Sarwar Beg, Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India. E-mail: email@example.com
Objectives Carbon nanotubes (CNTs) have attracted much attention by researchers worldwide in recent years for their small dimensions and unique architecture, and for having immense potential in nanomedicine as biocompatible and supportive substrates, as a novel tool for the delivery of therapeutic molecules including peptides, RNA and DNA, and also as sensors, actuators and composites.
Key findings CNTs have been employed in the development of molecular electronic, composite materials and others due to their unique atomic structure, high surface area-to-volume ratio and excellent electronic, mechanical and thermal properties. Recently they have been exploited as novel nanocarriers in drug delivery systems and biomedical applications. Their larger inner volume as compared with the dimensions of the tube and easy immobilization of their outer surface with biocompatible materials make CNTs a superior nanomaterial for drug delivery. Literature reveals that CNTs are versatile carriers for controlled and targeted drug delivery, especially for cancer cells, because of their cell membrane penetrability.
Summary This review enlightens the biomedical application of CNTs with special emphasis on utilization in controlled and targeted drug delivery, as a diagnostics tool and other possible uses in therapeutic systems. The review also focuses on the toxicity aspects of CNTs, and revealed that genotoxic potential, mutagenic and carcinogenic effects of different types of CNTs must be explored and overcome by formulating safe biomaterial for drug delivery. The review also describes the regulatory aspects and clinical and market status of CNTs.
Nanotechnology is a branch of science that is based on the applied principles of physics, electronics, engineering and material science at a molecular or submicron level. The term ‘nanotechnology’ is derived from a Greek word ‘nano’ meaning ‘dwarf’, hence it relates to materials of very small size ranges (0.1–100 nm). It covers an entire range of composite materials, including nanoparticles, nanoemulsions, dendrimers, quantum dots, nanocells, Xpclad nanoparticles, etc. Among these, carbon nanotubes (CNTs) have evolved as a novel nanocarrier system having a wide variety of applications. As the name indicates, these are hollow tube-shape substances of a polymeric nature made from monomers upon polymerization to give a tube-shaped structure. CNTs were discovered by Iijima in 1991 as an allotrope of carbon which rolled into cylindrical single-walled tubes. Further, multi-walled CNTs were made from graphitic rods by subjecting them to a discharge of an electric arc.[2,3] The higher surface area, conductivity, high tensile strength, typical length-to-diameter ratio up to 28 000 000:1 as well as potentially greater absorption abilities due to their cylindrical structure make them a novel nanomaterial for drug delivery and biomedical application.
CNTs are basically made up of pure carbon and belong to the family of fullerenes (C60), and are available in various geometrical shapes such as spherical, ellipsoidal or tubes.[5,6] Nowadays several members of fullerenes are being synthesized and categorized as a specific family according their carbon number as C20, C30, C36, C70 and C78 and are called graphenes. CNTs are graphenes with a hollow cylindrical tube-like structure, where sp2 hybridized carbon atoms are arranged in a specific pattern to form hexagonal structural units. This imparts higher C–C bond stiffness, tensile strength of 150 Gpa (Giga Pascal) and Young's modulus of approximately 1, which is a measure of nanotube stiffness. Chemically, CNTs originate from synthetic graphites on exposure to an electric arc or laser beam source.[8,9]
Recently, CNTs have been considered as an ideal nanocarrier in the field of nanomedicine, which is an applicative field that uses concepts of nanotechnology, biology and medicine. The various applications of these include: controlled drug delivery; targeted delivery of drug molecules to a specific site, to cancer cells; delivery of bionanotechnology products; as an additive to improve the solubility of poorly water soluble drugs; vaccines delivery, hormone and enzyme delivery; and as a nanofluidic device in drug delivery. CNTs have also been used in several biomedical applications, such as diagnostic tools, like nanosensors, nanorobots, nanoprobes and actuators for identifying various diseases. The literature survey on CNTs suggests that this novel nanocarrier has multiple biomedical applications. However, proven success in uses for different purposes may not translate to the use of CNTs in medicine for humans. For this purpose, it must be proven safe, albeit little data on cytotoxicity and limited control over functionalized-CNT behaviour have put a question mark on its success. Therefore, this review documents the advancement in biomedical application of these novel nanotubes as carriers, and also highlights the toxicity and regulatory aspects and current market status of CNTs.
Classification of CNTs
Nanotubes are mainly classified into two types depending upon the structure: (1) single-walled carbon nanotubes (SWNTs) and (2) multi-walled carbon nanotubes (MWNTs). In SWNTs only one sheet of graphene is arranged to give a cylindrical structure which is one atom thick, having a radius of up to 1 nm. The SWNTs are closed at both ends with cap-like structures during the process of synthesis and the rings form ends by C–C bonds. MWNTs consist of a few layers of graphene sheets (2–10), more than one atom thick, having an external diameter of >10 nm (Table 1). Also the SWNTs are structurally different from MWNTs by having different basic arrangements of the carbon atoms to give three different structural configurations: armchair arrangement where the chiral vector is characterized by the presence of chairs perpendicular to the tube axis; zigzag arrangement where the tube is characterized by having a V-shape perpendicular to the tube axis; and chiral or helical arrangement, which is unidentical from the above two types of arrangement. The degree of chirality in CNTs is a representative measure of their electrical as well as conductivity properties and helps in designing a wide variety of nanoelectronics instruments. Apart from this, chirality also determines the diameter of nanotubes, and metallic or semi-metallic characteristics.[7,12]
Table 1. Basic differences between single-walled and multi-walled carbon nanotubes
Granular or fluffy black powder, sometimes with a shiny metallic appearance
Mostly granular and fluffy black powder
Appearance on electron microscopy
Appears as aggregated bundles
Qualitative analysis using Raman spectroscopy
Gives characteristic peaks
There are no such characteristic peaks observed
Soluble in water and ethanol, form aggregates soon after sonication
Quite soluble in water but form slightly transluscent dispersions
Good electrical conductivity
Bad conductors of electricity
MWNTs are of two types depending upon the pattern of arrangement of the graphitic sheets. One is a Russian-doll model structural arrangement where graphite sheets are arranged in concentric layers, for example a sheet of (0, 8) SWNT enclaved within a large diameter (0, 10) SWNT. The second model is known as a parchment model in which a single sheet of graphite is rolled around itself, resembling a scroll of parchment or a rolled newspaper. Apart from these, there is another type of nanotube resembling SWNTs, known as double-walled nanotubes (DWNTs), having structural similarity with SWNTs; they are of considerable interest in the pharmaceutical field.
In addition CNTs can be classified into three different prototypic structures depending upon their shapes (Figure 1). These includes carbon nanohorns (CNHs), nanobuds and nanotorus.
CNHs are identical to CNTs in structure, prepared from graphites by a laser ablation method. These are single walled nanomaterials made from sheets of graphene (2–3 nm diameter) with the tip capped by a five-member ring. Unlike CNTs, a CNH resembles a horn-like structure having a cone shape with one broad open end and a narrow open end. They look like petals of a dahlia and are often described as ‘dahlia-like’ aggregates.[15,16] These have the unique property of changing diameter as their length increases. These have several applications in drug delivery and are used in drug delivery to cancer cells.[17–20] Nanotorus are theoretically described carbon nanotubes of structures bent into a torus (doughnut shape). Carbon nanobuds are newly discovered materials synthesized from CNTs and fullerenes having properties intermediate between them. They have a shape and structure similar to CNTs, but are characterized by the presence of an external outgrowth (bud) on the external surface.
Apart from these, CNTs are also classified in several ways depending upon their structural modifications, method of preparation and solubility properties, for example functionalized CNTs, solvent dispersed CNTs, surfactant assisted CNTs, nanotubes assisted with biomolecules.
Methods of preparation of CNTs
CNTs are widely accepted nanocarriers in the field of drug delivery and in biomedical application. For pharmaceutical use, the CNTs produced must be of good quality, free from impurities and carbonaceous matter and should not have damaged structures.
