INTRODUCTION:

Nanomaterials are chemical substances having dimensions in nanometers (10^-9m). Their extremely small size is attributed with enhanced properties when compared to the same materials in normal scale. This includes physical, electrical, chemical, thermal, magnetic optical properties – that make them the materials of the future. Some of the known nanoparticles currently under study are, fullerenes- the buckyballs and Carbon Nanotubes which are carbon based and lattice like molecules. Liposomes are nanoparticles used widely in pharmaceutical and cosmetic industries. They were the first class of nanomaterials to be engineered to deliver drugs but they tend fuse together in aqueous environment hence it has been replaced by other Nanomaterials. Liquid crystals are another class of Nanomaterials having organic liquid crystals which resembles the natural biomolecules like proteins and lipids.

Other nanomaterials include the nanoshells, quantum dots-semiconductors that can emit light in various colors, Superparamagnetic nanoparticles –that get attracted to magnetic field but lose their magnetism when the field is removed, dendrimers- the branched structure nanomaterials, and nanorods.

Carbon Nanotubes (CNT) – A revolution in Nanotechnology

Carbon Nanotubes are a highly efficient and useful class of nanomaterials. They are tube shaped materials, made of carbon, with diameter on a nanometric scale. In fact they are a roll of graphite sheet, rolled up to form a single layer of tube with a continuous uninterrupted hexagonal mesh and the carbon molecules are at the apexes of these hexagons. Carbon Nanotubes have a diameter of the range <1nm to 50 nm and a length in the range of microns. They fall under three categories: single-walled Nanotubes (SWNT), double-walled Nanotubes (DWNT) and multi-walled Nanotubes (MWNT). Carbon Nanotubes are known for their rigidity, strength, tenacity, high thermal and electrical conductivity etc. They have a low specific density, high strength and percentage strain at break. Their thermal conductivity is >3000 W/m.k and possess even very high electrical conductivity. Their rigidity is due to very high tensile strength (as high as 63 gigapascals for MWNT) and elastic modulus. They have a low density of 1.4 g/cm^3.The SWNT can withstand a pressure of even 25GPa. Notably, its bulk modulus is greater than diamond. Also, in a Nanotubes say (n,m) if n=m, it is metallic and armchair. If n-m is a multiple of 3, then it is semiconducting. Interestingly, metallic Nanotubes have a current density of 4x10⁹A/cm², which is thousand times higher than metals like copper. When it comes to thermal conductivity, CNT are very good conductors of heat, the property they own is called ballistic conduction. This excellent conductivity is only along the tube but they are insulators across the tube axis. Some of the unique properties in CNTs are mentioned in table 1. These admirable properties in CNT make them obvious choice in many applications^[^1][2].

Table 1 Mechanical properties of CNTs: A comparison

Materials	Young’s modulus (TPa)	Tensile strength (GPa)	% Elongation at break
Stainless steel	0.186–0.214	0.38–1.55	15–50
SWNT	~1 (from 1 to 5)	13–53	16
MWNT	0.2–0.8-0.95	11–63–150

Functionalization of Carbon Nanotubes

The Single-walled Nanotubes (SWNT) are insoluble in organic solvents and water. This is a major challenge that has to be met in order to make these carbon Nanotubes facilitate interaction and delivery of biomolecules, especially it affects drug delivery. Many strategies have evolved so far to functionalize the carbon Nanotubes. There are many strategies for functionalization of carbon Nanotubes, notably noncovalent functionalization of carbon Nanotubes, Polymer functionalization of multi-walled carbon Nanotubes, Aromatic small-molecule-based noncovalent functionalization, Bio-molecule-based noncovalent functionalization, covalent functionalization of carbon Nanotubes. In general functionalization of CNTs involves attaching appropriate chemical groups to their sp²hybridized carbon scaffold to form nanostructures. This results in improved properties, mainly the solubility and dispersion^[^7]. Some known functional groups are polyethylene glycol (PEG) and ammonium-terminated triethylene glycol, a reactive intermediate which allows the synthesis of several functionalized CNT mixtures. These functionalized Nanotubes possess a larger volume inside them which serves as a large container to carry drugs, and large surface to attach numerous functional groups. The CNT may or may not have end caps. This is because the lack of end caps can facilitate easy accessibility of the drug inside the nanotubes. The covalent functionalization has enhanced the utility of the nanostructures. In this process, the SWNT react faster than the semiconductors. Moreover the process involves mixing of the functional groups with the polymer matrix resulting in a composite nanotube formation. A major method is functionalization with solvophilic molecules and non-covalent surface coating by amphiphilic molecules like low molecular weight surfactants and polymeric amphiphilic.

