**3.1 Historical view**

Previously, it is mentioned that the first use of calcium phosphate in gene delivery application was conducted by Graham and Van Der EB in 1973. In this study, calcium phosphate was used for transfecting cells with Adenovirus 5 DNA to assay infectivity. (Graham & Van Der EB, 1973a). They diluted Adenovirus 5 DNA in a buffer containing Na2HPO4. Then, calcium chloride was added and the mixture was incubated with KB Cells. Using labeled DNA they concluded that by adding the calcium precursor in the experiment, the uptake of DNA increased and DNA showed a better stability against enzymatic degradation (Fig. 2). It was reported that this technique gave a 100 fold increase in efficiency over the DEAE-dextran method for human adenovirus DNA.

**Phases Solubility at 25 °C, -log (Ksp) pH Stability Range in aqueous** 

TCP) 28.9 Cannot be precipitated from

TCP) 25.5 Cannot be precipitated from

(TTCP) 38-44 Cannot be precipitated from

Anhydrate (DCPA) 6.90 Stable at temperatures above

Table 3. Solubility and pH stability of different phases of calcium phosphates (Reprinted

With the introduction of smaller calcium phosphate particles, it has become possible to use them in advanced fields of biomedicine. Calcium phosphate nanoparticles, with a size about 100 nm, are highly biocompatible. These particles are able to penetrate the outer membrane of cells and bacteria. Calcium phosphate nanoparticles could be utilized in different fields of biomedicine such as drug delivery, gene delivery, and imaging (Epple et al., 2010). Also, to produce high quality HAp bioceramics for artificial bone substitution, ultrafine HAp powder is usually employed. Nano-HAp powder results in easy handling, casting, and sintering leading to an excellent sintered body in the bioceramics preparation process (Cao

Previously, it is mentioned that the first use of calcium phosphate in gene delivery application was conducted by Graham and Van Der EB in 1973. In this study, calcium phosphate was used for transfecting cells with Adenovirus 5 DNA to assay infectivity. (Graham & Van Der EB, 1973a). They diluted Adenovirus 5 DNA in a buffer containing Na2HPO4. Then, calcium chloride was added and the mixture was incubated with KB Cells. Using labeled DNA they concluded that by adding the calcium precursor in the experiment, the uptake of DNA increased and DNA showed a better stability against enzymatic degradation (Fig. 2). It was reported that this technique gave a 100 fold increase in efficiency

Cannot be measured precisely. However, the following values were reported: 25.7 ± 0.1 (pH 7.40), 29.9 ± 0.1 (pH 6.00), 32.7 ±

0.1 (pH 5.28)

Hydroxyapatie (CDHA) <sup>≈</sup> 85.1 6.5-9.5

**3. Calcium phosphate nanoparticles as gene delivery vector** 

over the DEAE-dextran method for human adenovirus DNA.

Hydroxyapatite (HAp) 116.8 9.5-12

Dehydrate (DCPD) 6.59 2.0 – 6.0

β-Tricalcium Phosphate (β-

α-Tricalcium Phosphate (α-

Tetracalcium Phosphate

Dicalcium Phosphate

Dicalcium Phosphate

Amorphous Calcium Phosphate (ACP)

Calcium-deficient

et al., 2005).

**3.1 Historical view** 

from (Kalita et al., 2007)).

**2.3 Calcium phosphate nanoparticles** 

**solution at 25 °C** 

aqueous solutions.

aqueous solutions.

aqueous solutions.

Always metastable. The composition of a precipitate depends on the solution pH value and composition.

100 °C

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

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

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

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 & Wilkie, 1976).

Nano-Particulate Calcium Phosphate as a Gene Delivery System 585

Regarding the formation of DNA/calcium phosphate precipitates, they found that it is critical to add the solution of DNA/CaCl2 to the HEPES-phosphate buffer rather than in the reverse order. Also, they claimed that it is important to add the solution drop-wise, rather

In 1982, a research group at Yale University conducted research on the mechanism for entry of DNA/calcium phosphate complex in to mammalian cells by electron microscopy and

Electron microscopy and filter hybridization studies revealed that most of the DNA strands enter by phagocytosis. The effect of different drugs and respiratory inhibitors on the entry of DNA was also investigated (Table 4, Fig. 5). Results showed phagocytosis of DNA is inhibited both by respiratory inhibitors and drugs, such as Colcemid, which disassemble microtubules. They concluded that the uptake of DNA/calcium phosphate resembles "receptor mediated" phagocytosis. Also it was seen that ATP-depleted and cold treated cells were not able to adsorb the complex. Thus the authors claimed that the phagocytosis of DNA/calcium phosphate complex is an energy- and temperature-dependent process (Loyter et al., 1982a).