CNTs can be synthesized naturally by heating carbon black and graphites in a controlled flame environment. However, nanotubes produced by this method are mostly irregular in size, shape, mechanical strength, quality and purity because of the uncontrollable natural environment. Nowadays CNTs are synthesized by artificially developed methods of considerable interest to the pharmaceutical scientist, which include electric arc discharge (EAD), laser ablation technique (LA) and catalytic chemical vapour deposition (CVD) methods. In addition, several other techniques such as plasma enhanced chemical vapour deposition (PE-CVD) and high pressure carbon monoxide disproportionation process (HiPCO) technique are of recent interest. The HiPCO technique can be used for the catalytic production of SWNTs in a continuous-flow gas phase, using carbon monoxide (CO) as the carbon feedstock and Fe(CO)5 (iron pentacarbonyl) as the iron-containing catalyst precursor. The size and diameter distribution of the nanotubes can be roughly selected by controlling the pressure of the CO. This process is promising for bulk production of CNTs. Different methods of preparation produce CNTs with different physical and mechanical properties. The type of CNTs produced, solubility, mechanical properties, quality, purity and yield usually differ from one method to another (Table 2).
Table 2. Comparison of various techniques for preparation of carbon nanotubes
CNTs, carbon nanotubes; CVD, chemical vapor deposition method; EAD, electric arc discharge technique; HiPCO, high pressure carbon monoxide; LA, laser ablation technique; MWNTs, multi-walled carbon nanotubes; SWNTs, single-walled carbon nanotubes.
CNTs prepared from this method have higher Young's modulus with least defects as compared with other methods
As compared with EAD technique, LA can yield both SWNTs and MWNTs
The most economical method for production of CNTs, forming nanotubes with least production variables
Used for the catalytic production of SWNTs, and helps in production of nanotubes of any diameter
Inability to produce SWNT/MWNTs with uniform diameter, and mostly a time-intensive method
CNTs obtained are very narrow in diameter and form tangled ropes and bundles along with impurities
This method is found to have broad spectrum of advantages, hence advantages outweigh disadvantages
A costly technique
Method of choice
Not useful for industrial scale
Mostly used for laboratory scale and rarely for industrial scale
This is regarded as the method of choice for industrial scale
Promising for bulk production of carbon nanotubes
In the electric arc discharge method, two different types of electrodes (anode and cathode) are used. CNTs are produced at the end of the anode, which consists of pure graphite. The nanotubes are produced by high voltage beams (around 100 amp) of electrons produced by the electric arc, which bombards the graphite surface. The electric arc is a plasmon setup made across CNTs resulting in the formation of CNTs on a substrate.[8,24,25]
In the laser ablation technique, the nanotubes are produced by allowing a specific spectrum of laser beam to strike on the graphitic target using transition metal as catalyst, which produces both SWNTs and MWNTs. This method uses two different laser sources, as primary laser and secondary laser beam. The initial bombardment is done with the primary laser followed by a secondary laser beam to finally produce CNTs of high quality. This method has the advantage of producing nanotubes desired for particular applications.[26–28] However, this method has the drawback of being time consuming and costly.
The catalytic chemical vapour deposition (CVD) technique works on a principle completely different from the above methods. Here, the feed material used is present in the form of a mixed vapour phase (vaporized carbon along with an inert gas). This feed material is passed through a hot furnace where it decomposes to give CNTs deposited on the surface of a substrate. The substrate is made by embedding nanometre-sized nickel or cobalt particles, or a combination of both, as a catalyst on its surface and is generally heated to approximately 700°C.[26,29,30] The variables, including nanotube diameter and tensile strength, depend on the size of the metal particles. These can be controlled by masked deposition of the metal, by annealing or by plasma etching of a metal layer. For commercial production, the nano-sized metal particles are mixed with MgO or Al2O3 to increase catalyst support and increase the surface area for higher yield. Additionally, several newer techniques namely plasma-enhanced chemical vapour deposition (PECVD), CoMoCat process, thermal CVD, laser-assisted CVD and high pressure CVD have been developed for high quality CNT production.[32–36]
Characterization of CNTs
CNTs used in biomedical and drug delivery applications need to be characterized extensively to determine their fundamental properties. The characteristic properties include diametric size, shape, purity, solubility, electromechanical properties and thermal conductivity. Several instrumental and analytical techniques have been developed for characterizing nanotubes: scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), infrared spectroscopy (IR), nuclear magnetic resonance (NMR), Raman spectroscopy and atomic force microscopy (AFM) (Table 3). Each of the techniques has its own identity and advantages to help in determining CNT features. These methods have been used for several years, but there is no standard procedure developed in industry and no strict regulation.[7,38] The purity of the CNTs is the most important property, because commercially available CNTs are mostly contentious. Hence safety data sheets are used which help in indicating the purity of CNTs.
Table 3. A brief account of various techniques for characterization of carbon nanotubes
Instrumental methods of analysis
CNTs, carbon nanotubes; IR, Infra-red spectroscopy; MWNTs, multi-walled nanotubes; NMR, nuclear magnetic resonance; SWNTs, single-walled nanotubes.
Thermo gravimetric analysis (TGA)
Quantitative determination of the amount of carbon and non-carbon matter in CNTs, helps in assessment of purity, thermal stability and nanotubes homogeneity
Easy estimation of quality is possible
Unable to identify the metallic impurities present in CNT sample
(a) Novel technique for characterization and evaluation of SWNTs(b) Gives information about radial breathing mode (RBM) of nanotubes, which includes various vibrational transitions like radial movements, expansions and contractions
Gives RBM peaks, which help in distinguishing SWNTs from other carbonaceous material and also identifies the dispersion state of SWNTs in solution
Unable to evaluate the impurities like graphites, fullerenes, amorphous carbon
As described earlier, CNTs have emerged as efficient drug delivery carriers in the biomedical and drug delivery field. Several applications of CNTs are listed below.
CNTs in controlled drug delivery
In the 21st century, the concept of practising safe and effective medicine with a high therapeutic potential is of great importance. Inspired by the ‘magic bullet’ concept proposed by Paul Erlich, devices for drug delivery to a targeted site in a controlled manner were developed and are still under extensive research. Various types of carrier systems already used for controlled drug delivery include polymeric systems, dissolution controlled, diffusion controlled and osmotic pressure controlled systems, hydrogels, liposomes, microspheres and nanoparticles.[40,41] Recently CNTs have attracted much attention due to their ability to deliver drug molecules to a specific site in a controlled manner.[17,42–44] They are used in the controlled release of drugs as well as delivery of genetic material such as DNA, genes and antibodies. Drugs or biomolecules can be loaded inside the hollow tube or can be directly attached to the walls of CNTs. It has been postulated that the tubes develop a combined Van der Waals force as well as a hydrophobic interaction force, where the Van der Waals force is consistently more important for insertion of drug molecules. The special characteristic that makes nanotubes promising drug delivery carriers is their hollow monolithic structure having an outer and inner core, which can be modified by the method of functionalization with desired groups on the outer and inner areas. This helps in insertion of required drug molecules in the inner core environment while the outer surface can be modified for achieving biocompatibility and biodegradation. Functionalization is a process of chemical synthesis where desired functional groups can be introduced onto the wall of CNTs for various applications. The functionalized CNTs can be used for the purpose of enhanced biocompatibility within the body, enhancement of the encapsulation tendency and solubility, and multimodal drug delivery and imaging.[47–50]
Several studies on the fate of nanotubes in the body suggested that CNTs loaded with drug molecules could easily pass into cells and also have the tendency to enter the cell nucleus, thus both cellular and nuclear levels of targeted delivery can be achieved. Pantoratto et al.[52,53] examined the cellular and nuclear uptake of CNTs by TEM, which showed better uptake of CNTs with targeted action after functionalization due to the attachment of surface functional groups on cellular structures. They suggested that the most important factor that affects the fate of a CNT in the body is its dimension. Hence CNTs must be of nano size to prevent cellular opsonization as a harmful microbial intruder.
