Khuloud T Al-Jamal. American Scientist. Volume 103, Issue 2. Mar/Apr 2015.
Despite all of the amazing advances in drug development, no treatment can be effective unless it reaches its intended site. New delivery systems have emerged over the past 40 years, and are improving in their ability to accumulate therapeutic agents at a desired target, such as a tumor. By increasing this specificity of the active ingredient, the efficacy of a treatment can be improved, and its toxicity-and associated side effects-reduced. Still, there is enormous room for improvement, by making the systems smaller and more precise. Numerous carriers with sizes ranging from 10 to 1,000 nanometers have been investigated, most notably fat-based liposomes, highly branching molecules called dendrimers, and even tiny semiconductor crystals called quantum dots. Now carbon nanotubes have joined the list of possible delivery vehicles.
A carbon nanotube is a hollow cylinder made from a continuous unbroken hexagonal mesh of carbon molecules. In 1991, electron microscopist Sumió Iijima of NEC Corporation was the first to show how to consistently produce carbon nanotubes, and interest in them has exploded ever since. Today, different preparation methods allow the production of large amounts of nanotubes as well as more control over their dimensions. There are two main families: single-walled and multi-walled, the latter composed of many concentric layers. Both types generally have diameters of a few nanometers and lengths ranging from 50 nanometers to several micrometers.
What makes carbon nanotubes useful for a wide range of nanotechnology applications, including drug delivery, are their physical properties: large surface area, high length-to-diameter ratio, good electrical conductivity, high tensile strength, and thermal and chemical stability. Their elongated structure makes them much like nano-sized needles, so they are more efficient than spherical particles at crossing cell membranes. Moreover, their large surface area allows for the attachment of drugs or genes, which often have problems on their own-such as insolubility or inability to cross cellular barriers-that the nanotubes can help overcome.
A major challenge is that the surface chemistry of carbon nanotubes usually makes them aggregate in bundles, which leads to poor dispersion in the aqueous environment of the body. A process called functionalization helps tackle this issue. In one version of this process, nanotubes are connected to hydrophilic molecules, increasing their distribution in water. These molecules, usually biosafe polymers, use relatively weak electrostatic or van der Waals forces to wrap the nanotubes, but the adsorption method is not affected by the saline conditions in biological tissues.
Another variant of functionalization uses stronger, covalent bonding to connect chemical groups to the carbon atoms in the walls of the nanotubes. These reactions tend to require strong acidic conditions, which are used purposely to shorten the nanotubes, making them less toxic and more easily taken into cells. The reactions form carboxylic acid groups (-C(O)OH), which can be subsequently attached to different compounds, customizing the design.
Taking in Nanotubes
Current interest in nanotubes dates back to 2004, when Maurizio Prato of the University of Trieste in Italy and his colleagues showed for the first time that the high length-to-diameter ratio of nanotubes allowed them to effectively cross cell membranes. Following this discovery, nanotubes were shown to enter into mammalian cells by two means, either endocytosis or passive diffusion. During endocytosis, the cell membrane forms an indentation that then engulfs a substance; this mechanism takes energy to perform. Conversely, in passive diffusion a substance crosses the cell membrane without using energy.
My colleagues and I have also explored this ability of nanotubes. For example, in one study, we used nanotubes chemically functionalized with an ammonium group (NH3+) on both human lung cancer cells (designated A549 cells) and healthy human monocyte-derived macrophages. The latter take up foreign bodies, such as bacteria, through an energy-dependent mechanism named phagocytosis, which resembles endocytosis but can only engulf larger particles.
Our investigations with two- and three-dimensional transmission electron microscopy, which offer high contrast between nanotubes and tissues, found that A549 cells showed a substantial uptake after four hours of incubation. In contact with cancer cells, clusters of nanotubes tended to accumulate into vesicles more likely taken up by endocytosis, whereas single nanotubes were seen in the cytosol and believed to enter the cell by passive diffusion. We strengthened this conclusion when we incubated the cells with the nanotubes at cold temperatures, which blocked cell metabolism and inhibited endocytosis, but did not affect levels of passive diffusion of nanotubes.
We then carried out the same experiment using the macrophages, where the majority of nanotubes ended up being internalized inside vesicles or phagosomes. Again, cold temperatures blocked this uptake and allowed only passive diffusion. These experiments confirmed the ability of nanotubes to penetrate cells using both energy-dependent and energy-independent mechanisms and to localize in intracellular compartments.
However, a subsequent study that followed these cells for two weeks found a dramatic change in distribution, with the nanotubes widespread and not localized in a particular compartment. This outcome therefore provided evidence that nanotubes were able to translocate from the vesicular compartment into the cytosol of the cells.
Now that the cell internalization properties of nanotubes were proven, we could start loading them with therapeutic molecules to deliver into cells. One of our first cargoes was siRNAs (small interfering RNAs), which are able to inhibit gene expression, and have shown great promise in cancer therapy by silencing genes associated with cancer development. However, one major limitation is their poor ability to cross cell membranes.
