Phosphonopropionic acid coating as platform for the efficient grafting of (bio)molecules to hydroxyapatite nanoparticles

Phosphonopropionic acid coating as platform for the efficient grafting of (bio)molecules to hydroxyapatite nanoparticles

https://doi.org/10.33263/BioMed21.030048

María Laura Dittler 1, Hernán B. Rodríguez 1,*, Mariano Cipollone 2, Claudia A. Grillo 1 and Mónica C. Gonzalez 1,*

1   Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), CCT-La Plata-CONICET, Universidad Nacional de La Plata, La Plata, Argentina; mlauradittler@quimica.unlp.edu.ar; cgrillo@inifta.unlp.edu.ar

2   YPF Tecnología S.A. Baradero S/N (1925) Ensenada – Bs As., Argentina; mariano.cipollone@ypftecnologia.com

*  Correspondence: hernanrodriguez@inifta.unlp.edu.ar Scopus ID 12766535500

gonzalez@inifta.unlp.edu.ar Scopus ID 26661104400

Abstract

A new synthesis strategy intended to increase apatite available surface groups for the covalent functionalization with (bio)molecules was developed. To that purpose, hydroxyapatite nanoparticles (Ap) were synthesized and terminated with 3-phosphonopropionic acid (PPA) yielding Ap with surface bound propionic acid through P-C bonds (ApCOO). A thorough characterization of ApCOO by IR spectroscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, thermal gravimetric analysis, and specific surface area determination (BET model), strongly support an efficient surface grafting of the organophosphonates and a hydroxyapatite core of similar morphology and phase composition to that of synthetic Ap. ApCOO was able to adsorb bovine serum albumin  (BSA) up to 0.20 mg m–2 and showed no cytotoxic effects towards Balb/C 3T3 cells. ApCOO abundant carboxyl surface groups facilitated the particles grafting through stable amide bonds with basic fuchsin (Fu) and tyrosine (Ty) yielding well covered surfaces. Fu-bound hydroxyapatite powders showed ten times stronger fluorescence than free Fu in ethanol, thus expanding the potential uses of Ap as fluorescent sensors in the red region of the visible spectrum. On the other hand, Ty-bound hydroxyapatite powders showed negative surface charges and high stability of the coating in aqueous suspensions. In contrast, Ty-physically adsorbed hydroxyapatite powders rendered positively charged surfaces and an unstable coating.

Keywords: hydroxyapatite nanoparticles; propionic acid-terminated; tyrosine-grafted; fuchsin-grafted; fluorescence.

1. Introduction

Because of their high biocompatibility and similar chemical structure and biomimetic features to that of the mineral phase of hard biological tissues, hydroxyapatite nanoparticles (Ap) is used in a number of different tissue engineering applications [1,2] and as a vehicle for drug delivery [3]. Surface functionalization is a widely used strategy to enhance the positive biological response of synthetic apatite [3-6]. To date, the main effort towards the covalent functionalization of synthetic apatite is directed to overcome the available surface groups of crystalline apatite. Among the different reported strategies are the synthesis of apatite in the presence of amino acids [7], polycondensation of substituted alkoxysilanes on the apatite surface [8], grafting of organophosphonates (RP(O)(OH)2 with R = n-alkyl chain) [9,10], and apatite synthesis in the presence of carboxylate-bearing organic molecules [11].

In particular, surface modification of pre-formed apatite materials with low concentrations of organophosphonic acids has been reported to form disordered monolayers of covalently attached organophosphonic groups via P−O−P bonds, while solutions of higher concentrations led to a bulk modification of apatite with a high loading of organics [9]. Moreover, the “one pot” mixture of inorganic phosphates and alkylphosphonates during apatite synthesis is reported to generate mesoporous materials with high specific surface area and significant loss of crystallinity [10]. These studies are in line with early investigations reporting an inhibiting effect of low concentrations of phosphonates towards hydroxyapatite crystallites growth [12].

Among the molecules used to coat hydroxyapatite materials, those bearing COO functional groups deserve special attention as carboxylates have been shown to promote important biological processes in bone tissue engineering [13] and the induction of calcium phosphate mineralization [14,15]. Also, carboxyl functional groups may be easily activated to form peptide bonds [16] with amino acids and proteins to yield apatite materials with enhanced biological activity [17]. Formation of peptide bonds between the COOH bearing apatite and amino acids assures a constant and stable composite formulation upon mild changes in pH, temperature, ionic strength, and other (bio)molecules present in suspension, which is not expected in systems where the amino acid is physically adsorbed [18]. Also, the nature of the remaining surface charge might be different, since in adsorbed composites the α-carboxylate of the amino acid is preferentially bound to the apatite structure rendering more positively charged surfaces [19], while formation of peptide bonds between COOH bearing apatite and amino acids leaves free α-carboxylates groups which support a more negative surface charge.