In 2009, Yang and coworkers found that amine-functionalized mesoporous silica nanotubes (NH2-MSNTs), synthesized from needle-like nanoparticles of calcium carbonate (CaCO3) as inorganic templates and post-modification with 3-aminopropyltriethoxysilane via a sol–gel route having additional functionalization with blue fluorescent CdS quantum dots, were able to deliver the anti-inflammatory drug ibuprofen in controlled manner. Furthermore, ibuprofen release in simulated body fluid revealed that the drug was released from amine-functionalized systems at a significantly lower rate compared with that from amine-free systems and the incorporation of CdS quantum dots had almost no effect on ibuprofen release.
Similarly, Zhang et al. prepared nanohybrid hydrogels for controlled drug delivery. These nanohybrid hydrogels were obtained from the hydrogen bond self-assembly of poly (methacrylic acid) networks and carboxyl-functionalized multi-walled carbon nanotubes (MWNT-COOH). Such hydrogels showed low micropore densities and large mesh sizes with an increase in MWNT-COOH content. Upon contact with water, these nanogels swelled due to pH responsiveness and produced controlled release profiles of delivery for theophylline and aminophylline by modulating pH values.
Recently, a new generation of nanomaterials known as ‘smart bio-nanotubes’ was developed by the University of California.[57,58] The smart bio-nanotubes were in a trilayered structure made with the help of a microtubular protein called tubulin coated with a bilayer of lipid followed by coat of tubulin protein in the form of rings or spirals where proteins had either open ends (negatively overcharged) or closed ends (positively overcharged with lipid caps). The important formulation variable for smart bio-nanotubes that regulate the release profile is thickness of protein lipid versus protein coats, which leads to a basis for controlled release of drug molecules (Table 4).
Table 4. Overview of applications of carbon nanotubes in drug delivery
Functionalization of nanotubes
Double functionalization made on its side wall by amino groups by 1,3-dipolar cycloaddition reaction
POM attached with surface of SWNTs and complexed with chitosan
Initial burst release of POMs from chitosan-modified carbon nanotubes (CNT/chitosan complex) as compared with functionalization-free CNTs, increased stability of POM in physiologically relevant pH environment and showed high loading capacity and prolonged drug release
Nanotubes are being investigated as a good carrier for targeted delivery of drug molecules to various parts of body that could help in treating several diseases. Many techniques have been applied to achieve targeting of drug molecules.
Targeted drug delivery with chitosan–nanotube complexes
Chitosan is a polymer of marine origin, derived from molluscs and prawns. Chemically, it is a polysaccharide which has several applications, with a wide application in controlled drug delivery because of its safe and nontoxic nature. The basic mechanism through which this system achieves its targeted action is its mucoadhesive nature. When the chitosan is functionalized on the surface of CNTs, the cells become attached to the sidewalls of the nanotubes, resulting in the desired targeted release to the cells, with improved drug absorption. Animal studies of targeted drug delivery revealed that the CNT–chitosan complexes had better release and targeting properties than chitosan-free CNTs. The system may have several applications in drug delivery, transmucosal drug delivery, delivery of peptides and nucleic acids to targeted cells, and it was found to have a broad spectrum of applications in drug delivery to the lungs for the treatment of lung cancers.
Targeted delivery with functionalized CNTs
Functionalized nanotubes are a growing area of research for targeting malignant cells by virtue of better uptake by a specific population of malignant cells without affecting other collateral healthy tissues, which is a basic guiding principle of chemotherapy and targeted delivery.[60,61] Additionally, functionalized nanotubes are most preciously used for targeted delivery of nucleic acids, proteins, antibodies, drugs and other therapeutics agents to their site of execution. CNTs use for targeted delivery is widely accepted in treating malignant disorders such as choriocarcinoma, Burkitt's lymphoma, carcinoma of cervix, breast cancer, and testicular tumors. For example, methotrexate in cancer chemotherapy usually shows a low level of cellular uptake due to improper absorption from the gastrointestinal tract. However methotrexate administration using CNTs double functionalized by amino groups showed better cellular uptake. The functionalization was performed on MWNTs by 1,3-dipolar cycloaddition reaction of azomethine ylides which introduced amino groups in the orthogonal positions. Finally, methotrexate was attached with amino groups at the external surface. On administration, when the carriers reached the targeted tissues, cleavage of bonds took place to yield the drug molecules, with better cellular uptake. Several covalent functionalizations of CNTs with anticancer, antiviral and antibacterial drugs have been employed and are of considerable interest for research.[65,66]
Recent findings have shown that drug-loaded functionalized CNTs face some problems in release of their contents. Hence to overcome this problem, Kulamarva et al. encapsulated the CNTs in a novel membrane microcapsule made up of an alginate–poly-l-lysine-alginate (APA) membrane to form a polymeric membrane targeted drug delivery device. The nanotubes were either embedded in the core or attached to the surface of the alginate capsules. When such systems were delivered to the body they showed a promising drug release profile, safely and effectively, due to protection from the external harsh environment provided to the nanotubes by the polymeric membrane.
Targeted delivery with the help of nanotube-based antibody therapy
Earlier, the concept of antibody-mediated drug delivery was found to be the best approach for targeted drug delivery. However, loss of specificity of antibodies on binding with drug molecules is its biggest disadvantage. Nanotubes do not alter the specificity of antibodies on attachment and can deliver the drug at the targeted site. McDevit et al. delivered anticancer agents using SWNTs covalently functionalized by monoclonal antibodies for tumour targeting, and made radiation ion chelates and fluorescent compounds for diagnostic imaging to identify the site of target achieved. The drug molecules to be delivered with CNTs must have similar properties, with both targeting antibodies as well as nanotubes, because the antigenicity of antibodies must remain after attachment of the drug to the nanotubes and, subsequently, nanotubes with antibodies for their desired site of action. Therefore, they should match with each other and need to be compatible. These CNTs are capable of delivering several drug molecules (such as taxol) and more than one type of drug to the tumour site in different nanotubes attached to the surface of a single antibody. Ashcroft et al. revealed that using a specific type of single skin cancer antibody (ZME-108) enabled delivery of more than 40 nanotube-loaded drugs to the targeted site. This approach has the advantage of delivering both hydrophilic and lipophilic drugs. Welsher et al. reported that SWNTs with PEG functionalization and conjugated with the monoclonal antibody rituxan were able to target selectively the CD20 cell surface receptor on B-cells with little nonspecific binding to negative T-cells, and with Herceptin to recognize HER2/neu positive breast cancer cells. This antibody mediated approach has thus proved to be an ideal alternative in targeting drug molecules to cancer cells (Figure 2).
Nanotubes in lymphatic targeting
Lymphatic targeting is required for curing lymphatic diseases along with targeting drug molecules to the reticuloendothelial system (liver, spleen, kidney, etc.). Previously, many approaches have been tried for drug targeting to the lymphatic system (such as controlled-release microspheres, magnetic microspheres, etc). The major drawback of microspheres is their larger size, which causes capillary blockade and leads to ‘chemoembolism’-like problems. Inspired by the earlier concept, researchers have now shown that lymphatic targeting of drugs with the use of magnetic nanotubes proves to be better accepted.[73,74] Yang et al. suggested that controlling the size of CNTs could allow effective take up into the lymphatics. Functionalization of CNTs with folic acid and entrapped magnetic nanoparticles along with drugs showed better delivery to cancer cells in the lymph nodes. Using an externally placed magnet, the MWNTs could be retained in the targeted draining lymph nodes for several days and continuously released chemotherapeutic drugs. Selective killing of tumour cells overexpressing the folate receptor in the lymph nodes was achieved; the folate receptor is overexpressed across a broad spectrum of human tumours.
