) and noncomplementary strand (NS) on surface density of ASafter desorption (immobilization conditions discounted).

Figure 8. Binding ratio of GRGDSP-CS-conjugate compared to non-conjugated CS (400 nM AS in 40 mM acetate buffer, pH � 4.0, ps 8VSCE; hybridization: 400 nM CS or GRGDSP-CS in 50 mM Tris–HCl,pH � 7.5).

Figure 9. Cell adhesion of rat calvaria osteoblasts after incubationwith GRGDSP-peptide and GRGDSP-CS-conjugate, respectively.


150 MICHAEL ET AL.

alone and GRGDSP-CS, activity decreased to (68 � 7)% and(61 � 7)%, respectively.

DISCUSSION

The aim of the presented work was the stable and regiospe-cific immobilization of ODN-strands on titanium alloy sur-faces via oriented electrochemical entrapment of their phos-phorylated termini as key step for a new immobilizationsystem for biologically active molecules. These anchorstrands had to be accessible to hybridization with comple-mentary strands and with a conjugate of a complementarystrand and a bioactive molecule synthesized for this purpose.The influences of parameters such as concentration of anchorstrand or polarization potential and surface density of anchorstrand on immobilization and on hybridization, respectively,were objects of investigation. Additionally, the bioactivity ofthe synthesized conjugate had to be proven to exclude neg-ative interference from the nucleic acid moiety of the conju-gate.

Immobilization

The immobilization process begins with adsorption of theODN. Adsorption and desorption processes are based on anumber of physicochemical phenomena, including van derWaals forces, dipole-dipole interactions, hydrophilic versushydrophobic interactions, entropic effects, and electrostaticinteractions. In the investigated system, electrostatic interac-tions between adsorbate and adsorbent are important. Theyare controlled by the isoelectric point (IEP) of the surface andthe pKa-values of the adsorbate, that is, by the charge inter-action between them at a certain pH value. For chemicallypure Ti and Ti6Al4V similar IEP values of 4.5 and 4.3,respectively, were determined by Ro�ler et al.19 from elec-trokinetic measurements. Because the Ti6Al4V alloy showedonly very small differences in the IEP compared to puretitanium a value in the range of 4.3–4.5 is also assumed forTi6Al7Nb. From literature, pKa-values of 2.1, 7.2, and 12.7are known for phosphoric acid.20 Hence, at the applied pHvalue of 4.0 it could be expected that the titanium alloysurface is positively charged (at least partially) and the ter-minal phosphate group of the anchor strand negatively, whilethe phosphate groups of the sugar-phosphate-backbone arestill protonated. Under physiological conditions (pH 7.4),both surface and ODN should be charged negatively, result-ing in desorption of the ODN as shown in Figure 4. Thisassumption is supported by investigations of our group onTi6Al4V using fluorimetric methods and XPS.21 Those ex-periments showed that ODN cannot be stably fixed at neutralpH. At pH 4.0 terminal phosphate groups are essential forstable entrapment into an anodically grown oxide layer ontitanium alloys. This dependence of adsorption behavior onconditions in solution makes it possible to control the inter-action process between the ODNs and the titanium alloysurface by adjusting the pH values. The accessibility of such

immobilized anchor strands was proven by hybridization witha complementary strand carrying a fluorophore.21

Anodic polarization of titanium-based alloys leads to athickening of the air-formed passive layer. The growth pro-cess is caused by migration of anions from the electrolytetowards the phase boundary oxide/metal and of metalliccations towards the phase boundary oxide/electrolyte solu-tion, producing new oxide at both interfaces, electrolyte/oxide as well as oxide/metal. This results in a two-layersystem consisting of a dense inner (barrier) and a porousouter layer.22,23 At the phase boundary oxide/electrolyte ad-sorbed electrolyte species23–26 and even macromolecular ag-gregates27 can be incorporated into the outer part of thegrowing oxide layer. Thickness of the layer can be controlledby the applied potential, with an overall growth of 1.4–2.3nm/V.28–30 Consequently, immobilization stability of the an-chor strand by regioselective incorporation in the growingoxide layer should increase with rising potential, which isconsistent with the results presented in Figure 4(b). However,even at potentials as high as 14.5 VSCE, the anchor strand(AS) partly desorbs. The major reason for that effect isthought to be adsorption of AS on the newly formed anodicoxide layer during and following the electrochemical process.Furthermore, the formation of the surface has to be consid-ered as a dynamic process. Hence, a certain depth distributionof incorporated ODN has to be taken into account, resultingin a stability distribution.

