Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Metalated nucleotide chemisorption on hydroxyapatite Michele Benedetti a,⁎, Daniela Antonucci a, Federica De Castro a, Chiara R. Girelli a, Marco Lelli b, Norberto Roveri b, Francesco P. Fanizzi a,⁎ a b
University of Salento, Department of Biological and Environmental Sciences and Technologies, Via Monteroni, 73100, Lecce, Italy University of Bologna, Department of Chemistry “G. Ciamician”, Via Selmi 2, 40126, Bologna Italy
a r t i c l e
i n f o
Article history: Received 25 February 2015 Received in revised form 7 April 2015 Accepted 9 April 2015 Available online 19 April 2015 Keywords: Hydroxyapatite nanocrystals Nucleotide adsorption Nanomaterials Platinum complex Antitumor drug Antiviral drug
a b s t r a c t The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hydroxyapatite [Ca10(PO4)6(OH)2], is the main inorganic constituent of bones and teeth [1]. For this reason, it is extremely biocompatible and the applications in many medical fields are greatly expanding. Relevant examples are the artificial protheses, able to substitute bones and, in some cases, to be completely integrated in the natural tissues. Some attempts to impregnate these protheses, with drugs to be released in situ, are documented in the literature [2]. Recently the use of hydroxyapatite nanoparticles, in the drug delivery, has been suggested for the peculiar properties (biocompatibility and biodegradability) of this kind of aggregates able, in several cases, to avoid some general problems arising from the systemic administration of drugs [3,4]. Due to their recent availability, even in controlled morphologies [5], the nanoparticles of hydroxyapatite have been studied, to develop nanocarriers for drug delivery and for biomedical applications [6]. Nowadays, hydroxyapatite nanocrystals (nHAP) are experimented as carriers for the transport of proteins [7], antibiotics [8], antitumor drugs [9], radioisotopes [10], genes [11] and antigens for vaccines [12]. In all these applications, the main recognized nHAP internalization pathways in tissues and cells seem to be passive diffusion, active transport and endocytosis. In particular, in the case of particles greater than 10 nm, the internalization in eukaryotic cells can be mediated by phagocytosis, macropinocytosis and endocytosis (simple or ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Benedetti), [email protected] (F.P. Fanizzi).
clathrine mediated, via caveoles) [13–15]. Macromolecules and nanocrystals can bind specific receptors on the cell surface, inducing formation of cavities decorated by clathrines, internalized by endocytosis. In the cytoplasm, the vesicles lose the clathrines and fuse with other vesicles (endosomes). In this context, it is to be underlined that the nHAP were previously exploited as delivery systems for genes, drugs, and coadjuvants (which destabilize the endosomal membrane). Generally, nHAP improves the release of drugs and genes directly in the cytoplasm [16]. The importance of these techniques in the modulation of the activity of pharmacologically active molecules and in the reduction of possible side effects has also been recognized. For this reason, a lot of research has been devoted to the development of new nHAP based materials for the controlled release of drugs. This aspect is important in the case of antitumor and/or antiviral drugs because often very serious side effects may develop, causing the discontinuance of the chemotherapeutic treatment. In this context, the possible use of hydroxyapatite nanoparticles, as vectors for drugs, inducing a more efficient, localized and gradual delivery could be extremely useful. Indeed, the reduction of the peak concentration of administered drugs, reached in tissues, during the treatment, could prevent harmful side effects. In drug release applications, the chemical–physical properties of nHAP and their intrinsic high biocompatibility are very attracting [17]. For this reason, it could be relevant to clarify the factors affecting the known chemical adsorption (chemisorption) [6] of some substrates on nHAP and the possible controlled release in vitro and in vivo. For example, studies were conducted on the factors governing adsorption/release of some cisplatin derivatives and 5-fluorouracyl [18–20] on/from nHAP
http://dx.doi.org/10.1016/j.jinorgbio.2015.04.006 0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
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M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
solid matrices. In these cases it has been highlighted that the cytotoxicity of the drugs adsorbed on nHAP is different, with respect to the same drugs, when administered in the free form. In previous works, we studied many platinum complexes [21–25] even with metal bonded purine nucleotides [26–28], to better understand the mechanism of action of platinum based antitumor drugs. In this work, we focused our attention on model molecules with a variable number of phosphates, based on simple or metalated (Pt-bonded) nucleotide derivatives, Fig. 1. The possible adsorption of these molecules on hydroxyapatite nanoparticles is the topic of the present study. Molecules bearing phosphate groups have a variety of pharmacological, biological and technological applications [29–32]. In particular, nucleotide derivatives are very interesting in this context, because widely used, as experimental tools to modify nucleic acids, or as antimetabolites acting as powerful drugs. For instance, nucleotide analogues are molecules that generally can substitute the normal nucleotides, in the DNA/RNA synthesis and, for this reason, can be used as both antitumor and antiviral drugs (5-fluorouracyl, acyclovir, AZT, etc.) [20,30,33,34]. It is known that nucleotide, oligonucleotide, DNA and RNA derivatives can be adsorbed on hydroxyapatite [35–42]. For this reason, in this work, we wanted to enlighten the effect of the presence of a variable number of phosphates, in single simple guanine nucleotide derivatives, on the adsorption on nHAP. Moreover, we focused our attention on the evaluation of the molecular charge effects on the chemical adsorption, by studying model complexes, obtained when the guanine nucleotide derivatives are coordinated, by the purinic N7 donor, to a model bis-positive Pt(II) complex. This study may be relevant when considering the adsorption, transport and delivery of drugs, in specific tissues and/or cell compartments, with a possible modulation of both pharmacological activity and side effects. Previous work demonstrated high biocompatibility specifically for fully characterized nanometric hydroxyapatite [2,6]. Since this work is addressed to the study of nucleotide analogues adsorption on biomimetic hydroxyapatite nanocrystals, for their possible use as nanocarriers, we used the same biocompatible nanometric material.