Nanotubes in brain targeting
The blood–brain barrier (BBB) restricts the entry of substances so as to maintain the internal milieu of the brain. Many diseases remain untreated because of restriction in entry of therapeutic substances. The restriction in entry of therapeutic molecules to the brain is not only because of the BBB but also due to the presence of enzymes that restrict entry by degrading the neuropharmaceutical agents, food nutrients and minerals.[77–79] Conventional drug delivery systems that release drugs into the general circulation fail to deliver drugs effectively into the brain. Diseases that directly affect the central nervous ststem include Alzheimer's disease, dementia, parkinsonism, mood disorder, AIDS and viral and bacterial meningitis. These brain disorders require the development of an effective targeted delivery system. Therefore, drug delivery systems for the brain need to be re-configured. CNTs have attracted much attention for delivery of drug molecules to the brain, having an ability to cross the BBB. Kateb et al. reported that MWNTs are quite effective in delivering neuropharmaceutical agents to the inner environment of brain microglial cells. Similar findings were reported by VanHandel et al. They administered MWNTs by intratumoral injection to GL261 murine intracranial glioma cells for 24 h. Observation showed that 75% of MWNTs were taken up by macrophages of brain tumour cells. These findings suggested that the CNT-loaded drug can invade the BBB effectively compared with existing techniques. Additionally, nanotubes also have a potential role in treating neurodegenerative disorders due to their magnetic properties. Nanotubes in combination with nerve growth factors help in enabling specific cells to differentiate into neurons and thus treat neurological disorders.
Nanotubes in ocular drug targeting
CNTs have also been used for ocular delivery of therapeutic agents. They are found to help in local targeting of drug molecules to the retinal site. They also have the ability to cross the blood–retinal barrier, which is a semi-permeable tough membrane restricting the entry of drugs such as antibiotics, anticholinergics and mydriatics. However, little research has been done so far and thus this area needs to be further explored for ocular delivery.
CNTs in cancer therapy
It is very difficult to treat malignant cells or eradicate them from the body without ‘spillover’ to the normal cells. In more than 99% of cases chemotherapy destroys cancer cells along with killing of normal cells, with serious side effects. Nanotubes thus help greatly in treating the cancer cells in a safe manner.[84,85] Chemotherapeutic agents delivered with CNTs help in achieving better uptake by malignant cells without affecting collateral tissues. Consequently, nanotubes potentially lower the dose of drug by localizing its distribution at the tumour site only. In addition to chemotherapy, nanotubes also act as carriers for gene delivery, in treatment of carcinoma of cells.[48,87] Recently, Stanford University disclosed the newer applicative advantages of CNTs that have their own cancer-curing property. Results showed that nanotubes exposed to an infrared (IR) light source tend to heat up to 70°C to 160°C in a very few seconds (<120 s), and when placed at a tumour site easily destroy malignant cells of a specific population and seem to act like a tumoricidal agent. MWNTs specially prepared by the CoMoCAT process are of considerable interest for their use in chemotherapy due to their quick infrared absorbing properties. Specifically, CoMoCAT nanotubes with a uniform size (about 0.81 nm) and a narrow absorption peak at 980 nm are ideal candidates for such a novel approach. In a study for achieving better targeting ability, such nanotubes were attached to a tumour marker, folic acid, which bound to its specific folate receptor, and a radiation of specific wavelength with a 980 nm laser caused programmed death of malignant cells. Several works describe this process of cancer treatment as ‘photothermal therapy for cancer treatment’.[60,88] Levi-Polyachenko and group conducted an experiment on MWNTs and showed that nanotubes exposed to IR radiation for a period of < 2 s caused hyperthermia (heating of cellular environment) up to 42°C. This study showed that MWNTs exposed to strong IR wavelengths (700–1100 nm) caused hyperthermia (42°C) in peritoneal cells for up to 2 h, which helps in the treatment of colorectal carcinoma. Drugs like mitomycin C or oxaliplatin given to colorectal cancer cells lead to effective reduction of the malignant cell population due to hyperthermia-mediated increased cellular uptake of drugs by increased cell membrane permeability. Similarly Torti et al. reported that MWNTs doped with nitrogen gas induce thermal ablation causing death of cancer cells upon irradiation with an infrared beam. This technique is used in the treatment of kidney tumour cells. It was concluded that the anti-tumour activity of CNTs might be due to heat transduction, which leads to cellular cytotoxicity. Several variables were reported that make this technique effective, including the length of the nanotube. The longer the CNT, the more effective would be the therapy as less time of exposure would be required with a minimum dose of radiation due to the larger surface area.[91,92]
Similar to the above findings, MWNTs coated with DNA showed better tumoricidal action than non-DNA-encased MWNTs. Guenzel studied the tumoricidal action of DNA-coated nanotubes on 12 prostate cancer affected mice which received treatment with DNA-encased MWNTs, non DNA-encased MWNTs and MWNTs with and without laser beam separately. The results showed a better tumour cure rate with DNA-encased MWNTs within 70 s from a laser beam of 3 W power for up to six days. The suggested underlying basic mechanism might be that increased heat production around two- to three-fold of the threshold leads to death of malignant cells. This approach has an advantage over simple radiation/heat therapy to treat human tumours because of the selective tumoricidal action of DNA-encased nanotubes. Apart from the above, amino-functionalized MWNTs complexed with siRNA showed successful suppression of tumour in a human lung carcinoma model in vivo. It is not only MWNTs—researchers have also worked extensively with SWNTs for delivery of many anti-cancer agents: paclitaxel, doxorubicin, polyoxometalate, carboplatin, cisplatin, taxoids and siRNA.[101,102] It is the functionalization of CNTs which made easy attachment of an even layer of drug molecules or bioactive agents for tumour targeting.[95,96] SWNT–siRNA complex or siRNA attached with functionalized SWNTs also produced effective and prolonged suppression of tumour growth compared with the older techniques of siRNA delivery.[101,102] (Table 4).
Wang and coworkers observed that functionalized SWNTs are best in reducing the progression of human myelogenous leukaemia. The disorder is usually caused by a critical mediator cyclin A2 found in human body cells; a higher level is found in tumour cells and this plays a critical role in disrupting DNA replication, transcription and cell cycle regulation. Overexpression of cyclin A2 leads to development of several types of cancer, including leukaemia. Hence reduction or suppression of cyclin A2 level in the body is a suggested measure to prevent tumour progression. Functionalized SWNTs carrying small interfering siRNA into K562 myelogenous leukaemia cells showed inhibition of cyclin A2-dependent development of leukaemia, inhibition of cell proliferation and high apoptosis. This provides a new site of application for the field of nanotubes apart from chronic myelogenous leukaemia-related chemotherapy, towards reduction of multidrug resistance during chemotherapy of several diseases. Apart from the application of nanotubes in delivering pharmaceuticals and nucleic acids to malignant cells, recent literature recounts their important use in radiotherapy for treating cancer by increasing the rate of oxygen uptake to malignant cells as compared with the normal rate of uptake. This makes the radiotherapy more effective.[103,104] Apart from the CNTs, carbon nanohorns (CNHs) are now under investigation for exploring their application in chemotherapy. Water-soluble CNHs have been used for delivery of anti-cancer agents, such as doxorubicin administered intratumorally to human non-small cell lung cancer cell (NCI-H460)-bearing mice, and showed significant retardation of tumour growth associated with prolonged doxorubicin retention in the tumour cavity.[19,105,106]
CNTs in imaging
Nanotubes play a vital role in diagnostic procedures by helping in the imaging of organs and help in identifying the site of action of drugs in targeted delivery systems. CNTs are shown to have greater potential to act as a contrast agent in imaging and identification of cancer cells. Methotrexate (an anti-cancer agent), when given with fluoroscein probe functionalized nanotubes showed better visibility in the body due to the fluorescence produced by the drug-carrying probe on its surface. Other examples of drugs, such as amphotericin B (an antifungal used in dermatophytosis), were used for their action on reticuloendothelial organs along with considerable reduction of their unwanted toxic effects. CNTs functionalized with fluorescent compounds are used as a radio-opaque substance which produces images of the desired in-vivo organs. On administration of functionalized nanoformulations into the body, the nanoformulations are attach on the specific site for which they have been designed and can be studied with special imaging techniques such as gamma scintigraphy, radiolabelling.
