BSA incorporation with CDHA

Figure 4-4a shows the HR-TEM nanostructure of sample B-9.5 (in-situ process) and corresponding color-enhanced image is illustrated in Figure 4-4b. As it can be seen, the nanostructure of the BSA-CDHA hybrid may be divided into 4 regions as indicated with different colors. In the region A, the lattice fringes with parallel to (100) CDHA were clearly observed in the long needle-like crystallites, indicating a well-crystallized region. The region B, i.e., the layer nearest to the highly crystalline inner layer, is formed with a lattice

development strongly affected by the nearest region of highly-order structure. The region B is a result of "growth inhibition". In other words, much poor crystallization was observed [106].

As the crystal continuously grows, the effect of BSA molecule, becomes stronger in distributing the deposition of Ca-P materials, and in a certain case, conjugate as a complex and integrate into "outer area of the lattice" (the so-called BSA-CDHA complex), resulting in an amorphous region C. However, from its nanostructure, the crystallization is prohibited, and this may be caused by, possibly, a regulation of lattice mismatch resulting in somewhat

"gradient crystallinity". The outer region D can be considered as a transition region from BSA-CDHA complex to absorption BSA. An adsorbed BSA layer is essentially tangled with the underlying CDHA-BSA complex (region C).

It was also found that both nano-sized CDHA crystal and BSA-loaded CDHA samples prepared via the ex-situ process were more susceptible to electronic beam bombardment than that prepared via in-situ process, thus causing radiolysis under HR-TEM analysis [107]. This probably reveals that different extent of interaction exists between CDHA and BSA for in-situ and ex-situ processes, which could be further explored via derivative thermogravimetry (DTG) analysis as shown in Figure 4-5. For in-situ processes of A-9.5 and B-9.5, a broad band at 400^oC were detected, which is probably attributed to the thermal decomposition of the BSA-CDHA complex. Due to the incorporation of BSA molecules into regions C and D through electrostatic force (C sites) and/or Ca-bridging (P-sites), a higher thermal decomposition temperature was observed for BSA-CDHA complex compared to that of pristine BSA (350^oC). For the sample prepared in a lower pH value (A-7.5 and B-7.5), this band at 400^oC was not only detected but also the other strong band at 350^oC was detected.

The latter band can be primarily attributed to the thermal decomposition of adsorbed BSA molecules. The adsorption of BSA onto CDHA nano-crystals can be considered as a type of pseudo-Langmuir adsorption [104]. In other words, a number of the BSA layers are adsorbed on CDHA surface which are not tightly bound with CDHA crystals. Therefore, thermal

decomposition temperature of adsorbed BSA molecule is close to that of pristine BSA.

However, the band at 350 ^oC was not detected for both samples of A-9.5 and B-9.5, which is probably due to the fact that the outer-layered BSA molecules are easily eluted in the solution with a higher pH value upon synthesis [91]. In contrast, for the samples prepared via ex-situ processes, only a band at 350^oC was observed. This reflects that no strong interaction exists in the interface of the BSA molecules and CDHA crystals for the samples prepared via ex-situ process.

The TEM images in Figures 4-6(a) and (c) show that the BSA-loaded CDHA nano-crystals synthesized in different conditions present needle-like morphology with dimensions of 4-12 nm in diameter and 40-70 nm in length. The particle size was measured from the TEM images and each datum was averaged from at least 10 TEM photographs.

Furthermore, it was observed that crystal morphology was changed with an increase of solution pH for the in-situ processes (A and B). The dependence of the solution pH value on the aspect ratio of CDHA nano-crystals is further illustrated in Figure 4-7(a). It is interesting to note that the resulting BSA-loaded CDHA nano-crystals derived from the higher pH solution (A-9.5 and B-9.5) showed a smaller aspect ratio compared to that synthesized at a lower pH solution (A-7.5 and B-7.5). Such a preferential growth in the solution at a lower pH can be correlated with preferential adsorption. It means that more BSA should be adsorbed onto the C-sites of the CDHA nano-crystals at a lower pH value, which causes considerable preferential inhibition effect on the growth of nano-crystals along the ab axis, resulting in a high aspect ratio [108]. In contrast, at a higher solution pH, O^--Ca²⁺-COO^- (Ca-bridging) is prominent due to the stability of (PO4)3-. Therefore, crystal growth of in both C-sites and P-sites are inhibited, resulting in a lower aspect ratio. Moreover, it is known that the conformation of protein could be more linear (un-folding) in the solution with strong base and ionic strength [109]. The resulting unfolding BSA molecules and/or amino acid residues strongly attracted Ca²⁺ through the formation of Ca-bridging [110], which kinetically lead to

the inhibition growth on the P-sites of CDHA crystals at the earlier crystallization stage.

Therefore, the aspect ratio for nano-carriers prepared via the in-situ process at higher pH is reduced.

The corresponding amount of the BSA uptake by the CDHA particles (after calibration by subtracting water content) determined from the TGA is shown in Figure 4-7(b). Obviously, a lower pH resulted in a higher BSA incorporation, and as high as 17.5 wt% of the BSA associated with the CDHA was detected, which is about 75% higher than that reported in the literature [99]. In contrast, since the isoelectric point (IEP) of CDHA and BSA is around 7 and 4.7, respectively [111, 112]. It is then expected that the electrostatic repulsive force between the BSA and CDHA nano-crystal becomes dominant for a solution with a higher pH (9.5) compared to that with a lower pH (7.5). Therefore, an increase in solution pH resulted in a decreased amount of BSA.

Considerable differences in the amount of the BSA uptake associated with the CDHA at pH 9.5 can be seen from Figure 7(b), where the CDHA carries a higher amount of the BSA from the ex-situ process (C-9.5) than that from the in-situ processes (A-9.5 and B-9.5). This may be explained by the aspect ratio of the CDHA carries, as illustrated in Figure 7(a) which showed the aspect ratio of the nano-carriers prepared via the ex-situ process (C-9.5) was higher than that prepared via the in-situ process (A-9.5, B-9.5). The CDHA nano-particles with a higher aspect ratio indicates a higher population of the positively-charged C-sites for BSA adsorption and therefore, the total effective areas of adsorption sites (C-site) are much larger for the high aspect-ratio nano-particles compared to that of low aspect-ratio nano-particles per unit weight. If the above argument is correct, then, it is conceivable to realize that the ex-situ CDHA carries higher amount of the BSA than those in-situ CDHA at pH 9.5. However, at pH 7.5, the preferential growth along c-axis for carriers prepared via in-situ process (A-7.5, B-7.5) resulted in a larger aspect ratio compared to that prepared via ex-situ process (C-7.5). Consequently, in contrast to that at pH 9.5, a higher amount of BSA

(about 10 wt%) was detected in the in-situ CDHA than the ex-situ CDHA at pH 7.5.