Based on the previous discussion and with the aim of obtaining Ap with a plethora of surface groups but conserving Ap morphological properties, we developed a new synthetic strategy using 3-phosphonopropionic acid (PPA) to obtain versatile propionate-coated nanohydroxyapatite (ApCOO), capable of being used as building blocks for the preparation of bioinorganic nanocomposites. ApCOO cell viability was tested and its interaction with bovine serum albumin (BSA) proteins was evaluated, comparing results with those of Ap, in order to assess propionate-coating effects on protein adsorption and cytotoxicity. To probe the versatility of ApCOO particles as precursors in the synthesis of more complex surface-functionalized apatite-based materials, further derivatizations with basic fuchsin (Fu), a fluorescent dye commonly used for staining purposes in biomedical research, and with tyrosine (Ty), a fluorescent amino acid, were performed. Fluorescent Ap are of interest [20–22] in fields such as cell labeling, animal imaging and therapy, and drug delivery and release. On the other hand, Ty is a versatile amino acid that plays several important roles in biological systems [23]. The phenolic side chain of Ty and its unique chemical reactivity enable its involvement in many molecular interactions and biosynthetic transformations as interactions with metal ions in the active centers of enzymes and hydrogen bonding of Ty to surround residues and further concerted proton and electron transfer. Moreover, exploitation of the oxidation chemistry of the phenolic side chain of Ty to various forms, such as dityrosine cross‐linking endows the systems with enhanced structural properties, such as resilience and a high fatigue lifetime. Therefore, coating of Ap with Ty may open potential properties to hydroxyapatite-based materials.

2. Materials and Methods

The list of Materials, details of the standard Equipment and Physicochemical assays used for particle characterization, and BSA adsorption and release on Ap and ApCOO(10%) are provided in the Supplementary Material under the titles “Materials”, “Equipment”, and “Loading of BSA”, respectively. Biological standard procedures, including cell cultures used and cell viability assays (AO staining and MTT assay) are also depicted as Supplementary Material.

2.1. Synthesis of Ap

15 mL of a solution containing 0.981 g H3PO4 was added drop-wise into 10 mL Ca(OH)2 suspension (125 g L-1) over a period of 30 minutes under constant stirring and heating at 80°C. The mixture was then allowed to stand stirring at room temperature for one hour. The obtained Ap was separated from the mother liquor by filtration with hydrophilic 0.22 µm pore membranes, washed several times with deionized water, and dried at 80°C for 24 h.

2.2. Synthesis of ApCOO

The synthesis of ApCOO involved a one-pot procedure where the molar Ca:inorganic P = 1.67 ratio was maintained. Briefly, 15 mL of a solution containing 0.981 g H3PO4 was added drop-wise into 10 mL of 125 g L-1 Ca(OH)2 suspension to initiate Ap formation over a period of 30 minutes under constant stirring and heating at 80°C. Subsequently, 0.165 (or 0.083) g of PPA were added to the mixture, the suspension was stirred at room temperature for 3 h, and the particles separated from the mother liquor by filtration with a 0.22 µm hydrophilic membrane and washed with deionized water until no absorbance between 250-300 nm was observed in the washing liquors. To differentiate the particles obtained from each synthesis batch, the mole percent fraction of organic P to total P used in the synthesis procedure is indicated following the particle name, i.e. ApCOO(10%) and ApCOO(5%). Particles stored in aqueous suspensions at 40°C for 48 h showed constant pH and absorbance between 250-300 nm in the liquid phase (see Supplementary Material under the title “ApCOO Stability”), strongly supporting a high stability towards PPA elimination from the apatite surface.