Fig. 5. Effect of increasing concentration of Colcemid on the entry of DAPI-stained

cytoplasmic florescence was drastically reduced (Fig. 6B) (Loyter et al., 1982b).

These researchers also claimed that the pH of the formation of the DNA/calcium phosphate complexes is crucial for successful gene transfer. Studies on the effect of pH and DNA concentration on the entry of fluorescent dye-labeled DNA into cells showed that only during the calcium phosphate complexes formation in the pH rang of 7.1 to 7.5 could fluorescent spots be visualized in the cytoplasm of recipient cells. For the complexes formed

On the other hand, the DNA/calcium phosphate ratio is important on the adsorption of the complexes. When higher concentrations of DNA was utilized with the constant concentration of calcium phosphate, adsorption was not affected, whereas the appearance of

DNA/calcium phosphate complexes into *Ltk-* cells (Loyter et al., 1982a).

above pH = 7.5 no entry to cells could be detected (Fig. 6A).

than directly (Corsaro & Pearson, 1981).

fluorescent dyes (Loyter et al., 1982a; Loyter et al., 1982b).

Fig. 3. Part of transformed colony resulting from exposure of primary rat kidney cells to Adenovirus 5 DNA+ CaCl2 22 days previously. Three normal cells can be seen to the right of the photograph. Giemsa stain (Graham & Van Der EB, 1973b).

During the 1980's, the calcium phosphate method for in-vitro gene delivery had become a common method. In 1981, some of the parameters that affect the transformation procedure by calcium phosphate system had been investigated by Corsaro and Pearson (Corsaro & Pearson, 1981). First, to confirm the work of Stow and Wilkie in 1976, they performed a study on the effect of rinsing the complex of DNA/calcium phosphate with DMSO. They also added an additional variable to this experiment which was the exposure time of DNA/calcium phosphate complex to cells. They claimed that when suboptimal DNA exposure time is applied (e.g. 4-12 hours), the DMSO rinse increases the transformation frequency. However, rinsing with DMSO had no effect when the optimal condition was utilized. They concluded that exposure to DMSO offers no significant advantage.

Fig. 4. The effect of DMSO concentration on the enhancement of HSV-I DNA infectivity. Varying concentration of DMSO dissolved in HeBS () or eagle's medium () (Stow & Wilkie, 1976).

Fig. 3. Part of transformed colony resulting from exposure of primary rat kidney cells to Adenovirus 5 DNA+ CaCl2 22 days previously. Three normal cells can be seen to the right of

utilized. They concluded that exposure to DMSO offers no significant advantage.

Fig. 4. The effect of DMSO concentration on the enhancement of HSV-I DNA infectivity. Varying concentration of DMSO dissolved in HeBS () or eagle's medium () (Stow &

Wilkie, 1976).

During the 1980's, the calcium phosphate method for in-vitro gene delivery had become a common method. In 1981, some of the parameters that affect the transformation procedure by calcium phosphate system had been investigated by Corsaro and Pearson (Corsaro & Pearson, 1981). First, to confirm the work of Stow and Wilkie in 1976, they performed a study on the effect of rinsing the complex of DNA/calcium phosphate with DMSO. They also added an additional variable to this experiment which was the exposure time of DNA/calcium phosphate complex to cells. They claimed that when suboptimal DNA exposure time is applied (e.g. 4-12 hours), the DMSO rinse increases the transformation frequency. However, rinsing with DMSO had no effect when the optimal condition was

the photograph. Giemsa stain (Graham & Van Der EB, 1973b).

Regarding the formation of DNA/calcium phosphate precipitates, they found that it is critical to add the solution of DNA/CaCl2 to the HEPES-phosphate buffer rather than in the reverse order. Also, they claimed that it is important to add the solution drop-wise, rather than directly (Corsaro & Pearson, 1981).

In 1982, a research group at Yale University conducted research on the mechanism for entry of DNA/calcium phosphate complex in to mammalian cells by electron microscopy and fluorescent dyes (Loyter et al., 1982a; Loyter et al., 1982b).

Electron microscopy and filter hybridization studies revealed that most of the DNA strands enter by phagocytosis. The effect of different drugs and respiratory inhibitors on the entry of DNA was also investigated (Table 4, Fig. 5). Results showed phagocytosis of DNA is inhibited both by respiratory inhibitors and drugs, such as Colcemid, which disassemble microtubules. They concluded that the uptake of DNA/calcium phosphate resembles "receptor mediated" phagocytosis. Also it was seen that ATP-depleted and cold treated cells were not able to adsorb the complex. Thus the authors claimed that the phagocytosis of DNA/calcium phosphate complex is an energy- and temperature-dependent process (Loyter et al., 1982a).