The amounts of AS on the surface after immobilizationand desorption are furthermore controlled by its concentra-tion in the electrolyte as shown in Figure 5, reaching satura-tion between 400 and 800 nmol/L. The maximum fixedamount of AS was obtained with about 4 pmol/cm2 at 400and 800 nmol/L of AS.

To evaluate the achieved surface coverage, data werecompared with surface densities reported by other au-thors,31–42 even though they used a variety of immobilizationprocedures, for example formation of self-assembled mono-layers on gold using biotinylated mercaptans, streptavidin,and biotinylated ODN31 or binding of silanized nucleic acidson glass.32 Oligonucleotides in those investigation were12mers,33 15mers,31,34 17mers,34 18mers,35 20mers,32,36

25mers,37 26mers,38 and 30mers.39

Irrespective of the kind of immobilization procedure usedand the length of the oligonucleotides, surface densitiesranged from 1 to 50 pmol/cm2 and surface saturation wasoften attained in this range. Comparison of our results withthe aforementioned ones indicates that reasonable conditionsfor ODN-immobilization were found in the investigated tita-nium-based system.

Hybridization

Surface hybridization studies are of great interest in all fieldsof nucleic acid immobilization, since there are distinct dif-ferences compared to hybridization in solution. Immobilizednucleic acids are in much closer proximity than dissolvedones, which can lead to sterical hindrance. The anchorage of



a terminus restricts free movement of the strands. Both steri-cal effects can influence the association of double helices. Inaddition, the concentration within the anchor strand layer isvery high. The volume above the reported 4 pmol/cm2 ofimmobilized 60mer AS is confined by the height of the 60merwith an estimated length of 30 nm if a base-to-base distanceof 0.5 nm is assumed. Length estimation had to be based ondata known from dsDNA structure determination. Dimen-sions of Z-DNA were thought to be close to a noncurledssDNA since Z-DNA is the most stretched DNA doublehelix. If the DNA single strand is seen as a completelyunwound molecule, the aforementioned distance of 0.5 nm isestimated based on Z-DNA with a slope of 0.38 nm per basepair.43 Belosludtsev et al.33 used the same base-to-base dis-tance of 0.5 nm for their calculations. Thus, the resultingconcentration in the volume near the surface is about 2.3mmol/l or 42 g/l, which is above the concentrations usuallyemployed in homogeneous solutions. A comparison withhybridization in solution is therefore complicated. Further-more, at hybridization conditions (pH 7.5) the titanium-basedsurfaces as well as the nucleic acids’ sugar-phosphate-back-bones are negatively charged, which increases repulsionforces between the strands and between strands and surface.

These considerations implicate a certain surface densitylimit for the anchor strand, above which hybridization effi-ciency, considered as the hybridized fraction of the totalamount of anchor strand at the surface, will be below 1.0because of sterical hindrance and repulsion. This is supportedby the experimental results. Figure 7 shows declining bindingratio (BR) with rising surface densities. However, with de-creasing surface densities of AS nonspecific binding in-creases, resulting in an optimum, at which nonspecific inter-actions can be disregarded and hybridization is still sufficient.At the lowest obtained surface densities of AS, BRs of CSand NS are �3.5 and 2.5, respectively. This implies that, asthe hybridization efficiency (HE) reaches its maximum of 1.0,the remainder of 2.5 originates from nonspecific binding. AsFigure 7 demonstrates, the optimum surface density is about2.5 pmol/cm2 of AS with a HE of about 0.5.