2.3. Ty immobilization on ApCOO(10%)

Ty covalent grafting to ApCOO(10%) nanoparticles was performed through the coupling reaction between the particles carboxylates and the amine groups of the amino acid catalized by N,N´-dicyclohexylcarbodiimide (DCC) [24]. Secondary reactions were avoided protecting Ty carboxyl group by esterification with benzyl alcohol. To that purpose, 1.5 g of L-Ty was mixed with 4 mL HCl and 40 mL benzyl alcohol and the resulting solution maintained at 81°C for ca. 1 hour before product extraction with ethyl ether and 10% HCl aqueous solutions. The pH of the remaining aqueous phase was adjusted to 9.1 by NH4OH addition. The precipitated Ty benzyl ester solid was isolated by filtration with 0.22 µm hydrophilic membranes and washed with ethanol.

To activate the particles carboxylic groups, 4 mg DCC and 4 mg N-hydroxysuccinimide (NHS) were added to 10 mL of an ethanol suspension of ApCOO (10%) (1 g L–1) and the mixture was left stirring at room temperature in the dark for 18 h. Subsequently, 5 mg of Ty benzyl ester was added, and the coupling reaction left to proceed for 72 h under constant stirring. The particles were separated from the reactive mixture by filtration with a 0.22 µm hydrophilic membrane and washed with ethanol until no absorbance at 350 nm was observed in the washing liquors. The benzyl ester group was then hydrolyzed in a phosphate buffer of pH 6.8 and 0.125 M ionic strength, at 60°C for 24 h [25]. The obtained surface-modified particles, denoted as ApCOOTy, were kiln-dried at 60°C during 24 h.

On the other hand, Ap covered with Ty by physical adsorption were obtained for comparison purposes. To that purpose, 5 mg of Ty were mixed with 10 mL of an aqueous suspension of Ap (1 g L-1) and the mixture was left stirring at room temperature in the dark for 48 h. The coated particles were separated from the reaction mixture by filtration with a 0.22 µm hydrophilic membrane and washed with water. Ty is observed to slowly desorb with time when Ty-adsorbed apatite powders were resuspended in pure water, as observed from variations in the Z-potential of the particles and an increased absorbance of the suspension filtrate.

2.3. Fu immobilization on ApCOO (10%)

Freshly prepared ApCOO (10%) particles (vide supra) were surface derivatized with Fu involving amide bonding, following a similar procedure as described for Ty derivatization but adding 2 mg Fu. To eliminate adsorbed Fu traces, the conjugated nanoparticles, ApCOOFu, were resuspended in ethanol, filtered with 0.22 µm filters, and washed. The latter cycle was repeated until washing liquids did not show traces of Fu by UV-vis absorption spectroscopy. ApCOOFu particles remained as a red solid filtrate on 0.22 µm membranes, as shown in the Supplementary Material under the title “ApCOOFu coloured filtrates”. Blank experiments performed with Fu and ApCOO in the absence of DCC and NHS, showed that Fu does not remain physically adsorbed on ApCOO after thorough washing. Altogether these observations support an efficient Fu covalent grafting to ApCOO. Figure 1 illustrates the particular surface chemistry of the obtained hydroxyapatite-based particles, as a guide to help understand the following results.

Figure 1. Illustrative surface representation of Ap, ApCOO, ApCOOFu, and ApCOOTy.

3. Results and Discussion

3.1. Ap and ApCOO characterization

High Resolution Transmission Electron Microscopy (HRTEM) images of Ap, ApCOO (5%), and ApCOO (10%) depicted in Figure 2 revealed the formation of highly crystalline rod-shaped Ap with lattice spacings of ca. 0.34 nm, align along the particle long axis, in line with those reported for hydroxyapatite (002) planes [26]. Rods are of 10-20 nm width and between 50-100 nm long. Because of agglomeration, it was not possible to determine the size distribution of the particles.

Corresponding XRD diffractograms of Ap, ApCOO (5%) and ApCOO (10%) are shown in Figure 3. All spectra showed patterns and peak widths characteristic to hydroxyapatite powders [27]. Impurity phases such as α- and β-tricalcium phosphate (TCP), if present, are at very low levels as no significant contribution of TCP most intense peaks at 2θ = 30.5-31.6 are observed [28]. These observations clearly suggest that mainly hydroxyapatite crystalline domains are formed upon the late addition of 5 and 10% PPA during synthesis.

