2.2.3. Nano-Diamond

Nano-diamond is considered to be another type of carbon-based nanomaterial and is rarely used in cancer treatment. ND is considered an allotrope of carbon with an average particle size between 4 to 6 nm and which in aggregation could be recorded as having particle sizes of 100 to 200 nm. The surface of each ND possesses functional groups which allow for a different series of active molecules to be conjugated to it, including anticancer drugs [39,40]. The appearance of functional groups on ND surfaces works in the form of a covalent bond between the ND and other material, such as a polymer, which improves the solubility of the ND powder in various solvents [39]. NDs have an excellent biocompatibility, meaning they can be used in chemotherapy. For example, the biocompatibility of a fluorescent ND powder with a particle size of 100 nm has been confirmed for kidney cells by Yu et al. [41].

Some trials which have reported the utilization of ND in biomedical applications are summarized below. Carboxylated NDs have been utilized to improve the solubility in aqueous media of some anticancer drugs. Purvalanol A and 4-hydroxytamoxifan, known to be effective drugs for breast and liver cancers, have been conjugated with NDs [42]. Moreover, efficient drug loading capacity has been confirmed for NDs with negative charge which are accompanied by both NaCl and cationic DOX ions to achieve better treatment for HT-29 colorectal cancer via a more efficient method, in comparison to other carbon-based materials [43].

Upal et al. [44] have studied, using *invitro* testing, modified and surface-unmodified (–NH2 and –COOH) ND for its ability to carry the anti-HIV-1 drug efavirenz and its cytotoxicity. It was found that there was a highly significant drug loading capacity for the unmodified ND conjugated drug formulation in comparison to the surface-modified ND, which had a very low toxic effect. NDs have shown few successful trials on an in vitro basis but animal studies need to be carried out in the near future to figure out their effect on the vital organs and their clearance mechanism. However, NDs suffer from the same restrictions as carbon-based materials, as mentioned above.

### *2.3. Mesoporous Silica Nanoparticles (MSNPs)*

One common types of NP which is used as a drug delivery carrier is the solid mesoporous silica nanoparticle. Mesoporous NPs are particles in which the core of the particle is solid in nature while the shell or the particle's surface is porous in nature. Alternatively, the particle itself is porous in nature. MSNPs are inorganic particles comprising of a honeycomb-like porous structure with empty channels (pores) of nanosized dimensions [45]. There are two types of mesoporous silica according to their particle size: mesoporous silica microspheres and mesoporous silica nanospheres. Although the use of mesoporous silica microspheres is important in numerous non-biological applications, they are not appropriate for use in many biomedical applications. However, the average particle diameter of a typical MSNP is ~100 nm with pore volumes ~0.9 cm<sup>3</sup>/g, pore volume surface areas ~900 m<sup>2</sup>/g, and pore sizes ~2 nm [46]. A hexagonal crystal structure has been recorded for MSNP porous channels (Figure 5).

Slowing et al. [47] have studied the implications of surface functionalization of MCM-41-Type MSNPs on the mechanism and efficiency of endocytosis different charge profiles on HeLa. The study concluded that the ability of MSNPs to escape endosomal entrapment was influenced by surface functionalities as this is considered to be a key factor in the designing of compounds that are effective for intracellular delivery.

**Figure 5.** TEM images of mesoporous silica nanoparticle (MSNP) material recorded from the direction (**a**) parallel or (**b**) perpendicular to the long axis of the meso-channels [45]. Reproduced with copyright permission from Elsevier, 2008.

### *2.4. Gold Nanoparticles (GNPs)*

Gold nanoparticles are considered excellent carriers for anticancer drug delivery because of their many unique features, such as their tunability in surface characteristics and particle size, their inertness, and their biocompatibility. Sabu et al. [48] have demonstrated that gold NPs can be loaded with anticancer agents. Paclitaxel (an anticancer drug) has been conjugated covalently by Gibson et al. [49] to gold NPs of size 2 nm, and the system has been found suitable to be used as a self-therapeutic. Dhar et al. [50] have conjugated gold NPs with DNA (producing DNA-AuNPs) to be employed as carriers of platinum compounds for the treatment of cancer. Cellular internalization of the tethered platinum was more effective than free cisplatin in killing cancer cells.