The effect of surface density on hybridization efficiencywas also studied by other authors. Chrisey et al.36 whoimmobilized mercapto-modified ODN on aminosilanizedglass surfaces, found a dependence of HE on surface density.Peterson et al.37 used surface plasmon resonance (SPR) spec-troscopy to investigate surface density and hybridization ki-netics. Mercapto-modified ODNs were immobilized co-valently on gold. Surface density was controlled by ionicstrength, interfacial electrostatic potential, or whether ssDNAor dsDNA was used. HE and kinetics were almost exclusivelycontrolled by surface density, irrespective of the immobiliza-tion procedure applied. At a surface density of 4.65 pmol/cm2

HE was nearly 1.0 with kinetics similar to a Langmuir iso-therm while at higher surface densities (18.27 pmol/cm2) HEfell to 0.1 and the hybridization rate was extremely low.Peterson et al.37 demonstrated excellent HE and fast hybrid-ization kinetics when dsDNA was immobilized und subse-quently dissociated by heating up to 95°C. Thus, the resulting

ssDNA-surface was perfectly structured for hybridization,because of the sterical and electrostatic spacer effect ofdsDNA. This optimal surface density was reached at 4.65pmol/cm2, which is close to the optimum in the presentedsystem.

In spite of all the differences between hybridization onsurfaces and in solution, parallels exist. Magnesium cationsdistinctly accelerate hybridization in solution as reported byMichael44 and Michael et al.45 This acceleration could also beobserved at the electrochemically modified titanium alloysurfaces since a distinct additional increase of bound amountsof substance is reached during the 2nd hybridization step inthe presence of MgCl2 (Figure 6).

The observed influence of Mg2� ions on hybridization isthought to have its basis in a variety of effects—mainlyconformational, sterical, and electrostatic. Like monovalentmetal ions (Na�, K�, etc.), Mg2� can reduce electrostaticrepulsion between the sugar-phosphate-backbones of thecomplementary strands.46 However, the main kinetic effectsseem to originate from conformational benefits induced bycoordinative binding of divalent metal cations46–50 as dis-cussed for hybridization in solution.44,45 Additionally, mag-nesium cations can adsorb on the negatively charged samplesurface, thus reducing repulsion between ODNs and the sur-face. However, the increased nonspecific binding in the pres-ence of Mg2� demonstrates negative influences of magne-sium cations on the surface. Most likely, there is a formationof complex-like assemblies with Mg2� as a central ion andhydroxyl groups of the titanium oxide surface as well asfunctional groups of the ODN as ligands. Besides optimalsurface densities of anchor strands, adjustment of the con-centration of Mg2� seems to be another optimization param-eter to obtain high hybridization efficiencies and low nonspe-cific binding.

Summarized Description of the Immobilization andHybridization System Investigated

Immobilization and hybridization behavior of the investi-gated system can be summarized according to Figure 10.

Figure 10. Experimental workflow and definition of resulting amountsof ODN detected on sample surface.


152 MICHAEL ET AL.

Immobilization of the anchor strands begins with their ad-sorption. Because of the oxide layer thickening, a fraction ofODN is partially incorporated into the anodic oxide layer toa sufficient depth, hence fixed, while others remain and be-come unstably entrapped as well as adsorbed. This overallamount of substance, detected after the immobilization pro-cedure, can be defined as bound amount. During desorptionprocedure (second stage), adsorbed and unstably entrappedODN are removed. Consequently the remaining amount ofsubstance after desorption is defined as fixed.

During hybridization experiments the ODN-modified tita-nium alloy surface is exposed to CS and NS, respectively.Amounts of CS or NS on the surface after the hybridizationexperiment are defined as bound amount (Figure 10). How-ever, in the case of NS bound amount is defined as nonspe-cifically bound amount, while in the case of CS boundamount is the sum of hybridized and nonspecifically boundamount (Figure 10). Deduced from this definition are theterms binding ratio (BR) and hybridization efficiency (HE) asshown in Eqs. (1)–(3):

BRCS or NS �bound amount (CS or NS)

fixed amount AS(1)

HE �hybridized amount CS

fixed amount AS(2)

For equal amounts of fixed AS the terms HE and BR arerelated:

HE � BRCS � BRNS (3)

The above equations are valid under the premise that theextent of nonspecific binding is the same for CS and NS andthat the interaction of AS and CS is based on duplex forma-tion.