Figure 4A shows the ATR-IR spectra of Ap, ApCOO (5%), and ApCOO (10%). The spectrum of Ap shows the fundamental vibration modes of PO4 groups of the apatitic nanocrystalline structure at about 960, and 1010-1100 cm-1 [29]. Small bands at 875 and 1460-1420 cm-1 attributed to residual carbonate from the synthesis process are also observed [30]. The ATR-IR spectrum of both ApCOO (5%) and ApCOO (10%) also depict Ap characteristic bands. However, while ApCOO (5%) mainly shows these bands, the spectrum of ApCOO (10%) also depicts the presence of new peaks at 2925-2850, 1440-1410, 1320, and 800 cm-1 due to methylene symmetric and asymmetric stretching, CO stretching in carboxylic acid salts [31], P-CH2R, and C-O vibrations [32], respectively, thus confirming the presence of P-bonded propionate groups on ApCOO (10%) surface.

Considering that ApCOO (10%) shows the presence of abundant surface P-bonded propionate groups while maintaining the crystalline structure of Ap, all further studies will be described for these particles.

Figure 2. HRTEM micrographs of Ap, ApCOO (5%), and ApCOO (10%).
Figure 3. X-ray diffractograms of Ap, ApCOO- (5%), and ApCOO- (10%), from bottom to top, respectively. Bars stand for synthetic hydroxyapatite principal reference pattern [27].

TGA analysis of Ap (Figure 4B) obtained in an O2 environment in the temperature range from 100 to 650°C yield information on adsorbed substrates, as decomposition of the Ap structure is expected to occur at temperatures over 800°C when the dehydration processes start taking place [29]. The about 6.3% total mass loss of Ap in the temperature range from 100 to 520°C may be attributed to loss of adsorbed and lattice water and the 1.7% loss between 450-650°C to the elimination of surface carbonate-like groups, in good agreement with literature reports for nano-sized Ap powders [33].

ApCOO (10%) shows a different loss pattern than Ap (Figure 4B) at temperatures between 120 and 440°C, which may be attributed to the presence of organic matter. The mass loss of ~7% observed in this temperature range may be assigned to the oxidation of the propyl carboxylate groups to CO2. However, the contribution to the mass loss due to lattice water cannot be neglected. Moreover, a further loss of 1.4% is observed in the temperature range between 450-650°C, attributed to the elimination of surface carbonate-like groups, in agreement with that of Ap. Therefore, considering that lattice water mass might be on the same order as that of Ap, ca. 0.055 moles of propionic acid groups per 100 g of ApCOO (10%) is estimated. Considering the measured specific surface area of 67.5 m2 g-1 for ApCOO (10%), an area of ~20 Å2 per propionic acid group is estimated, which is in the order of the cross sectional area of aliphatic carboxylic acids in a water/air film interface [34], thus indicating a well-covered surface with PPA.

Figure 4. (A) ATR-IR spectra of Ap (black lines, curve a), ApCOO (5%) (blue lines, curve b), and ApCOO (10%) (red lines, curve c). (B) TGA performed in oxygen atmosphere of Ap (black line, curve a), ApCOO (10%) (red lines, curve b). Dotted lines stand for Ap (black lines, curve c) and ApCOO (10%) (red lines, curve d) corresponding derivative curves.

The XPS survey spectra of Ap and ApCOO  (10%) depict the main lines for Ca, O, C, and P, as shown in the Supplementary Material under the title “XPS”. Considering the experimental sensitivity factors relative to the different elements, surface stoichiometric ratios of Ca1P0.6O2.8 and Ca1P0.55O2.7 were determined for Ap and ApCOO (10%), respectively. The Ca:P ratio of Ap is in line with the expected theoretical hydroxyapatite stoichiometric ratio Ca1P0.6O2.6 [35], while that of ApCOO (10%) is ca. 10% higher thus suggesting a slightly Ca-enriched surface.

XPS signals for the individual elements are also shown in the Supplementary Material under the title “XPS”. Both, Ap and ApCOO (10%) powders show Ca2p1/2 and Ca2p3/2 lines at 347.0 eV separated by 3.5 eV, and P2p1/2 and P2p3/2 lines at 133 eV separated by 0.9 eV, characteristic of the apatite moieties. O1s XPS signals for Ap and ApCOO (10%) show the major contribution (> 98%) of a band at 531.5 eV assigned to oxygen atoms in phosphates [36]. Because O1s peaks tend to be broad and the binding energy of the surface groups of interest fall within a very narrow range, it is not possible to unambiguously discriminate organic oxygen contribution. However, despite organic contamination may mask the analysis of the C1s peak, O-C=O components appear at 288.5 eV and may be easily identified. The C1s peak of ApCOO (10%) clearly shows a higher contribution of carboxylates (ca. 21% of total C) than Ap (< 7%), thus also supporting the presence of surface propionic acid groups in the ApCOO(10%) samples.

