3.1. Physico-chemical characterization
The X-ray powder diffraction patterns of pure Furosemide drug and HAPs, as well as those of HAPs@Fur hybrids are shown in Figure 1. Furosemide is a crystalline material (Figure 1A), whose pattern well agrees with that reported in the literature and attributed to Form I [7, 31, 32]. The HAP-Pure sample reflections (Figure 1B) are in very good agreement with the peak positions expected for Ca10(PO4)6(OH)2 compound (Card N. 74-0565), with a hexagonal lattice and the P63/m space group. The same is true for HAP-Sr sample: its pattern is well comparable with that of HAP-Pure, apart a shift to lower angles of all the peak positions. This is due to the difference of the ionic radii of Sr and Ca ions (1.18 and 1.31 Å with respect to 1 and 1.18 Å for the six and nine coordination environments respectively for the two cationic sites in HAP), justifying the lattice expansion [33]. In addition, the pattern of HAP-Sr, with broader and less defined peaks with respect to HAP-Pure, is suggestive of a low crystallinity, notwithstanding the same synthesis conditions. An effect of the Sr ions on the kinetic of the synthesis can be supposed. The XRPD patterns of the hybrids are practically over imposable to the corresponding patterns of HAPs: all the peaks pertain to the HAP phase alone, no peaks due to Furosemide can be evidenced. This can be due to the presence of Furosemide in amorphous form. However, from these evidences, it is clear that the only XRPD data are not able to confirm the hybrids formation, but other kind of analysis, particularly spectroscopic or compositional ones, are required to prove the Furosemide presence on HAP surfaces. However, an indirect proof of the presence of Furosemide on the HAP particles could be represented by the reduction of the intensities of the reflections of the entire hybrids’ patterns with respect to the corresponding HAPs: this evidence could be related to the coverage of HAP particles with a sort of a coating layer of drug.
The spectroscopic analysis could provide useful insights into the Furosemide adsorption onto the HAP nanoparticles. The FT-IR spectra of Fur, HAP-Pure, HAP-Sr and the hybrids HAP-Pure-Fur and HAP-Sr-Fur are reported in the ranges 4000-2000 cm−1 and 2000-650 cm−1 in Figures 2A and 2B, respectively.
The spectrum of the pure Furosemide drug well agrees with the literature data of the polymorphic Form I, in agreement also with XRPD findings [32]. Fur exhibits the main vibrational frequencies at 3396 and 3278 cm−1 (sulphonamide primary amine stretching), 3348 cm−1 (secondary amine stretching), 1667 cm−1 (carboxyl stretching), 1590 and 1559 cm−1 (N-H bending vibrations), and 1315 and 1139 cm−1 (SO2 asymmetric and symmetric stretching).
The HAP-Pure FT-IR spectrum too well agrees with those reported in the literature for Hydroxyapatite [34]. In this case, few bands are observed in the analysed FT-IR range: the sharp band at 3568 cm−1 is assigned to the OH stretching mode, the bands at 1088 and 1022 cm−1 are due to P-O antisymmetric stretching modes and that at 962 cm−1 to P-O symmetric stretching mode. The HAP-Sr spectrum is like that of HAP-Pure: the bands due to P-O bonds slightly shift to lower wavenumbers, due to the higher reduced mass of the harmonic oscillator related to the Sr presence. In addition, the band at 3568 cm−1 is very low, substituted by a broader band however attributable to OH vibration. This can be due to the increase of lattice parameters because of Sr substitution and the formation of an open structure about the OH groups, reducing the crystal force field and increasing the mixing of the OH vibrational and translational motions.
In the FT-IR spectra of the hybrids (Figure 2A and B), together with the bands previously commented of HAP-Pure and HAP-Sr samples, some small peaks attributable to the drug are visible particularly in the range 1600-1200 cm−1. The band at about 1610 cm−1, well evident in both the samples, is compatible with the hydrogen bonds formation between HAP and Fur. Other low bands typical of the drug are also shifted with respect to those of the Fur alone, suggesting a complex hydrogen bonding scheme in the hybrid compounds.
Thermal data, in particular the melting or decomposition temperatures of drugs, can be employed to prove the formation of new compounds, because of drug adsorption [3–5]. The DSC curves of Fur, HAP-Pure-Fur, and HAP-Sr-Fur are reported in Figure 3. The curve of Fur agrees with the literature, the following thermal effects are present: a small endothermic peak at about 135°C, due to the solid-solid phase transition between Form I, the low temperature stable form, and a high temperature stable form [31], in enantiotropic relationship; a complex endothermic-exothermic thermal signal which indicates a melting-decomposition process of the drug at about 217°C.
HAP, as other anologous inorganic compounds, has a high structural stability in this temperature range and, as expected, no thermal events are present [35].
The DSC traces of both hybrids are similar (Figure 3): a flat curve, without any thermal event, apart a broad band at low temperature due to water desorption, is observed, proving that the Furosemide turned out to be amorphous and stable to decomposition, because of its adsorption onto the HAP surfaces.
