# Biodegradable and Biocompatible Systems Based on Hydroxapatite Nanoparticles
## 4 Functional HAp Biocomposites through Modification/Encapsulation Techniques
### Hydroxyapatite as Non-Viral Gene Delivery System
Silicate substitution higher than 60 mol % resulted in considerable reduction in transfection. Transfection efficiency of agents based on CaPs can be increased by protecting DNA with successive layers of CaP (Figure 9) through a multi-step precipitation process [121]. These multi-shell particles showed higher transfection efficiency than single cell particles since DNA became more protected against intracellular biochemical degradation. Moreover, these multilayered particles were capable to retain their activity during 2-3 months of storage. It was also demonstrated that the inclusion of a cationic peptide or polymer on such multi-shell nanoparticles could further enhance the transfection efficiency [122]. Specifically, a protamine sulfate-coated CaP  
Figure 8: Flow chart showing the formation of nanoparticles encapsulating pDNA in the aqueous core of the bis (2-ethylhexyl) sulfosuccinate surfactant (AOT) microemulsion in hexane. The strategy involved the precipitation of the CaP in the aqueous core of the microemulsion droplets in the presence of pDNA. Adapted from [111], Elsevier, 2004.  
showed higher efficiency to transfect human embryonic kidney cells after one week of storage at 4 \({}^{\circ}\)C than conventional CaP particles. Cationic peptides rich on arginine are able to condense DNA improving transfection efficiency and protecting the polynucleotide from DNases. [PERSON] et al. [123] have proposed the use of scaffolds based on collagen and loaded with multi-shell nanoparticles incorporating a gene transfection agent (e.g., plasmid DNA) to allow invasion of cells that were subsequently transfected to first produce and then release growth factor proteins in a local area. In this way, typical problems of growth-factor releasing systems can be avoided: (a) a deficient tissue repair process due to a non-sustained release; and (b) a low local activity and concentration of growth factors at the last stages of the process. Molecular dynamic simulations have been performed in order to demonstrate the ability of the B double helix of DNA to be encapsulated inside HAp nanopores without causing a significant distortion in both the inter-strand hydrogen bonds and the intra-strand molecular stacking (Figure 10) [124]. Figure 10: Simulated structure showing the double helix of B-DNA as a nucleating agent for the development of a HAp crystal. Reproduced with permission from [124; 125], Springer-Creative Commons Attribution License, 2013; John Wiley & Sons, Inc., 2014. Figure 9: CaP/DNA nanoparticles with encapsulation of DNA by deposition of successive CaP layers, and their sedimentation coefficient distributions. Adapted from [121], John Wiley & Sons, Inc., 2006. The high stabilization between calcium ions of HAp and phosphate groups of DNA allowed the maintenance of the DNA secondary structure independently of its sequence. In contrast, similar calculations performed with fluoroapatite indicated the loss of DNA characteristics when the biomolecule was embedded in the mineral pores due to the lack of favorable electrostatic interactions. Molecular dynamic calculations showed that CaP clusters could be formed arising from DNA polyphosphate backbone. In fact, the strong electrostatic interactions (i.e., Ca\({}^{2+}\)-PO\({}_{4}\)[3]-) were essential to form ion complexes that served as nucleation sites of the indicated clusters. Moreover, temporal evolution of radial distribution functions derived from atomistic molecular dynamics simulations of aqueous solutions containing Ca\({}^{2+}\), PO\({}_{4}\)[3]- and OH\({}^{-}\) in the presence and absence of B-DNA demonstrated that DNA promoted mineral growth [126]. Effective encapsulation of DNA for both amorphous and crystalline HAp was unambiguously demonstrated by TEM, UV, XPS and gel electrophoresis (Figure 11) after removal of all superficially adsorbed DNA by digestion with deoxyribonuclease. Theoretical calculations demonstrated that the exposed crystalline surface of HAp has a determinant influence on the adsorption capacity of DNA (Figure 12).