**1. Introduction**

576 Non-Viral Gene Therapy

Young, A. (2007). Structural insights into the clathrin coat. *Semin.Cell Dev.Biol.*, Vol.18,

Zhang, X.; Pan, S.R.; Hu, H.M.; Wu, G.F.; Feng, M.; Zhang, W. & Luo, X. (2008).

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No.4, (August 2007), pp. 448-458, ISSN 1084-9521

Four decades ago, calcium phosphate systems were introduced for in-vitro gene delivery applications. Recently, many studies have been conducted regarding the different applications of these systems in delivering genes to different cell types for therapeutic purposes. Although there are important limitations of using calcium phosphates in gene delivery, there is a high interest in using this type of gene delivery system. This is because of the significant biocompatibility of calcium phosphates, easy synthesis methods of this system, and intrinsic characteristics of calcium phosphates that increase the transfection efficiency. The combination of these properties are rarely seen in other gene delivery systems.

This chapter aims to localise calcium phosphate nanoparticles among the most common non-viral gene delivery systems. It also reviews the history of using calcium phosphates in gene delivery applications and the efforts made to make this system suitable for further clinical applications.

#### **1.1 Non-viral gene delivery**

The application of non-viral systems increased considerably after it was shown that using viral systems can result in several problems including difficulty in production, limited opportunity for repeated administrations due to acute inflammatory response, and delayed humoral or cellular immune responses. Insertional mutagenesis is also a potential issue for

Nano-Particulate Calcium Phosphate as a Gene Delivery System 579

targeted cells. Silica nanotubes (Namgung et al., 2011), zirconia (Tan et al., 2007), carbon nanotubes (Pantarotto et al., 2004) and layered double hydroxides (Choy et al., 2008) are some examples of these inorganic systems. However, their low transfection efficacy limits

The following sections discuss calcium phosphates; one of the most important groups of

The work of Graham and Van Der Eb completed in 1973 shows the first application of calcium phosphate in condensation of genetic materials. The brilliant results of their research were that calcium phosphate could condense DNA and increase the transfection efficiency with a relatively simple procedure (Graham & Van Der EB, 1973a). This first research led to vast application of this technology in in-vitro gene delivery because of the

In order to have a better understanding of calcium phosphate gene delivery properties, first we shall have a look at the structure and characteristics of the calcium phosphate family.

Calcium phosphate-based bioceramics have been used in medicine and dentistry for decades. Applications include dental implants, percutaneous devices, periodontal treatment, alveolar ridge augmentation, orthopedics, maxillofacial surgery, otolaryngology and spinal

Bone is a natural nano-composite composed of organic (40%) and inorganic (60%) components. The inorganic constituent of bone is made up of biological apatites, which provide strength to the skeleton and act as a storehouse for calcium, phosphorus, sodium, and magnesium. These biological apatites are structurally similar, though not identical, to the mineral apatite hydroxyapatite (HAp, Ca10(PO4)6(OH)2). Hydroxyapatite is the most ubiquitous and well-known phase of calcium phosphate. It has the Ca/P ratio of 1.67 (Narayan et al., 2004). Different phases of calcium phosphate ceramics are used depending upon whether a resorbable or bioactive material is desired. The stable phases of calcium phosphate ceramics depend considerably upon temperature and the presence of water,

Going through aforementioned properties, it can be realized that the calcium phosphates family includes several members with different characteristics. Calcium phosphate ratio, Ca/P, has been found as the best way to distinguish among these members. In table 2 these

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

either during processing or use in the environment (See Fig. 1) (Hench, 1991).

their use. Table 1 summarizes inorganic nanoparticles properties.

demonstrated easy preparation method and proper results.

inorganic non-viral gene delivery systems.

**2. Calcium phosphate** 

**2.1 Calcium phosphates family** 

members are shown based on their Ca/P ratio.

surgery (Hench, 1991).

**2.2 Properties** 

some viral systems that integrate foreign DNA into the genome (Al-Dosari & Gao, 2009). Although viral systems such as retrovirus, adenovirus, and adeno-associated virus are potentially efficient, non-viral systems have some advantages in that they are less toxic, less immunogenic, and easier to prepare (Nishikawa & Huang, 2001).

A lot of research has been conducted to find suitable non-viral systems. An ideal gene delivery method needs to meet 3 major criteria:


Recently, various materials have been introduced as potential gene delivery systems. Three groups of substances are more advantageous in this application. These three groups are:


However, some limitations accompany the use of most of these systems including cell toxicity, immune response and low tranfection efficiency.

#### **1.2 Inorganic vectors**

Inorganic systems have been used in in-vitro gene delivery for many years, but their clinical application has been developed mostly in the last decade when aminofunctionalized silica was introduced. Researchers at Saarland University showed that amino-functionalized silica exhibits good gene tranfection efficiency in addition to its suitable biocompatibility (Kneuer et al., 2000; Csogor et al., 2003; Sameti et al., 2003). Because of this, several studies have been conducted on using amino-funtionalized silica as a gene delivery system (Bharali et al., 2005; Roy et al., 2005; Klejbor et al., 2007; Choi et al., 2008). Research was also conducted on using silica in combination with other polymers for gene delivery. Results demonstrated that making composites of certain polymers with silica nanoparticles could enhance transfection efficiency due to the dense nature of silica nanoparticles (Luo et al., 2004).

There is an increasing interest in mesoporous silica for drug/gene delivery applications because of their higher capacity and of the potential for tailored release of the active molecule. Some studies have been conducted on functionalized or non-functionalized mesoporous silica but the research on using this type of inorganic systems is still ongoing (Park et al., 2008; Slowing et al., 2008).

Some studies have been done on using functionalized gold nanoparticles as a gene delivery system. The results demonstrated the feasibility of using this approach, but further research is needed in this new area (Liang et al., 2010; Niidome et al., 2011).

In addition to calcium phosphate, (their gene delivery application is reviewed in this chapter), other inorganic systems have also been studied regarding in-vitro gene delivery to targeted cells. Silica nanotubes (Namgung et al., 2011), zirconia (Tan et al., 2007), carbon nanotubes (Pantarotto et al., 2004) and layered double hydroxides (Choy et al., 2008) are some examples of these inorganic systems. However, their low transfection efficacy limits their use. Table 1 summarizes inorganic nanoparticles properties.

The following sections discuss calcium phosphates; one of the most important groups of inorganic non-viral gene delivery systems.