We functionalized nanotubes with polymers that had a large number of branches made from amino groups, called dendron groups, to enhance their ability to carry siRNA molecules. Amino functional groups are positively charged and thus can bind the negatively charged siRNAs by electrostatic interaction.
We first showed that these functionalized nanotubes were able to penetrate A549 cells, with or without siRNA attached. To demonstrate siRNA uptake, we used fluorescently labeled siRNA that could be tracked by fluorescence microscopy. After 24 hours of incubation, results showed that siRNA was indeed internalized in A549 cells when dendron-nanotubes were used as a carrier. In contrast, when we used another carrier, called a cationic liposome, to carry siRNA by electrostatic interactions, it demonstrated much lower siRNA uptake.
Our next step was to repeat the process with a form of siRNA that is engineered to be toxic to a different type of lung tumor cell, called Calu 6. After positive results in cells in vitro that showed that complexing this siRNA with nanotubes increased its toxicity, we carried out a series of experiments in mice growing tumors of this type. Again we compared the siRNA alone to it being carried by both nanotubes and liposomes, and also to the functionalized nanotubes alone. After five injections directly into the tumors of the different complexes over 27 days, tumors treated with the nanotubesiRNA were significantly smaller compared to the others.
We then stained sections of tumor to show that extended areas of dead tissue were associated with the nanotubesiRNA injection site. Labeling methods that highlight fragmented DNA present when cells initiate a death process named apoptosis were only positive for tumors injected with the nanotubes and siRNA, not nanotubes alone, confirming that the toxicity in the tumor was due to the drug and not the vector itself. Untreated tumors or tumors treated with the liposome complex showed predominantly healthy cells with limited areas of tissue positive for apoptosis.
Other types of anticancer agents can also be loaded onto carbon nanotubes. In another study, we first functionalized multi-walled nanotube surfaces with a substance that enhances their water dispersibility, then we attached a widely used anticancer agent called doxorubicin, which works by blocking DNA replication in cancer cells. We showed the increased therapeutic efficiency of the complex versus doxorubicin alone in breast cancer cells (called MCF-7) after 24 hours of incubation, concluding that nanotube delivery helped the drug to enter the cell and reach its target, the nucleus. Thus nanotubes have proven to be efficient carriers for small anticancer drugs and larger molecules such as siRNA for the treatment of cancer.
Follow Those Nanotubes
To use carbon nanotubes in drug delivery, ones injected intravenously must circulate in the patient’s bloodstream for enough time to be able to reach the tumor. However, when injected intravenously, most nanoparticles larger than 100 nanometers have a tendency to be captured by phagocytic cells constituting the reticuloendothelial system (RES). This system is composed of cells mostly found in the liver and spleen, and thus nanotubes tend to accumulate in these organs, limiting their therapeutic effect. Ones that are not captured by the RES circulate in the blood and are progressively excreted in the urine via the kidneys.
My colleagues and I wanted to better determine why nanotubes were being captured and how the process might be mitigated. We needed to image where the nanotubes went after injection. We used three types of functionalized multi-walled nanotubes, two that we gave a low density of functional groups, and one a higher degree of functionalization. To follow the organ distribution of the nanotubes, we also attached the radionuclide indium-111 (niIn) to the surface of carbon nanotubes using a caging molecule, called DTPA. We were then able to follow their localization in organs with single-photon emission computed tomography (SPECT), a nuclear medicine imaging technique using gamma rays that provides three-dimensional views. The SPECT imaging is coupled to an x-ray computed tomography (CT) scanner, which creates images of dense tissues as white (mostly bones) and soft tissue as gray, therefore giving additional anatomical information to localize radiolabeled nanotubes.
After intravenous injection in mice, both the low-functionalized types showed higher accumulation into the RES compared to the higher one. Results suggested that a low density of functionalization could be responsible for the bundling and aggregation of nanotubes in the blood and their accumulation into the RES system. In contrast, higher density of functionalization led to more individualized nanotubes and reduced the RES accumulation. Such studies can guide the future preparation of nanotubes to make them more effective delivery vectors.
But an additional outcome of this work is to show that functionalized nanotubes can be used both for imaging, and also potentially to deliver therapeutic radioactive material. The latter work only used a small amount of radionuclide at the surface, but to create a dose high enough for a treatment effect, we encapsulated them inside the inner cavity of nanotubes.
The radio-emitter we chose in this experiment was iodine-125, commonly used in nuclear medicine and imaging. We had to heat single-walled nanotubes to 900 degrees Celsius to get iodine-125 salts to fill the inner cavity by a process based on the capillary effect. As the nanotubes returned to room temperature, the cooling also closed their two ends, forming a sealed capsule containing a high dose of radionuclide. We used a solvent to dissolve away any excess material external to the nanotube.