Fig. 5. Effect of increasing concentration of Colcemid on the entry of DAPI-stained DNA/calcium phosphate complexes into *Ltk-* cells (Loyter et al., 1982a).

These researchers also claimed that the pH of the formation of the DNA/calcium phosphate complexes is crucial for successful gene transfer. Studies on the effect of pH and DNA concentration on the entry of fluorescent dye-labeled DNA into cells showed that only during the calcium phosphate complexes formation in the pH rang of 7.1 to 7.5 could fluorescent spots be visualized in the cytoplasm of recipient cells. For the complexes formed above pH = 7.5 no entry to cells could be detected (Fig. 6A).

On the other hand, the DNA/calcium phosphate ratio is important on the adsorption of the complexes. When higher concentrations of DNA was utilized with the constant concentration of calcium phosphate, adsorption was not affected, whereas the appearance of cytoplasmic florescence was drastically reduced (Fig. 6B) (Loyter et al., 1982b).

Nano-Particulate Calcium Phosphate as a Gene Delivery System 587

concentration on transfection efficiency using DNA/calcium phosphate complexes can be seen. The authors concluded that when Chloroquine treatment was effective, it increased the fraction of cells that could be successfully transfected. They claimed that this conclusion was supported by the results of experiments in which cells were transfected with linear forms of viral DNA. In that case, in Chloroquine treated cells, the number of DNA molecules which had re-circularized and were able to replicate was much larger

With the same approach, in 1984 a research group in Norway used different inhibitors of intracellular degradation (such as 3-methyl adenine, NH4Cl, FCPP and etc.) and claimed that the frequency of transformation was increased due to increasing the cytoplasmic level

In 1987, Chen and Okayama introduced a new method for gene delivery with calcium phosphate systems. The aim of their work was the formation of DNA/calcium phosphate complexes gradually in medium during incubation with cells. They found that in this method the crucial factors that affect the transfection efficiency are the pH of the buffer used for calcium phosphate precipitation (optimized pH was 6.95) and the CO2 level during the incubation of DNA with cells. They also found that the amount and the form of DNA are important factors. It was observed that circular DNA has better efficiency than linear DNA but, the reason for this phenomenon was not clear at that time. The authors claimed that the efficiency of their method is comparable to the efficiency of other common transfection

In 1990 Orrantia and Chang investigated the intracellular distribution of DNA after the DNA/calcium phosphate complexes move into the cells. Results showed that only a small fraction of internalized DNA could be found in the nucleus, the target place for gene delivery. In the enriched nuclear fraction, the mouse cells retained 6.4% of internalized DNA

Fig. 7. Effect of Chloroquine concentration on transfection efficiency. Rat-1 cultures were transfected by co-precipitating calcium phosphate and polyoma DNA, 20 ng () and 100 ng

than untreated cells (Luthman & Magnusson, 1983).

of exogenous DNA (Table 5) (Ege et al., 1984).

systems of that time (Chen & Okayama, 1987).

while the human cells retained only 2.2% (Fig. 8).

(○) (Luthman & Magnusson, 1983).


Table 4. The effect of various drugs and respiratory inhibitors on introduction of DNA into *Ltk- Aprt-* cells (Reproduced from (Loyter et al., 1982a)).

Fig. 6. Adsorption (cells containing adsorbed fluorescent dots) and uptake (cells containing more than 10 intracellular fluorescent dots) of DAPI-stained DNA as a function of the pH of the DNA/calcium phosphate complex (A) and DNA concentration in DNA/calcium phosphate complex (B) (Loyter et al., 1982b).

One of the limitations of calcium phosphate systems in gene delivery applications is that most of the input DNA is degraded before it reaches the nucleus of the cell, where gene expression and DNA replication take place. In 1983, Luthman and Magnusson conducted research on increasing the efficiency of transfection by inhibiting the lysosomal degradation using Chloroquine as a lysosomotropic compound. For this purpose they used a conventional procedure for transfection with calcium phosphate, but they added Chloroquine to the growth medium of the cells. In Fig. 7 the effect of Chloroquine

**System Effect of DNA Entry** 

Colcemid (5 μg/ml) Complete inhibition

 2 deoxyglucose Partial inhibition NaN3 Partial inhibition NaF Partial inhibition NaF + 2 deoxyglucose Complete inhibition NaN3 + 2 deoxyglucose Complete inhibition Table 4. The effect of various drugs and respiratory inhibitors on introduction of DNA into

