As it is essential to tailor the characteristics of HAP nanoparticles to control the affinity of the cargo with the delivery system, several authors have studied the relationship between different physicochemical properties and protein absorption on the surface
[11][12][13][14]. CaPs are able to adsorb more protein than other materials, as calcium and phosphate ions are present as preferential binding sites for proteins. Several authors studied the protein adsorption potential of HAP powders treated with heat, where it was found that there are two main correlations between the superficial surface area and the protein adsorption capacity of HAP: the higher the superficial surface area, the higher the protein adsorption; however, when HAP is sintered, intergranular microporosity is formed and less proteins can be adsorbed
[9][15]. In a study by Rouahi et al., an HAP powder with agglomerated granules and a low value of surface area due to the partial fusion of the particles was synthesized. For the FTIR characterization, although the HAP composition was confirmed, the formation of TCP was not observed even though the sample was treated with high heat, which is contrary to other studies displayed in
Table 1, where Sofronia et al. obtained TCP after sintering at 1400 °C
[5]. However, the carbonate peak located around 1500 cm
−1 disappeared after the heat treatment. Nevertheless, the authors explained that the heat treatment did affect the protein adsorption potential and that the slight difference between samples was due to the surface area values, where the original samples that were not sintered had higher surface area values than those that underwent heat treatment. Finally, the ceramics prepared from the sintered samples had higher microporosity and intergranular microporosity, which explained the higher values of cells attaching on the surface. According to the previous study, increasing the surface area results in higher protein adsorption. Furthermore, as the microporosity decreases, the lower the protein adsorption and cell attachment rates become
[5].
Another study that discussed the relationship between the calcination temperature and the adsorption and controlled release of bovine serum albumin (BSA) was carried out by Dasgupta et al.
[19]. The authors reported a process of synthesis of nano-HAP using a reverse micelle template system and protein loading through electrostatic interactions between the nanoparticle surface and the charged amino acids of BSA. The release profiles were evaluated at three different pH solutions. During the physicochemical characterization, when the particles were calcined at 600 °C, pure HAP was formed instead of TCP, which forms at higher temperatures and is explained by the absence of the OH
− bending frequency in FTIR spectra. This phase transformation happening at higher temperatures was noticed because of the disappearance of the HPO
42− at 870 cm
−1 and the OH
− band at 3569 cm
−1. The authors also mentioned that this change was reflected in the XRD pattern
[19]. In the case of particle size analysis, it was found that at higher calcination temperatures, the particle size increased as well. The HAP nanocrystals calcined at 600 °C showed the highest surface area (73 m
2g
−1) and aspect ratio. For the loading experiments, those studied at a pH higher than 7.5 maintained high stability because at a lower pH, the dissolution of nano-HAP could destroy the stable interface between the BSA and the nanoparticles. This interface was formed by the positive calcium ions and the negative polar heads of the BSA molecule. Since the pH of the BSA and CaPs suspension was above the isoelectric point of each BSA, TCP, and HAP, both the BSA and the nano-HAP carried negative charges on their surface. The stern layer of anions [A-, (H
2PO
4−, OH
−] attached to Ca
2+ ions was the source of negative charges on the CaP nanoparticle surface. This interaction gradually decreased as the pH increased from 7.5 to 8.5 and 9, due to an enhanced electrostatic repulsion force between the particle surface and the BSA. The surface area was the main factor of interaction, as the higher the surface area, higher the surface charge density of the nanoparticles, resulting in a higher degree of electrostatic interactions. Since the surface of HAP materials cannot be easily modified through surface treatments to form hydroxyl-, amino-, or carboxyl- groups as is possible with metals and polymers, peptides can be adsorbed on HAP by modifying the surface
[15]. This was also the case in a study by Kojima et al., where several peptide-HAP complexes were made to adsorb cytochrome c, myoglobin, and BSA
[17]. Firstly, the complexes were made by adding peptides during the HAP synthesis process along with the calcium ions source. The peptides contained amino groups on their structures (glutamic acid and lysine) and were easily detected through FTIR spectroscopy, where two major bands of amide I and amide II stretching groups appeared at 1650 and 1560 cm
−1, signals that according to
Table 1 are not found among other HAP examples. Then, selective adsorption was proven with a lysine–HAP complex and the acidic protein BSA. The authors report that the selective adsorption was due to electrostatic interactions between the peptides on HAP surface and the proteins, as adding the peptides changed the surface potential
[17]. As carbonate ions can be found in HAP samples sintered at low temperatures, the authors did not discuss whether the small signal they found at 1650 cm
−1 was due to a possible carbonate substitution on the HAP structure. Even though it is not discussed in the paper, as the signal is smaller than those HAP samples with high carbonate content, it can be assumed that the complex was made between the peptide and HAP. As seen in
Table 1, increasing the sintering temperature can reduce the carbonate concentration, thus affecting peptide and protein adsorption as there are less ions for the biomolecules to interact with
[20]. In the case of the study by Kojima et al., protein and peptide adsorption was still possible because of the electrostatic interactions between the side chains of the peptide and the proteins
[17]. For these reasons, it is important to take into consideration which groups are meant to interact via electrostatic forces and set an appropriate pH to set the expected charges of each groups.