O
A
N 8
9
N
OH
1 2
6 3
N
NH2
O
4'
n
3'
2'
OH
R
B
1'
2.1. Synthesis of complexes All solvents and reagents, except otherwise stated, were purchased from Aldrich Chemical Company and used as received. [Pt(N7-dGUO) (dien)] (1a), dien = diethylenetriamine, [Pt(N7-dGMP)(dien)] (1a′), [Pt(N7-dGDP)(dien)] (1a″), [Pt(N7-dGTP)(dien)] (1a‴), [Pt(N7-GUO) (dien)] (1b), [Pt(N7-GMP)(dien)] (1b′), [Pt(N7-GDP)(dien)] (1b″), and [Pt(N7-GTP)(dien)] (1b‴) were synthesized as previously described [29,30]. NMR spectra were recorded on a Bruker Avance III HD 600 or on a Bruker Avance DPX 400 using deuterated solvents. Spectra were referenced to the residual HOD signal (1H) or 85% H3PO4 (31P) as internal or external standard, respectively.
2.2. Synthesis of hydroxyapatite nanocrystals Plate shaped hydroxyapatite nanocrystals (Figs. 2, S1 and S2) were synthesized according to a previously published method [43], with some modifications, according to a recent procedure, as reported by some of us [2].
2.3. Adsorption tests on hydroxyapatite nanocrystals In a typical experiment 50 μL of an nHAP suspension (hydroxyapatite concentration = 71 mg/mL) was diluted in about 500 μL of water, at room temperature and the pH value was adjusted to ≈7, if required, by addition of HCl or NaOH. Then the necessary volume of the tested sample solution and water were added to obtain a final volume of 600 μL and a final concentration of the nucleotide derivative in the nHAP suspension of ≈7.0 mM. The obtained solution was left under stirring for about 20 h (T = 295 K), to favor the sample adsorption on the nHAP and equilibrium reaching. The final suspension was then centrifuged (at 3000 rpm, for 10 min) and therefore the mother solution was removed. The solid nHAP, was then resuspended in 1 mL of distilled water, centrifuged again by removing the surnatant water. The last operation was repeated three times, following a standard procedure [6], to guarantee a complete removal of both the initial mother solution and the physically adsorbed (physisorbed) nucleotide derivatives. Only the eventual nucleotide derivative present at this stage, on the solid phase, was considered strongly chemically adsorbed (chemisorbed) on the nHAP. The nHAP samples were then resuspended in D2O (600 μL) and dissolved, by addition of the minimum amount of DCl, to induce the dissolution of the solid phase and the release of the nucleotide derivative. These latter were quantified by NMR spectroscopy, after addition of a known amount of absolute ethanol, as internal standard, Table 1.
NH Pt
H2N
NH2 O
N N
OH HO P O O
5NH 4
5'
HO P O O
7
2. Experimental section
NH N
NH2
O
n OH
R
Fig. 1. In the figure are represented (A) the 2′-deoxy-nucleoside (R = H) and nucleoside (R = OH), n = 0–3, derivatives a–a‴ and b–b‴, respectively, and (B) the corresponding model platinum complexes, 1a–1a‴ and 1b–1b‴, respectively. The atom numbering scheme, for nucleotides, is also reported.
Fig. 2. TEM image of synthetic hydroxyapatite nanocrystals (nHAP) with plate-shaped morphology (scale bar 100 nm).
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Table 1 Chemical adsorption of nucleotide derivatives on nHAP. Nucleotide derivative
Amount of chemical adsorption (μmol/mg)a
dGUO dGMP− dGDP2− dGTP3− [Pt(N7-dGUO)(dien)]2+ [Pt(N7-dGMP)(dien)]+ [Pt(N7-dGDP)(dien)] [Pt(N7-dGTP)(dien)]−
0.01 0.17 0.39 0.17 0.04 0.00 0.00 0.22
The reported amounts of chemical adsorption, were quantified by NMR, after dissolution in water of the treated nHAP. The nucleotide derivatives, were adsorbed on nHAP in controlled conditions (water solution; T = 295 K; pH ≈ 7; nucleotide concentration ≈ 7 mM; nHAP concentration ≈ 6.5 mg/mL). The nHAP was than washed three times by water, to eliminate the physically adsorbed nucleotides. The chemically adsorbed nucleotides were released, after acidic dissolution of the treated nHAP in D2O. The amounts were calculated measuring the nucleotide concentration by 1H NMR, with respect to an internal standard. Similar values were obtained in the case of corresponding guanosine derivatives. a The reported data are average values of two sets of parallel independent measurements (±SD ≈ 0.02 μmol/mg).