Recent developments have shown that nanoimaging of several body parts can be done by administering nanotubes encapsulating miniaturized video systems in the form of a pill. This system can be easily swallowed, which helps in imaging the disease area in a nanoscale segment of a particular tissue or organ, where techniques such as endoscopy and colonoscopy fails due to their macro size imaging.
CNTs in solubility enhancement
CNTs prepared synthetically are hydrophobic in nature and thus in soluble in water. Therefore, they create a problem in drug delivery and targeting. However, functionalization introduces the required functional groups by covalent functionalization on their sidewalls and tips to make them hydrophilic.[110,111] These functionalized nano carriers are able to deliver several hydrophobic biomolecules (proteins, peptides, nucleic acids, enzymes) to the target site. Several poorly water-soluble drug molecules can be delivered, such as doxorubicin, fluorescein, etc. In addition to primary functionalization of nanotubes, secondary or double functionalization can be done by coupling with amino acids and bioactive peptides, allowing enhanced solubility characteristics. This forms the basis for the treatment of several diseases, like cancers, and vaccine and gene delivery where the carrier system enters damaged cells and releases enzymes for accelerating the autodestruction signaling for malignant cells or may repair the cells for their normal functions. The basic mechanism by which they improved solubility as well as release is by ‘functionalized-partitioning’.[43,113]
CNTs as a drug delivery nanocapsule
Nanocapsules are the small nano-sized particulate system which acts as a carrier for loading drug molecules. They are usually transported to a targeted tumour site and release their contents on change in environmental conditions in close proximity to the targeted area. A major advantage of the nanocapsule delivery system is the protection of the drug from the external environment. After release, it enters into the cell and is prevented from being ejected by attaching with the help of specialized surface structures such as ligands, antibodies and functional groups. Though nanocapsules have some similarity with nanoparticles, they differ in the sense that nanoparticles are hollow spherical particles while nanocapsules are core-containing solid particles. Therefore, CNTs are identical to nanocapsules in their property of encapsulation and their capability of not being ejected from the targeted cell, which makes them an ideal drug delivery carrier for the treatment of diseases like cancer, tuberculosis and bacterial and viral disease where the chance of drug resistance is more common due to efflux of drug molecules from cells by the action of of P-glycoprotein.[51,116,117]
CNTs as a tool in drug discovery
In the 21st century, pharmaceutical drug discovery is a most powerful area of research in finding new drug molecules for therapeutic use. The major drawback in drug discovery is time consumption due to the trial and error involved in the methods. CNTs are now used for drug discovery in the form of sensors along with several advanced information technology systems such as data mining and computer-aided drug design for identification of genes and genetic material for drug discovery and development.[118,119] Functionalized CNTs also have great importance in drug discovery.
CNTs for biomedical application
Nanotubes have become of interest in their application as an accessory tool for various allied fields along with biomedical uses.[120–122] The applications of CNTs as biomedical tools are discussed in the following sections.
Diagnostic tests are those which help in determining the level of diseases, pathological conditions, etc. Nanotubes can assist such diagnosis, where they are used in the form of artificial smart intelligence devices such as nanosensors and nanorobots. These nanosensors and nanorobots are nano-size devices meant for implantation in the body.[123,124]
Nanosensors are devices that detect small physiological changes qualitatively and quantitatively. There are various types of nanosensor that have been studied for application in biomedical devices. CNTs have several physical properties of which the temperature coefficient is exploited in designing pressure nanosensors. This works on the concept that when temperature increases by two orders of magnitude, this leads to twice the rate of pressure developed, which is detected by a pressure-sensing device.
Liu and Dai discovered an advanced form of pressure-sensing device, called a piezoelectric pressure sensor, made up of CNTs. These are grown on SWNT surfaces suspended in square polysilicon membranes. They work by the mechanism that when uniform pressure is applied on the membrane this leads to a change in resistance in the SWNTs. It has several applications including use in patient monitoring, hospital beds, respiratory devices, inhalers, eye surgery and kidney dialysis machines.[126,127]
During eye surgery they help to reduce operation difficulties by reducing fluid turnover and control the vacuum that is used to remove the fluid. In addition to this they are quite useful in hospital bed-ridden patients and burn victims for preparing mattresses consisting of sensors that regulate a series of inflatable air-filled chambers that provide a smooth sensation to the patient with less friction, pain and burning and promote healing. Pressure sensors can also be used for detecting sleep apnoea where, if there is no movement in sleep for a certain period, the sleeper is awakened by an alarm. Likewise, pressure sensors also help in monitoring the operation of dialysis systems by measuring the inlet and outlet pressures of both the blood and the solution during kidney dialysis. Recent advances in pressure sensor devices led to the development of intelligent pressure sensing systems which play a vital role in manufacturing portable respiratory devices, including both diagnostic devices (e.g. spirometers, ergometers and plethysmographs) and therapeutic equipments (e.g. ventilators, humidifiers, nebulizers and oxygenometers). They serve patients with disorders of asthma, sleep apnoea and chronic obstructive pulmonary disease.
Biosensors are the nanosized materialistic devices which have applications in detecting biological disorders of the human body. Sotiropoulou and Chaniotakis used CNTs as an immobilization matrix for the development of an amperometric biosensor. The biosensor was developed by growing aligned MWNTs on platinum substrates.
CNT-based nanobiosensors are now used to detect DNA sequences in the body and help in the detection of very specific pieces of DNA related to cancer production, and identification of genes and biomolecules such as antibodies associated with human autoimmune diseases.[130–132] These sensors have also been used during space missions due to their advantage of transdermal administration through skin. Other important applications of biosensors include glucose sensing and blood pressure monitoring. Use of CNTs as chemical sensors also has potential applications in liquid chemistry for the detection of sodium, potassium and other mineral elements in blood samples and measurement of pH value. Due to their small size and less power consumption, nanotubes are used in various other biomedical applications, such as monitoring of pulse, temperature, repair of damaged cells, monitoring the activity of the heart, heart beat regulation, treatment of retinal diseases and in cochlear impairment-related hearing problems.[135,136]
CNTs can also be used as flow sensors for precise measurements of respiratory gases. Ghosh et al. showed that when liquid flows on the surface of bundles of SWNTs they induce a voltage in the direction of fluid flow, which helps in designing micromachines, such as heart pacemakers, that do not need heavy battery packs or recharging.
Early cancer detection could reduce disease progression to other parts of the body. Therefore, diagnostic procedures must be sensitive to detect malignant cells so that cancer can be cured in its early stages by radiation or other chemotherapeutic treatment. Among several types of cancers, prostate cancer can be a cause of death due to delayed diagnosis. Recently, nanotube-based bionanosensors have been helpful in detecting such type of cancers in their early stage. Small needle cellular bionanosensors are devices made up of nanotube constructs that have the sensitivity to differentiate between metastatic and non-metastatic cancer when inserted directly into blood by detecting change in hormonal levels. Initial testing with a cell biosensor showed that a nanotube electrode can characterize different solutions of LNCaP prostate cancer cells. The nano biosensor device basically consists of three different parts, a responsive material, a nanoneedle system and a coil antenna. Responsive materials are said to be the heart of the sensor, which help in sensing and detecting the difference between cancer and non-cancer cells. The responsive material is made up of inductors and solenoids prepared from nanowires and coiled nanotubes. Nanotubes provides real selectivity to the sensor in detecting cells and thus their sensitivity can be further enhanced by coating or functionalization with gold material, by bioconjugation using antibodies, or with special receptors for easy detection. MWNTs act as a telescope and measure the change in electrical resistance by the strain produced due to electrochemical bond expansion and this change in resistance sends signals to the detector and amplifier. Nanowire responsive materials have an intrinsic ability to sense and respond to external stimuli. Nanoneedle systems help in the injection of the sensor into the body and the coil antenna helps in transmitting the signals recorded by nanotubes. In addition to these, other parts include a power-generating system and piezoelectric nanobelts.
