Key words

ceramic nanoparticles; drug delivery; transfection; gene silencing; tissue engineering

21.1 Introduction

The stimulation of cellular attachment and proliferation is a major challenge in tissue engineering. In addition to using a suitable scaffold to provide a suitable environment for cells, it is possible to enhance cell proliferation by the addition of biomolecules, either attached to the scaffold surface or incorporated into the scaffold.

Nanomaterials can be used to compensate some scaffold limitations such as the absence of cellular adhesion molecules and the inability of cells to self-assemble into 3D tissues. In addition, nanoparticles present a convenient way to attach biomolecules to their surface which can then be transferred into cells. For example, bioceramic nanoparticles are commonly applied in regenerative therapy and tissue engineering in bone, where nanoparticles interact with bone cells and tissues to induce osteogenesis and mineralization (Tautzenberger et al., 2012). One of the main applications for nanoparticles in tissue engineering is the incorporation of controlled release systems, such as growth factors and cytokines into scaffold, which are important for the support of tissue morphogenesis, viability and functionality (Epple et al., 2010; Giljohann et al., 2010; Goesmann and Feldmann, 2010; Stark, 2011; Tautzenberger et al., 2012).

Nanoparticles and their application in tissue engineering and drug delivery have been the focus of many research groups in the past decades (Niemeyer and Mirkin, 2004; Schmid, 2004; Riehemann et al., 2009; Giljohann et al., 2010; Kim et al., 2010; Laroui et al., 2011; Stark, 2011). Due to their small size, nanoparticles can pass through the cell membrane and deliver therapeutic molecules into living systems (Rolland, 1999; Langer and Peppas, 2003; Dietz and Bahr, 2004; Heiser, 2004; Sokolova et al., 2012). Different kinds of nanoparticles have been developed, and many have been tested in vitro, in vivo or even applied into pre-clinical trials or clinical application. In this chapter, the application of ceramic nanoparticles in tissue engineering and drug delivery is presented (Table 21.1).

21.2 Ceramic nanoparticles

Ceramic nanoparticles consist of inorganic compounds, typically stabilized by suitable molecules to achieve their colloidal stability. In general, only few systems are used in biomedicine. We can distinguish between non-biodegradable nanoparticles (e.g. aluminum oxide, titanium oxide) and biodegradable nanoparticles (e.g. calcium phosphate and silica). For biomolecule delivery, typically calcium phosphate and silica are used. It should be noted that there is also a very large range of nanoparticles and nanoscopic structures that consist of organic polymers and can be used to carry biomolecules.

Calcium phosphate is the inorganic component of biological hard tissue, i.e. of bone and teeth (Dorozhkin and Epple, 2002). It is soluble under acidic conditions (pH of 5 and less) which is exploited during bone remodeling (resorption of bone mineral by osteoclasts). After cellular uptake, calcium phosphate is soluble under the conditions of lysosomal degradation. Therefore, calcium phosphate nanoparticles are well suited to deliver biomolecules. In addition, calcium phosphate ceramics are often used as solid bone substitution materials or as scaffolds for cell growth. In this case, ceramics like hydroxyapatite (HAP) or β-tricalcium phosphate (β-TCP) are typically used. In addition, calcium phosphate cements and highly concentrated dispersions of calcium phosphate nanoparticles are used (Bohner, 2010).

21.3 Nanoparticles for drug delivery

Nanocarriers are important in medicine as a drug delivery system (Riehemann et al., 2009). Nanoparticles, applied in biological studies, can be used for different purposes: as a drug delivery system, for imaging, and for therapeutic application (Liong et al., 2008). Biomolecules of interest can be directly injected at the respective site, where they are usually rapidly cleared from the body or they can become distributed in the body, bringing unwanted side effects. Therefore, a local and cell specific drug delivery by nanoparticles is favored over free dissolved biomolecules as nanoparticles are less mobile than dissolved molecules. Furthermore, biomolecules can be incorporated into nanoparticles to protect them against biodegradation, e.g. by enzymes.