3. Results and discussion In this work, we evaluated, at neutral pH, the effect of the number of phosphates increasing, on the adsorption on nHAP, for a series of water soluble purine nucleotide derivatives: dGUO (a), dGMP (a′), dGDP (a″), dGTP (a‴), GUO (b), GMP (b′), GDP (b″), and GTP (b‴), Fig. 1. Moreover, the effect of the coordination of a bis-positive Pt(II) complex, to the N7 position of the nucleotide derivatives has been evaluated, in the following water soluble model complexes: [Pt(N7-dGUO)(dien)] (1a), [Pt(N75′-dGMP)(dien)] (1a′), [Pt(N7-5′-dGDP)(dien)] (1a″), [Pt(N7-5′dGTP)(dien)] (1a‴), [Pt(N7-GUO)(dien)] (1b), [Pt(N7-5′-GMP)(dien)] (1b′), [Pt(N7-5′-dGDP)(dien)] (1b″), and [Pt(N7-5′-dGTP)(dien)] (1b‴), Fig. 1. In order to quantify the chemical adsorption (chemisorption) of the nucleotide derivatives on nHAP, we performed specific experiments. The quantification of the tested nucleotides adsorption was assessed by 1H NMR spectroscopy. The adsorption tests, were generally conducted by direct interaction of the purine derivative, dissolved in water, at a given concentration, with a water suspension of nHAP. The interaction between the purine derivative and the nHAP, was generally allowed for about 20 h. Preliminary tests guaranteed this time being enough for reaching the equilibrium. The suspension was then separated from the mother solution by centrifugation and washed with D2O, to eliminate the physically adsorbed (physisorbed) nucleotide derivatives (a variable physically adsorbed fraction was observed in all tested nucleotide derivatives). The resulting solid suspended in D2O was treated with the minimum amount of DCl, in order to dissolve nHAP and release in solution the purine derivative. The resulting solutions were then analyzed by 1H NMR in order to assess and quantify the eventual presence of the previously chemically adsorbed nucleotide derivatives, after addition of an internal standard.
A
A H8 H1
B 9.0
8.0
7.0
6.0
5.0
4.0
3.0
ppm
Fig. 3. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP (the minimum amount of DCl was added), previously treated at neutral pH with water solutions of 5′dGMP (a′) and [Pt(dien)(N7-5′dGMP)] (1a′), respectively. Strong adsorption (chemisorption) was evidenced in the case of 5′dGMP (A), but not in the case of the complex [Pt(dien)(N7-5′dGMP)] (B). A similar trend was also observed in the case of 5′GMP (b′) and [Pt(dien)(N7-5′GMP)] (1b′).
adsorbed nucleobases, due to the partial protonation of the phosphate moiety and/or partial dissolution of the solid nHAP matrices. By 31P NMR, we could also observe in last cases the parallel partial hydrolysis of the phosphate moiety in the released nucleotide derivatives. 3.2. Adsorption of N7-platinated nucleotide derivatives on hydroxyapatite nanocrystals The 1H NMR analysis, demonstrated that in the case of complexes 1a, 1b, 1a′, 1b′, 1a″, and 1b″, the chemisorption on nHAP, in water solution at pH ≈ 7, is very low, falling in the range 0.00–0.04 μmol/mg of hydroxyapatite, Figs. 3 and 4 and Table 1. On the other hand, if the same experiment is conducted with complexes 1a‴ and 1b‴, the adsorption on nHAP is comparable with respect to that observed in the case of simple nucleotides, falling in the range 0.20–0.22 μmol/mg of hydroxyapatite,
A H8
H1
3.1. Adsorption of nucleotide derivatives on hydroxyapatite nanocrystals The 1H NMR analysis demonstrated that, in the adopted experimental conditions, in the case of not phosphorylated nucleotide derivatives dGUO and GUO, the adsorption on nHAP, in water solution at pH ≈ 7, is negligible, falling in the range 0.00–0.01 μmol/mg of hydroxyapatite, Table 1. On the other side, the 1H NMR analysis, demonstrated that in the case of simple phosphorylated nucleotide derivatives dGMP, dGDP, dGTP, GMP, GDP and GTP the adsorption on nHAP, in water solution at pH ≈ 7, is much more pronounced, ranging from 0.15 to 0.40 μmol/mg of hydroxyapatite, see Figs. 3–5 and Table 1. Interestingly, in the case of phosphorylated nucleotide derivatives, the acidification to a pH value in the range 3–5, by washing with CH3COOH water solutions (0.1 M), induced the partial release of the
B
9
8
7
6
5
4
3
ppm
Fig. 4. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP (the minimum amount of DCl was added), previously treated at neutral pH with water solutions of 5′dGDP (a″) and [Pt(dien)(N7-5′dGDP)] (1a″), respectively. Strong adsorption (chemisorption) was evidenced in the case of 5′dGDP (A), but not in the case of the complex [Pt(dien)(N7-5′dGDP)] (B). A similar trend was also observed in the case of 5′GDP (b″) and [Pt(dien)(N7-5′GDP)] (1b″).
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
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M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
A H8 H1
dien H1
H8
B 9
8
7
6
5
4
3
ppm
Fig. 5. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP, (the minimum amount of DCl was added) previously treated at neutral pH with water solutions of 5′dGTP (a‴) and [Pt(dien)(N7-5′dGTP)] (1a‴), respectively. A strong adsorption (chemisorption) was evidenced in the case of both 5′dGTP (A) and the complex [Pt(dien)(N7-5′dGDP)] (B). A similar trend was also observed in the case of 5′GTP (b‴) and [Pt(dien)(N7-5′GTP)] (1b‴).