The morphological characterization was performed by SEM microscopy. The micrographs of Fur, HAP-Pure and HAP-Sr and those of the hybrids are shown in Figures 4A-E. Fur is made of flattened sticks with length ranging from one micron to a few tens of micron (Fig. 4A). HAP-Pure occurs in the form of dense aggregates of small, rounded particles (Fig. 4B) and the same is true for HAP-Sr (Fig. 4C), whose particles seem smaller and less compact with respect to the pure HAP. The adsorption measurements, in the BET portion, allowed determining a surface area and pore volume of 357 m2/g and 0.24 cm3/g for HAP-Sr with respect to 143 m2/g and 0.08 cm3/g for HAP-Pure. The areas are very high, if compared to analogous samples prepared by similar synthesis routes [23, 24], suggesting the formation of highly microporous hydroxyapatites. An effect of Sr substitution is obviously evident on both surface area and pore volume, due to the same kind of synthesis. These features could be positive for the subsequent drug loading, which could be, in principle, expected to be higher for HAP-Sr sample.
The hybrids’ morphologies resemble those of the corresponding HAP samples (Fig. 4D, E): aggregates of small, rounded particles are again well evident, confirming that the adsorption of furosemide does not change the external morphology. No particles attributable to pure drug are visible.
The EDS microanalysis, combined with SEM, is a widespread tool to check the homogeneity of cations distribution, and therefore, in our case, to support the hybrids formation by analysing the presence of the characteristic elements of HAP (Ca and P ions, and Sr substituent) and Furosemide (Cl and/or S ions). The elemental maps obtained for the hybrid compounds show a good homogeneous distribution of all the elements and show that Cl and S (not shown) ions are located in the same points of the powders, where also Ca and P are mainly concentrated (Figure 5). This observation obviously supports the hybrids’ formation. For all the samples, the EDS spectra allowed to determine the chemical compositions, which were found in very good agreement with the expected stoichiometries: Ca/P ratio is near to the ideal value 1.67, and the Sr substituent is present in the expected amount (Table 1). The peaks of S and Cl, both elements present in the Furosemide molecule, confirm the existence of the drug in the analysed samples. Their amount, in both the hybrids, is the same, due to the presence of 1 Cl and 1 S atoms in the furosemide molecule. From the atomic ratios (Table 1) we can calculate a drug loading of about 9.1 wt% and 8.3 wt% for HAP-Sr-Fur and HAP-Pure-Fur respectively. These data will be compared with the amount of drug loading determined by UV-Vis spectroscopy (see par. 3.2).
Table 1
– Atomic percentages of the elements obtained by EDS analysis
|
Ca
|
P
|
Sr
|
S
|
Cl
|
HAP-Pure
|
17.89
|
11.55
|
-
|
-
|
-
|
HAP-Sr
|
8.99
|
8.04
|
6.01
|
-
|
-
|
HAP-Pure-Fur
|
10.17
|
6.70
|
-
|
0.27
|
0.28
|
HAP-Sr-Fur
|
4.82
|
5.05
|
3.41
|
0.26
|
0.29
|
3.2. Pharmaceutical characterization
The drug loading was determined to be 8.2 ± 1.0% for HAP-Sr-Fur and 7.2 ± 0.8% for HAP-Pure-Fur and these values are in good agreement with those obtained from the EDS microanalyses (Table 1).
From the solubility experiment, it was possible to determine that, for both hybrids, the Fur solubility is greater than four times the dose (corresponding to 100 mg L−1), and much higher than the solubility of Fur alone (6 mg L−1) [6].
The contact angles measured for HAP-Pure-Fur andHAP-Sr-Fur in the different fluids show an increased wetting ability compared to Fur alone. This effect is immediately evident at 1 sec after deposition (Figure 6), but increases more significantly over time (Figure 7) in all the media considered. HAP-Sr-Fur starts from values of 45-55° θ, which are lower compared to HAP-Pure-Fur, 55-65° θ, and both are at least half the values obtained from Fur alone. Moreover, the contact angles of the two hybrids decrease quickly with time to 10-20° θ in few seconds, showing a fast wetting ability, also explaining their more rapid dissolution behaviour (see Figure 8). On the other side, the contact angle of the drug remains constant throughout all the experiment (300 sec) (Figure 7).
The dissolution tests (Figure 8) confirm the improved performances of the two hybrid compounds in comparison with the drug alone in all the fluids considered. Fur is quite insoluble at pH=1 (simulating fasted gastric environment), but even in this condition HAP-Pure-Fur and HAP-Sr-Fur showed a very fast dissolution rate of the drug reaching 90% of the dose dissolved in about 120 min. Even more evident is the improvement of the drug dissolution rate from the two hybrids in the fluid prescribed by the US Pharmacopoeia, pH=5.8 buffer, in which the dose is delivered completely in less than 20 min. Only in the unbuffered condition (deionised water) a slight difference in the dissolution profile between HAP-Pure-Fur and HAP-Sr-Fur can be noticed, probably due to the faster wettability of this last compound. In any cases, both hybrids are able to greatly improve the Fur dissolution rate even in the neutral condition compared to the drug alone.
Our results represent a great improvement on the state of the art of furosemide drug delivery systems. In fact, our dissolution profiles in acidic medium (pH=1) are similar or even better than those reported for inorganic-organic hybrids based on silica particles [10–12], which however do not report tests in the conditions suggested by Pharmacopoeia (pH=5.8).
Furosemide, as already discussed, is characterized by a considerable variability in the intensity of the effect, due to its low solubility linked to the medium pH and to a very variable oral bioavailability. For this reason, the enhanced dissolution rate of Fur in the different fluids is a great advantage especially in the therapy of critical diseases when the effect should be reached as soon as possible.