Similar to nanosensors, nanorobots have shown application in the build up of immunity by protecting from harmful bacteria and viruses, etc., in protection of skin by curing diseases, protection from teeth decay, surgical procedures for preparing nanosyringes, electromechanical nanothermometers and nanojet engines. Another advanced application of nanorobots is to protect the skin from external unwanted stimuli by removing pathogens and accelerating the new skin recovery process by optimal moisturization due to its self-intelligence system. Similarly, a mouthwash containing such a system has been shown to protect teeth from bacteria and to lift foods and plaques.
Besides the application of nanotubes in diagnosis, they are now used in many surgical operating procedures. In earlier days surgical procedures were carried out using large-size macro-scale instruments that were quite difficult to handle and hence made the surgical procedures more complicated. The major disadvantages of these macro instruments were difficulty of handling by physician/surgeon, chances of larger wounds leading to severe pain, scarring of patients and higher operating time, which may result in fatigue of the surgeon and could lead to errors in surgery. Additionally such types of instrument are unable to operate in sophisticated procedures like ophthalmic, ear and nasal surgery and in colonoscopy. Hence, to overcome the above difficulties, nanosized small artificially intelligent equipment is of considerable interest. Being a nano-size material, nanotubes are now used for preparing the nanoscale surgical instrument devices which have now made surgery easier and more sophisticated. The nanosized equipment, including forceps, scalpels and grippers, have sensor-type embedded systems, which guide the physician to cut a particular area of tissue.[44,142,143] Nanorobots are also used in surgery for several diseases including oesophageal, colorectal, gynaecological, prostatic, cardiac, liver, gallbladder and bypass surgery.
Briefly, nanoprobes are devices that are designed to access information from a remote or unknown region of a body cavity like the stomach, uterus, ear or heart. There have been several reports on the use of CNTs in preparing probes.[144–147] For example, Stevens et al.  used the nanotubes and attached them with an atomic force microscope (AFM) tip made up of silicon or silicon nitride by electric arc-discharge method and were able to image a protein filament using these tips. The high mechanical robustness and low buckling force of CNTs help in reducing the force exerted on the sample during imaging and so could be applied for imaging soft materials like biological samples.
Nanotweezers are instruments that have the capacity to modify cell physiology. Basically they are a kind of probe that is driven by electrostatic interaction between two nanotubes on a probe tip. They work on the principle of balancing elastic restoring force with electrostatic force. Nanotweezers are used for manipulation and modification of biological systems, like changing the physiology, shape, size and morphology of the cell, DNA sequence and assembling cellular structures, and have many other applications in treatment of diseases.[136,150]
CNTs have now emerged as an excellent material for the manufacturing of actuators. These are widely used nowadays in the field of biomedical engineering where they are used in preparing biomedical equipment such as artificial limbs, artificial ocular ciliary muscles, irises, optical display units and pulsating hearts. Baughman et al. described the use of CNTs for preparing actuators. Nanotubes are found to be the best material for actuators because of their excellent physicochemical properties such as the ability to withstand higher stress-strain forces, electromechanical properties and higher electrochemical coefficients. The variables that affect the actuator property of CNTs include the type of nanotube used (SWNT/MWNT), size, method of preparation, purity and chirality. Actuators work by converting electrical energy to mechanical energy. After energy conversion, the actuator utilizes mechanical energy in performing useful work. Baughman et al. showed that CNTs, after conversion of electrical energy into mechanical energy, behave like an artificial muscle which generates higher mechanical stress compared with natural muscle. Both the single-walled and multi-walled nanotubes were found to be good candidates for several biomedical surgical devices.
Nanofluidic systems in drug delivery
Nanotubes are also employed in delivering drug through injectable routes. Generally this route is considered to be a route of emergency; hence drug formulations that are given by this route must favour certain conditions, such as particle size control, syringeability, sterility, zeta-potential and apyrogenicity, for their safe and effective fate in the body. The most vital problem seen with this route is the chance of blockage of blood vessels due to large particle size that leads to tissue toxicity. Hence, nanotubes are designed as a small nanofluidic system carrying drugs for injectable use, which make safer drug delivery possible. Among the nanotubes, MWNTs are most widely used because of their special properties such as higher mechanical strength. These systems are shown to have wide application in drug delivery through intravenous routes in chemotherapy for tumour targeting and also in treating diseases like lupus, diabetes and AIDS.[155,156]
CNTs as quantum dots for therapeutic purpose
Quantum dots (Q-dots) are defined as nanoparticles with size in the range of 2–10 nm. Chemically these are semiconductor and diode substances capable of emitting light of various colours. Due to their light-emitting property, they have potential application in imaging of various body parts like X-rays. In recent years, research revealed that CNTs can also generate Q-dots, or may behave like a Q-dot, due to their mechanism of coulomb blockade and hence the phenomena of electron quantization leads to formation of such types of light-emitting nanoparticles. Q-dots emerged as a special tool in imaging because of their continuous light-emitting characteristics and this does not fade when exposed to UV light. In addition, Q-dots also help in drug targeting and diagnosing the site of action of the drug molecules attached on it. Example include the delivery of antisense agents and Q-dots attached on MWNTs. Bhirde and co-scientists delivered cisplatin and epidermal growth factor (EGF) attached on SWNTs specifically to target squamous cell cancer and compared them with the non-targeted control SWNT–cisplatin without EGF. Imaging studies in head and neck squamous carcinoma cells (HNSCC) overexpressing EGF receptors (EGFR) using Q-dot luminescence and confocal microscopy showed that SWNT-Qdot–EGF bioconjugates internalized rapidly into the cancer cells and vice-versa. Also, regression of tumour growth was rapid in mice treated with targeted SWNT–cisplatin–EGF relative to non targeted SWNT–cisplatin.
CNTs as a medium for biotechnological/microbiological products
Microbiological products include substances derived from microbial origin. However, the natural process of preparing these products is quite time consuming and costly. Recently, nanotubes have emerged as an excellent carrier for producing such products by acting as a growing medium for microorganisms. Czaja et al. used this method in the production of cellulose which was found to have several biomedical applications. Cellulose is generally produced by the bacteria Gluconobactrium xylinum, but the rate of production does not fulfill the rate of demand. For this purpose, MWNTs were used with Hestrin and Schramm (HS) medium and produced higher yield than conventional methods of production. Tissue engineering is a field of biotechnology that helps in regular monitoring and evaluation of engineered tissues along with other biomaterial for improving growth function. Nanotubes help a lot in tissue engineering by tracking of cells, sensing of microenvironments, in delivery of transfection agents and in scaffolding of them. In addition they help in examination of the engineered tissue by optical means, magnetic resonancing and by radiotracer contrast agents.
CNTs in vaccine delivery
Vaccines are biological substances used for imparting immunization against foreign disease-causing pathogenic microorganisms. They usually impart active immunity to the human body by inducing antibody production in a most natural way. Major problems associated with vaccine delivery include improper absorption, chances of antigen-induced hypersensitivity, anaphylactic reactions and hypersensitivity due to vaccine adjuvants. Several novel approaches have been tried for improving vaccine delivery, including liposomes, microspheres, nanoparticles and bone delivery systems. Similarly, CNTs have also been tried for vaccine delivery, where they help in improving vaccine action due to their adjuvant-like action in increasing the body to antigenic exposure.[164,165] CNTs, when conjugated with antigenic peptides, can act as a new system for safe and effective delivery of synthetic vaccines.
CNTs in gene therapy
Gene therapy can be improved by using CNTs that help in the replacement of damaged or missing genes. The major problem associated with gene delivery is the complication of DNA passing through the cell membrane.[166,167] CNTs help in transportation of DNA into cells.[168–170] Researchers have made nanotubes with dendrimers grafted onto the surface for treating gene defects by delivering the grafted genes. Recently, the potential of CNTs as matrices to support and stimulate neural growth has been reported. Prato et al. reported ammonium-functionalized CNTs as a vector for gene-encoding nucleic acids and plasmid DNA to demonstrated the enhancement of gene therapeutic capacity in comparison with DNA alone. Yang et al. delivered siRNA complexed with SWNTs for efficient gene delivery. The siRNA and SWNT complex can be easily taken up by splenic immune recognizing cells such as CD11c+ cells, CD11b+ cells and Gr-1+CD11b+ cells to induce the immune response for the particular gene.