Fig. 5 and Table 1. Interestingly also in these cases, as observed for simple nucleotides, the acidification to a pH value in the range 3–5, by washing with CH3COOH water solutions (0.1 M), induced the partial release of the adsorbed complexes, probably for the partial dissolution of the solid nHAP matrices. Moreover, as observed for simple nucleotides, 31P NMR experiments showed again a partial hydrolysis of the triphosphate moiety, in the released complexes, as a consequence of acidification in the presence of nHAP. 3.3. Adsorption as a function of the electric charge of the nucleotide derivative If we look in the literature from the typical Ka values of nucleotide derivatives [44,45], we can evaluate the most abundant species present in solution and their residual electric charge, in water solution at pH ≈ 7. On this basis, in the case of nucleotides and for the corresponding platinum complexes, the expected residual electric charge values for the most abundant species, in water solution, at neutral pH, have been indicated here following: GUO (a), dGUO (b), GMP− (a′), dGMP− (b′), GDP2− (a″), dGDP2 − (b″), GTP3 − (a‴), dGTP3 − (b‴), [Pt(N7-dGUO)(dien)]2 + (1a), [Pt(N7-GUO)(dien)]2 + (1b), [Pt(N7-dGMP)(dien)]+ (1a′), [Pt(N7-GMP)(dien)]+ (1b′), [Pt(N7-dGDP)(dien)] (1a″), [Pt(N7GDP)(dien)] (1b″), [Pt(N7-dGTP)(dien)]− (1a‴), [Pt(N7-GTP)(dien)]− (1b‴), see Fig. 1. If we examine the previously described adsorption experiments, we can evidence a direct influence of the residual electric charge for the specific nucleotide derivative, in determining adsorption. In fact, we experimentally found that the cationic and neutral nucleotide derivatives: [Pt(N7-dGUO)(dien)]2+, 1a; [Pt(N7-GUO)(dien)]2+, 1b; [Pt(N7-dGMP)(dien)]+ (1a′), [Pt(N7-GMP)(dien)]+ (1b′), GUO (a), dGUO (b), [Pt(N7-dGDP)(dien)] (1a″), and [Pt(N7-GDP)(dien)] (1b″), are not or only slightly chemically adsorbed on nHAP, in water solution at neutral pH, see Table 1. On the contrary, the derivatives with a negative charge: GMP− (a′), dGMP− (b′), GDP2− (a″), dGDP2− (b ″), GTP3 − (a‴), dGTP3 − (b‴), [Pt(N7-dGTP)(dien)]− (1a‴), [Pt(N7GTP)(dien)]− (1b‴), are strongly adsorbed on nHAP in water solution, at neutral pH, see Table 1. In this latter case, the chemical adsorption can be reverted only by considerably reducing the pH of the solution, with consequent protonation of phosphates. In other words, the considered nucleotide derivatives can be clustered into two groups showing a different adsorption behavior. The first of them (neutral or positively
charged) giving only a reversible physical adsorption and unable to give irreversible chemical adsorption and the second (negatively charged) prone to give irreversible chemical adsorption, at neutral pH. The previous considerations, are consistent with the presence of positive charges (Ca2+ and H+ cations), localized on the surface of the nHAP at neutral pH, as previously suggested [40–42]. Indeed, being these adsorption phenomena a mainly electrostatic interaction, only the surface interactions between the nucleotide derivatives and the nHAP, could fully explain the observed experimental results. Moreover, in principle we could expect general attractive interactions between the phosphate residues of nucleotide derivatives and nHAP but this occurs only in the case of nucleotide derivatives with a net negative charge. The zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. In the reported experiments, an increase of the adsorption efficiency, of the nucleotide derivatives on the nHAP, with the increasing number of the sole phosphates was not observed but, according to previous discussion, a net negative charge of the tested compounds seems to be required for chemical adsorption. The proposed mechanism is consistent with the previously studied effects of negative charge density on oligonucleotides, in determining the amount of adsorption on hydroxyapatite, stating that the adsorption of oligonucleotides takes place because of the electrostatic interaction between the negative phosphate groups and the positive ions on the hydroxyapatite crystals surface [41,36]. 4. Conclusions The use of nHAP bonded to modified nucleotides could have a variety of pharmacological, biological and technological applications. For this reason, it is of fundamental importance the rationalization of the phenomena at the basis of the adsorption of a nucleotide derivative on nHAP, in water solution. The experiments here reported evidence the importance of the residual charge of a nucleotide derivative in the modulation of the adsorption efficiency of molecules, on nHAP dispersed in water. We demonstrated that a cationic or neutral charge on a nucleotide derivative produces an adsorption absent or strongly reduced. On the contrary, the presence of a net negative charge produces a relevant chemical adsorption on nHAP, which can be reverted by acidification. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give a stable chemisorption. For this reason, at neutral pH, the presence of phosphates is not a sufficient prerequisite to guarantee adsorption on nHAP, and the number of phosphates can only slightly modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The proposed mechanism is in agreement with the previously proposed mechanism for the adsorption of oligonucleotides on hydroxyapatite [36,41]. Previous considerations are important to preview the binding ability of drugs, based on simple nucleotide derivatives or molecules bearing phosphate groups, on hydroxyapatite. Acknowledgments The University of Salento, Italy; the MIUR, Ministero dell’Istruzione, dell’Università e della Ricerca, (PON 254/Ric. Potenziamento del “Centro Ricerche Per La Salute Dell’uomo E Dell’ambiente” Cod. PONa3_00334); the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Bari (Italy) and the RBAP114AMK, RINAME Project, “Rete integrata per la nano medicina” (funds for selected research topics), are acknowledged for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2015.04.006.
