**2.2 Properties**

Calcium phosphates being light in weight, chemically stable and compositionally similar to the mineral phase of the bone are preferred as bone graft materials in hard tissue engineering. They are composed of ions commonly found in physiological environment, which make them highly biocompatible. Many research works demonstrated the biocompatibility of calcium phosphates in-vitro and in-vivo. In addition, these bioceramics are also resistant to microbial attack, pH changes, and solvent conditions (Thamaraiselvi & Rajeswari, 2004; Kalita et al., 2007). Degradation properties are very important, especially in the application of calcium phosphates related to drug delivery. It has been shown that

Nano-Particulate Calcium Phosphate as a Gene Delivery System 581

different crystalline phases of calcium phosphate present different degradation properties. Table 3 summarizes the solubility properties and stability pH range of calcium phosphate.

Fig. 1. Calcium phosphate phase equilibrium diagram with 500 mmHg partial pressure of

water. Shaded area is the processing range to yield HAp (Hench, 1991).

Ca/P Name Formula 2 Tetracalcium phosphate Ca4O(PO4)2 1.67 Hydroxyapatite Ca10O(PO4)6(OH)2 N/A\* Amorphous calcium phosphate Ca10-xH2x(PO4)6(OH)2

1.50 Tricalcium phosphate (α, β, γ) Ca3(PO4)2

0.5 Calcium metaphosphate (α, β, γ) Ca(PO3)2

\*

N/A = not applicable

from (Vallet-Regi & Gonzalez-Calbet, 2004)).

1.33 Octacalcium phosphate Ca8H2(PO4)6.5H2O 1 Dicalcium phosphate dihydrate CaHPO4.2H2O 1 Dicalcium phosphate CaHPO4 1 Calcium pyrophosphate (α, β, γ) Ca2P2O7 1 Calcium pyrophosphate dihydrate Ca2P2O7.2H2O 0.7 Heptacalcium phosphate Ca7(P5O16)2 0.67 Tetracalcium dihydrogen phosphate Ca4H2P6O20 0.5 Monocalcium phosphate monohydrate Ca(H2PO4)2.H2O

Table 2. Various calcium phosphates with their respective Ca/P atomic ratios (Reprinted


Table 1. Some key properties of inorganic nanoparticles which are used for transfection in cell biology (Reprinted from (Sokolova & Epple, 2008)).

**Solubility in** 

**<sup>μ</sup>gL-1 Comments** 

semiconducting

biodegradable, biocompatible;

may be made fluorescent by incorporation of lanthanides; cations and anions may be substituted

covalently functionalized to improve solubility

Not biodegradable, hollow; may be

may be loaded with

ferromagnetic or superparamagnetic; toxic in uncoated form

easily covalently functionalized, for example, with thiols

ferromagnetic or superparamagnetic; harmful for cells in uncoated form; solubility increases with falling pH

high selective anion exchange capacity; biodegradable in slightly acidic environment; cations can be substituted

Biodegradable; available also in micro- or mesoporous form (e.g., zeolites); easily functionalizable, for example, by chlorosilanes

Bactericidal; dissolution product (Ag+) potentially harmful for cells

fluorescent, semiconducting

semiconducting

and

molecules

**Typical Size Range** 

Sulfide CdS 2–5 nm 0.69 ngL-1 toxic, fluorescent,

diameter of a few nm and length of a few

50–200 nm

Zinc Sulfide ZnS 3–50 nm 67 ngL-1 fluorescent,

Table 1. Some key properties of inorganic nanoparticles which are used for transfection in

Nickel Ni 5–100 nm ≈ 0 immunogenic, toxic

0

moderate, increases below pH 5–6

ca. 120 mg SiO2 L-1 (for silica particles)

5 mg L-1

(hydroxyapatite) 10–100 nm 6.1 mg L-1

mm

Platinum CoPt3 3–10 nm ≈<sup>0</sup>

Gold Au 1–50 nm ≈ 0

(Magnetite) Fe3O4 5–20 nm ≈<sup>0</sup>

Mg6Al2(CO3)(OH)16·4

(hydrotalcite)

Silica SiO2·nH2O 3–100 nm

Silver Ag 5–100 nm ≈ 0

Zinc Oxide ZnO 3–60 nm 1.6 to

cell biology (Reprinted from (Sokolova & Epple, 2008)).

H2O

**Kind of nanoparticle** 

Cadmium

Calcium Phosphate

Carbon

Cobalt-

Iron Oxide

Layered Double Hydroxide

Nanotubes Cn

**Chemical Composition** 

Ca10(PO4)6OH2

different crystalline phases of calcium phosphate present different degradation properties. Table 3 summarizes the solubility properties and stability pH range of calcium phosphate.

Fig. 1. Calcium phosphate phase equilibrium diagram with 500 mmHg partial pressure of water. Shaded area is the processing range to yield HAp (Hench, 1991).


\* N/A = not applicable

Table 2. Various calcium phosphates with their respective Ca/P atomic ratios (Reprinted from (Vallet-Regi & Gonzalez-Calbet, 2004)).

Nano-Particulate Calcium Phosphate as a Gene Delivery System 583

Fig. 2. Effect of CaCl2 on adsorption of 14C-Ad5 DNA to KB cells. KB cells were exposed to MEM-Tris containing DNA plus CaCl2 at various concentrations. The curves represent the fraction of radioactivity recovered in the medium (●), in the DNase digest (○), or in the SDS

With the same methodology, this group conducted another study to transform rat kidney cells with the DNA of human adenovirus 5. In Fig. 3 the transfected area is clearly visible as contained small, round, densely packed cells characteristic of adenovirus transformation. This work claimed that the "calcium technique" was a suitable system to study transformation by adenovirus DNA and the efficiency of transformation, though not high,

In another study, Graham, Veldhuisen and Wilkie used the aforementioned technique to investigate the infectivity of herpes simplex virus type I (HSV-I) (Graham et al., 1973). In 1975, Abrahams and Van Der EB made a transformation of rat kidney cells and mouse 3T3 cells by DNA from Simian Virus 40 using "calcium technique". They stated that this technique for in-vitro transformation was reproducible (Abrahams & Van Der Eb, 1975). Later, Van Der EB and Graham successfully used "calcium technique" to determine the ability to transform primary baby rat kidney (BRK) cells with specific fragments of human

In 1976, Stow and Wilkie reported that treatment of cells with dimethyl sulphoxide (DMSO) after injection with "Herpes Simplex Virus DNA"/calcium phosphate complex could lead to a significant increase in the number of plaques obtained. These researchers proposed that DMSO could initiate the plaque formation. It was interesting that in other method (DEAE-dextran) using DMSO did not exhibit that significant enhancement (Fig. 4) (Stow &

appeared to be reasonably reproducible (Graham & Van Der EB, 1973b).

lysate of the cells () (Graham & Van Der EB, 1973a).

adenoviruses 2 and 5 DNAs (Van Der EB et al., 1977).

Wilkie, 1976).


Table 3. Solubility and pH stability of different phases of calcium phosphates (Reprinted from (Kalita et al., 2007)).