Depending on the intended application and target of nanoparticles, they are composed of different compounds as a complex and well-organized system (Fig. 21.1) (Sinha et al., 2006; Gil and Parak, 2008; Liong et al., 2008):

Nanoparticles are also a promising tool for application in photodynamic therapy (PDT) or vaccination. PDT is a well-established method in the treatment of tumours and bacterial biofilms using a combination of a photosensititive dye, light, and oxygen to generate singlet oxygen (MacDonald and Dougherty, 2001). A photosensitive dye is administered into or onto the tissue, exposed to light and excited to a triplet state which leads to the generation of singlet oxygen that destroys cancer cells or bacteria. Calcium phosphate nanoparticles can serve as a carrier for the photosensitive dye into tumour cells (Schwiertz et al., 2009).

Nanoparticles can be potentially applied for vaccination again viral infections. Traditional vaccination strategies are based either on live attenuated viruses or on inactivated pathogens. Mostly these strategies result in a humoral but not in a cellular immune response. Nanoparticles, applied for vaccination, may contain adjutants (e.g. immunoactive oligonucleotides, such as CpG or Poly (I : C)) to induce the maturation of antigen-presenting cells in vivo by interacting with elements of the innate immune system and virus specific antigens to trigger the immune response (Epple et al., 2010).

21.4 Nanoparticles for gene transfer (transfection)

The application of nanoparticles for gene transfer represents a wide and promising field, because it allows the long-term expression of certain proteins and a longer and more effective therapeutic effect. Gene transfer or transfection is the introduction of DNA into eukaryotic cells (Azzam and Domb, 2004). This process involves the transport of extracellular nucleic acids through the cell membrane all the way to the nucleus (Fig. 21.2). If DNA is brought into the nucleus, it can induce the production of specific proteins (Azzam and Domb, 2004; Kodama et al., 2006; McNeil and Perrie, 2006). There are two types of transfection: transient transfection, where plasmid DNA stays outside of the host chromosome, and a stable transfection, where new DNA is integrated into the chromosome and passed over to new generation.

The application of transfection in gene therapy is based on the introduction of genetic material into specific target cells or tissues to treat human disorders like restoring missing functionality or eradication pathogenic dysfunction. In molecular biology, transfection has become an important tool to analyze gene structure, function and regulation. It is a major challenge in gene therapy to develop an efficient carrier for the introduction of a therapeutic gene into desired cells (Racz and Hamar, 2006), as DNA alone cannot successfully enter cells (Wolff and Budker, 2005). As delivery systems, we can distinguish between viral and non-viral systems (Kurreck, 2009; Reischl and Zimmer, 2009). Non-viral delivery systems for gene therapy have been widely proposed as safer alternatives to viral vectors. Inorganic nanoparticles present a useful method to deliver nucleic acids into cells (see Sokolova and Epple, 2008, for a review).

Many studies have proven the applicability of ceramic nanoparticles as efficient carriers of nucleic acids. For such nanoparticles, size, morphology, surface charge and the ability to protect biomolecules from the degradation inside the cells are important parameters which also play a significant role in the success of gene delivery. Furthermore, the chemical composition of the nanoparticles is of importance to avoid an adverse cellular response (e.g. towards non-biodegradable particles like carbon nanotubes).

The cell membrane is a permeable phospholipid bilayer which constitutes the outer layer of a cell (Castella and Cremer, 2006; Sato and Feix, 2006). Different proteins are also found in the membrane bilayer, e.g. receptor proteins, recognition proteins and transport proteins. Small molecules can move into a cell by diffusion through channels or with the help of specific transport proteins (Chrispeels and Agre, 1994; Noskov and Roux, 2006).

There are different mechanisms for nanoparticle uptake into the cells, such as clathrin- or caveolae-mediated endocytosis and macropinocytosis (Sahay et al., 2010). Studies with inhibitors of endocytosis have shown that nanoparticles can be transported into a cell either by one or multiple mechanisms at the same time (Lorenz et al., 2006).