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Metalated nucleotide chemisorption on hydroxyapatite Michele Benedetti a,⁎, Daniela Antonucci a, Federica De Castro a, Chiara R. Girelli a, Marco Lelli b, Norberto Roveri b, Francesco P. Fanizzi a,⁎ a b
University of Salento, Department of Biological and Environmental Sciences and Technologies, Via Monteroni, 73100, Lecce, Italy University of Bologna, Department of Chemistry “G. Ciamician”, Via Selmi 2, 40126, Bologna Italy
a r t i c l e
i n f o
Article history: Received 25 February 2015 Received in revised form 7 April 2015 Accepted 9 April 2015 Available online 19 April 2015 Keywords: Hydroxyapatite nanocrystals Nucleotide adsorption Nanomaterials Platinum complex Antitumor drug Antiviral drug
a b s t r a c t The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hydroxyapatite [Ca10(PO4)6(OH)2], is the main inorganic constituent of bones and teeth [1]. For this reason, it is extremely biocompatible and the applications in many medical fields are greatly expanding. Relevant examples are the artificial protheses, able to substitute bones and, in some cases, to be completely integrated in the natural tissues. Some attempts to impregnate these protheses, with drugs to be released in situ, are documented in the literature [2]. Recently the use of hydroxyapatite nanoparticles, in the drug delivery, has been suggested for the peculiar properties (biocompatibility and biodegradability) of this kind of aggregates able, in several cases, to avoid some general problems arising from the systemic administration of drugs [3,4]. Due to their recent availability, even in controlled morphologies [5], the nanoparticles of hydroxyapatite have been studied, to develop nanocarriers for drug delivery and for biomedical applications [6]. Nowadays, hydroxyapatite nanocrystals (nHAP) are experimented as carriers for the transport of proteins [7], antibiotics [8], antitumor drugs [9], radioisotopes [10], genes [11] and antigens for vaccines [12]. In all these applications, the main recognized nHAP internalization pathways in tissues and cells seem to be passive diffusion, active transport and endocytosis. In particular, in the case of particles greater than 10 nm, the internalization in eukaryotic cells can be mediated by phagocytosis, macropinocytosis and endocytosis (simple or ⁎ Corresponding authors. E-mail addresses: [email protected] (M. Benedetti), [email protected] (F.P. Fanizzi).
clathrine mediated, via caveoles) [13–15]. Macromolecules and nanocrystals can bind specific receptors on the cell surface, inducing formation of cavities decorated by clathrines, internalized by endocytosis. In the cytoplasm, the vesicles lose the clathrines and fuse with other vesicles (endosomes). In this context, it is to be underlined that the nHAP were previously exploited as delivery systems for genes, drugs, and coadjuvants (which destabilize the endosomal membrane). Generally, nHAP improves the release of drugs and genes directly in the cytoplasm [16]. The importance of these techniques in the modulation of the activity of pharmacologically active molecules and in the reduction of possible side effects has also been recognized. For this reason, a lot of research has been devoted to the development of new nHAP based materials for the controlled release of drugs. This aspect is important in the case of antitumor and/or antiviral drugs because often very serious side effects may develop, causing the discontinuance of the chemotherapeutic treatment. In this context, the possible use of hydroxyapatite nanoparticles, as vectors for drugs, inducing a more efficient, localized and gradual delivery could be extremely useful. Indeed, the reduction of the peak concentration of administered drugs, reached in tissues, during the treatment, could prevent harmful side effects. In drug release applications, the chemical–physical properties of nHAP and their intrinsic high biocompatibility are very attracting [17]. For this reason, it could be relevant to clarify the factors affecting the known chemical adsorption (chemisorption) [6] of some substrates on nHAP and the possible controlled release in vitro and in vivo. For example, studies were conducted on the factors governing adsorption/release of some cisplatin derivatives and 5-fluorouracyl [18–20] on/from nHAP
http://dx.doi.org/10.1016/j.jinorgbio.2015.04.006 0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
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M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
solid matrices. In these cases it has been highlighted that the cytotoxicity of the drugs adsorbed on nHAP is different, with respect to the same drugs, when administered in the free form. In previous works, we studied many platinum complexes [21–25] even with metal bonded purine nucleotides [26–28], to better understand the mechanism of action of platinum based antitumor drugs. In this work, we focused our attention on model molecules with a variable number of phosphates, based on simple or metalated (Pt-bonded) nucleotide derivatives, Fig. 1. The possible adsorption of these molecules on hydroxyapatite nanoparticles is the topic of the present study. Molecules bearing phosphate groups have a variety of pharmacological, biological and technological applications [29–32]. In particular, nucleotide derivatives are very interesting in this context, because widely used, as experimental tools to modify nucleic acids, or as antimetabolites acting as powerful drugs. For instance, nucleotide analogues are molecules that generally can substitute the normal nucleotides, in the DNA/RNA synthesis and, for this reason, can be used as both antitumor and antiviral drugs (5-fluorouracyl, acyclovir, AZT, etc.) [20,30,33,34]. It is known that nucleotide, oligonucleotide, DNA and RNA derivatives can be adsorbed on hydroxyapatite [35–42]. For this reason, in this work, we wanted to enlighten the effect of the presence of a variable number of phosphates, in single simple guanine nucleotide derivatives, on the adsorption on nHAP. Moreover, we focused our attention on the evaluation of the molecular charge effects on the chemical adsorption, by studying model complexes, obtained when the guanine nucleotide derivatives are coordinated, by the purinic N7 donor, to a model bis-positive Pt(II) complex. This study may be relevant when considering the adsorption, transport and delivery of drugs, in specific tissues and/or cell compartments, with a possible modulation of both pharmacological activity and side effects. Previous work demonstrated high biocompatibility specifically for fully characterized nanometric hydroxyapatite [2,6]. Since this work is addressed to the study of nucleotide analogues adsorption on biomimetic hydroxyapatite nanocrystals, for their possible use as nanocarriers, we used the same biocompatible nanometric material.