Incorporating Biomolecules into CNTs

It is a well established fact that CNTs cross membranes easily because of their enhanced properties and facilitate delivery of biomolecules like peptides, proteins, amino acids, and drugs into the cells. For this, firstly these biomolecules have to be attached to the CNTs. These biomolecules can be connected to the CNTs by a covalent linkage or non-covalent linkage^[^3]. There are methods, whereby, the SWNTs are linked covalently to peptide nucleic acid and these macromolecular wires were hybridized with complementary DNA. The DNA was added predominantly at or near the ends of the functionalized nanotubes. The MWNTs were prepared by the spray pyrolysis method. A green method was proposed by Yang et al, wherein problems of environmental pollution, equipment corrosion, and health problems were countered well. Some examples of CNT constructs are the flourescein probe in combination with the antifungal drug amphotericin B or flourescein and the methotrexate (antitumor agent). CNTs help reducing toxicity of the drug administered alone.

Delivery of therapeutic molecules into the cells

The nanotube facilitated oligonucleotide transport inside living cells and plasmid DNA gene delivery, both have yielded good results, which can make the CNT as non-viral delivery vectors. Using CNT systems some of the limitations of other non-viral vectors can be overcome, especially the poor pharmacokinetic profiles of the administered oligonucleotide and plasmid DNA conjugates, and the low levels of gene expression obtained. The systems that are currently used for delivering drugs are- dendrimers, polymers, liposomes but CNT is the most effective because of their high-loading capacities and their ability to be taken up by the cells. The drug encapsulation into CNTs improves the dispersion in water, bioavailability of the drug, and reduces toxicity. The CNTs are rendered in such a manner that they carry the drug in them, move towards the cells, penetrate into them and reach specific locations in the cell. Finally they break apart to deliver the drug as desired. Several cells take up CNT, including the immune cells, such as macrophages, monocytes, natural killer cells, dendritic cells, T and B cells. CNT does not affect the functions of these cells and are thus less toxic. CNTs can induce an innate immune response which is dependent on the type of functionalization as well as the size^[20][21].

CNT in therapeutics: targeted drug delivery

Once the CNTs are synthesized, functionalization is carried out to make them biocompatible. Once they become biocompatible and ready to carry load of drugs, they are treated with the desired biomolecules and introduced into the body. The CNT specifically targets the cells because of its composition and delivers the therapeutic biomolecules into the cell. The therapeutic value of the delivered drug is determined some time after the delivery. In the preliminary stages of the research, B-cell epitope of Foot and Mouth Disease Virus (FMDV) were covalently attached to the amine groups on CNT, using a linker. Immunization of mice with FMDV peptide-nanotubes conjugates provoked high antibody response. Moreover these antibodies were peptide-specific and surprisingly antibodies were not produced against the CNTs.

Fighting cancer with CNTs

As a treatment to cancer mediated by the CNT, the carbon Nanotubes are coated with folic acid (vitamin B). The cancer cells have a lot of folic acid receptors, proteins on surface of a cell that binds the folic acid. The cancer cells bind folic acid coated carbon Nanotubes and then the carbon Nanotubes gets through the cancer cell. Once inside, by using an infrared laser carbon Nanotubes are heated up. Carbon Nanotubes absorb infrared light and heat up. Thus by shining a laser, the cancer cells with the carbon Nanotubes gets selectively heated up, while the normal cells don’t heat up at all. It doesn’t take much heat to kill a cell, within only a few degrees the cell begins to die. Hence the cancerous growth is stopped^[11][17]. Tumor antigens are characteristics of cancers. There are two types of tumor antigens – Tumor Specific Antigens (TSA) present exclusively on tumor cells and Tumor-Associated Antigens (TAA), present in some normal cells also. Tumor specific antigens include even products of the ras and p53 genes. Mutation in these genes produce products which are tumor associated proteins. Table 2 represents the various tumor antigens of corresponding tumors.

