**3.2 Current studies**

588 Non-Viral Gene Therapy

**Transformation Frequency after** 

cells

**6 hours** 

**Addition Concentration**

DNA + 3-methyl

cells (reproduced from (Ege et al., 1984)).

efficiency (Omahoney & Adams, 1994).

(Orrantia & Chang, 1990).

excreted to the cytosol (Orrantia & Chang, 1990).

None, no DNA 0 None, DNA alone 4 DNA + glycerol 17 % 10 DNA + DMSO 20 % 22 DNA + NH4Cl 20 mM 64 DNA + FCPP 1 μM 50 DNA + Procaine 10 mM 3 DNA + chloroquine 100 μM 5 DNA + monensin 5 mM 1

adenine 5 mM 46 Table 5. Effect of different compounds on the transformation frequency of rat 2 *tk-*

transfected with pAGO DNA 6 hours after incubation of the indicated compounds with the

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

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%

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

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 some of these studies in this chapter.

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 multishell calcium phosphate as illustrated in Fig. 9.

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

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

Nano-Particulate Calcium Phosphate as a Gene Delivery System 591

Recently there has been an approach to incorporate other agents or materials with calcium phosphate to improve its function as a gene delivery system. Stabilizing with bisphosphonate (Giger et al., 2011), coating with lipids (Zhou et al., 2010), incorporating in alginate hydrogel (Krebs et al., 2010) and association with Adenovirus (Toyoda et al., 2000)

(a)

(b) Fig. 11. Concentration of Ca++ ions in SBF solution after predicted period of time. A: Sr-CaP,

are some examples for this approach.

B: Mg-CaP (Not Published).

surface area, and a high dissolution rate (Fig. 11). The zeta potential (Table 6) was increased in comparison with simple calcium phosphate. They concluded that due to increased surface charge and solubility, these novel systems could increase the gene transfection efficiency (Hanifi et al., 2010a; Hanifi et al., 2010b).

Fig. 9. Schematic set-up of the apparatus used for preparation of DNA-functionalized calcium phosphate nanoparticles. Calcium nitrate and diammonium hydrogen phosphate solutions are mixed in a vessel to form a precipitate. A part of the dispersion is taken with a syringe and mixed with DNA solution in an Eppendorf tube (Sokolova et al., 2006).

Fig. 10. Comparison of the transfection efficiency of multi-shell calcium phosphate/DNA by different methods. There are significant differences between single-shell and triple-shell (P<0.01) and triple-shell and the standard calcium phosphate methods (P<0.05) (Sokolova et al., 2006).

surface area, and a high dissolution rate (Fig. 11). The zeta potential (Table 6) was increased in comparison with simple calcium phosphate. They concluded that due to increased surface charge and solubility, these novel systems could increase the gene transfection

Fig. 9. Schematic set-up of the apparatus used for preparation of DNA-functionalized calcium phosphate nanoparticles. Calcium nitrate and diammonium hydrogen phosphate solutions are mixed in a vessel to form a precipitate. A part of the dispersion is taken with a

syringe and mixed with DNA solution in an Eppendorf tube (Sokolova et al., 2006).

Fig. 10. Comparison of the transfection efficiency of multi-shell calcium phosphate/DNA by different methods. There are significant differences between single-shell and triple-shell (P<0.01) and triple-shell and the standard calcium phosphate methods (P<0.05) (Sokolova et

al., 2006).

efficiency (Hanifi et al., 2010a; Hanifi et al., 2010b).

Recently there has been an approach to incorporate other agents or materials with calcium phosphate to improve its function as a gene delivery system. Stabilizing with bisphosphonate (Giger et al., 2011), coating with lipids (Zhou et al., 2010), incorporating in alginate hydrogel (Krebs et al., 2010) and association with Adenovirus (Toyoda et al., 2000) are some examples for this approach.

Fig. 11. Concentration of Ca++ ions in SBF solution after predicted period of time. A: Sr-CaP, B: Mg-CaP (Not Published).

Nano-Particulate Calcium Phosphate as a Gene Delivery System 593

Alton, E. W. F. W., Middleton, P. G., Caplen, N. J., Smith, S. N., Steel, D. M., Munkonge, F. M.,

Bharali, D. J., Klejbor, I., Stachowiak, E. K., Dutta, P., Roy, I., Kaur, N., Bergey, E. J., Prasad, P.