O
A
N 8
9
N
OH
1 2
6 3
N
NH2
O
4'
n
3'
2'
OH
R
B
1'
2.1. Synthesis of complexes All solvents and reagents, except otherwise stated, were purchased from Aldrich Chemical Company and used as received. [Pt(N7-dGUO) (dien)] (1a), dien = diethylenetriamine, [Pt(N7-dGMP)(dien)] (1a′), [Pt(N7-dGDP)(dien)] (1a″), [Pt(N7-dGTP)(dien)] (1a‴), [Pt(N7-GUO) (dien)] (1b), [Pt(N7-GMP)(dien)] (1b′), [Pt(N7-GDP)(dien)] (1b″), and [Pt(N7-GTP)(dien)] (1b‴) were synthesized as previously described [29,30]. NMR spectra were recorded on a Bruker Avance III HD 600 or on a Bruker Avance DPX 400 using deuterated solvents. Spectra were referenced to the residual HOD signal (1H) or 85% H3PO4 (31P) as internal or external standard, respectively.
2.2. Synthesis of hydroxyapatite nanocrystals Plate shaped hydroxyapatite nanocrystals (Figs. 2, S1 and S2) were synthesized according to a previously published method [43], with some modifications, according to a recent procedure, as reported by some of us [2].
2.3. Adsorption tests on hydroxyapatite nanocrystals In a typical experiment 50 μL of an nHAP suspension (hydroxyapatite concentration = 71 mg/mL) was diluted in about 500 μL of water, at room temperature and the pH value was adjusted to ≈7, if required, by addition of HCl or NaOH. Then the necessary volume of the tested sample solution and water were added to obtain a final volume of 600 μL and a final concentration of the nucleotide derivative in the nHAP suspension of ≈7.0 mM. The obtained solution was left under stirring for about 20 h (T = 295 K), to favor the sample adsorption on the nHAP and equilibrium reaching. The final suspension was then centrifuged (at 3000 rpm, for 10 min) and therefore the mother solution was removed. The solid nHAP, was then resuspended in 1 mL of distilled water, centrifuged again by removing the surnatant water. The last operation was repeated three times, following a standard procedure [6], to guarantee a complete removal of both the initial mother solution and the physically adsorbed (physisorbed) nucleotide derivatives. Only the eventual nucleotide derivative present at this stage, on the solid phase, was considered strongly chemically adsorbed (chemisorbed) on the nHAP. The nHAP samples were then resuspended in D2O (600 μL) and dissolved, by addition of the minimum amount of DCl, to induce the dissolution of the solid phase and the release of the nucleotide derivative. These latter were quantified by NMR spectroscopy, after addition of a known amount of absolute ethanol, as internal standard, Table 1.
NH Pt
H2N
NH2 O
N N
OH HO P O O
5NH 4
5'
HO P O O
7
2. Experimental section
NH N
NH2
O
n OH
R
Fig. 1. In the figure are represented (A) the 2′-deoxy-nucleoside (R = H) and nucleoside (R = OH), n = 0–3, derivatives a–a‴ and b–b‴, respectively, and (B) the corresponding model platinum complexes, 1a–1a‴ and 1b–1b‴, respectively. The atom numbering scheme, for nucleotides, is also reported.
Fig. 2. TEM image of synthetic hydroxyapatite nanocrystals (nHAP) with plate-shaped morphology (scale bar 100 nm).
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Table 1 Chemical adsorption of nucleotide derivatives on nHAP. Nucleotide derivative
Amount of chemical adsorption (μmol/mg)a
dGUO dGMP− dGDP2− dGTP3− [Pt(N7-dGUO)(dien)]2+ [Pt(N7-dGMP)(dien)]+ [Pt(N7-dGDP)(dien)] [Pt(N7-dGTP)(dien)]−
0.01 0.17 0.39 0.17 0.04 0.00 0.00 0.22
The reported amounts of chemical adsorption, were quantified by NMR, after dissolution in water of the treated nHAP. The nucleotide derivatives, were adsorbed on nHAP in controlled conditions (water solution; T = 295 K; pH ≈ 7; nucleotide concentration ≈ 7 mM; nHAP concentration ≈ 6.5 mg/mL). The nHAP was than washed three times by water, to eliminate the physically adsorbed nucleotides. The chemically adsorbed nucleotides were released, after acidic dissolution of the treated nHAP in D2O. The amounts were calculated measuring the nucleotide concentration by 1H NMR, with respect to an internal standard. Similar values were obtained in the case of corresponding guanosine derivatives. a The reported data are average values of two sets of parallel independent measurements (±SD ≈ 0.02 μmol/mg).