Fig. 6. Adsorption (cells containing adsorbed fluorescent dots) and uptake (cells containing more than 10 intracellular fluorescent dots) of DAPI-stained DNA as a function of the pH of the DNA/calcium phosphate complex (A) and DNA concentration in DNA/calcium

One of the limitations of calcium phosphate systems in gene delivery applications is that most of the input DNA is degraded before it reaches the nucleus of the cell, where gene expression and DNA replication take place. In 1983, Luthman and Magnusson conducted research on increasing the efficiency of transfection by inhibiting the lysosomal degradation using Chloroquine as a lysosomotropic compound. For this purpose they used a conventional procedure for transfection with calcium phosphate, but they added Chloroquine to the growth medium of the cells. In Fig. 7 the effect of Chloroquine

<sup>μ</sup>g/ml) No effect

DMSO (10%, 10-30 min) No effect

**Drugs** 

Cytochalasin B (1-4

**Respiratory inhibitors** 

*Ltk- Aprt-* cells (Reproduced from (Loyter et al., 1982a)).

phosphate complex (B) (Loyter et al., 1982b).

concentration on transfection efficiency using DNA/calcium phosphate complexes can be seen. The authors concluded that when Chloroquine treatment was effective, it increased the fraction of cells that could be successfully transfected. They claimed that this conclusion was supported by the results of experiments in which cells were transfected with linear forms of viral DNA. In that case, in Chloroquine treated cells, the number of DNA molecules which had re-circularized and were able to replicate was much larger than untreated cells (Luthman & Magnusson, 1983).

With the same approach, in 1984 a research group in Norway used different inhibitors of intracellular degradation (such as 3-methyl adenine, NH4Cl, FCPP and etc.) and claimed that the frequency of transformation was increased due to increasing the cytoplasmic level of exogenous DNA (Table 5) (Ege et al., 1984).

In 1987, Chen and Okayama introduced a new method for gene delivery with calcium phosphate systems. The aim of their work was the formation of DNA/calcium phosphate complexes gradually in medium during incubation with cells. They found that in this method the crucial factors that affect the transfection efficiency are the pH of the buffer used for calcium phosphate precipitation (optimized pH was 6.95) and the CO2 level during the incubation of DNA with cells. They also found that the amount and the form of DNA are important factors. It was observed that circular DNA has better efficiency than linear DNA but, the reason for this phenomenon was not clear at that time. The authors claimed that the efficiency of their method is comparable to the efficiency of other common transfection systems of that time (Chen & Okayama, 1987).

In 1990 Orrantia and Chang investigated the intracellular distribution of DNA after the DNA/calcium phosphate complexes move into the cells. Results showed that only a small fraction of internalized DNA could be found in the nucleus, the target place for gene delivery. In the enriched nuclear fraction, the mouse cells retained 6.4% of internalized DNA while the human cells retained only 2.2% (Fig. 8).

Fig. 7. Effect of Chloroquine concentration on transfection efficiency. Rat-1 cultures were transfected by co-precipitating calcium phosphate and polyoma DNA, 20 ng () and 100 ng (○) (Luthman & Magnusson, 1983).

Nano-Particulate Calcium Phosphate as a Gene Delivery System 589

In 1996, a research group in Taiwan conducted some research works on electrochemical properties of DNA/calcium phosphate complexes. The study focused on the variation of zeta potential with changes in pH for calcium phosphate and DNA/calcium phosphate complexes. The point of zero charge (pzc) and isoelectric point (iep) were found to be at pH 7.09 and 7.0, respectively. With addition of plasmid DNA, both pzc and iep points shifted to

In their other research on this topic, they revealed that the pH of the formation of DNA/calcium phosphate complexes and the concentration of DNA within the complexes were the crucial factor for the entry of these complexes to cells. The results of their study showed that optimum transfection efficiency occurred in the region close to the iep of DNAcalcium phosphate co-precipitates of pH 7.15 and close to the maximum flocculation of this colloidal system. The enhanced cell transformation efficiency occurred at pH 7.01. The zeta potentials of the DNA co-precipitates prepared in the absence of DMEM and calf serum were determined to lie between 11 and 21 mV. Preparation within these limits resulted in an efficient internalization of the DNA/calcium phosphate complexes, and for endocytosis

In 2004, Jordan and Wurm investigated the methods that were applied previously for gene delivery with calcium phosphate particles by different authors. They stated that all of the numerous variations of the protocol found in the literature are based on the same principle—a spontaneous precipitation that occurs in supersaturated solutions. Although a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of optimized parameters that assure certain properties of the precipitate. Finally, they concluded that despite a rapidly growing choice of efficient transfection reagents, this method remains highly attractive due to its highly biocompatible

