# Biodegradable and Biocompatible Systems Based on Hydroxapatite Nanoparticles
## 4 Functional HAp Biocomposites through Modification/Encapsulation Techniques
### Hydroxyapatite as Non-Viral Gene Delivery System  
Non-viral vectors have some advantages with respect to viral systems despite showing usually lower transfection efficiency. Low cost, adaptability for large scale production, high reproducibility and potential non-immunogenicity are positive characteristics that make non-viral vectors a promising alternative [106]. CaP based systems are highly interesting due to the capacity of calcium ions to form ionic interactions with the phosphates of DNA, leading to complexes able to be transported across cell membranes through a ion-channel mediated endocytosis [91]. In addition, CaPs have excellent biocompatibility and bioresorbability. Main disadvantages are associated to lower levels of gene expression [107] due to endosomal escape and partial protection of DNA from nuclease degradation [108]. CaP co-precipitation is a routine procedure applied for in vitro DNA transfection [109]. Nevertheless, its application in vivo is hampered due to the difficulty to guarantee the delivery of DNA to any cell type [110] and also the lack of control of the final particle size that lead to a poor experimental reproducibility [111]. Co-precipitation of CaP and DNA in the aqueous core of reverse micellar droplets has been proposed as good alternative since monodispersed particles with a size close to 80 nm in diameter could be obtained (Figure 8) [111]. Note that particles with sizes lower than 100 nm have been reported as appropriated to be endocytosed by cells [112]. The indicated methodology is also able to encapsulate 85% of DNA, which obviously become protected from an external DNase environment [113]. Plasmid DNA (pDNA) was for example effectively encapsulated in CaP nanoparticles that were specifically targeted to liver cells after appropriate surface modification [110]. Although the entrapped pDNA was highly protected from enzymatic degradation, the transfection efficiency was around 80%. This limited value was attributed to a partial disintegration of DNA molecules during the ultra-sonication performed as prerequisite for re-dispersion of particles in aqueous buffered medium. The use of soft preparative methods that avoided ultrasonication clearly improved in vitro transfection efficiency in HeLa cell lines [110]. Different improvements concerning encapsulation have been formulated: (a) surface adsorption or biopolymers such as CHI in order to prevent particle aggregation [114]; (b) substitution of  
Figure 7: Scheme showing the synthesis of super paramagnetic mesoporous HAp nanoparticles as a carrier for DOX. (**a**) SPION conjugation for the formation of super paramagnetic Hap; (**b**) Formation of a mesoporous HAp with nanometer pores in combination with SPIONs as a core; (**c**) DOX molecules loaded into the magnetic nanoparticles coated with mesoporous HAp by a diffusion process. Adapted from [105], Elsevier, 2016.  
calcium cations by magnesium and/or strontium ions; and (c) anionic substitution of phosphates and hydroxyl ions by carbonate and/or fluoride ions [115; 116; 117]. In fact, ionic substitution may be beneficial because can hinder aggregation, may increase loading efficiency as a consequence of a higher charge density, and may enhance dissolution of the endocytosed particles [106]. Silicon substitution of HAp has also been explored for non-viral gene delivery because great changes of properties (e.g., morphology, cell parameters, surface properties, crystallinity and kinetics of dissolution) have been reported [118; 119; 120]. [PERSON] et al. [106] demonstrated that the structure of HAp could be retained up to a 18.6 mol % of substitution of phosphates by silicates but an amorphous phase (i.e., for silicate contents between 16.6 and 33.3 mol %) and even new hydrated calcium silicate phases were formed at higher degrees of substitution. An additional 20%-50% increase in transfection was observed for particles containing 8.3-50 mol % of silicate, owing to its rapid dissolution properties that makes feasible the escape of nanoparticles from lysosomal degradation.