Similar specific surface areas of (66.3 ± 0.1) and (67.5 ± 0.1) m2 g-1 were determined from the fitting to the BET model of Ap and ApCOO (10%) N2-adsorption isotherms, respectively.

3.2. ApCOOFu and ApCOOTy characterization

The ATR-IR spectrum of ApCOOFu and ApCOOTy depicted in Figure 5A curves a and b, respectively, show peaks at 2930-2840, 1595, 1440-1420, 1320, and 800 cm-1 already attributed to the presence of P-bonded propionate groups on the Ap surface. The spectrum of ApCOOFu also shows well defined peaks at 1320 and 760 cm-1 due to aromatic C-N and C-H bending and ring torsion, respectively, observed in Fu spectrum. Peaks at 1600 and 1560 cm-1, characteristic of CO and NH bend in amides, may be assigned to the amide bonds linking propionic acid carboxylic groups with Fu amines. On the other hand, ApCOOTy shows peaks appearing at 1212, 1670-1630, and 1150 cm-1, characteristic of stretching vibrations of C-O in phenols, C=O in amides, C-C in aromatic rings, and C-O in aliphatic esters and carboxylic acids, respectively, in line with the attachment of Ty through amide bonds to the carboxyl groups of ApCOO (10%).

TGA curves of ApCOOFu powders (Figure 5B) show ~11% total mass loss between 150 and 550°C, assigned to the loss of surface bound organic matter (propionic acid and Fu) and lattice water. Around 4% mass Fu is estimated from the difference in mass loss between ApCOO (10%) and ApCOOFu in that temperature range. Thus, ca. 0.013 moles Fu/100 g particles is estimated, almost one fourth that of attached propionic acid groups. ApCOOFu positive surface charge (ς = +2.8 ± 0.1, Zeta potential at pH 6.6 and 0.05 M ionic strength) compared to that of ApCOO (10%) (ς = -21 ± 2) strongly suggest an efficient grafting of one of Fu amine groups to ApCOO (10%) surface carboxyl, leaving the second Fu amine group free and protonated at pH 6.6 (Figure 1).

TGA curves of ApCOOTy, (Figure 5B), show an overall mass loss of 19% between 150 to 640°C, indicating that ca. 12% mass of Ty (0.042 moles, 75% that of surface carboxylates) is attached to 100 g of particles, thus supporting an efficient coverage with Ty.

Figure 5. (A) ATR-IR spectra of ApCOOFu (blue lines, curve a), and ApCOOTy (green lines, curve b). (B) TGA performed in oxygen atmosphere of ApCOOFu (blue lines, curve a), ApCOOTy (green lines, curve b). Dotted lines stand for ApCOOFu (blue lines, curve c) and ApCOOTy (green lines, curve d) corresponding derivative curves.

Zeta potential values (ς = -15.2 ± 0.5) of ApCOOTy in aqueous suspensions of pH 6.6 and 0.05 M ionic strength compared to that of ApCOO (10%) under identical conditions, vide supra, further supports the formation of peptide bonds between ApCOO (10%) surface carboxyl groups and the amino group of Ty. Interestingly, ς values measured for physisorbed Ty on Ap (prepared in a way similar to ApCOOTy, but using Ap and Ty, without DCC/NHS) are +2.76 ± 0.08, in agreement with literature reports suggesting that the α-carboxylate of the amino acid is preferentially bound to the apatite structure leading to positive surface charge [19]. Thus, these results support chemical linkage of Ty to the carboxylate-functionalized Ap surface via amide bonding.

3.3. BSA loading to Ap and ApCOO (10%)

Since surface chemistry has great effects on protein adsorption, BSA loading and releasing on ApCOO (10%) and Ap at 37ºC were investigated and compared. Figure 6A shows the time-dependent adsorption of BSA on both, Ap and ApCOO (10%) at two initial solution concentrations, [BSA]0 = 0.05 and 0.6 g L-1, respectively. In the presence of low [BSA]0, both, Ap and ApCOO (10%) rapidly adsorb BSA reaching similar loads after 5 h, in agreement with literature reports on BSA adsorption on hydroxyapatite crystallites [38]. A tenfold increase in [BSA]0 leads, in both cases, to an early fast adsorption and a subsequent slower process. While the early fast regime is attributed to the surface acting as a sink to the proteins due to strong attractions, the sharp slowdown after a certain amount of protein adsorption is attributed to potential barrier crossing caused by the already adsorbed proteins [39].