Chen, C. & Okayama, H. (1987). High-Efficiency Transformation of Mammalian-Cells by

Choi, E. W., Shin, I. S., Lee, C. W. & Youn, H. Y. (2008). The effect of gene therapy using

Choy, J.-H., Park, M. & Oh, J.-M. (2008). Gene and Drug Delivery System with Soluble

Corsaro, C. M. & Pearson, M. L. (1981). Enhancing the Efficiency of DNA-Mediated Gene-

Csogor, Z., Nacken, M., Sameti, M., Lehr, C. M. & Schmidt, H. (2003). Modified silica particles

Dufes, C., Uchegbu, I. F. & Schatzlein, A. G. (2005). Dendrimers in gene delivery. *Advanced* 

Ege, T., Reisbig, R. R. & Rogne, S. (1984). Enhancement of DNA-Mediated Gene-Transfer by

Epple, M., Ganesan, K., Heumann, R., Klesing, J., Kovtun, A., Neumann, S. & Sokolova, V.

Gao, X., Kim, K. S. & Liu, D. X. (2007). Nonviral gene delivery: What we know and what is

Giger, E. V., Puigmarti-Luis, J., Schlatter, R., Castagner, B., Dittrich, P. S. & Leroux, J. C. (2011).

Graham, F. L. & Van Der EB, A. J. (1973a). New Technique for Assay of Infectivity of Human

Graham, F. L. & Van Der EB, A. J. (1973b). Transformation of Rat Cells by DNA of Human

CTLA4Ig/silica-nanoparticles on canine experimental autoimmune thyroiditis.

Inorganic Carriers, In: *NanoBioTechnology : bioinspired devices and materials of the future*,

for gene delivery. *Materials Science & Engineering C-Biomimetic and Supramolecular* 

Inhibitors of Autophagic-Lysosomal Function. *Experimental Cell Research*, 155, 1, pp. 9-

(2010). Application of calcium phosphate nanoparticles in biomedicine. *Journal of* 

Gene delivery with bisphosphonate-stabilized calcium phosphate nanoparticles. *J* 

*Academy of Sciences of the United States of America*, 102, 32, pp. 11539-11544 Cao, L. Y., Zhang, C. B. & Huang, H. F. (2005). Synthesis of hydroxyapatite nanoparticles in

ultrasonic precipitation. *Ceramics International*, 31, 8, pp. 1041-1044

O. Shoseyov and I. Levy, pp. 349-367, Humana Press, Totowa, N.J.

Transfer in Mammalian-Cells. *Somatic Cell Genetics*, 7, 5, pp. 603-616

Plasmid DNA. *Molecular and Cellular Biology*, 7, 8, pp. 2745-2752

*Journal of Gene Medicine*, 10, 7, pp. 795-804

*Drug Delivery Reviews*, 57, 15, pp. 2177-2202

*Materials Chemistry*, 20, 1, pp. 18-23

next. *Aaps Journal*, 9, 1, pp. E92-E104

Adenovirus 5 DNA. *Virology*, 52, 2, pp. 456-467

Adenovirus-5. *Virology*, 54, 2, pp. 536-539

*Control Release*, 150, 1, pp. 87-93

*Systems*, 23, 1-2, pp. 93-97

16

Defect in Cystic-Fibrosis Mutant Mice. *Nature Genetics*, 5, 2, pp. 135-142 Benns, J. M., Choi, J. S., Mahato, R. I., Park, J. S. & Kim, S. W. (2000). pH-sensitive cationic

shaped polymer. *Bioconjugate Chemistry*, 11, 5, pp. 637-645

Jeffery, P. K., Geddes, D. M., Hart, S. L., Williamson, R., Fasold, K. I., Miller, A. D., Dickinson, P., Stevenson, B. J., Mclachlan, G., Dorin, J. R. & Porteous, D. J. (1993). Noninvasive Liposome-Mediated Gene Delivery Can Correct the Ion-Transport

polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb

N. & Stachowiak, M. K. (2005). Organically modified silica nanoparticles: A nonviral vector for in vivo gene delivery and expression in the brain. *Proceedings of the National* 


Table 6. Surface charge of Sr and Mg substituted calcium phosphate nanoparticles (Reproduce from (Hanifi et al., 2010a; Hanifi et al., 2010b)).