Wu et al. reported that when single-stranded DNA bound to SWNTs, DNA probes were protected from enzymatic cleavage and interference from nucleic acid binding proteins. Study showed that an SWNT modified DNA probe could target a specific mRNA inside living cells and cause increased self-delivery capability and intracellular biostability compared with free DNA probes. Hence, this new conjugate provides great potential for applications in the field of genetic engineering.
CNTs in anti-sporal applications
Wang et al. at Clemson University reported application of nanotubes in treating anthrax infection. Earlier, during civil wars, anthrax spores were used as a biological weapon. Their common mode of transmission is through inhalation which causes respiratory problems. CNTs have been shown to help in protection against bioterrorism. Galactose-a, b and mannose-c sugar functionalized SWNTs molecules stick on the spore surface to form a coat and neutralize their toxicity and prevent infection. The basic mechanism of disinfection is prevention from inhalation and absorption from the bronchiole surface. Study showed that nanotubes play several identical roles in disease treatment by minimizing toxicity with dose reduction of standard therapeutics and allowing a multiple payload capacity to achieve both targeted activity and combating infectious strains and resistant strains.[82,175]
Toxicity of CNTs
Nanomaterials have unique properties in comparison with bulk materials, such as a high surface area to volume ratio that also leads to unique mechanisms of toxicity from xenobiotics. In general, researchers found that the toxicity originated from the nanomaterial size and surface area, composition and shape. Reduction of size leads to an exponential increase in surface area relative to volume, consequently making the nanomaterial surface more reactive towards itself (aggregation) and to its surrounding environment (biological components).[177–179] Accumulation of nano-sized material may also cause increased uptake into tissues, which alters the critical biological function of cells. Recent studies on the toxicity of CNTs performed by nanotechnologists are presented in Table 5.
Table 5. A brief account of the toxicity of carbon nanotubes during in-vitro and in-vivo studies with different models
Tested cardiopulmonary toxicity on mice for acid functionalization (AF)-SWNT and carbon black particles
Intrapulmonary instillation of (AF)-SWNTs enhanced cardiac ischaemia/reperfusion injury and caused myocardial degeneration, no significant ischaemia/reperfusion injury on exposure to non-functionalized SWNTs or ultrafine carbon particles
Might be due to enhanced translocation, or via systemic vascular response generated in association with the AF-SWNT-mediated pulmonary injury
Investigated the effect of various physicochemical features of MWNTs on toxicity and biocompatibility with cultured human neuroblastoma cells by using MTT, WST-1, Hoechst and oxidative stress assays
Human neuroblastoma cells
Loss of cell viability was minimal for pure MWNTs (99% purity), but cell proliferation was decreased significantly for 97% purity MWNTs and AT- MWNTs (97% purity, surface oxidation 8%); no intracellular ROS were detected
Purity, concentration and functionalization affects the cell viability, 5–10 µg/ml MWNTs was supposed to be ideal for for gene and drug therapy against cancer
Investigated adverse effects of SWNT using a cell culture
Immortalized human epidermal keratinocytes (HaCaT)
Oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability
Cell viability was due to accumulation in cells
Recently, Qu et al. demonstrated temporary organ injury in lungs and heart due to delayed clearance of carboxylated MWNTs in mice. The delayed clearance was a result of the strong tendency of MWNTs to agglomerate. Persistent accumulation of agglomerated MWNTs in the lungs even caused inflammatory responses. Another group studied the influences of SWNTs with different degrees of agglomeration on primary cultures derived from chicken embryonic spinal cord (SPC) or dorsal root ganglia (DRG). SWNTs' significant decrease in DNA content was more pronounced if cells were exposed to highly agglomerated SWNTs as compared with better dispersed SWNT-bundles. In addition, cell-based ELISA tests showed that the amount of glial cells was reduced in both peripheral nervous system (PNS) and central nervous system (CNS) derived cultures. This suggested that, on SWNTs introduction at high concentrations, it is likely that adverse effects on glial cells and neurons might occur.
CNTs were shown to impair cell viability, which could be caused by the strong tendency of CNTs to agglomerate.[182,183] Bottini et al. compared the toxicity of pristine and oxidized MWNTs on human T cells and found that the MWNTs were more toxic and induced massive loss of cell viability through programmed cell death. Chiaretti et al. disclosed that MWNTs on intraperitoneal administration produce neither significant neurovegetative nor behavioural effects up to 10 mg/kg of body weight. Similarly, neither modification of immunoglobulins nor of humoral immunity was observed. Rotoli et al. described an alteration of the permeability of human airway epithelial cells after treatment with MWNTs, whereas, Poland et al. revealed that MWNTs resulted in asbestos-like, length-dependent, pathogenic behaviour on introduction into the abdominal cavity of mice. Shvedova et al. investigated the adverse effects of SWNTs using a cell culture of immortalized human epidermal keratinocytes (HaCaT). They observed oxidative stress and cellular toxicity due to formation of free radicals, accumulation of peroxidative products, antioxidant depletion and loss of cell viability. Exposure to SWNTs also resulted in ultrastructural and morphological changes in cultured skin cells. Metal traces available in nanotubes are also another causative agent for toxicity. Pulskamp et al. did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all CNT products. However, increase in ROS was observed due to the presence of metal particles as well as metalloids in commercial nanotubes, while treatment with a highly ultra-purified form of CNTs had no biological adverse effects. Thus, it could be concluded that presence of metal in commercial nanotubes might be a major toxicity inducer.
Genotoxic potential and mutagenic and carcinogenic effects of different types of CNTs were reported recently but very little work has been published. This could be primary genotoxicity (direct interactions of particles with the cells) or secondary genotoxicity (generation of an excess of reactive oxygen species, ROS). Zhu et al. developed MWNTs and studied their genotoxic effects. They reported DNA damage through ROS and also an increase in the mutation frequency in mice. Similarly, Muller et al. found genotoxic effects, such as increase in micronuclei frequency, in rats during in vivo as well as in vitro studies. Also, SWNT treatment did not show mutagenic effects in Salmonella typhimurium YG1024 and YG1029 strains and caused a non-significant micronuclei increase in the V79 lung fibroblast cell line. However, the genotoxic effect of MWNTs is not universal. Dissimilar results were reported by Szendi and Varga. However, Takagi et al. suggested a carcinogenic effect in peritoneal cells after intraperitoneal injection MWNTs on p53 heterozygous mice and observed mesothelioma in peritoneal cells. Similar to the above findings, Sakamoto et al. observed that the carcinogenetic potential of MWNTs in scrotal cells led to the development of mesothelioma in rats, confirming the toxicity profile of MWNTs. There is much in the literature reporting the carcinogenicity of MWNTs in different organs of humans and animals. Such toxicity includes pulmonary toxicity, lung inflammation, granulomas, fibrotic reactions and mutations in epithelial cells and cardiopulmonary toxicity.[195,196]
Recently many conflicting reports concerning the safety and biocompatibility of carbon nanotubes (CNTs) have been published. Long-term accumulation and low toxicity of SWNTs in intravenously exposed mice was reported by Yang et al. However, no acute toxicity was reported by Zeni et al. on administration of SWNTs. Similarly, other authors have reported low or no cytotoxic effects due to exposure to CNTs.[188,197–199] Huczko and Lange reported null risk of skin irritation and allergy on dermatological trials of CNTs. Recently, Bardi et al. suggested that PF127-coated MWNTs do not induce apoptosis of cortical neurons on administration to mouse cerebral cortex. Moreover, the presence of MWNTs can reduce PF127 toxicity. The mutagenic effect of MWNTs of small surface/volume ratio, high diameter and less than 0.1% of metal contaminants have been evaluated for mutagenic activity by the bacterial reverse mutation assay (Ames test). These MWNTs were devoid of mutagenic effect in the bacterial cellular systems tested in that they did not significantly increase the number of revertant colonies. Thus, it was concluded that the MWNTs studied appeared to be devoid of mutagenic effects and the lack of mutagenicity might be related to their structure or to their purity. Correspondingly, Vittorio et al. studied the influence of purity and surface oxidation on the cytotoxicity of multi-walled CNTs in human neuroblastoma cells. In-vitro studies corroborated that cell viability was not affected and apoptosis and ROS were not induced in the SH-SY5Y cells after a three-day incubation period with three different types of CNT dispersed in Pluronic F127 solution. Purity of MWNTs and the concentration was found to affect the cell viability and proliferation. Results indicated that a concentration of 5–10 µg/ml MWNTs and 99% purity seem ideal for studies on the design and development of artificial MWNT nanovectors for gene and drug therapy against cancer. We suggest studying the manuscript of Smart et al. for better understanding of CNT toxicity.