Brown et al. [51] have reported that oxaliplatin in its active form conjugated with gold NPs has possessed an effective killing ability with regard to cancer cells compared to pure gold NPs. It has also been proven that BSA-capped gold NPs can effectively be used as a nanocarrier for anticancer drug MTX and has been considered to be an anticancer for MCF-7 breast cancer cells when compared to non-capped NPs, as earlier declared by Yu-Hung et al. [52]. Furthermore, core/shell gold NPs have been fabricated and evaluated in order to be used as carriers for dual treatment where a radiosensitizer was presented by GNPs and the anticancer drug (DOX) [53].

Manivasagan et al. [54] have demonstrated a new method for the production of gold NPs to be used as a drug carrier or to be useful in photo-acoustic imaging (PAI). In particular, they have synthesized multifunctional doxorubicin-loaded fucoidan-capped gold NPs (DOX-Fu AuNPs). Dhamecha et al. [55] have also reported that GNPs work as an effective drug carrier for targeting DOX to fibrosarcoma cancer cells. They have been observed to subsequently reduce DOX-associated cardiac toxicity and myelo-suppression. DOX was seen to be absorbed onto GNPs with a high drug loading capacity, and industrial scalability of the process was demonstrated in their research [55]. Scientists working with GNPs face very challenging issues related to their removal from the human body along with their accumulation in the liver and kidney.

#### *2.5. Other NPs Used as Drug Delivery Carriers*

Some other inorganic materials (Table 1) have been used as drug delivery systems, and include functional multilayer fluorescent nonporous silica SNPs with an external shell, e.g., those containing primary amino groups [56]. AL-Ajmi et al. [57] have also prepared crystalline ZnO NPs using organic precursor techniques. These ZnO NPs were used as a drug carrier to deliver 5-Fluorouracil (5 Fu).


**Table 1.** Inorganic nanocarriers for drug delivery.

#### **3. Inorganic NPs for Hard Tissue Regeneration**

The ideal nanostructured composite presented in the human is bone, which is considered the typical model of a hierarchical microstructure [58]. Nonstoichiometric nanocrystalline hydroxyapatite (HAP), (Ca10(PO4)6(OH)2), which is 20–40 nm in length, is the major inorganic component of bone, and Type-I collagen, which is ~300 nm in length, is its major organic counterpart. It has been revealed that HAP nanocrystals cover bone collagen molecules to form nanocomposites fibers. HAP C-axes are generally aligned along the collagen molecules in these nanocomposites [59].

Recently, the synthesis of nanomaterials for bone repair has been explored based on the simulation of various bone properties. One significant characteristic of materials used for bone regeneration is their mechanical property, and this property is possessed by di fferent nanomaterials [60,61]. The other approach is to carry out surface modification at the nano-level, as this can provide an improved matrix for osteoblasts to grow and function (Figure 6) [62]. Several types of materials are suitable as biocompatible materials for bone healing with applications possible for metallic oxides (aluminium oxide, zirconium dioxide, and titanium dioxide), the CaPs family (hydroxyapatite, tricalcium phosphate (TCP), and calcium tetraphosphate) and glass ceramics (bioglass and ceravital) [63].

In addition, nano-sca ffolds have superior properties in comparison to micro-sca ffolds, owing to their higher porous microstructures that can mimic a real extracellular matrix (ECM), their porosity, and their other physical properties. This in turn improves their osteoblast adherence and viability [60,64,65]. Moreover, NPs enable the delivery of drugs and growth factors to promote healing and functional recovery. Modifying the surface of these biocompatible materials at the nano-scale for bone healing purposes has also been studied [66].

**Figure 6.** NPs used to repair a bone fracture: in cases of bone fracture, nanomaterials have been implanted into the target area (adapted from [62]).

## *3.1. Carbon Nanotubes*

Owing to the impressive mechanical characteristics of carbon nanotubes, the idea of reinforcing brittle ceramic such as HAP using CNTs for the treatment of bone disease and fractures has been considered in the last three decades. Zhang and Kwok [67] have investigated the bone healing ability of HAP-TiO2-CNT NPs. They noted the formation of an apatite layer on the surfaces of monolithic HAP and HAP-CNT and HAP-TiO2-CNT NP coatings after 4 weeks of soaking in Hanks' solution. In addition, no remarkable effect was recorded on the formation of the apatite layer on the surfaces of TiO2 and CNT when added to HAP. Furthermore, it has been reported that the addition of CNTs to HAP has improved the mechanical properties of the final composite as recorded by nano-indentation technique. These results revealed that the higher the addition of CNTs the greater the mechanical properties which were achieved compared to pure HAP [68].