Figure 6. (A) Adsorption of BSA (xa mg protein per m2 of particle area) on Ap (circles) and ApCOO (10%) (triangles) as a function of time for initial protein concentrations [BSA]0 = 0.05 (opened symbols) and 0.6 g L-1 (closed symbols) in aqueous solutions of pH 6.6-6.7 at 37 ºC. (B) Plot of the adsorbed BSA (xa) vs. the BSA solution concentration, [BSA]sn, at 37 ºC onto Ap (˜) and ApCOO (10%) (D), respectively. Full and dashed lines stand for the fitting to eq. 1. Grey symbols stand for the values obtained from the desorption experiments shown in the inset. Inset B: %BSA desorption in phosphate buffer of pH 7.3 from a BSA load of 0.25 mg m-2 for Ap (˜) and 0.20 mg m-2 for ApCOO (10%) (D), respectively, as a function of time.

Figure 6B shows Ap and ApCOO (10%) adsorption isotherms, where xa is the amount of adsorbed BSA per unit surface area of substrate and [BSA]sn is the remaining BSA concentration in solution after no more protein is absorbed in the time-dependent adsorption experiments. Protein desorption experiments (Figure 6B inset) performed for the highest BSA loads investigated (0.25 mg m-2 Ap and 0.20 mg m-2 ApCOO (10%)) indicate that ca. 10% desorption takes place after three hours leading to new xa and [BSA]sn values (grey symbols in Figure 5B). These values do not match those of the adsorption isotherm even in the presence of competitively adsorbed inorganic phosphates, in line with an irreversible BSA adsorption [38]. The experimental points of the respective isotherms can be reasonably fitted (r2> 0.95) to a Langmuir-type form of isotherm given by eq. 1, where xm stands for the saturation mass of BSA capable of covering 1 m2 of the particles surface, and K is a constant related to the adsorption affinity.

x_\mathrm{a}=\frac{\mathrm{K}\cdot x_{\mathrm{m}}\cdot\mathrm{[BSA]}{\mathrm{sn}}}{1+\mathrm{K}\cdot\mathrm{[BSA]}{\mathrm{sn}}}

(1)

From the fitting of the data in Figure 6B to eq. 1, xm = (0.27 ± 0.05) mg m-2 and (0.21 ± 0.04) mg m-2 are obtained for Ap and ApCOO (10%), respectively, within the range reported for negatively charged hydroxyapatite surfaces at 37°C, either as obtained or amino acid-coated [7]. The low internal stability of BSA allowing the protein to expose its NH3+ groups on reaching the negatively charged surface has been reported to enhance BSA binding in surfaces of negative Z-potentials [40–44].

3.4. ApCOOFu and ApCOOTy optical properties

Ethanol suspensions of ApCOOFu of ca. 0.1 attenuation and Fu ethanol solutions of 0.1 absorbance, both at 510 nm, show similar fluorescence spectra as shown in Figure 7A. However, the fwhm fluorescence band of covalently bonded Fu in ApCOOFu is somewhat broader and of much higher intensity than free Fu in ethanol solutions, in agreement with the reported behavior of Fu in solid matrix media [45]. An intense red fluorescence is also observed by fluorescence microscopy of ApCOOFu powders deposited on a glass slide, as shown in the fluorescence image in Figure 7A inset.

Figure 7. (A) Fluorescence spectra of an ethanol suspension of ApCOOFu (full line) and Fu solution in ethanol (.dotted line) obtained upon excitation at 510 nm. Inset: Epifluorescence microscopy image of dried ApCOOFu powder on a glass slide. (B) ApCOOTy (full line) and Ty (.dashed line) fluorescence spectra obtained upon excitation at 270 nm. The same attenuation/absorbance of ca. 0.1 at the corresponding excitation wavelengths was used in all cases.

It is well known from the literature [46-48], that the fluorescence quantum yield of triarylmethane dyes as Fu is higher in solid matrix than in liquid medium. The molecular rigidity of the dye in solid medium, together with conformational restrictions associated with the chemical linkage [49], appears to reduce the non-radiative relaxation processes that compete with fluorescence, and may explain the fluorescence increase observed here in ethanolic suspensions of ApCOOFu compared with the free dye.