The present knowledge of CNT toxicity is inadequate and contradictory, and thus still requires more extensive toxicity, safety and efficacy studies on animal models and in humans. The effects of CNTs' aggregation, size, length, functionalization, metal impurities and polymers on safety require more thorough research. Functionalization of SWNTs and MWNTs and its effects on aggregation and consequently genotoxicity needs to be evaluated.
Regulatory aspects of CNTs
Awareness of nanotechnology has dramatically risen in recent years among lawmakers, regulators and environmental activists. However, the question of whether, and how well, to regulate nanotechnology is not new. To date increasing interest in the field of development and nanotechnology applications of nanomaterials has had an impact on development of strict regulatory norms in their production and use in animals as well as in humans. The major guideline involves the related environmental health and safety issues of existing nanomaterial. As per the reports of the US Environmental Protection Agency (EPA), CNTs require major regulatory concern over toxicity as well as environmental safety.
CNTs can enter the human body after inhalation, dermal exposure or ingestion. They have a high bioavailability and can penetrate the bloodstream, digestive tract, surface tissues and blood–brain barrier without difficulty. Depending on whether nanomaterials are toxic or not, exposure to nanomaterials could trigger negative health effects. In contrast, carbon nanotubes can be coated and functionalized to prevent them from physical aggregation problems, resulting in long circulation in the air being inhaled and deposition deep into the respiratory tract. Studies in rodents and in cells have shown that ultrafine CNTs also escape detection and clearance by macrophages to a greater extent than fine particles, and may to a greater degree travel from alveolar regions to the blood circulation, where they may migrate to vital organs of the body. Ultrafine particles can produce greater inflammatory effects via free radical generation due to their small particle size and shape, surface charge, coatings and functionality, leading to toxicity, surface reactivity and influence on the cytotoxicity. CNTs could also facilitate transport of toxins deeper into the soil due to their larger surface area, bind with pollutants and transport them through the soil causing pollutants to be absorbed faster and deeper than normal, leading to formation of possibly newer toxic compounds due to strong catalytic actions.
Another significant regulatory concern is the potential effects of bioaccumulation due to the high bioavailability of nanotubes. A recent study performed in Rice University and Georgia Institute of Technology reported that CNTs formed aggregates that lead to alteration of their general physicochemical properties. Thus, they suggested further consideration of bulk behaviour of these nanomaterials and their counterparts in developing regulatory norms. Apart from these, a definitive knowledge of the development and framework of regulatory guidelines for the manufacture and handling of these nanomaterials will help in marketing in the near future.
Clinical trials and market status of CNTs
Despite the several regulatory issues regarding production, handling, toxicity and environmental safety of CNTs, they are still accepted in the field of nanotechnology due to their immense biomedical and drug delivery potential. Hence as successful as the alliance has been seen in the use of these nanomaterials, to address and solve many problems in the laboratory, the ultimate measure of the program's success lies in the translation of research discoveries to the clinic. To date several products based on carbon nanotubes have reached the clinical trial stage while several are already being marketed.
Recently, Ensysce Biosciences Inc. received regulatory approval for the development and conducting a clinical trial on SWNTs for siRNA delivery into tumours for treatment of cancer cells. Similarly, Calando Pharmaceuticals, Inc. has received clinical approval to carry out Phase-1 clinical trials on its patented nanoparticle-based drug delivery technology for transfer and delivery of small interfering ribonucleic acid (siRNA) for its own first therapeutic candidate, IT-101. Also, Tego BioSciences Corporation (Tego) has obtained its first approval on patented technology based on the antioxidant properties of fullerenes, a member of the CNT family, and signed agreements with Bronx Project, Inc. (TBP) for development and commercialization of the patented product carboxylated fullerenes (C3) in the treatment of Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, brain trauma and schizophrenia. Nanotopes are a bioactive fibre system used to regenerate specific tissues, including neurons, vasculature, bone, myocardium, cartilage and spinal cord, and for the treatment of peripheral arterial, which is a nanotube-based product developed by Nanotope, Inc.
Thus, for better monitoring of outcomes of clinical trials on nanomaterials for their safe use, several global regulatory authorities have come into the frontline, such as the US FDA, the EMEA and the TGA. In a recent report, the FDA has approved clinical trials on CNTs for screening of their imaging property in colorectal cancers.
In addition to the clinical-trial stage, several CNT-based products are being marketed, such as Graphistrength by Arkema Corporation used to optimize the performance of CNTs in polymer matrices such as thermoplastic, elastomeric and thermosetting plastics. While many MWNT products have been developed, such as Sizicyl, EpoCyl, PregCyl and Nanocyl, by Nanocyl S.A. for sizing of fibres, MWNTs containing 15 graphene sheets (Baytubes by Bayer Material Science) are used in polymer matrix to strengthen the material. However, to date no CNT-based drug delivery system has received approval for marketing.
Nano delivery systems hold great potential to overcome many of the present obstacles of drug delivery, of which carbon nanotubes (CNTs) have been proposed and actively explored as multipurpose innovative carriers for drug delivery and diagnostic applications. They are extremely versatile nanocarriers that can be included in numerous different fields because of their great material properties. They have many unique physical, mechanical and electronic properties. These distinct and exceptional properties have made it possible to exploit CNTs for numerous applications, such as sensors, probes, actuators, composites, nanoelectronics devices and drug delivery systems within biomedical applications. As a result, in a very short time, CNTs appear to have drawn the attention of nanotechnologists from both industry and academia.
In the last two decades, remarkable work has been done in the field of CNTs, including biomedical application. Functionalization of CNTs has opened new perspectives in the study of their biological properties. Attachment of an organic moiety to nanosized tubes has made possible their use in diagnostics for imaging as well as for targeting purposes, especially in cancer therapy and infectious disease treatment. Nanotube drug delivery holds future promise for high treatment efficacy combined with minimal side effects for cancer therapy with low drug doses. In addition, it is the most promising non-viral nanocarrier in human gene therapy and in nucleic acid, peptide, vaccine and protein delivery. As much work is under progress, it is expected that plenty of applications of CNTs will be explored in the near future.
Even though CNTs are playing a larger and most promising role in the field of nanomedicine, more research is required to guarantee safety in drug delivery. Toxicity studies are critical to establish the full in-vivo potential of CNTs for drug delivery before their actual application and marketing. Physiological, physicochemical and molecular processes need to be considered for understanding of clinical and pre-clinical toxicity of CNTs. As there have been conflicting results regarding toxicity, more comprehensive studies are required. Regardless of the knowledge gained in recent years in nanotoxicology, scientists still are not able to precisely anticipate the behaviour and biokinetics of CNTs. Cytotoxicity and genotoxicity are major concerns, and antigenicity remains poorly characterized and understood. Strict nano regulation is compulsory and needs amending to cover environmental, health, pharmaceutical and safety issues. The commercialization of CNTs requires clear-headed and thoughtful environmental, health and safety research and meaningful and open discussion of broader societal impacts of pharmaceutical and toxicological issues.
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest to disclose.
This review received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.