Hybridization Behavior and Biological Activity of theCell-Adhesion Peptide–ODN–Conjugate

Hybridization efficiency (HE) of the CS–GRGDSP–conju-gate without addition of MgCl2 was distinctly lower than thatof CS under the same conditions. However, in the presence ofMgCl2, HE reached the same absolute extent (Figure 8). Thisbehavior can be ascribed to several factors. Sterical hindranceand diminished motility of the complementary strand due tocoupling of the cell-adhesion peptide to the ODN is certainlyone cause. Another reason might be interactions betweenODN and the peptide possibly occurring within one conjugateor between two conjugates.

Such interactions are known from aptamers, which arenucleic acids with specific binding sites for amino acids suchas arginine or citrulline, as reported by Famulok.51 Theseeffects can obviously be overcome by optimization of param-eters that favor the hybridization of the two complementary

nucleic acid strands, such as concentration of MgCl2, expo-sure time, or surface density.

For the success of the whole immobilization system, pres-ervation of the activity of biomolecules coupled to DNAsingle strands is important. As the results of the cell adhesionexperiment (Figure 9) show, the binding ability of the pep-tide–DNA–conjugate to integrins in solution is similar to thatof the nonconjugated peptide. It clearly implies preservationof the biological activity of the peptide when conjugated toDNA.

CONCLUSIONS

The results presented here clearly demonstrate the feasibilityof the immobilization and hybridization system as shownschematically in Figure 10. Terminally phosphorylated ODNcan be immobilized by partial entrapment into anodicallygrown oxide layers on titanium materials after an electrostat-ically driven oriented (terminal) adsorption. The stability ofsuch a fixation is much better compared to pure adsorptiveimmobilization.

Accessibility of immobilized anchor strands for hybridiza-tion was successfully proven and is the prerequisite for amodular system intended to be built up from different con-jugates with varying hybrid stabilities at an oligonucleotide-modified titanium surface. The hybridization ability of thesystem also supports the thesis of predominantly verticalorientation of the anchor strands. Obviously, there is anoptimum of surface density and entrapment depth at whichaccessibility of the anchor strand is sufficiently high forhybridization while the titanium alloy surface is still pro-tected against nonspecific binding of dissolved ODN. Forsubsequent modularization by hybridization, this optimumshould be adjusted, mainly via the concentration of the an-chor strand.

Further optimization will be carried out in future experi-ments, which will comprise the following: long term stabilitytests of AS fixed on the surface; influence of hybridizationtime, pH, ionic strength, and temperature as well as concen-tration of CS on hybridization; stability of formed hybrids asfunction of experimental and structural parameters; kineticsof surface hybridization; and other titanium materials, forexample chemically pure Ti.

Biomolecular coatings of implants are always endangeredby degradation processes in the body. DNA and RNA aredegraded by nucleases, which can be prevented or deceler-ated by the modification of strands with LNA or by the use ofPNA. Therefore, future investigations will also include theuse of LNA-modified strands and PNA.

Hybridization of cell-adhesion-peptide-DNA-conjugates,as model system for biologically active molecules coupled tothe complementary strand, is possible to the same extent asfor the complementary strand alone. Furthermore, it could beshown in a first cell-adhesion experiment that the peptidepreserves its binding ability to integrins in solution. Cellresponse to conjugates hybridized with immobilized anchor



strands (i.e. the complete system) will be part of futureexperiments. These investigations will be extended to conju-gates of ODN and bioactive molecules such as other cell-adhesion peptides, growth factors, or anti-inflammatorydrugs.

The authors would like to thank Dr. Steffen Taut and his staff ofthe Central Radionuclide Laboratory of Dresden University of Tech-nology for providing the analysis facilities and methodical support.The authors are very grateful to G. Graubner and M. Schuhmann fortheir assistance in the laboratory.

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