3. Results and discussion In this work, we evaluated, at neutral pH, the effect of the number of phosphates increasing, on the adsorption on nHAP, for a series of water soluble purine nucleotide derivatives: dGUO (a), dGMP (a′), dGDP (a″), dGTP (a‴), GUO (b), GMP (b′), GDP (b″), and GTP (b‴), Fig. 1. Moreover, the effect of the coordination of a bis-positive Pt(II) complex, to the N7 position of the nucleotide derivatives has been evaluated, in the following water soluble model complexes: [Pt(N7-dGUO)(dien)] (1a), [Pt(N75′-dGMP)(dien)] (1a′), [Pt(N7-5′-dGDP)(dien)] (1a″), [Pt(N7-5′dGTP)(dien)] (1a‴), [Pt(N7-GUO)(dien)] (1b), [Pt(N7-5′-GMP)(dien)] (1b′), [Pt(N7-5′-dGDP)(dien)] (1b″), and [Pt(N7-5′-dGTP)(dien)] (1b‴), Fig. 1. In order to quantify the chemical adsorption (chemisorption) of the nucleotide derivatives on nHAP, we performed specific experiments. The quantification of the tested nucleotides adsorption was assessed by 1H NMR spectroscopy. The adsorption tests, were generally conducted by direct interaction of the purine derivative, dissolved in water, at a given concentration, with a water suspension of nHAP. The interaction between the purine derivative and the nHAP, was generally allowed for about 20 h. Preliminary tests guaranteed this time being enough for reaching the equilibrium. The suspension was then separated from the mother solution by centrifugation and washed with D2O, to eliminate the physically adsorbed (physisorbed) nucleotide derivatives (a variable physically adsorbed fraction was observed in all tested nucleotide derivatives). The resulting solid suspended in D2O was treated with the minimum amount of DCl, in order to dissolve nHAP and release in solution the purine derivative. The resulting solutions were then analyzed by 1H NMR in order to assess and quantify the eventual presence of the previously chemically adsorbed nucleotide derivatives, after addition of an internal standard.
A
A H8 H1
B 9.0
8.0
7.0
6.0
5.0
4.0
3.0
ppm
Fig. 3. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP (the minimum amount of DCl was added), previously treated at neutral pH with water solutions of 5′dGMP (a′) and [Pt(dien)(N7-5′dGMP)] (1a′), respectively. Strong adsorption (chemisorption) was evidenced in the case of 5′dGMP (A), but not in the case of the complex [Pt(dien)(N7-5′dGMP)] (B). A similar trend was also observed in the case of 5′GMP (b′) and [Pt(dien)(N7-5′GMP)] (1b′).
adsorbed nucleobases, due to the partial protonation of the phosphate moiety and/or partial dissolution of the solid nHAP matrices. By 31P NMR, we could also observe in last cases the parallel partial hydrolysis of the phosphate moiety in the released nucleotide derivatives. 3.2. Adsorption of N7-platinated nucleotide derivatives on hydroxyapatite nanocrystals The 1H NMR analysis, demonstrated that in the case of complexes 1a, 1b, 1a′, 1b′, 1a″, and 1b″, the chemisorption on nHAP, in water solution at pH ≈ 7, is very low, falling in the range 0.00–0.04 μmol/mg of hydroxyapatite, Figs. 3 and 4 and Table 1. On the other hand, if the same experiment is conducted with complexes 1a‴ and 1b‴, the adsorption on nHAP is comparable with respect to that observed in the case of simple nucleotides, falling in the range 0.20–0.22 μmol/mg of hydroxyapatite,
A H8
H1
3.1. Adsorption of nucleotide derivatives on hydroxyapatite nanocrystals The 1H NMR analysis demonstrated that, in the adopted experimental conditions, in the case of not phosphorylated nucleotide derivatives dGUO and GUO, the adsorption on nHAP, in water solution at pH ≈ 7, is negligible, falling in the range 0.00–0.01 μmol/mg of hydroxyapatite, Table 1. On the other side, the 1H NMR analysis, demonstrated that in the case of simple phosphorylated nucleotide derivatives dGMP, dGDP, dGTP, GMP, GDP and GTP the adsorption on nHAP, in water solution at pH ≈ 7, is much more pronounced, ranging from 0.15 to 0.40 μmol/mg of hydroxyapatite, see Figs. 3–5 and Table 1. Interestingly, in the case of phosphorylated nucleotide derivatives, the acidification to a pH value in the range 3–5, by washing with CH3COOH water solutions (0.1 M), induced the partial release of the
B
9
8
7
6
5
4
3
ppm
Fig. 4. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP (the minimum amount of DCl was added), previously treated at neutral pH with water solutions of 5′dGDP (a″) and [Pt(dien)(N7-5′dGDP)] (1a″), respectively. Strong adsorption (chemisorption) was evidenced in the case of 5′dGDP (A), but not in the case of the complex [Pt(dien)(N7-5′dGDP)] (B). A similar trend was also observed in the case of 5′GDP (b″) and [Pt(dien)(N7-5′GDP)] (1b″).
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
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M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
A H8 H1
dien H1
H8
B 9
8
7
6
5
4
3
ppm
Fig. 5. The reported 1H NMR spectra were obtained by dissolving in D2O hydroxyapatite nanoparticles, nHAP, (the minimum amount of DCl was added) previously treated at neutral pH with water solutions of 5′dGTP (a‴) and [Pt(dien)(N7-5′dGTP)] (1a‴), respectively. A strong adsorption (chemisorption) was evidenced in the case of both 5′dGTP (A) and the complex [Pt(dien)(N7-5′dGDP)] (B). A similar trend was also observed in the case of 5′GTP (b‴) and [Pt(dien)(N7-5′GTP)] (1b‴).