Research on using calcium phosphate nanoparticles for gene delivery application is still continuing. Researchers perform a lot of new experiments to optimize the parameters involved in gene delivery with calcium phosphate nanoparticles. We have tried to review

A research group in the University of Duisburg-Essen, proposed a method to prepare multishell calcium phosphate/DNA particles. They utilized a simple method to prepare multi-

They prepared different nanoparticles and showed that with multi-shell calcium phosphate/DNA nanoparticles the transfection efficiency is increased due to the protection of DNA against nuclease enzymes (Fig. 10). Moreover, the authors claimed that in contrast with conventional calcium phosphate, these particles could be stored for weeks without loss

They also showed that the standard calcium phosphate method selectively unbalanced intracellular calcium homeostasis while it remained at low control levels after transfection using nanoparticles. They concluded that with using DNA-functionalized calcium phosphate nanoparticles, cells are able to cope with the associated calcium uptake and therefore proved their method to be a superior transfection method (Neumann et al., 2009). Hanifi et al. conducted some research on the feasibility of using strontium and magnesium substituted calcium phosphate in gene delivery applications. They prepared the particles via a simple sol-gel route. They obtained some particles with nano-size structure, high specific

higher values of 7.18 and 7.15, respectively (Yang & Yang, 1996a).

to occur (Yang & Yang, 1996b).

nature (Jordan & Wurm, 2004).

some of these studies in this chapter.

shell calcium phosphate as illustrated in Fig. 9.

of their transfection efficiency (Sokolova et al., 2006).

**3.2 Current studies** 


Table 5. Effect of different compounds on the transformation frequency of rat 2 *tk* cells transfected with pAGO DNA 6 hours after incubation of the indicated compounds with the cells (reproduced from (Ege et al., 1984)).

The authors concluded that transfection with DNA/calcium phosphate is a procedure with low efficiency partly because most of the endocytosed DNA is quickly degraded and excreted to the cytosol (Orrantia & Chang, 1990).

In 1994, O'Mahoney and Adams modified the calcium phosphate transfection procedure described by Chen and Okayama in 1987 and claimed that they reached a reliable and reproducible method with high transfection efficiency. They claimed that the critical factor in this method is the standing time of the DNA/CaCl2/BES-buffered saline prior to addition to cultured cells. They concluded that in the optimal condition it is possible to reach 100% efficiency (Omahoney & Adams, 1994).

Fig. 8. Distribution of internalized DNA in subcellular fractions from human and mouse cells. Cultured Cells were transfected with 32P-labeled high-molecular-weight DNA/calcium phosphate for 4 h. : Human primary fibroblast cells, : Transformed mouse Ltk- cells (Orrantia & Chang, 1990).

In 1996, a research group in Taiwan conducted some research works on electrochemical properties of DNA/calcium phosphate complexes. The study focused on the variation of zeta potential with changes in pH for calcium phosphate and DNA/calcium phosphate complexes. The point of zero charge (pzc) and isoelectric point (iep) were found to be at pH 7.09 and 7.0, respectively. With addition of plasmid DNA, both pzc and iep points shifted to higher values of 7.18 and 7.15, respectively (Yang & Yang, 1996a).

In their other research on this topic, they revealed that the pH of the formation of DNA/calcium phosphate complexes and the concentration of DNA within the complexes were the crucial factor for the entry of these complexes to cells. The results of their study showed that optimum transfection efficiency occurred in the region close to the iep of DNAcalcium phosphate co-precipitates of pH 7.15 and close to the maximum flocculation of this colloidal system. The enhanced cell transformation efficiency occurred at pH 7.01. The zeta potentials of the DNA co-precipitates prepared in the absence of DMEM and calf serum were determined to lie between 11 and 21 mV. Preparation within these limits resulted in an efficient internalization of the DNA/calcium phosphate complexes, and for endocytosis to occur (Yang & Yang, 1996b).

In 2004, Jordan and Wurm investigated the methods that were applied previously for gene delivery with calcium phosphate particles by different authors. They stated that all of the numerous variations of the protocol found in the literature are based on the same principle—a spontaneous precipitation that occurs in supersaturated solutions. Although a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of optimized parameters that assure certain properties of the precipitate. Finally, they concluded that despite a rapidly growing choice of efficient transfection reagents, this method remains highly attractive due to its highly biocompatible nature (Jordan & Wurm, 2004).