21.5 Nanoparticles for gene silencing

Nanoparticle systems can be also successfully applied for gene silencing. Gene silencing, RNA interference (RNAi) or so called antisense technology is the introduction of small-interfering RNA (siRNA) into the cytoplasm of the cells that specifically turn off the production of proteins (Meister and Tuschl, 2004; Mello and Conte, 2004; Mitterauer, 2004; Leung and Whittaker, 2005; Lu et al., 2005; Gilmore et al., 2006; Kurreck, 2009). It has been widely used as a powerful tool to inhibit a specific gene function for disease treatment (Kurreck, 2009).

Long double-stranded RNA (dsRNA) is processed inside a cell by the endonuclease Dicer into short fragments of about 21 nucleotides, siRNAs (Brummelkamp et al., 2002; Vermeulen et al., 2005). Then the siRNA is incorporated into the RNA-induced silencing complex (RISC) which is guided by the antisense strand of the siRNA to the complementary target messenger RNA (mRNA). Afterwards, the mRNA is enzymatically cleaved and protein synthesis is inhibited, as target mRNA is no longer available (Fig. 21.3).

Like DNA, siRNA alone cannot cross the cell membrane due to its negative charge (Reischl and Zimmer, 2009). There has been intensive work on developing effective delivery approaches for a therapeutic application of siRNA (Krishnamachari and Salem, 2009; Reischl and Zimmer, 2009). Calcium phosphate nanoparticles are very suitable for cellular delivery of oligonucleotides due to their high biocompatibility and good biodegradability (Sokolova et al., 2007). They can be prepared with a multi-shell structure to protect the siRNA layer from enzymatic degradation. Highly efficient gene silencing was shown in vitro (Sokolova et al., 2007; Zhang et al., 2010; Hu et al., 2012; Klesing et al., 2012).

21.6 Fluorescent nanoparticles for imaging

Fluorescence detection plays an important role in molecular biology and medicine. There are different types of fluorescent labeling agents, such as organic dyes, mainly based on fluorescein, rhodamine and cyanine, or fluorescing nanoparticles that often improve optical properties, such as an enhanced photostability and a larger Stokes shift, of organic fluorophores (Wang et al., 2006; Goesmann and Feldmann, 2010; Sokolova and Epple, 2011; Tabakovic et al., 2012). Fluorophores and other molecules can be covalently coupled to the surface of silica nanoparticles due to the presence of silanol groups (Ow et al., 2005; Ohulchanskyy et al., 2007; Li and Binder, 2011). Attractive possibilities are also offered by mesoporous silica-based nanoparticles which can also be functionalized on the inner surface (Vallet-Regi et al., 2007; Trewyn et al., 2007; Rosenholm et al., 2010). For instance, the selective functionalization of the inner and outer surface of mesoporous silica nanoparticles was used to develop a pH-sensitive fluorescent sensor (Slowing et al., 2007).

Due to ionic nature of calcium phosphate, a covalent functionalization of its surface as in the case of silica nanoparticles is not possible (Dorozhkin and Epple, 2002). However, HAP accepts many ionic substitutions both in anion and cation lattice positions. The introduction of lanthanoide cations leads to a fluorescent material (Buehler and Feldmann, 2006). Some research groups prepared europium-doped apatite by co-precipitating a mixture of Ca^2 + and Eu^3 + by phosphate in a water–ethanol mixture and used them as a luminescent probe in cell culture experiments (Doat et al., 2005). It was also possible to prepare fluorescent calcium phosphate nanoparticles by precipitation, followed by surface functionalization for colloidal stabilization, by incorporating small amounts of terbium and europium into HAP (Padilla Mondejar et al., 2007; Neumeier et al., 2011). Another way to prepare fluorescent calcium phosphate nanoparticles is the adsorption of fluorescing molecules on their surface. The precipitation method may be conveniently used for such purposes (Sokolova and Epple, 2011).

The incorporation of dyes into the nanoparticles usually increases their brightness and the quantum efficiency compared with that of dissolved free dye up to several times. By microemulsion-coprecipitation, the cyanine dye was incorporated into Cy3 into calcium phosphate nanoparticles. The molecular brightness was 17 times higher, and the quantum efficiency was 4.5 times higher in the case of Cy3-encapsulated calcium phosphate nanoparticles compared to free Cy3 molecules in the solution (Muddana et al., 2009).