Fig. 5 and Table 1. Interestingly also in these cases, as observed for simple nucleotides, the acidification to a pH value in the range 3–5, by washing with CH3COOH water solutions (0.1 M), induced the partial release of the adsorbed complexes, probably for the partial dissolution of the solid nHAP matrices. Moreover, as observed for simple nucleotides, 31P NMR experiments showed again a partial hydrolysis of the triphosphate moiety, in the released complexes, as a consequence of acidification in the presence of nHAP. 3.3. Adsorption as a function of the electric charge of the nucleotide derivative If we look in the literature from the typical Ka values of nucleotide derivatives [44,45], we can evaluate the most abundant species present in solution and their residual electric charge, in water solution at pH ≈ 7. On this basis, in the case of nucleotides and for the corresponding platinum complexes, the expected residual electric charge values for the most abundant species, in water solution, at neutral pH, have been indicated here following: GUO (a), dGUO (b), GMP− (a′), dGMP− (b′), GDP2− (a″), dGDP2 − (b″), GTP3 − (a‴), dGTP3 − (b‴), [Pt(N7-dGUO)(dien)]2 + (1a), [Pt(N7-GUO)(dien)]2 + (1b), [Pt(N7-dGMP)(dien)]+ (1a′), [Pt(N7-GMP)(dien)]+ (1b′), [Pt(N7-dGDP)(dien)] (1a″), [Pt(N7GDP)(dien)] (1b″), [Pt(N7-dGTP)(dien)]− (1a‴), [Pt(N7-GTP)(dien)]− (1b‴), see Fig. 1. If we examine the previously described adsorption experiments, we can evidence a direct influence of the residual electric charge for the specific nucleotide derivative, in determining adsorption. In fact, we experimentally found that the cationic and neutral nucleotide derivatives: [Pt(N7-dGUO)(dien)]2+, 1a; [Pt(N7-GUO)(dien)]2+, 1b; [Pt(N7-dGMP)(dien)]+ (1a′), [Pt(N7-GMP)(dien)]+ (1b′), GUO (a), dGUO (b), [Pt(N7-dGDP)(dien)] (1a″), and [Pt(N7-GDP)(dien)] (1b″), are not or only slightly chemically adsorbed on nHAP, in water solution at neutral pH, see Table 1. On the contrary, the derivatives with a negative charge: GMP− (a′), dGMP− (b′), GDP2− (a″), dGDP2− (b ″), GTP3 − (a‴), dGTP3 − (b‴), [Pt(N7-dGTP)(dien)]− (1a‴), [Pt(N7GTP)(dien)]− (1b‴), are strongly adsorbed on nHAP in water solution, at neutral pH, see Table 1. In this latter case, the chemical adsorption can be reverted only by considerably reducing the pH of the solution, with consequent protonation of phosphates. In other words, the considered nucleotide derivatives can be clustered into two groups showing a different adsorption behavior. The first of them (neutral or positively
charged) giving only a reversible physical adsorption and unable to give irreversible chemical adsorption and the second (negatively charged) prone to give irreversible chemical adsorption, at neutral pH. The previous considerations, are consistent with the presence of positive charges (Ca2+ and H+ cations), localized on the surface of the nHAP at neutral pH, as previously suggested [40–42]. Indeed, being these adsorption phenomena a mainly electrostatic interaction, only the surface interactions between the nucleotide derivatives and the nHAP, could fully explain the observed experimental results. Moreover, in principle we could expect general attractive interactions between the phosphate residues of nucleotide derivatives and nHAP but this occurs only in the case of nucleotide derivatives with a net negative charge. The zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. In the reported experiments, an increase of the adsorption efficiency, of the nucleotide derivatives on the nHAP, with the increasing number of the sole phosphates was not observed but, according to previous discussion, a net negative charge of the tested compounds seems to be required for chemical adsorption. The proposed mechanism is consistent with the previously studied effects of negative charge density on oligonucleotides, in determining the amount of adsorption on hydroxyapatite, stating that the adsorption of oligonucleotides takes place because of the electrostatic interaction between the negative phosphate groups and the positive ions on the hydroxyapatite crystals surface [41,36]. 4. Conclusions The use of nHAP bonded to modified nucleotides could have a variety of pharmacological, biological and technological applications. For this reason, it is of fundamental importance the rationalization of the phenomena at the basis of the adsorption of a nucleotide derivative on nHAP, in water solution. The experiments here reported evidence the importance of the residual charge of a nucleotide derivative in the modulation of the adsorption efficiency of molecules, on nHAP dispersed in water. We demonstrated that a cationic or neutral charge on a nucleotide derivative produces an adsorption absent or strongly reduced. On the contrary, the presence of a net negative charge produces a relevant chemical adsorption on nHAP, which can be reverted by acidification. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give a stable chemisorption. For this reason, at neutral pH, the presence of phosphates is not a sufficient prerequisite to guarantee adsorption on nHAP, and the number of phosphates can only slightly modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The proposed mechanism is in agreement with the previously proposed mechanism for the adsorption of oligonucleotides on hydroxyapatite [36,41]. Previous considerations are important to preview the binding ability of drugs, based on simple nucleotide derivatives or molecules bearing phosphate groups, on hydroxyapatite. Acknowledgments The University of Salento, Italy; the MIUR, Ministero dell’Istruzione, dell’Università e della Ricerca, (PON 254/Ric. Potenziamento del “Centro Ricerche Per La Salute Dell’uomo E Dell’ambiente” Cod. PONa3_00334); the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Bari (Italy) and the RBAP114AMK, RINAME Project, “Rete integrata per la nano medicina” (funds for selected research topics), are acknowledged for financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2015.04.006.
Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
M. Benedetti et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
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Please cite this article as: M. Benedetti, et al., Metalated nucleotide chemisorption on hydroxyapatite, J. Inorg. Biochem. (2015), http://dx.doi.org/ 10.1016/j.jinorgbio.2015.04.006