Because the radionuclide was inside, the surface of the nanotube was still available to be externally decorated by chemical functionalization to make them biologically compatible and dispersible in water. We attached a carbohydrate molecule that is widely seen in nature and plays essential roles in many biological processes. This carbohydrate can act as a messenger inside the cell and can terminate detrimental reactions induced by microbial and immune attack. Interestingly, they also appear to behave as specific agents in case of infection or immune response. We used this last property to create targeted nanotubes that could accumulate at a desired site after injection in the bloodstream, and avoid healthy organs. In this case, the carbohydrate targeted lung tissue.
To demonstrate the integrity of the tilled nanotubes, we compared the biodistribution of free and encapsulated iodine-125. When injected intravenously, nonencapsulated iodine-125 accumulated into the thyroid, as this organ is responsible for capturing circulating iodine. Once encapsulated into the singlewalled nanotubes, we observed a relocalization of the signal from the thyroid to the lung. This study established that radionuclides can be encapsulated within the nanotubes for radiotherapy with controlled localization. Cancer treatment could benefit from such nanocapsules by targeting radio-sensitive tumors.
Putting all the pieces together, carbon nanotubes’ ability to carry therapeutic agents can be used in conjunction with modem medical scanning techniques to create nanoparticles displaying both therapeutic and imaging properties. Such entities are called theranostic agents, as they combine therapeutic and diagnostic properties, constituting an ideal tool for image-guided delivery.
By the use of these agents in cancer therapy, it became possible to treat and monitor the disease simultaneously, and gain valuable information about the localization of the nanoparticles after injection into the patient so an accurate trigger for drug release can be achieved. In a recent study, we developed nanotube hybrids by conjugating two different types of imaging agents to their surface. This approach let us see nanotubes using conventional imaging techniques and offered superior performance by combining the two imaging methods.
Magnetic resonance imaging (MRI) and SPECT are currently mainstream clinical diagnostic approaches used in cancer diagnosis. MRI is a technique based on the application of a strong magnetic field to form images of the body, whereas SPECT is a nuclear medicine imaging technique. The combination of MRI and SPECT is particularly interesting as each of these imaging techniques has major advantages that compensate their respective drawbacks.
A good imaging technique should present two main properties: sensitivity and resolution. A sensitive imaging technique will be able to detect an imaging agent, even in small quantity. The higher the sensitivity of the technique, the less dose of imaging agent is required to be injected. But using a sensitive technique is not sufficient. The imaging technique must display excellent spatial resolution that is characterized by the ability to distinctively differentiate two particles close to each other. A technique displaying a high resolution will be able to differentiate two particles with less than 1 millimeter between them.
SPECT displays high sensitivity compared to other imaging techniques. However, the spatial resolution of SPECT is low (on the order of several millimeters). On the contrary, MRI offers high spatial resolution (below 1 millimeter) but offers a low sensitivity. Therefore, each technique compensates the limitation of the other and, by combining both techniques in one machine, imaging performances can be greatly enhanced. Multimodal imaging techniques combining SPECT and MRI can be found in clinics, but one remaining challenge is to develop imaging probes or contrast agents that can be detected by both techniques.
In our study, we developed dual imaging multi-walled nanotube contrast agents, achieved by decorating them with superparamagnetic iron oxide nanoparticles (abbreviated SPION) and also conjugating them with radiolabeled agents. SPION can be detected using MRI, whereas radio-labeled agents can be detected by SPECT.
To prepare the nanotubes, we oxidized them in the presence of nitric acid (HNO3), leading to the formation of carboxylic groups onto the tip and sidewall of the nanotubes. Iron acetate (C4H6Fe04), a precursor for the formation of the SPION, was decomposed by thermal annealing. The metal ions interacted strongly with the surface groups present on the nanotubes, leading to the formation of the SPION nanoparticles. Then, to link radionuclides to the surface of the nanotubes, we used a chemical compound called biphosphonate, which displays a strong binding affinity to the surface of SPION. The biphosphonate-SPION hybrid was also functionalized with a linker that binds the radioisotope technetium-99m. Thus, the first carbon nanotubes for dual SPECT and MRI imaging were prepared.
To establish the stability of the hybrid after intravenous injection, we compared the biodistribution of radiolabeled functionalized bisphosphonate alone and the radio-labeled nanotubes. Whereas the former accumulated quickly in bones, as bisphosphonate molecules have high affinity for bone tissues, radio-labeled nanotubes showed an accumulation in the lungs and some in the liver and spleen, but not in the bones. Therefore these results demonstrated the stable binding of the radionuclide to the nanotubes.
Our team also measured the MRI contrast properties of the complex in the liver. By increasing the concentration of the hybrid, we observed a higher MRI effect, which supported dose-dependent imaging properties of the hybrids. In consequence, we established the dual imaging properties of the nanotube complex in vivo, offering promising applications for the development of carbon nanotubes as theranostic agents.
This work shows that the attractive features of carbon nanotubes allow various therapy and imaging applications. However, before bringing them to patients, their efficacy and safety will need to be further investigated. Modifying nanotubes to make them biocompatible is also a research focus in our lab. We hope that patients will one day take profit from this tremendous therapeutic and imaging platform.