# Biodegradable and Biocompatible Systems Based on Hydroxapatite Nanoparticles
## 4 Functional HAp Biocomposites through Modification/Encapsulation Techniques
### Hydroxyapatite Nanoparticles As Carriers for Drug Delivery Systems  
HAp has extensive applications as a carrier for delivery systems of a large variety of therapeutic agents (e.g., drugs, genes, antigens, enzymes, proteins) [148; 149]. HAp allows a control of texturalparameters, pore size, surface area and surface functionalization, which have a primordial role in the design of drug delivery systems, as recently reviewed by [PERSON] et al. [150]. Different examples of recently published works can be explicitly indicated. HAp coated liposomes have been demonstrated as an effective local drug delivery system for hydrophobic compounds like indomethacin, a nonsteroidal anti-inflammatory drug [151]. The coating of HAp decreased the release rate and specifically 20 h were necessary to deliver the 70% of the drug while only 5 h were required for the uncoated liposome [151]. Alendronate (Alen) is a bisphosphonate with a recognized therapeutic effect against osteoporosis due to its ability to suppress bone resorption by inhibiting osteoclast activity [152]. Combination of the osteocoductive properties of HAp and the therapeutic effect of Alen was achieved by conjugating it into the outmost layer of a layer-by-layer (LbL) HAp coating based on poly(allylamine) and alginate (ALG) (Figure 13). nHAp particles have also been considered for achieving pH-responsive drug delivery taking advantage of the dissolution of HAp in acidic conditions [154]. For example, rhodamine 6G, a positively charged dye, was encapsulated in HAp nanoparticles coated with ALG (a biopolymer that provides excellent biocompatibility and surface functionality through their COOH groups) with a loading efficiency close to 63.0%. The release at pH 2.0 was found to be 2.5 times higher than the amount delivered after a similar period of 8 h [154]. Recent works have indicated that high concentration of HAp nanoparticles may show an antiproliferative and proapoptotic activity on cancer cells [155; 156]. Specifically, adverse effects on the metabolic viability of several types of cancer cells (e.g., hepatoma, colon cancer, osteosarcoma, and melanoma) have been detected [157; 158]. Apoptotic rate of human hepatoma BEL-7402 cells was 18.68%, 22.27%, 33.49%, 49.03%, and 57.16% at 50, 75, 100, 150 and 200 mg/L concentration of HAp nanoparticles, respectively. It was postulated that the main mechanism of growth inhibition by HAp nanoparticles in cancer cells was a consequence of the high calcium concentration and nuclear localization of the particles. Calcium ions can stimulate intracellular apoptotic signals (i.e., activation of protein digesting enzymes named caspases), which allow replacing old, damaged or dysfunctional cells. The anticancer activity of HAp has been specifically related to caspase activation and cytochrome C release from the mitochondria [159]. In fact, majority of anticancer drugs are designed to induce apoptosis through a mitochondrion-mediated pathway [160]. This favors the release of apoptogenic factors such as cytochrome C as well as the activation of caspases. The size of nanoparticles usually has a significant effect on the apoptotic efficacy, although this effect is not well established in the case of HAp nanoparticles. [PERSON] et al. [159] evaluated the influence of the size of HAp nanoparticles in human hepatoma HepG2 model cells, being detected that apoptosis induction efficacy decreased in the order of 45 nm \(>\) 26 nm \(>\) 78 nm \(>\) 175 nm. Furthermore, particles with sizes between 20 and 80 nm were able to  
Figure 13: Scheme owing the preparation of nHAp particles coated with a multilayer able to conjugate Alen. Adapted from [153], The Polymer Society of Korea and Springer Sciene + Business Media Dordrecht, 2016.  
activate caspases and the release of cytochrome C from mitochondria. The best efficiency was observed for 45 nm-sized particles. Cellular response was therefore correlated with the cellular internalization of particles.