A different application for the CNTs that has been reported aside from their being used as reinforcing material during bone regeneration is as a biosensor for bone regeneration [69]. Using NPs as a delivery system for bio-active molecules from bone healing, for example, both genes and proteins can be delivered to enhance propagation of osteoblasts, angiogenesis, and a reservoir for the necessary calcium salts [69]. Furthermore, one study has been conducted using multi-walled carbon nanotubes (MWCNTs) impregnated into HAP and the dip coating of a nanocomposite onto a titanium alloy (Ti-6Al-4V) plate to enhance the surface roughness of the implant. The authors noted that the mechanical properties of the HAP coating were enhanced and the surface roughness was reduced upon the addition of low concentrations of MWCNTs in comparison with pure HAP. Moreover, normal cell attachment and the growth process were confirmed by cell studies [70].

Other studies have discussed the cytotoxicity of titanium alloy implants coated with plasma-sprayed CNT-reinforced HAP embedded in rodents' bones, and it has been observed that there is no adverse effect or cytotoxicity associated with the addition of CNTs to bone tissues [71]. Balani and Agarwal have also confirmed the non-toxicity of a HAP-CNT coating on Ti-6Al-4V implants and have observed the ultimate growth of human osteoblast cells near the CNT regions [72].

CNT-based 3D networks have been found to be suitable for cell growth as a biomatrix scaffold [73]. In [73] a 3D network was produced by exerting chemically-induced capillary forces in order to manipulate the vertically aligned CNT array and the authors found that those networks seemed to be suitable for cell growth as a scaffold (Figure 7).

**Figure 7.** Scanning electron micrographs of L929 mouse fibroblasts growing on a multi-walled carbon nanotube (MWCNT)-based network [73]. Reproduced with copyright permission from the American Chemical Society, 2004.

Another scaffold based on MWCNTs has been reported by Abarrategi et al. [74]. Here, the MWCNTs were functionalized by chitosan (MWNT/CHI) and these nanocomposites were affirmed to be a scaffold for tissue engineering. In addition, this study showed that the MWNT/CHI scaffolds implanted subcutaneously accompanied by recombinant human bone morphogenetic protein-2 (rhBMP-2) for 3 weeks had increased the growth of C2C12 cell lines. Afterwards, the MWNT/CHI scaffold composite was replaced by cells and bone regeneration was observed. In this study the authors confirmed the suitability of the MWNT/CHI scaffolds for utilization in bone reconstruction. Apart from hard (bone) tissue regeneration, CNTs have also been utilized for soft tissue regeneration (nerve) in order to assess cells behavior [75]. For this purpose, Keefer et al. [76] designed a neural network made of tungsten and stainless-steel wires and the electrode's wires were coated with CNTs by electrochemical methods.

### *3.2. Titanium Dioxide (TiO2)*

Titanium dioxide or titania (TiO2) has been used as a bioactive coating and there has been consideration given to using TiO2 within the coating as reinforcement as a technique for improving the mechanical reliability of HAP. Also, it has been proven that TiO2 has the ability to induce osteoblast cell adhesion and growth [77,78]. It has been determined that the smaller the titanium grain size the greater the osteoblast adhesion. This may be because of the higher surface area attributed to the TiO2 nanoparticles. Various techniques have been used to improve the TiO2-HAP nanocomposite coatings for biomedical applications in order to enhance the strengths of coating and adhesion, reducing a loss of implants because of potential failure at the metal-coating interfaces and achieving an increase in biological response [79,80].

Kuwabara et al. [80] tested the bone formation ability on TiO2-HAP nanocomposite coatings with a thickness of 100 nm. They cultured the osteoblast cells, which were derived from rat bone marrow, on the surface of coatings with different electrical charges and found that the HAP- and TiO2-coated surfaces demonstrated a higher influence with regard to adhering to and propagation of cellsin comparison to pure HAP or pure TiO2-coated surfaces. Ahmed et al. [81] revealed that the proliferation of mesenchymal stem cells (MSCs) was enhanced after using a Se/Ti nanocomposite. Furthermore, another study has unveiled a new composite which was generated using HAP/chitosan bioactive nanocomposites [82].