ApCOOTy acetonitrile suspensions of ca. 0.1 attenuation and Ty acetonitrile solutions of 0.1 absorbance, both at 270 nm, show similar fluorescence spectra and fluorescence intensity, as shown in Figure 7B, supporting an efficient grafting of Ty to ApCOO (10%).

3.5. Cytotoxicity evaluation

Cell viability of Ap and ApCOO (10%) on Balb/C 3T3 cells after 24 h exposure was analyzed by microscopic images with AO staining [50]. Figure 8A shows the microscopic images of Balb/C 3T3 cells stained with AO grown on glass slides coated with Ap, and ApCOO (10%) and on control glass without particle coating. Cell viability given as percentage of cell density with respect to the control shows a (42 ± 9)% (p<0.001) higher proliferation for culture cells grown on ApCOO (10%) than those grown on Ap surface. A lower cell proliferation than controls is also reported in the literature for bare Ap, in line with the behavior observed here for Ap [51-53]. Reported in vitro assays with MC3T3-E1 osteoblast cell cultures and apatite surfaces [52,53] suggest that cells may be subject to changing levels of Ca2+ and PO43− ions due to a rapid “pull-down” of extracellular ions onto the “bare” apatite surface. Thus, apatite coating with serum proteins is critical in making less detrimental the mediation of cell–apatite interaction, possibly through the modulation of phosphate-mediated cell death. Also, adsorption of bisphonates like tiludronate on bare apatite surfaces [53] is reported to stimulate preosteoblastic HOPs and HBMSCs cell proliferation. Our results showing higher proliferation for Balb/C 3T3 cells grown on ApCOO (10%) than on “bare” Ap are in line with these observations, thus suggesting an increment in cell viability in ApCOO (10%) microenvironment.

Cell viability of Ap and ApCOO (10%) on Balb/C 3T3 cells after 24 h exposure was also analyzed by the MTT assay, measuring the reduction of MTT to formazan by dehydrogenase enzymes of intact mitochondria in living cells [54]. Figure 8B revealed ca. 15% increase (p<0.001) of mitochondrial activity for Ap with respect to the control (cells without treatment), while no significant differences were observed for ApCOO (10%). The fact that bare-apatite nanoparticles show less cell proliferation on Ap-deposited glasses than controls, while observing an enhanced proliferation than controls by MTT assays, may be assigned to a different surface distribution of the particles due to the composition of the supporting material (glass or plastic). Also, it is reported in the literature that assays directly counting viable cells are more sensitive than MTT as a test for the antiproliferative effect of hydroxyapatite [55]. Despite these differences, both viability assays seem to indicate that the use of PPA for the surface modification of Ap does not bring into negative effects towards Balb/C 3T3 cells.

Figure 8. (A) Microscopic images of Balb/C 3T3 cells stained with AO grown on glass slides covered with Ap, ApCOO (10%), and control, respectively, after 24 h incubation. (B) MTT assays on Balb/C 3T3 cells after 24 h exposure to 0.53 mg Ap and ApCOO (10%). *** indicate significant differences at p < 0.001 with respect to the controls.

4. Conclusions

Herein we describe the one pot synthesis of modified Ap by surface grafting of organophosphonic acids involving P-O-P bonds and yielding propionic acid-terminated Ap (ApCOO). This new synthesis strategy was developed to increase apatite available surface groups for the covalent functionalization with (bio)molecules of interest. The estimated coverage of a propionic acid group per 20 Å2 and the observed surface charge inversion of the coated apatite strongly suggest an efficient organophosphonic surface grafting. The obtained particles do not show additional cytotoxic effects on surface functionalization towards Balb/C 3T3 cells.

ApCOO abundant carboxyl surface groups facilitated the efficient particles grafting with Fu and Ty involving stable amide bonds. These new material hybrids showed particular properties with potential technological uses: ApCOOFu is a highly fluorescent apatite (510 nm maximum excitation, 570-650 nm emission) suitable for optical sensing purposes; ApCOOTy is a highly stable, negatively-charged aminoacid-covered apatite whose potential properties as filling material in bone tissue engineering needs still being explored. Moreover, a similar grafting strategy may be used to incorporate other bioactive molecules to the Ap surface, such as osteogenic-related peptides, to produce peptide-modified composites with enhanced functions as bone repair materials [56]. Irrespective of these particular examples, it was demonstrated that ApCOO might constitutes a versatile platform suitable for bone tissue engineering.

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