Table 2 Antigens associated with tumors

Tumor antigen	Tumor type
Carcinoembryonic antigen (CEA)	Bowel cancers
Alphafetoprotein (AFP)	Germ cell tumors
tyrosinase	Malignant melanoma
Ras and p53 products	Various tumors

These tumor antigens are therefore tumor markers in most cases against which the CNT mediated lysis can be done, wherein the CNT are designed to identify the cellular target, penetrate and kill the cell by heating mechanism.

Epidermal growth factors and targeted drug delivery

The family of epidermal growth factor receptor (EGFR) plays an important role in determination of the cell lineage, and in cell survival in the adult. Activating mutants and over-expression of these EGFR results in oncogenesis by inducing cells to proliferate. The EGFR family of receptor tyrosine kinase comprises the EGFR (ErbB1), ErbB2/HER2/neu, ErbB3/HER3 andErbB4/HER4. There are various alterations that affect ErbB receptors in human cancers. In about 25 % of breast cancer ErbB2 expression is high. ErbB2 over-expression is also associated with ovarian, gastric and bladder cancers. In glioblastomamultiforme tumors ErbB1 is often mutated in the intracellular domain rendering the tyrosine kinase constitutively active. Furthermore, some tumors increase the production of EGF-related growth factors leading to the persistent activation of ErbB receptors. Viruses also utilize the EGF signaling pathway in many ways Hepatitis B virus and Epstein-Barr virus both activate EGF receptor expression during invasion. The avian erythoblastosis virus encodes an active form of the EGFR,, while the human papilloma virus E5 appears to block the degradation of activated receptors resulting in the internalized receptor being returned to the plasma membrane. Therapies that target the EGFR in cancers include the use of suramin that prevents binding of EGF with their corresponding receptors. By using functionalized carbon Nanotubes, the EGFR on cancerous cells can be prevented from bind the EGF.

Ensuring a safe targeted delivery- an important aspect in therapeutics

For a safe delivery of drugs, a careful measure should be taken as respiratory diseases or systemic immune responses may be caused. It is still unknown how long SWCNTs and MWCNTs persist in the tissues after inhalation exposures. Studies in mice showed that the MWCNTs remain in tissues for several months. For an effective therapy the nanoparticles have to be in tissues for sustained drug delivery but the persistence will prove toxic to the tissues consequently^[^18]. Biodegradable nanoparticles are considered to be the best for drug delivery to tissues but they are still valuable when they are cleared quickly from tissues. These nanoparticles are termed as biopersistent nanoparticles. An ideal design is constructing functionalized Nanotubes having limited half life in tissues- which render them less potential to cause chronic side effects like tissue fibrosis. Tumor-targeting SWCNTs have been constructed from water soluble CNTs which involves covalently attaching monoclonal antibodies to the functionalized CNTs. A safe and effective delivery can be ensured with low dosage of drugs combined with high dispersability of the CNTs, effective functionalization and the biodegradability that can prevent chronic side effects.

CONCLUSION:

From this review of the potential and ongoing utilization of Carbon Nanotubes to target specific cells in the body and delivering therapeutic molecules to cure diseases is studied. Carbon Nanotubes (CNT), which have high strength, load carrying capacity and very small size (in nanometers), are subjects of interest in medicinal field for penetrating cells and delivering therapeutic biomolecules. Recent developments have shown clearly that the drug delivery using CNTs is far more beneficial because of reduced toxicity and effectiveness in targeting drugs to the cells. Cancer being a very invasive and difficult disease to treat, can now be treated effectively using the nanobiotechnological concept. The major success in treatment of any disease is targeting diseased cells while sparing the healthy cells without any damages and side effects. But in case of cancers, most treatment like radiotherapy, chemotherapy or combinational therapies, all targets cancer cells but only at the cost of the neighboring healthy cells. Thus the CNT mediated drug delivery that specifically targets cells is a novel strategy that will have widespread applications in the near future.

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Received on 26.08.2013 Accepted on 12.09.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 209-212