In a different approach, the multi-shell calcium phosphate nanoparticles with fluorescent-labeled oligonucleotides can be synthesized. Uptake and transport of fluorescent nanoparticles were visualized by laser scanning confocal microscopy (Plate XIII, between pages 354 and 355) (Sokolova et al., 2012).

21.7 Nanoparticles in tissue engineering

Tissue engineering is a promising tool for regenerative medicine, but its application has been limited by the lack of suitable scaffolds (bioactive degradable substrate) which permit cellular ingrowth into large-scale objects (Armentano et al., 2010). Nanoparticles play a prominent role in biodegradable and biocompatible polymer matrixes to obtain nanocomposites with specific properties. The primary materials for scaffold fabrication in tissue engineering are biodegradable polymers, which can be divided into two groups: natural polymers, such as polysaccharides and synthetic polymers, such as poly(lactic acid) (PLA) or poly(glycolic acid) (PGA) (Boccaccini et al., 2006).

For nanocomposites the commonly applied nanoparticles are HAP (Woodard et al., 2007), carbon nanotubes (Shi et al., 2006), gold nanoparticles (Carotenuto and Nicolais, 2004), alumina (Al₂O₃), titania (TiO₂) or Bioglass® particles (Boccaccini et al., 2006), which improve the mechanical properties of the polymer scaffolds. It is now accepted that cells need a suitable environment to proliferate inside a scaffold. Nanotechnology and nanoparticles can be used in different ways to improve the scaffolds for tissue engineering. First, they can act as carriers for biomolecules which help to provide a cell-friendly environment and may also deliver biomolecules into cells (Dvir et al., 2011). Second, they may be a part of the scaffold to improve its mechanical or biological properties. In the case of HAP, this is especially interesting with respect to tissue engineering of bone and cartilage (Lode et al., 2009; Schliephake et al., 2009; Zhou and Lee, 2011; Uskokovic and Uskokovic, 2011). As nanoparticles can also be attached to material surfaces and act as delivery agents to the surrounding tissue, they may also be useful for a local gene therapy to stimulate bone growth (Keeney et al., 2010; Kovtun et al., 2012; Tautzen-berger et al., 2012). In general, cells react to the nanostructure of a scaffold material, and this can be exploited to enhance the cellular reaction and proliferation without changing the material itself (Navarro et al., 2008; Smith et al., 2009; Nuffer and Siegel, 2010; Dvir et al., 2011). Of course, fluorescent nanoparticles are of interest to visualize tissue-engineered structures. Zhang et al. (2004) have shown that the rate of scaffold bioactivity can be varied by the amount of Bioglass®, which is incorporated in the polymer matrix. Webster et al. (2000) reported that nano-sized titania in a scaffold can enhance the adhesion of osteoblasts and decrease the adhesion of fibroblasts. Novel polymer matrix nanocomposites are expected to be adaptive, biofunctional and to contain active components, so that they can be designed for specific purposes by varying the type of nanoparticles and the polymeric systems.

21.8 Conclusions and future trends

Taking the knowledge about different nanoparticle systems together, we can summarize the main characteristics for optimal nanoparticulate carriers: They should be small (up to about 150 nm), chemically well defined, effectively carry specific molecules inside the cells and be protected from degradation by enzymes. For in vivo application, cell- or organ-specific targeting is preferable to avoid side effects from the drug and the nanoparticles delivered to other tissues. A local application of nanoparticles, possible immobilized into or onto a scaffold, instead of the systemic application of drug molecules alone, can also circumvent systemic side effects. However, many questions still remain concerning the reproducible synthesis of well-characterized nanoparticles (e.g. the doses of nanoparticles and active biomolecules must be exactly known before any clinical application) as well as a better understanding of their interaction with components of biological fluids (like proteins) and their final fate in the body (e.g. dissolution, biodegradation, excretion). This necessitates an intimate collaboration of scientists from chemistry, materials science, biology and clinical medicine.